101例重型β-地中海贫血基因谱及其临床表现

Effects of the hypoxia-induced human retinal pigment epithelial cells which transfected by adenovirus-mediated Slit2 and adenovirus-mediated Slit2 ShRNA on the proliferation of human choroidal microvascular endothelial cells

摘要:

目的：观察腺病毒介导的Slit2及Slit2 ShRNA转染缺氧诱导的人视网膜色素上皮（retinal pigment epithelial cells，RPE）细胞对人脉络膜微血管内皮细胞（human choroidal microvascular endothelial cell，HCMEC）增殖的影响，探讨Slit2在脉络膜新生血管中的可能作用，为脉络膜新生血管（choroidal neovascularization，CNV）提供新的治疗思路。方法：体外培养并鉴定人RPE细胞、HCMEC；200 ?滋mol/L 氯化钴建立化学缺氧模型，Transwell小室建立细胞共培养模型；将缺氧的RPE细胞随机分为Slit2组（加入Slit2）、Slit2 ShRNA组（加入Slit2 ShRNA）、空腺病毒组（加入空腺病毒）、缺氧组，12、24、48 h后采用CCK 8（Cell Counting Kit-8，CCK 8）法检测HCMEC的增殖。结果：不同组别存在组间差别，差异均有统计学意义（F=98.122，P=0.000），不同时间点存在差别（F=3388.913，P=0.000），组别与时间点的交互作用（F=82.863，P=0.000）。Slit2组吸光度（absorbance，A）值在24 h、48 h均高于其他组（与缺氧组P=0.001，其余P=0.000），Slit2 ShRNA组A值在24 h、48 h均低于其他组（48 h与缺氧组P=0.003，与空腺病毒组P=0.008，其余P=0.000）。结论：Slit2的高表达可明显促进HCMEC的增殖，沉默RPE细胞中的Slit2的表达后，会明显抑制HCMEC的增殖。

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.