大鼠脂肪源性干细胞PDE5基因沉默对肾小管上皮细胞缺血-复氧损伤的保护作用  

Protective effects of adipose-derived stem cells with phosphodiesterase 5 inhibition by lentivirus- mediated stable gene silencing on ischemia- reperfusion injury of renal tubular epithelial cells

作　　者：黄昭[1] 陈伟明[1] 于晓春[1] 刘继云[1] 

机构地区：[1]广州市第一人民医院危重症监护中心,510180

出　　处：《中华肾脏病杂志》2015年第9期693-700,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81301613）

摘　　要：目的探讨慢病毒介导抑制脂肪源性干细胞（adipose-derived stem cells，ADSCs）磷酸二酯酶5（phosphodiesterase5，PDE5）基因表达对缺血-复氧（ischemia／reo。ygenation，I／R）损伤的大鼠肾小管上皮细胞（NRK-52E）增殖和凋亡的影响，以及ADSCs自身分泌功能和上皮转化功能的改变。方法分离纯化大鼠ADSCs，传代培养及鉴定；构建PDE5-shRNA慢病毒表达载体及阴性对照（NCshRNA），采用慢病毒包装系统介导建立稳定PDE5低表达的ADSCs，Western印迹法检测PDE5蛋白表达；建立肾小管上皮细胞体外I／R模型，并建立ADSCs和NRK-52E细胞的Transwell非接触共培养体系；Edu法检测各组NRK．52E细胞增殖，流式细胞仪检测细胞凋亡；ELISA法检测各组上清中肝细胞生长因子（hepatocyte growth factor，HGF）和成纤维细胞生长因子（fibroblast growth factor，FGF）水平；实时荧光定量PCR法检测E钙黏蛋白（E—cadherin）mRNA表达，流式细胞仪检测细胞角蛋白18（Cytokeratin18，CK18）的表达。结果成功构建PDE5-shRNA慢病毒表达载体，筛选并成功建立PDE5稳定低表达的ADSCs细胞系；与正常对照组相比较，I／R组NRK．52E细胞增殖率受到显著抑制，总凋亡率明显增加（P〈0．05），ADSCs共培养上清液中HGF及FGF水平升高（均P〈0．05）；而与阴性对照组相比，与PDE5-shRNA转染组ADSCs共培养的NRK-52E细胞增殖显著增加，凋亡率明显下降，且HGF、FGF水平进一步升高，ADSCs上皮细胞标志物E—cadherin及CK18均表达显著增加，以上差异均有统计学意义（均P〈0．05）。结论本研究应用慢病毒载体介导ADSCs稳定低表达PDE5，可能通过促进干细胞分泌多种生长因子，有效改善肾小管细胞I／R状态下增殖及凋亡，同时促进自身向上皮细胞分化，为干细胞体内移植治疗肾脏缺血再灌注损伤中增加肾小管上皮细胞的生存活力、提升干细胞治疗效能提供了初Objective To explore the protective effects of adipose- derived stem cells （ADSCs） with phosphodiesterase 5 inhibition by lentivirus- mediated stable gene silencing on the proliferation and apoptosis of renal tubular epithelial cells induced by ischemia-reperfusion injury in vitro. Methods To isolate cultivate and indentify ADSCs from rats. Lentiviral expression vector of carrying PDE5 shRNA gene was transfected into ADSCs, and a negative control group was set up.Western blotting was used to detect PDE5 protein expression levels. ADSCs were co-cultured with NRK -52E in a transwell system, and NRK-52E cells were treated with ischemia/reoxygenation protocol. Edu assay was performed to evaluate the proliferation of NRK cells, flow cytometry to detect the apoptosis of NRK cells, and ELISA to quantify the protein expressions of fibroblast growth factor （FGF） and hepatocyte growth factor （HGF）. The expression of E- cadherin and cytokeratin 18 （CKI8） was quantified by real time PCR and flow cytometry. Results Western blotting for PDE5 protein indicated a significant reduction of PDE5 protein levels in PDE5 shRNA transduced population. After the treatment of ischemia/reoxygenation in vitro, the proliferative viability and apoptosis of NRK-52E cells co-cultured with ADSCs induced by PDE5 gene inhibition were significantly improved, compared to the normal group （all P 〈 0.05）. And the release of HGF, FGF were markedly enhanced （all P 〈 0.05）. Moreover, the NRK- 52E cells survival, the expression of E- cadherin and CK18 on PDE5 inhibited ADSCs co-cuhured with I/R injured NRK ceils was significantly increased compared to that in the negative control group （all P 〈 0.05）. Conclusion ADSCs preconditioned by inhibition of PDE5 can be a powerful novel approach to improve the survival of renal tubular cells following ischemia -reperfusion injury, and have an obvious tendency to transform epithelial cells.

关 键 词：环核苷酸磷酸二酯酶类 5型 再灌注损伤 干细胞 

分 类 号：R692[医药卫生—泌尿科学]