Abstract:Objective：To investigate the expression of phosphatidylinotidol 3-kinase/protein kinase B（PI3k/Akt） signal pathway，matrix metalloproteinase-2（MMP-2） and matrix metalloproteinase-9（MMP-9） in villi tissues of normal pregnancy and spontaneous abortion and their relationship. Methods：Real-time PCR，immunohistochemistry and Western blot were used to detect the expression and location of PI3k，Akt，p-Akt，PTEN，MMP-2，MMP-9 between normal artificial abortion（20 cases） and spontaneous abortion（20 cases） in villi tissues. Results：The mRNA expression of PI3k（P=0.000），Akt（P=0.005） and MMP-9（P=0.000） in the villi tissues was higher in normal artificial abortion（NOR group） than in spontaneous abortion（SPO group）. The mRNA expression of PTEN in the villi tissues was higher in SPO group than in NOR group（P=0.000）. The protein expression of PI3k（P=0.006），Akt（P=0.000），p-Akt（P=0.000） and MMP-9（P=0.000） in the villi tissues was higher in NOP group than in SPO group. The protein expression of PTEN was higher in SPO group than in NOR group（P=0.000）. MMP-2 had no significant change between two groups（P=0.092）. Western blot results were consistent with the immunohistochemical results. PI3k（P=0.047），Akt（P=0.011），p-Akt（P=0.001），MMP-2（P=0.893） and MMP-9（P=0.005）. Conclusion：PI3k/Akt signal pathway is involved in the spontaneous abortion during the first trimester，which may affect the expression of MMP-9 and reduce the invasion of tropholast. This study provides new evidence for clinical preven－tion and treatment of early abortion.

Abstract:Objective：To investigate the effects of growth arrest and DNA damage 45 alpha（Gadd45α） gene on pro1iferation，apopto－sis，migration and invasion of HTR8/SVneo cells and the role of Gadd45α in the pathogenesis of preeclampsia. Methods：The role of Gadd45α in HTR8/SVneo cells was examined by RNA interference technology using Gadd45α specific short hairpin RNA（siRNA）. The experiment was composed of two groups：si-Gadd45α group and si-negative control group（si-NC group）. mRNA and protein ex－pression of Gadd45α was identified by qRT-PCR and Western blot respectively. Changes of cell proliferation，apoptosis，migration and invasion were respectively detected by methyl thiazolyl tetrazolium（MTT），flow cytometry，transwell migration and Matrigel invasion assay. Placental villious explants culture model was employed to further verify the effect of Gadd45α on outgrowth and migration of extravillous trophoblast cells. Gelatin zymography was used to detect the expression of matrix metalloproteinase（MMP）-2/9 in culture medium. Western blot was used to detect the expressions of tissue inhibitors of MMPs（TIMP）-1 and TIMP-2. Results：（1）The trans－fection efficiency was about 90%. The mRNA and protein levels of Gadd45α in si-Gadd45α group were reduced by 80% and 70% respectively，with significant differences（P<0.01）. （2）The results of MTT and flow cytometry indicated that cell prolifer－ation and apoptosis were not affected by siRNA of Gadd45α（P＞0.05）. （3）Knocking-down Gadd45α expression by siRNA significantly promoted invasion（1.65 folds） and migration（2 folds） potentials of HTR8/SVneo cells（P<0.01）. （4）Gadd45α silencing significantly promoted outgrowth of villous explants in vitro（P=0.005）. （5）Gadd45α regulated migration and invasion through control－ling gelatinolytic activities of MMP-9，MMP-2（P<0.05） and the expressions of TIMP-1 and TIMP-2（P<0.01）. Conclusion：Gadd45α is probably involved in the onset of preeclampsia by regulating invasion and migration of trophoblast through controlling the activities of MMPs and the expressions of TIMPs.

Abstract:Objective：To investigate the effects of checkpoint kinase 1 siRNA transfection on the proliferation and cell cycle of human ovarian carcinoma HO8910 cells and its mechanisms. Methods：Three Chk1 siRNA targeting fragments were designed and synthe－sized. After the transfection，Western blot was conducted to determine the expression of Chk1 at protein level. The effective Chk1 siR－NA fragment was transfected into HO8910 cells，and MTT method and flow cytometry analysis were used for determining the influence of Chk1 siRNA on the proliferation and cell cycle，respectively. Then，Western blot was used to detect cell cycle of related genes Cy－clinA，CDK4 and Cdc25A at protein level. Results：One fragment among 3 Chk1 siRNAs was valid. In HO8910 cells，after transfec－tion with the effective Chk1 siRNA，the protein level of Chk1 was decreased about 75.3%，significantly lower than that in the non-transfected group and liposome transfection group（P=0.000）. At 48 h after transfection，the activity of cell proliferation was inhibited with an inhibition rate of （52.1±5.1）%，significantly higher than that in the liposome transfection group（7.6±3.8）%（P=0.000）. Flow cytometry showed that the proportion of HO8910 cells in G2/M phase was （5.03±2.62）%，significantly lower than that in the non-transfected group（19.35±2.19）% and liposome transfection group（19.76±2.67）%（P=0.000）. Western blot results showed that in the HO8910 cells transfected with Chk1 siRNA，the protein levels of CyclinA，CDK4 and Cdc25A were enhanced，which were statis－tically different compared with those of non-transfection group and liposome group（P=0.000）. Conclusion：After Chk1 gene silencing by siRNA，the proliferation of the human ovarian carcinoma HO8910 cells was inhibited，which was related with the block－ade of G2/M phase arrest.

Abstract:Objective：To compare the impact of different dosage regimens of cisplatin（CDDP） or/and paclitaxel（PTX） on the repopu－lation of ovarian cancer cells. Methods：（1）SKOV3 cells were treated with CDDP，PTX or CDDP combined with PTX respective－ly，each arm treated with fractional dose regimen（the total dosage was dividedly administration on d1 and d8） or single dose regimen（the total dosage was administration on d1）；（2）The apoptosis and repopulation of SKOV3 cells were observed under microscope；（3）The cell survival on d7 and d21 in each group was detected by CCK-8 method. Results：（1）In CDDP groups，no significant dif－ference was found between the two regimens（P=0.970）；（2）In PTX groups，the repopulation of SKOV3 cells in fractional dose regi－men was significantly inhibited compared with that of single dose regimen（P=0.000）；（3）Fractional schedule significantly inhibited the repopulation of SKOV3 cells in CDDP+PTX groups（P=0.000），and such suppressive efficiency was even more evident in CDDP+PTX group than in PTX monotherapy group（P=0.000）. Conclusion：CDDP+PTX with fractional dose regimen might prevent resistance or recurrence of ovarian cancer cells through significantly inhibiting the repopulation of ovarian cancer cells.

Abstract:Objective：To establish hypoxia drug resistance model（HDRM） of human ovarian cancer cell lines A2780 and SKOV3 by chemical induction method of Cobalt Chloride（CoCl2） and to compare the drug resistant characteristics of those two cell lines. Methods：CoCl2 with different concentrations（0，50，100，150，200，300 μmol/L） was added to A2780 and SKOV3 cells for 12，24，48，72 h. Cell proliferation activity（CPA） was detected by methyl thiazolyl tetrazolium（MTT）. MTT was used to detect the drug resistance multiple（DRM） of paclitaxel under the different concentrations of CoCl2. The same cell lines without CoCl2 were taken as normoxic control. CoCl2 with concentration of 150 μmol/L was added to A2780 and SKOV3 cells medium for 24 h respectively and the expression of HIF-1α mRNA was detected by the real time polymerase chain reaction. The same cell lines without CoCl2 were taken as normoxic control. Results：The proliferation activity of A2780 and SKOV3 cells was correlated with CoCl2 in a dose and time dependent manner（A2780 group：F=1 165.416，P＝0.000；SKOV3 group：F=2 802.394，P＝0.000）. Paclitaxel resistance fold of those two kinds of cells was increased with the increase of CoCl2 concentration（F=7 842.711，P＝0.000）. With the same concentration of CoCl2，DRM of SKOV3 cells was significantly higher than that of A2780 cells（50 μmol/L group：t=-48.287，P＝0.000；100 μmol/L group：t=-263.205，P＝0.000；150 μmol/L group：t=-143.305，P＝0.000）. With 150 μmol/L CoCl2 culturing for 24 h，DRM of A2780 and SKOV3 cells reached to 9.84±0.11 and 21.08±0.08，respectively. The increase of HIF-1α mRNA expression in SKOV3 cells was significantly higher than that of A2780 cells（t=-5.573，P＝0.000）. Conclusion：Ovarian cancer HDRM can be successfully established by CoCl2 induction method and DRM is related with cell characteristics. Under the same condition，SKOV3 cell line is easier to establish HDRM than A2780 cell line，and SKOV3 is an ideal cell line for hypoxia resistance reversal study.

Abstract:Objective：To investigate the expression of Rho-associated coiled-coil forming protein kinase（ROCK） in epithelial ovarian tumors and its effects on proliferation and apoptosis in ovarian carcinoma cells. Methods：Expression of ROCK in 80 patients with ep－ithelial ovarian tumors was assessed by immunohistochemical staining. After being treated with Fasudil，the inhibitor of ROCK，the proliferation and apoptosis of SKOV3 cells were tested by cell counting kit-8（CCK-8） and flow cytometry（FCM） respectively，the activity of cysteinyl aspartate specific proteinase－３（caspase-3），caspase－3 was detected by spectrophotometry，and the expression of p53 was detected by Western blot. Results：The expression of ROCK was higher in epithelial ovarian carcinoma tissues than in benign tumors（ ?字2=20.961，P=0.000），and higher in advanced carcinoma tissues than in early carcinoma tissues（ ?字2=16.675，P=0.000）. Com－pared with the untreated SKOV3 cells，the inhibition ratios and apoptosis rates in Fasudil treated cells were increased in an dose de－pendent relationship（F=402.537，P=0.000；F=124.251，P=0.000）. In addition，Fasudil significantly enhanced caspase-3 activity（F=32.423，P=0.000） and increased the expression of p53（F=31.599，P=0.000） in concentration-dependent manner. Conclusion：The expression of ROCK is higher in ovarian carcinoma than in benign tumor. ROCK may play a certain role in the proliferation and apoptosis of ovarian carcinoma cells.

Abstract:Objective：To discuss the strategies for treating refractory plycystic ovary syndrome（PCOS） by risk factors logistic regression analysis in clomiphene citrate（CC） resistant PCOS patients. Methods：A total of 92 PCOS patients were divided into CC resistant group and CC effective group. Patients' weight，height，age，glucose tolerance test（OGTT）（0’，120’），insulin（0’，120’），follicle-stimulating hormone（FSH），luteinizing hormone（LH），testosterone（T），estradiol（E2） and homocysteine（Hcy） were measured；the body mass in－dex（BMI） and homeostasis model assessment for insulin resistance（HOMA-IR） were calculated. The statistically significant differ－ence was analyzed by logistic regression analysis and ROC curve analysis. Results：There were statistically significant differences in age（P=0.000），BMI（P=0.005），Hcy（P=0.000），OGTT（0’）（P=0.000），INS（0’）（P=0.014），T（P=0.042），HOMA-index（P=0.000） be－tween CC resistant group and CC effective group. Hcy，OGTT（0’），INS（0’），T，HOMA-index，BMI，and age were negatively correlat－ed with CC resistance.The area under ROC curve was 0.981 Hcy，95%CI=0.972-0.990. Conclusion：Hcy is the best risk predictors in predicting CC resistant of refractory PCOS.

Abstract:Objective：To observe the effects of different moxibustion therapies on contents of superoxide dismutase（SOD），malonic dialdehyde（MDA） and reproductive endocrine in male rats with partial androgen deficiency of aging male（PADAM）. Methods：Totally 50 male rats were randomly divided into normal control group（n=10） and model group（n=40）. The rats in model group were injected with cyclophosphamide in abdominal cavities to make PADAM model. Thirty-two rats were randomly selected from model group and were randomized into mild-moxibustion group，scarring-moxibustion group，rolin group and control group. Before modeling，after modeling and at the end of experiments，all rats' serum total testosterone（TT） and free testosterone（FT） levels were detected and exhaustion swimming tests and tail suspension experiments were observed. The SOD and MDA levels in kidney and testis tissues as well as the serum luteinizing hormone（LH） and follicle-stimulating hormone（FSH） levels were determined at the end of the experiments. Results：（1）A successful modeling of PADAM was made by using cyclophosphamide. （2）After the treatments，the serum TT and FT levels in three treatment groups increased signifi－cantly compared with those pre-therapy and control group（P=0.000）. The serum LH and FSH levels decreased significantly（P=0.000）. The duration of immobility and the duration of ex－haustion swimming also improved significantly（P=0.000）. SOD activities in the kidney and testis increased obviously（P=0.000） and MDA contents decreased significantly（P=0.000）. The curative effect of mild-moxibustion group was better compared with that of the other two groups（P=0.004，P=0.009；P=0.000，P=0.000；P=0.000，P=0.003） and no significant difference was found between scarring-moxibustion group and androlin group（P=0.762，P=0.282，P=0.247）. Conclusion：（1）Two kinds of moxibustion therapies could improve the depression state，myodynamia and energy in the aging rats as well as increase TT and FT levels，with the similar effects of testosterone propionate. （2）Two kinds of moxibustion therapies could increase SOD activities and decrease MDA contents in the kidney，indicating a correlation between electro-acupuncture cured PADAM and improved free radical related systems in the body. （3）Mild-moxibustion had a better curative effect，providing experi－mental data and proof for clinical treatment.

Abstract:Objective：To detect the expression and location of Znf230 in mouse testis and spermatozoa by Znf230 polyclonal anti－serum. Methods：Expression and localization of Znf230 in mouse testis at different developmental stages and epididymal spermato－zoa were determined by RT-PCR，immunoblotting，immunohistochemistry and immunoluorescence. Results：Znf230 remained relatively stable and was expressed throughout postnatal development of the mouse testis. It was primarily expressed in the nuclei of spermato－gonia（from postnatal d 6 to postnatal d 12） and subsequently in the acrosome system and the entire tail of round spermatids，elongated spermatids and spermatozoa（from postnatal d 18 to mature sperm）. Conclusion：Znf230 may play an important role in mouse spermatogenesis.

Abstract:Objective：To investigate the effect of big conductance Ca2+-activated K+ channel（BKca）on RhoA/ROCK signaling pathway of diabetes mellitus-induced erectile dysfunction（DMED） rats. Methods：Rats were divided into control group（n=8），DMED group（n=8） and NS1619 group（treatment group，n=7）. The NS1619 group was treated with BKca specific opener（NS1619） for two weeks. Rats underwent cavernous nerve stimulation to determine the value of intracavernous pressure（ICP）/mean arterial pressure（MAP） and the number of erection was recorded. Corpus cavernosum smooth muscle was isolated for detection of contractile force. Western blot and real-time PCR were used to detect the expression of RhoA，ROCK1，ROCK2，which determined the differences of RhoA/ROCK signaling pathway and phosphorylation of myosin light chain phosphatase（MLCP）. Results：DMED rat models were successfully estab－lished. While erectile function and compliance of smooth muscle was decreased in rats with DMED，administration of NS1619 im－proved erectile responses（P=0.025，0.024） and compliance（P=0.031，0.024）. Treatment significantly decreased the expression of RhoA，ROCK2 and MYPT-1（P=0.029，0.003，0.002），but not ROCK1（P=0.533）. Compared with those in control group the NS1619 group have a higher expression levels of those protein（P=0.018，0.040，0.003）. Conclusion：Up-regulation of the RhoA/ROCK sig－naling pathway is harmful to erectile function. Activation of BKca can improve erectile function by down-regulation the level of RhoA/ROCK and phosphorylation of MLCP.

Abstract:Objective：To preliminarily assess the expression and significance of transforming growth factor-β1（TGF-β1） in the apop－tosis of rat spermatogenic cells induced by cytoxan. Methods：Cytoxan was applied for male Wistar rats aged 9 d. After 24 h，4 weeks and 8 weeks，apoptosis of spermatogonia was counted severally by TUNEL and TGF-β1 was detected severally by RT-PCR in testes of 10 rats in experimental group and control group. Results：（1）There was no difference in the apoptosis of spermatogenic cell and TGF-β1 expression between control group and experimental group at 24 h after the treatment at 24 h after the treatment. （Fbetween groups=1.25，P=0.924）（Fbetween groups=1.59，P=0.339）（2）Apoptosis of spermatogenic cell and TGF-β1 expression were significantly increased in experimental groups than in control group（Fbetween groups=126.73，P=0.005）（Fbetween groups=32.86，P=0.007）. Conclusion：Cytoxan not only increases apoptosis of spermatogenic cell in testes，but also results in elevated expression of TGF-β1 of testes in rat，indicating that increased expression of TGF-β1 in testes will lead to increased apoptosis of spermatogenic cell in testes.

Abstract:Objective：To analyze the features of chromosomal abnormalities and azoospermia factor（AZF） of males with severely im－paired spermatogenesis. Methods：G-banding and multi-PCR method were used to analyze the karyotypes of chromosome and AZF microdeletions respectively. Results：In 443 cases of azoospermia and 183 cases of severe oligozoospermia，79 cases（12.62%） of chro－mosomal abnormalities were found；68 cases（15.35%） in azoospermia group and 11 cases（6.01%） in severe oligozoospermia group. Meanwhile，the translocation karyotype 46，XY，t（4；8）（q35；q22） in severe oligozoospermia group was firstly reported. There were 68 cases（10.86%） of Y chromosome AZF microdeletions，accounting for 11.51% and 9.29% in azoospermia group and severe oligo－zoospermia group respectively. The deletion type AZFc was the most common，followed by AZFb and AZFd；the constituent ratio of the three types were 41.18%，20.59%，10.29%，respectively. In 68 cases of Y chromosome microdeletions，14 cases complicated with chro－mosomal abnormalities（10 cased in azoospermia group and 4 cases in severe oligozoospermia group）. Conclusion： Genetic abnormality is important for patients with severely impaired spermatogenesis. Detection chromosome abnormality and Y chromosome microdeletions is necessary before applying assisted reproductive technology in order to decrease the risk of abnormal gene inherited from the father.

Abstract:Objective：To explore the different clinicopathological characteristics and prognostic factors for patients with prostate cancer （PCa） in different age groups. Methods：All 105 patients diagnosed with PCa from January 2006 to May 2013 were divided into young group（＜60 years，45 patients） and old group（≥60 years，60 patients）. Clinical symptoms，international prostate symptom rating scale （IPSS），total prostate specific antigen（TPSA），clinical staging，Gleason score，incidence of bone metastasis，and treatment in both groups were explored. Results：Frequent micturition and dysuria were the primary symptoms in both groups; there were significant differences in the symptom of ostalgia between the two groups（Z=-2.436，P=0.015）. There were significant differences in the IPSS score，TPSA value，and Gleason score between the two groups（Z=-3.460，P=0.001）（Z=-2.830，P=0.005）（Z=-2.580，P=0.01）. There was no sig－nificant difference in clinical staging（Z=2.500，P=0.114） but there were statistical differences in bone metastasis incidence between the two groups（?字2=4.618，P=0.032）. Fifteen and 26 patients respective in young group and old group received radical prostatectomy；the others underwent endocrine therapy. Conclusion：Frequent micturition and dysuria are the primary symptoms in both groups；but more patients in young group suffer from ostalgia symptom. The symptoms are lighter in young group；but TPSA value，pathological malignant degree and bone metastasis are more severe in young group. There is no difference in clinical staging between the two groups.

Abstract:Objective：To explore cohort retention and associated factors in HIV pre-exposure prophylaxis（PrEP） clinical trial among men who have sex with men（MSM）. Methods：Totally 297 MSM were enrolled by non-probability sampling methods and then as－signed to daily dose，intermittent dose and blank control arms randomly. Clinical follow-up and questionnaire were carried out every 3 months. Cumulative cohort retention rates of different arms were described by Kaplan-Meier estimation. Univariate and multivariate Cox regression were used to analyze associated factors of cohort retention. Results：43.1%（128/297） of participants completed the fol－low-up in this study and the mean retention time was （5.85±3.99） months. Those who were in intermittent dose arm（AHR=0.62，95%CI＝0.42 to 0.91） and older than 30 years（AHR=0.65，95%CI＝0.47 to 0.90） showed higher cohort retention. Conclusion：The co－hort retention of PrEP clinical trial among MSM in this study is not high. Effective measures should be taken to improve the cohort re－tention of MSM in blank control arm and younger age.

Abstract:Objective：To investigate the effect of nucleophosmin 1（NPM1） on the apoptosis in leukemic cells and the relevant molec－ular mechanisms. Methods：OCI/AML3 cell lines（NPM1 mutant type） and HL60 cell lines（NPM1 wild type） were served as experi－mental group and negative control group. Mock group，pGIPZ group，shNPM group were established in the meantime. Expressions of the NPM1 mutant A（NPM1-mA） gene and protein were detected by qRT-PCR，Western blot and immune cytochemistry. Apoptosis analy－sis was determined by flow cytometry and Wright-Giemsa staining. The gene and protein expression of apoptosis related molecular（Bax/Bcl-2） was detected by qRT-PCR and Western blot and the activity of p-ERK was also assessed by Western blot. The percent－age of apoptotic cells was detected after treatment with MAPK/ERK pathway inhibitor（PD98059） by flow cytometry. Results：The re－sults showed significant down-regulation of NPM1-mA mRNA levels（F=20.078，P=0.000；PMock vs. shNPM=0.001，PpGIPZ vs. shNPM=0.003，PMock vs. pGIPZ=0.333） and protein levels after NPM1 silencing，accompanying by reduce of cytoplasm NPM mutant protein of OCI/AML3 cells. In OCI/AML3 cells，down-regulation of NPM1-mA resulted in increase of cell apoptotic rate（F=25.236，P=0.000；PMock vs. shNPM=0.001，PpGIPZ vs. shNPM=0.001，PMock vs. pGIPZ=0.729） and morphological changes including apoptosis bodies under light microscopy. NPM1 gene si－lencing led to increase of pro-apoptotic protein Bax expression（F=7.649，P=0.000；PMock vs. shNPM=0.015，PpGIPZ vs. shNPM=0.015，PMock vs. pGIPZ=0.984） and decrease of anti-apoptotic protein Bcl-2 expression at mRNA and protein levels（F=5.190，P=0.000，PMock vs. shNPM=0.034，PpGIPZ vs. shNPM=0.029，PMock vs. pGIPZ=0.909）. NPM1 gene silencing can also down-regulate the expression of p-ERK. Furthermore，PD98059 promoted OCI/AML3 leukemic cell apoptosis（F=25.643，P=0.007）. Conclusion：NPM1-mA plays an important role in leukemic cell anti-apoptosis，which involving MAPK signal pathway and Bax/Bcl-2 expression.

Abstract:Objective：To observe the effect of miR-451 on glomerular morphological changes in diabetic nephmpathy（DN） of type 2 diabetic db/db mice and to explore the role of miR-451 in pathogenesis of early DN. Methods：Thirty-two 7-week-old db/db and normal db/m male mice were divided into miR-451 group，empty plasmid group，untreated group and normal control group. After in－traperitoneal injection of plasmid for two weeks，the glomerular morphological changes were observed under the light microscope. Also，the expression of miR-451 was tested by real-time PCR and the tyrosine3-monooxy-genase/tryptophan 5-monooxygenase activation protein（Ywhaz），p38MAPK and MAPK kinase 3（MKK3） protein levels were examined by Western blot and immunofluorescence. Results：Elevation of urinary albumin excretion rate（UAE） was shown in the untreated group of 7-week-old db/db mice，suggesting that the 7-week-old db/db mice had the features similar to the diabetic nephropathy at early stage. Then plasmid injection ex－periment was executed on mice for two weeks. Real-time RT-PCR results showed that miR-451 was 1.79 folds high in miR-451 group than in empty plasmid group（P=0.002）. Also，glo-meruli acreage was smaller in miR-451-treated group than in the empty plasmid and untreated groups（P=0.000）. Moreover，Western blot and immunofluorescence results demonstrated that Ywhaz，p38MAPK and MKK3 proteins were reduced in miR-451 group（P=0.000）. Conclusion：miR-451 can repress glomerular hypertrophy by targeting Ywhaz gene and affecting MAPK signal pathway. Therefore，miR-451 might play an important role in the pathogenesis of DN.

Abstract:Objective：To explore the effect of the hypoxia-induced human retinal pigment epithelial cells（RPE） which transfected by adenovirus-mediated Slit2 and adenovirus-mediated Slit2 ShRNA on the proliferation of human choroidal microvascular endothelial cells（HCMEC），to explore the possible role of Slit2 in choroidal neovascularization（CNV） and to provide new ideas for the treat－ment of choroidal neovascularization. Methods：RPE cells and HCMEC cells were identified and cultured in vitro. Hypoxic conditions were achieved by adding 200 ?滋mol/l cobalt chloride to medium to mimic hypoxia. RPE cells and HCMEC cells were cultured in a contact co-culture system by Transwell chamber. The hypoxia RPE cells were randomly divided into Slit2 treated group（adding Slit2RNA），Slit2 shRNA treated group（adding Slit2 shRNA），empty adenovirus group（adding empty adenovirus），hypoxia group. CCK8 assay was carried out to determine the proliferation of HCMEC cells after 12，24，48 h. Results：There were statistically signifi－cant differences among different groups（F=98.122，P=0.000） and different time points（F=3388.913，P=0.000）. The interaction be－tween different groups and time points were also different（F=82.863，P=0.000）. The absorbance（A） value was higher in Slit2 treated group than in other groups（hypoxia group：P=0.001；other groups：P=0.000）. The A value was lower in Slit2 shRNA treated group than in other groups at 24 h and 48 h（at 48 h：hypoxia group，P=0.003；empty adenovirus group，P=0.008；other groups，P=0.000）. Conclusion：High-expression of Slit2 can significantly promote the proliferation of HCMEC cells. Silencing Slit2 shRNA expression in RPE cell can significantly inhibit the proliferation of HCMEC cells.

Abstract:Objective：To investigate the effect of Slit2 gene on vascular endothelial growth factor（VEGF）in retinal pigment epithelium（RPE）cells and the function of PI3K/AKT and MEK/ERK signaling pathways during the course. Methods：（1）human APRE-19 cells were cultured in vitro. （2）RPE cells were transfected by adenovirus-mediated Slit2（Ad-Slit2），then cultured for 12，24，48 h. （3）Before being transfected by Ad-Slit2，RPE cells were pretreated with MEK inhibitor PD98059 and PI3K inhibitor LY294002 for 1 h. （4）VEGF mRNA expression levels were detected by real-time PCR and the protein expression levels were detected by ELISA. The protein expression levels of p-ERK，p-AKT were detected by Western blot. Results：After RPE cells being transfected with Ad-Slit2，real-time PCR result showed that there were significant statistically differences in VEGF mRNA expression among different time points（F=12.789，P=0.018） and among different groups（F=16.855，P=0.015）；time points and groups were interrelated；at 24 h and 48 h，the VEGF mRNA expression was higher in Ad-slit2 group than in adenovirus group（P24 h=0.002 ，P48 h=0.018）. ELISA results showed that there were significant statistically differences in VEGF protein expression among different time points（F=24.886，P=0.000） and among different groups（F=28.250，P=0.006）；time points and groups were interrelated；at 48 h，the VEGF protein expression was higher in Ad-Slit2 group than in adenovirus group（P=0.003）. Western blot showed that p-ERK and p-AKT protein expressions increased in RPE cells transfected with Ad-Slit2；PD98059 decreased the activation of p-ERK and LY294002 decreased the activation of p-AKT. real-time PCR results showed that there were statistically significant differences in VEGF mRNA expression among different time points（F=32.202，P=0.000） and among different groups（F=15.062，P=0.001）；time points and groups were interrelated（F=8.372，P=0.006）. ELISA results showed there were statistically significant differences in VEGF protein expression among different time points（F=90.703，P=0.000） and among dif－ferent groups（F=69.636，P=0.000）；time points and groups were interrelated（F=4.968，P=0.005）. Conclusion：The expression of the VEGF in RPE cells transfected with Ad-Slit2 can be increased by MEK/ERK and P13K/Akt signaling pathways.

Abstract:Objective：To observe the protective effect of nerve growth factor（NGF） loaded ploy lactic-co-glycolic acid（PLGA） micro－spheres on retinal ganglion cells（RGCs） following the optic nerve injury. Methods：The NGF-PLGA microspheres were made using PLGA and the properties of microspheres were tested. Totally 69 SD adult male rats were randomly divided into 5 groups：normal control group（group A），sham operated group（group B），physiological saline injection group（group C），NGF injection group（group D） and NGF-PLGA injection group（group E）. Nine rats in groups A and B，17 rats in groups C，D and E. All rats in groups B-E underwent ultrasound irradiation. Samples of retinal section were collected for immunohistochemistry staining and HE staining to ob－serve the performance of glucose regulated protein 78 kD（GRP78） and the pathological morphology change of retina of each group at 12 h and on the 1st d，3rd d，7th d. Number of RGCs retrograde labeled by fluorogold（FG） before the operation was checked on the 7th d. Results：The mean size of microsheres was （3.17±0.60） μm. The encapsulation efficiency of NGF-PLGA microspheres was 68.4%，the loading rate was 0.0323% and the cumulative rate of drug-release on the 7th d was 82.6%.There were significant differ－ences among 5 groups in RGCs count（F=65.858，P=0.000）.There was no difference in RGCs count between group A and B（P=0.862>0.05），but apparent differences among other groups were observed（P<0.05）. Imunohistochemistry staining showed no positive cell in groups A or B. There were significant differences in GRP78 positive rate of RGCs among groups C-E at different time points（Fgroup=184.330，Pgroup=0.000）　and among each group at different time points（Ftime=21.472，Ptime=0.000）. HE staining showed the retinal ede－ma of group E was reduced on the 7th d compared with that of group C and D. Conclusion：NGF loaded PLGA microspheres com－bined with ultrasonic irradiation can improve the protective ef－fect on RGCs after the optic nerve injury.

Abstract:Objective：To research the effects of silencing tripartite motif containing 25（TRIM25） gene on cisplatin resistance and apop－tosis in cisplatin resistant lung cancer cell lines A549/DDP. Methods：Four types of short hairpin RNA were designed and prepared. The recombinant plasmids（pt1，pt2，pt3，pt4） were constructed respectively，which were transfected into A549/DDP cells using lipofectamine. Real-time PCR and Western blot were used to detect the inhibitory effects. The resistance of cisplatin to A549/DDP cells was detected by MTT assay. Cell apoptosis was tested by flow eytometry（FCM）. Results：The recombinant plasmid pt1 can inhibit mRNA and protein expression of TRIM25 specifically. MTT data showed that the cisplatin IC50 was decreased significantly in pt1 group than in pNC group（P=0.000） and control group（P=0.000） after the TRIM25 gene inhibition. According to the results of FCM，apoptotic cells was significantly decreased in pt1 group than in pNC group（P=0.005） and control group（P=0.004）. Conclusion：TRIM25 gene silencing in A549/DDP cells can effectively reduce cisplatin resistance and promote cells apoptosis.

Abstract:Objective：To investigate the route of gut bacterial translocation in a rat model after spinal cord injury（SCI）. Methods：Es－cherichia coli labeled with enhance green fluorescent protein was inoculated to track bacterial translocation. ②Twenty-four Wistar rats were divided into normal group（n=8） and injury 24 h group（n=8） and injury 48 h group（n=8）. ③Liver and mesenteric lymph nodes were analyzed with bacterial cultures；the portal vein was analyzed with endotoxin assays；the small intestines were observed un－der light microscope，fluorescence microscope and electron microscope；liver and mesenteric lymph nodes were observed under light microscope. Results：①SCI caused impaired intestinal epithelial barrier function. The escherichia coli translocated through the injured epithelial cell or the leakage to the lamina propria of intestinal villi. ②The lymph nodes were injured. Positive rates：injury 24 h group（100%，8/8），injury 48 h group（100%，8/8）. ③The histologic change of liver was apparent. Positive rates：injury 24 h group （87.5%，7/8），injury 48 h group（100%，8/8）. ④Endotoxin levels in injury 24 h group and injury 48 h group were significantly differ－ent from that of normal group（F=69.774，P=0.000）. Conclusion：After SCI，the intestinal barrier function is damaged and the bacteria and endotoxin are increased. Due to the increased permeability of intestinal mucosal，the bacteria translocate from the gut to other places through lymphatic and blood routes and form the endotoxemia，which amplifies the local and general inflammation and formu－lates a vicious circle.

Abstract:Objective：To identify the maintenance of an undifferentiated state of mouse alveolar epithelial typeⅡ（ATⅡ） cells by the co-culture of feeder cells（mouse embryonic fibroblast cells，MEFs）. Methods：The mouse lung single cells were prepared by the com－bination use of dispase，collagenase and DNase I. ATⅡ cells were purified by immune adherence. The culture medium was high glu－cose dulbecco modified eagle medium（HG/DMEM） addicted with 10% fetal bovine serum（FBS）and antibiotics. ATⅡ cells were co-cultured with MEFs and were identified by immunofluorescence staining of pulmonary surfactant protein C（SPC） and Aquaporins 5（AQP5）. Results：（1.7-2.0）×107 cells per mouse lung was obtained. ATⅡ cells can maintain their undifferentiated state until the fourth generation when being cultured on feeder cells，but will differentiate into alveolar AT Ⅰ cells when being cultured alone. Conclusion：MEF cells can maintain the undifferentiated state of ATⅡ.

Abstract:Objective：To explore the method of using 5-bromo-2-deoxyuridine（BrdU） to label human umbilical cord mesenchymal stem cells（hUC-MSCs），and to explore its effect on cell proliferation and apoptosis. Methods：HUC-MSCs were isolated and cultured by direct adherent method，then the surface marker and potential ability of multi-direction differentiation were analyzed .The fifth passage of cells were labeled with BrdU at concentrations of 10，15，20，25 ?滋mol/L，excluding BrdU as control group. Cells were culti－vated respectively for 24，48，72，96 h. Trypan blue testing was used to detect cell vitality，MTT method was used to analyze cell pro－liferation and AnnexinV-FITC assay was used to detect cell survival rate and apoptosis rate. Results：Trypan blue testing showed that the living cells was more than 96% and there was no statistical significance between experimental group and control group（compar－ison among different concentration groups，P=0.89；comparison among different time points，P=0.61；interaction between time and dif－ferent concentrations，P=0.89）. MTT method detected that there was no significant difference in proliferation activity between labeled cells and unlabeled cells（P >0.05）. There were differences between cell proliferation and apoptosis rate（proliferation rate，F=12.02，P<0.001；apoptosis rate，F=6.148，P=0.009）. Conclusion：The tracing method of BrdU labeling hUC-MSCs is safe and ef－fective. The best labeling concentration and time is 20 μmol/L and 72 h.

Abstract:Objective：To study the effects of phosphatase of regenerating liver cell-1（PRL-1） small interfering RNA（siRNA）on inva－sion of tongue cancer cells（TCA8113） and the mechanism. Methods：After tongue squamous cancer cell line TCA8113 being trans－fected by PRL-1 siRNA，the mRNA and protein of PRL-1 and matrix metalloproteinase-2（MMP-2） and matrix metalloproteinase-9 （MMP-9） were determined by real-time PCR and Western blot assay，respectively. TCA8113 cell invasiveness was detected through Transwell chamber using model test. Results：After transfection by PRL-1 siRNA，the level of mRNA（0.425 0±0.010 0） and pro－tein（2.720 0±0.110 0） of PRL-1 was down-regulated（P=0.000，P=0.000）. The mRNA level of MMP-2 and MMP-9（0.562 2±0.180 0，0.617 1±0.100 0）as well as the protein level of MMP-2 and MMP-9（0.592 4±0.010 0，0.476 2±0.020 0） were decreased obviously（P=0.024，P=0.010，P=0.000，P=0.000）. The invasion ability of human tongue squamous cancer cell was reduced（64.33±4.04）（P=0.000）. Conclusion：PRL-1 gene might play an important role in development of human oral squamous cell carcinoma and down-regulation of PRL-1 could inhibit invasion of human tongue squamous cancer cell. This may be related to the lower expression of MMP-2 and MMP-9.

Abstract:Objective：To study the effect of specially silencing of phosphatase of regenerating liver cell-1（PRL-1） gene expression on migration and invasion potencies of tongue carcinoma cells. Methods：Five interfering sequences of shRNA targeting PRL-1 gene were designed，synthesized and constructed in corresponding plasmids and were confirmed by PCR electrophoresis and DNA sequencing. The most effective sequence of shRNA was screened by Western blot and the lentivirus was produced and packaged. TCA8113 cells were infected with recombinant lentivirus and the stably transfected cells were filtered and obtained. The silencing efficiency of PRL-1 gene was assessed by real-time PCR. The migration and invasion potencies of PRL-1 gene silenced cells were analyzed by wound healing and Transwell invasion assay. Results：PCR electrophoresis and DNA sequencing revealed that 5 shRNA sequences were di－rectionally constructed in plasmids. PRL-1-shRNA-1 was the most effective interference sequence selected by Western blot. The sta－bly infected TCA8113 cells were established after interfering and filtering. The expression of PRL-1 gene mRNA was decreased sig－nificantly（F=809.120，P=0.000）. After silencing the PRL-1 gene expression，the migration and invasion potencies of TCA8113 cells were significantly suppressed（F=1 092.970，P=0.000）. Conclusion：Silencing of PRL-1 gene expression can inhibit the migration and invasion potencies of TCA8113 cells significantly.

Abstract:Objective：To investigate the effect of adipose-derived stem cells（ADSCs） on young and elderly derived fibroblasts in Tran－swell system. Methods：ADSCs were isolated from human adipose tissues and were cultured. Fibroblasts derived from young people and old people were isolated from human skin tissue and were cultured. ADSCs and fibroblasts were co-cultured in Transwell chamber. Fibroblasts proliferation was evaluated by cell proliferation and cytotoxicity assay kit. Expression of fibroblasts collagen typeⅠand matrix metalloproteinase-1（MMP-1） and β-galactosidase mRNA were evaluated by RT-PCR. Results：（1）The proliferation of youth experimental group and elderly experimental group were obviously higher than that of young control group and elderly control group（P=0.000），and the proliferation of youth experimental group is higher than that of elderly experimental group（P=0.000）. （2）The ex－pression of collagen typeⅠand β-galactosidase mRNA of youth experimental group and elderly experimental group was obviously higher than that of youth control group and elderly control group（P=0.000），and the expression of collagen typeⅠand β-galactosidase mRNA of youth experimental group was higher than that of elderly experimental group（P=0.002，P=0.000）. （3）The expression of MMP-1 mRNA of youth experimental group and elderly experimental group was obviously lower than that of youth control group and elderly control group（P=0.000），but the expression of MMP-1 mRNA of youth experimental group was lower than that of elderly experimental group（P=0.055）；the results may relate with the proliferation of fibroblasts and decreased production of MMP-1 in the elderly and the young after the co-culture. Conclusion：Fibroblasts cultured with ADSCs can promote fibroblasts proliferation and collagen secretion.

Abstract:Objective：To study the expression and significance of Fcγ receptor ⅡB（FcγRⅡB） of B cells in lupus-prone MRL-lpr mice. Methods：Peripheral blood of lupus-prone MRL-lpr mice at the age of 4，8，12，20 weeks were collected and serum levels of total IgG，anti-dsDNA，anti-ribosomal P0 and anti-smRNP were detected to analyze the progression of disease. The 20 weeks old MRL-lpr mice were sacrificed and the expression of FcγRⅡB and CD69 of B cells in blood and spleen were detected. The relationship between FcγRⅡB expression and B cell activation were analyzed. Results：The serum levels of total IgG，anti-dsDNA，anti-ribosomal P0 and anti-smRNP were increased with the increase of age. Expression of FcγRⅡB of B cells in blood and spleen was decreased obviously（P＜0.05） and expression of CD69 was increased obviously（P＜0.05） in MRL-lpr mice. What’s more，the CD69 positive activated B cells up-regulated the expression of FcγRⅡB in C57BL/6 mice，and this didn’t happen in MRL-lpr mice. Conclusion：The expression of FcγRⅡB is related to the activation of B cells and altered expression of FcγRⅡB may be one of the mechanisms of B cell over-activation in lupus-prone MRL-lpr mice.

Abstract:Objective：To investigate the effects of astragalus polysaccharides on methane dicarboxylic aldehyde（MDA），caspase-3 and intercellular adhesion molecule -1（ICAM-1） in mice with sepsis-induced acute hepatic injury by cecal ligation puncture（CLP）. Methods：The mice were randomly divided into sham group（SHAM group，n=25），sepsis group（SEP group，n=25），APS treatment group（APS group，n=25） and saline -treated group（NS group，n=25）. Mouse model of acute hepatic injury was copied by CLP. The mice in APS group were injected with APS at a dosage of 50 mg/（kg?d）；no special treatment was made in SEP group and SHAM group；the mice in NS group was injected with equal volume of normal saline. Pathological changes in liver tissues were observed by staining. MDA，caspase-3 and ICAM-1 changes in plasma and liver homogenates，and the expression of caspase-3 in liver tissues were detected. Results：In SEP group，hepatocyte swelling began to show progressive increase at 12 h and peaked at 48 h. The severity of liver damage improved significantly in APS than in SEP group. The concentration of MDA（（29.69±4.62） mol/ml，P=0.041；（23.95±4.10） mol/ml，P=0.003），caspase-3（（33.12±9.01） ?滋mol/L，P=0.000；（31.25±8.68） ?滋mol/L，P=0.000） and ICAM-1（（439.49±71.66） ?滋g/L，P=0.009；（321.87±68.64） ?滋g/L，P=0.000） in plasma of APS group decreased at 24 h and 48 h，after treat－ment by APS. The concentration of MDA and caspase-3 in liver homo-genates also began to decrease in the corresponding time points. The expression of caspase-3（（19.93±4.53） ?滋mol/L，P=0.005（16.91±4.76） ?滋mol/L，P=0.000） in liver tissue of APS group was sig－nificantly lower than that in SEP group at 24 h and 48 h. Conclusion：Acute hepatic injury can be reduced by APS；its effect may be associated with the decreased expression of MDA，ICAM-1and caspase-3.

Abstract:Objective：To explore the effects of altered expression of Gpc1 expression on the proliferation of glioma cells and to address the potential role of hedgehog signaling pathway in the mediation of Gpc1 action. Methods：One cell line having relatively high activa－tion in Hedgehog signaling pathway and higher Gpc1 expression from four glioma cell lines was selected via Western blot. Gpc1-RNAi stable cell line was constructed to detect the changes of Hedgehog pathway component. The activity of Hedgehog via luciferase reporter gene after altering Gpc1 expression was detected. Gpc1-RNAi stable cell line proliferation viability was detected by MTT. Results：The Hedgehog pathway was suppressed（F=8 407.34，P=0.000） and glioma cell viability was restricted（F=27.68，P=0.000） after knock－ing down Gpc1 expeession. Conclusion：Glioma cell proliferation can be regulated by Gpc1 via Hedgehog pathway

Abstract:Objective：To research the effects of diabetes on periodontitis model in rats. Methods：The animal model of periodontitis was established by ligating the right maxillary first molar of 48 male SD rats（6 weeks，160-180 g） through stainless steel wire. All rats were divided into two groups randomly：periodontitis combined with diabetes（G1）group and periodontitis（G2） group. Diabete’s models were established by injecting streptozocin intraperitoneally. Six rats were killed in each group after 1，2，3，4 weeks. Sulcus bleeding index（SBI），probing depth（PD）and alveolar bone loss（ABL） were measured. Periodontal histology was observed by micro－scope. Interleukin-1β（IL-1β） and tumor nectosis factor-α（TNF-α） levels were analyzed by immunohistochemistry. Results：（1）At the same time，SBI，PD，ABL were higher in G1 group than in G2 group with statistical significances（P<0.05）；（2）The inflammations of periodontal histology were more serious in G1 group than in G2 group；（3）At the same time，IL-1β and TNF-α levels were higher in G1 group than in G2 group（P<0.05）. Conclusion：Under the experiment condition，diabetes can aggravate the rat periodontitis.

Abstract:Objective：To discuss whether dihydroergotamine can improve the condition of headache sufferers with stroke-related risk factors and to observe changes of health-related quality of life（HRQoL） in patients. Methods：The patients with headache were ran－domly divided into two groups. Experimental group received oral dihydroergotamine tablets while the control group received oral pla-cebo. The brief diary was used to record patient’s condition of headache（including headache frequency，headache duration and degree of pain and the use of acute pain medication）. Results：A total of 77 patients completed the study，53 patients in experimental group and 24 patients in control group. HRQoL and headache condition were improved to varying degrees among patients in both exper－imental group and control group，and the effect was much better in experiment group than in control group. The comprehensive mental score and the comprehensive emotional and physical score were quite different between two groups（effective size：0.76 vs. 0.56，0.56 vs. 0.40，0.68 vs. 0.57）. The dosage of analgesic was different between two groups（effective size：0.65 vs. 0.43）. Conclusion：Dihydroergotamine can improve the patient’s condition and HRQoL score of headache sufferers with stroke-related risk factors，using less acute analgesic.

Abstract:Objective：To observe the clinical efficacy of CO2 laser combined with pingyangmycin for laryngeal contact granuloma. Methods：Clinical data of 18 cases of laryngeal contact granuloma diagnosed from January 2011 to December 2012 in our hospital were retrospectively analyzed. All 18 patients had 22 times treatment history. Among them，9 patients were treated by CO2 laser op－eration combined with pingyangmycin（group 1） while 13 patients were treated by single CO2 laser operation（group 2）. All patients were followed up over 6 months after the operation. Results：The one-time curative rate was 100% in group 1 and was 53.8% in group 2. 7 cases were cured and 6 cases experienced recurrence. The relapse rate of CO2 laser operation combined with pingyang-mycin was lower than that of single CO2 laser operation（P<0.05）. Conclusion：Laryngeal contact granuloma has a strong tendency to recur. The reason of occurrence and recurrence may relate with a large number of fiber cells and vascular proliferation. For recurrent patients，CO2 laser combined with pingyangmycin leads to vascular occlusion and granuloma growth inhibition；therefore can signifi－cantly reduce the recurrence rate.

Abstract:Objective：To assess the changes in visual function（VF） and quality of life（QOL） among patients following Blindness Prevention Surgery Program in a rural area of Chongqing. Methods：The prospective study selected cataract patients via mobile eye screening camps. VF and QOL questionnaires were completed prior and at 3 months after the surgery. Cataract surgery with posterior chamber intraocular lens（IOL） implantation was performed on patients by a blindness prevention surgery group. Results：Three hundred and thirty four patients were enrolled with a mean age of 70.45±9.78. The mean preoperative VF score was 20.00±36.67 and the mean postoperative VF was 70.00±30.00（P<0.001）. The mean preoperative QOL score was 68.06±44.44 and the mean postoperative was 94.44±27.78（P<0.001）. The elevated VF scores in severe blindness group were higher than those in vision impairment group and unilateral blindness group（PVI=0.008，PUL=0.042）. The elevated QOL scores in severe blindness group were higher than those in vision impairment group and unilateral blindness group（P＜0.001）. No significant difference was found in the elevated VF scores between four age groups（F=0.397，P=0.755）；but the elevated QOL score were lower in 60-69 age group than in < 60 year-old group and ≥81 year-old group（P＜60=0.038，P≥81=0.009）. Conclusion：The VF and QOL score of cataract patients increased significantly following surgery，suggesting that the surgery is an effective method for improving the QOL of cataract patients in rural areas. Blindness Prevention Surgery Program should be continued in Chongqing and the postoperative follow-up should be conducted carefully.

Abstract:Objective：To analyze the complications，recurrence and vicarious therapeutic dosage of total thyroidectomy（TT） and sub-total thyroidectomy（STT），and to explore the clinical value of total thyroidectomy in the treatment for patients with benign thyroid diseases. Methods：Totally 86 patients with bilateral multiple nodular goiter in our department from May 2009 to May 2010 were ana－lyzed retrospectively. Patients were divided into 2 groups： group A（48 patients underwent bilateral STT） and group B（38 patients underwent bilateral TT）. Results：All 86 patients were diagnosed as benign thyroid diseases by pathologic examination. Three patients in group A and 2 in group B experienced temporary postoperative laryngeal nerve injury（P >0.05）. Two patients in group A and 2 patients in group B had temporary postoperative hypoparathyreosis（P >0.05）. The operation time was （96.85±22.93） min in group A and （79.05±19.34） min in group B（P<0.05）. The intraoperative blood loss was （110.33±29.98） ml in group A and （58.03±20.09） ml in group B（P<0.05）. The vicarious therapeutic dosage was （131.77±39.19） μg/d in group A and （300.66±48.45） μg/d in group B（P<0.05）. Eight patients in group A and no patient in group B suffered recurrence. No patient had permanent laryngeal nerve injury and hypoparathyreosis. Conclusion：With the advantages of less intraoperative blood loss，shorter operation time，lower postoperative recurrence rate and complication rate，TT is superior to STT in the treatment of thyroid multi-nodular diseases，although higher vicarious therapeutic dosage is required for STT after the surgery.