miR-208a在大鼠心肌缺血再灌注损伤及缺血后处理中的表达及其作用

Expression and mechanism of miR-208a in rat myocardial ischemia reperfusion injury and ischemic postconditioning

摘要:

目的：探讨miR-208a及?琢-肌球蛋白重链（?琢-myosin heavy chain，a-MHC）、β-肌球蛋白重链（β-myosin heavy chain，β-MHC）在大鼠心肌缺血再灌注损伤（ischemia reperfusion injury，IR）及缺血后处理（ischemic postconditioning，IPO）中的表达变化及其作用。方法：取SD大鼠，使用改良垫扎球囊法建立IR模型，随机分为3组（每组6只），Sham组、IR组、IPO组。检测各组血流动力学指标；real-time PCR检测miR-208a、?琢-MHC、β-MHC基因表达；Western blot检测?琢-MHC、β-MHC 蛋白含量。结果：与IR组相比，IPO能明显降低左室舒张末期容积（left ventricular end diastolic volume，LVEDV）和左室收缩末期容积（left ven－tricular end systolic volume，LVESV），增加左室射血分数（left ventricular ejection fraction，LVEF）和左室短轴缩短率（left ventricu－lar fraction shorting，LVFS），改善心功能。miR-208a表达水平在IR组明显增加，IPO组明显降低。β-MHC表达在IR组明显上调，IPO组明显下调，且基因和蛋白表达水平一致。?琢-MHC基因和蛋白表达水平在3组无明显差异。结论：miR-208a和β-MHC在缺血再灌注组明显增加，缺血后处理能使miR-208a和β-MHC表达下降，并在一定程度上改善心功能。

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.