摘要
目的 观察解偶联蛋白-2（UCP-2）基因差异表达对人脐静脉内皮细胞（HUVEC）增生及凋亡的影响。 方法 构建UCP-2 rs660339位点碱基是胞嘧啶（C）或胸腺嘧啶（T）的慢病毒载体UCP-2C、UCP-2T。将HUVEC分为UCP-2C组、UCP-2T组、阴性对照（NC）组，分别转染UCP-2C、UCP-2T、无意义阴性病毒。荧光显微镜观察各组HUVEC病毒感染效率；实时定量聚合酶链反应检测各组HUVEC UCP-2 mRNA表达；细胞计数试剂盒、流式细胞仪检测各组HUVEC增生、凋亡情况；蛋白免疫印迹法、激光共聚焦显微镜检测各组HUVEC B细胞淋巴瘤/白血病-2（Bcl-2）蛋白的表达及荧光强度。 结果 各组HUVEC病毒感染效率均＞80%。UCP-2C组、UCP-2T组HUVEC UCP-2 mRNA表达较NC组显著增高,差异有统计学意义（F=29.183，P=0.001）。目的病毒感染后48、72、96 h，UCP-2C组、UCP-2T组HUVEC增生显著高于NC组，差异有统计学意义（F=15.970、16.738、5.414，P=0.004、0.004、0.045）。感染后72 h，与NC组比较，UCP-2T组、UCP-2C组HUVEC凋亡率均降低，差异有统计学意义（F=277.138，P=0.000）；UCP-2T组HUVEC凋亡率较UCP-2C组低（P=0.003）。UCP-2T组、UCP-2C组HUVEC Bcl-2蛋白表达显著高于NC组，差异有统计学意义（F=425.679，P=0.000）；UCP-2T组HUVEC Bcl-2蛋白表达高于UCP-2C组（P=0.002）。UCP-2T组、UCP-2C组HUVEC Bcl-2蛋白荧光强度显著高于NC组，差异有统计学意义（F=11.827，P=0.008）；UCP-2T组、UCP-2C组之间HUVEC Bcl-2蛋白荧光强度差异无统计学意义（P=0.404）。 结论 UCP-2 rs660339位点差异表达可能影响HUVEC增生及凋亡；UCP-2T较UCP-2C抑制凋亡作用更显著。
Objective To observe the influences of uncoupling protein 2 （UCP-2） rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell （HUVEC）. Methods Two UCP-2 green fluorescent protein （GFP） lentivirus constructs were created with the rs660339 locus carried C or T （UCP-2C or UCP-2T）, respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control （NC） group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. 〉80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased （F=29.183,P=0.001） in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two （F=15.970,P=0.004）, day three （F=16.738,P=0.004）, day four （F=5.414,P=0.045） post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group （F=277.138,P=0.000）, and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C （P=0.003）. The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group （F=425.679,P=0.000）, and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C （P=0.002）. The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group （F=11.827,P=0.008）, while there was no difference between UCP-2T and UCP-2C group （P=0.404）. Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2017年第1期52-56,共5页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金（81100679、81300771）
上海市科学技术委员会科研计划项目（16140901003）
关键词
内皮细胞/病理生理学
细胞增殖
细胞凋亡
基因表达
慢病毒感染
Endothelial cells/pathophysiology
Cell proliferation
Apoptosis
Gene expression
Lentivirus infections
