Cyr 61基因过表达对人肾小管上皮细胞凋亡的影响

摘　要　目的:观察Cyr61基因过表达对人肾小管上皮细胞 (HKC)凋亡的影响,探讨Cyr61在常染色体显性多囊肾病(ADPKD)发病中的作用及机制。　方法:RT PCR扩增Cyr61基因全长,构建pcDNA3. 1 Cyr61重组质粒,转染并筛选获得稳定转染pcDNA3. 1 Cyr61的HKC细胞。经RT PCR、Northern、Westernblot鉴定转染细胞系基因的整合及表达。对空白HKC、空质粒pcDNA3. 1转染的HKC、Cyr61转染的HKC和囊肿衬里上皮细胞去血清培养 72h后,流式细胞仪检测细胞凋亡指数,Westernblot检测磷酸化Akt和Bad含量。　结果:构建了pcDNA3. 1 Cyr61重组质粒并获得稳定转染该质粒的HKC细胞。空白HKC与质粒pcDNA3. 1转染组之间上述指标无显著差异(P>0 05),Cyr61基因转染组与空白组、pcDNA3. 1转染组相比,凋亡指数显著降低 (P<0. 01),磷酸化Akt和磷酸化Bad含量显著增高 (P<0 .01 ),Cyr61基因转染组与囊肿衬里上皮细胞之间上述指标无显著差异 (P>0. 05);在Cyr61抗体存在下,Cyr61基因转染组的上述指标与空白组无显著差异 (P>0 .05 )。　结论:过表达Cyr61的HKC对去血清诱导的凋亡特性与囊肿衬里上皮细胞相似,过表达的Cyr61可能通过自分泌途径,促进Akt和Bad磷酸化,抑制细胞凋亡,参与ADPKD囊肿形成和发展。

Cyr61 over-expression induce apoptosis on human renal tubular epithelial cells

Objective:To investigate the effect of Cyr61 on apoptosis of human renal tubular epithelial cells(HKC) and explore the role of Cyr61 in the morphogenesis and development of autosomal dominant polycystic kidney disease (ADPKD). Methodology:Cyr61 cDNA was amplified by RT PCR. A recombinant plasmid pcDNA3 1 Cyr61 was constructed by colning Cyr61 gene into pcDNA3 1 HKC cells were transfected with pcDNA3 1 Cyr61. Then the untransfected cells, pcDNA3 1 transfected cells, pcDNA3 1 Cyr61 transfected cells and cyst lining epithelial cells were all treated by serum free DMEM for 72h. The apoptosis exponent and phosphorylation of Akt and Bad were observed by flow cytometry and Western blot. Results:The apoptosis exponent of cells transfected with pcDNA3 1 Cyr61 was decreased significantly compared with those untranfected and transfected with pcDNA3 1( P <0 01), While phosphorylation of Akt and Bad were obviously increased ( P <0 01). There was no significant difference between pcDNA3 1 Cyr61 transfected cells and cyst lining epithelial cells ( P >0.05), the same result was observed between pcDNA3 1 Cyr61 transfected cells and normal HKC with the existence of Cyr61 antibody. Conclusion:The apoptosis propert of HKC with Cyr61 over expressed was similar to cyst lining epithelial cells. The Cyr61 protein over expressed in HKC can promote phosphorylation of Akt and Bad and inhibit apoptosis through autocrine, which may be relevant to the formation and development of renal cyst in ADPKD.