特异性小干扰RNA沉默CHOP对肾小管上皮细胞凋亡的影响

Effect of CHOP expression knock-down on apoptosis of renal tubular epithelial cells

目的:研究C/EBP同源蛋白(CHOP)对肾小管上皮HK2细胞凋亡的影响。方法:采用qPCR法检测急性肾损伤患者及健康对照者血清中CHOP的mRNA水平。体外培养肾小管上皮HK2细胞,随机分为对照组、阴性组和si-CHOP组,si-CHOP组和阴性组分别转染CHOP小干扰RNA(siRNA)和阴性对照siRNA,通过转化生长因子β1(TGF-β1)诱导细胞损伤。MTT法检测细胞的活力,流式细胞术检测细胞的凋亡率, Western blot检测细胞中细胞核抗原Ki-67、增殖细胞核抗原(PCNA)、caspase-3和cleaved caspase-3的蛋白水平。结果:与健康对照者相比,急性肾损伤患者血清中CHOP的表达量显著增加(P<0.05);转染CHOP siRNA显著降低肾小管上皮细胞HK2中CHOP的水平,与对照组相比差异具有统计学意义(P<0.05)。敲减CHOP的表达显著增加肾小管上皮HK2细胞的活力(P<0.05),降低其凋亡率(P<0.05),增加Ki-67和PCNA的表达量(P<0.05),下调cleaved caspase-3的蛋白水平(P<0.05)。结论:在急性肾损伤患者血清中CHOP的水平增加。敲减CHOP表达通过调节增殖和凋亡相关蛋白的表达抑制肾小管上皮HK2细胞的凋亡。

AIM: To study the effect of C/EBP homologous protein(CHOP) on the apoptosis of renal tubular epithelial HK2 cells. METHODS: The serum mRNA levels of CHOP in the patients with acute kidney injury and healthy controls were detected by qPCR. In vitro, renal tubular epithelial HK2 cells were divided into control group, negative group(transfected with negative control siRNA), si-CHOP group(transfected with CHOP siRNA), and induced by transforming growth factor-β1(TGF-β1). The viability of the cells was measured by MTT assay, and the apoptotic rate was analyzed by flow cytometry. The protein levels of nuclear antigen Ki-67, proliferating cell nuclear antigen(PCNA), caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS: Compared with the healthy controls, the serum mRNA levels of CHOP in the patients with acute kidney injury were increased significantly(P<0.05). Transfection with CHOP siRNA significantly decreased the expression of CHOP in the renal tubular epithelial HK2 cells(P<0.05). Knock-down of CHOP expression by siRNA significantly increased the viability of renal tubular epithelial HK2 cells(P<0.05), decreased the apoptotic rate(P<0.05), increased the expression of Ki-67 and PCNA(P<0.05), and down-regulated the protein level of cleaved caspase-3(P<0.05). CONCLUSION: The serum mRNA levels of CHOP were increased in the patients with acute kidney injury. Knock-down of CHOP expression inhibits the apoptosis of renal tubular epithelial cells by regulating the expression of proliferation-and apoptosis-related proteins.