组织蛋白酶B对人脐静脉血管内皮细胞增殖及凋亡的影响

Effect of cathepsin B on human umbilical vein endothelial cell proliferation and apoptosis

目的:探讨组织蛋白酶B(CTSB)对人脐静脉血管内皮细胞系(HUVEC)增殖及凋亡的影响。方法:慢病毒转染HUVEC,获得稳定过表达CTSB的HUVEC细胞系HU-CTSB。CCK-8法检测转染前后各组细胞的增殖情况;流式细胞法检测各组细胞的凋亡情况;实时定量PCR法检测各组细胞内凋亡基因Bax;蛋白质印迹法检测各组细胞被剪切的内caspase3表达差异。结果:CCK8法检测发现,与转染前及空病毒对照组HU-MOCK相比,HU-CTSB细胞的生存率明显下降(P<0.01);流式细胞法检测发现,HU-CTSB细胞的凋亡率明显增高(P<0.01);实时定量PCR法检测发现,HU-CTSB细胞中Bax mRNA水平增高(P<0.01);蛋白质印迹法检测发现,HU-CTSB细胞中Bcl蛋白表达下降,且被剪切的caspase3的量最多。结论:CTSB可以抑制HUVEC的生长,并通过上调Bax的表达,激活caspase3通路诱导HUVEC发生凋亡。

Objective：To study the roles of cathepsin B （CTSB） in the proliferation and apoptosis of human umbilical vein endothelial cell （HUVEC）. Method：Lentiviral CTSB vector was transfected into HUVEC cell line for the construction of stable HUVEC cell line expressing CTSB （HU-CTSB）. Proliferation of cells before and after the transfection were detected with CCK8 method; the apoptosis rates were assayed by flow cytometryl bax gene was determined with qPCR method; and the expression of cleaved caspase-3 protein of each group was detec- ted with Western Blot method. Result： Compared with those before transfection and empty virus control group HU-MOCK, the viability of HU-CTSB cells was significantly decreased （P〈0.01） and the apoptosis rate of HU- CTSB ceils was significantly increased （P〈0.01）; Results from qPCR and Western Blot showed that both the hax mRNA level and the expression of cleaved caspase-3 protein in HU-CTSB cells were obviously increased （P〈 0.01）. Conclusion：CTSB could inhibit the growth of HUVEC cells and induce the cellular apoptosis through the upregulation of bax gene and the activation of the caspase-3 pathway.