CAJ | 학술논문

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(phosphodiesterase 5,PDE5)基因表达对缺血?复氧(ischemia/reoxygenation,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(Cytokeratin 18 ,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步的实验基础.
목적 탐토만병독개도억제지방원성간세포(adipose?derived stem cells,ADSCs)린산이지매5(phosphodiesterase 5,PDE5)기인표체대결혈?복양(ischemia/reoxygenation,I/R)손상적대서신소관상피세포(NRK?52E)증식화조망적영향,이급ADSCs자신분비공능화상피전화공능적개변. 방법 분리순화대서ADSCs ,전대배양급감정;구건PDE5?shRNA만병독표체재체급음성대조(NCshRNA),채용만병독포장계통개도건립은정PDE5저표체적ADSCs , Western인적법검측PDE5단백표체;건립신소관상피세포체외I/R모형,병건립ADSCs화NRK?52E세포적Transwell비접촉공배양체계;Edu법검측각조NRK?52E세포증식,류식세포의검측세포조망;ELISA법검측각조상청중간세포생장인자(hepatocyte growth factor,HGF)화성섬유세포생장인자(fibroblast growth factor,FGF)수평;실시형광정량PCR법검측E개점단백(E?cadherin)mRNA표체,류식세포의검측세포각단백18(Cytokeratin 18 ,CK18)적표체. 결과 성공구건PDE5?shRNA만병독표체재체,사선병성공건립PDE5은정저표체적ADSCs세포계;여정상대조조상비교,I/R조NRK?52E세포증식솔수도현저억제,총조망솔명현증가(P<0.05),ADSCs공배양상청액중HGF급FGF수평승고(균P<0.05);이여음성대조조상비,여PDE5?shRNA전염조ADSCs공배양적NRK?52E세포증식현저증가,조망솔명현하강,차HGF、FGF수평진일보승고,ADSCs상피세포표지물E?cadherin급CK18균표체현저증가,이상차이균유통계학의의(균P<0.05). 결론 본연구응용만병독재체개도ADSCs은정저표체PDE5 ,가능통과촉진간세포분비다충생장인자,유효개선신소관세포I/R상태하증식급조망,동시촉진자신향상피세포분화,위간세포체내이식치료신장결혈재관주손상중증가신소관상피세포적생존활력、제승간세포치료효능제공료초보적실험기출.
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 (CK18) 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-cultured with I/R injured NRK cells 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.