慢病毒介导磷酸二酯酶5基因修饰对大鼠脂肪干细胞效能的影响  

Effect of phosphodiesterase 5 inhibition by lentivirus-mediated stable gene silencing on survival of adipose-derived stem cells

作　　者：黄昭[1] 陈裕胜[1] 吴晓琴[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：《中华神经医学杂志》2015年第11期1081-1088,共8页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81301613）

摘　　要：目的探讨慢病毒介导的磷酸二酯酶5（PDE5）基因抑制对大鼠脂肪干细胞（ADSCs）增殖、凋亡及分泌功能的影响及其可能机制。方法（1）分离纯化大鼠ADSCs，传代培养及鉴定。构建沉默PDE5基因不同位点的3组PDE5慢病毒表达载体（PDE5sbRNA1、PDE5shRNA2、PDE5shRNA3）并设置阴性对照（NCshRNA），采用慢病毒包装系统稳定转染ADSCs，应用RT-PCR检测ADSCs PDE5 mRNA表达，应用Western blotting检测ADSCsPDE5蛋白表达，以筛选出最高效的转染细胞株。（2）实验分为4组：正常细胞组、缺血一复氧（I／R）损伤细胞组（正常细胞＋I/R损伤）、阴性对照组（NC shRNA-ADSCs＋I／R损伤）、PDE5shRNA．ADSCs＋UR损伤组（PDE5 sh I／R组）。采用CCK-8法及Edu法检测各组细胞24h、48h增殖情况，采用流式细胞仪检测细胞凋亡情况，采用ELISA法检测上清液中血管内皮生长因子（VEGF）、表皮生长因子（FGF）及细胞内环磷鸟苷（cGMP）水平。结果成功构建了PDE5慢病毒表达载体，筛选并成功建立了稳定低表达PDE5基因的ADSCs细胞株PDE5 shRNA—ADSCs。与正常细胞组相比，UR损伤细胞组ADSCs增殖率明显降低，早期、晚期及总凋亡率均明显增加，上清液中VEGF、FGF水平及细胞内cGMP水平明显增高，差异均有统计学意义（P〈0．05）；阴性对照组与I／R损伤细胞组比较差异均无统计学意义（P〉0．05）；而与阴性对照组相比．PDE5shI／R组ADSCs增殖显著增加，凋亡率明显下降，且上清液中VEGF、FGF水平及细胞内cGMP水平进一步升高，差异均有统计学意义（P〈0．05）。结论PDE5基因表达被抑制后可能通过上调cGMP水平，有效改善ADSCs在I／R状态下的增殖、凋亡及分泌功能，优化其效能。Objective To explore the in vitro effect of lentiviral expressions vector carrying phosphodiesterase 5 （PDE5）-shRNA gene on proliferation, apoptosis and secretion of adipose-derived stem cells （ADSCs）. Methods （1） ADSCs were separated and purified from the rats, sub-cultivation and identification were performed. Lentiviral expression vectors carried different PDE5 shRNA genes （PDE5 shRNA1, PDE5 shRNA2 and PDE5 shRNA3） were established and negative controls （NC shRNA） were used. The above vectors were transfected into ADSCs with lentivirus package; Western blotting was carried out to detect the PDE5 protein expression level and real time-PCR was carried out to detect the mRNA expression level so as to select the most efficient cell line. （2） ADSCs were divided into four groups： normal control group, ischemia-re-oxygenation （I/R） injury group （normal cells＋I/R injury）, NC group （NC shRNA-ADSCs＋I/R injury） and experimental group （PDE5 shRNA-ADSCs＋I/R injury）; CCK-8 assay and Edu method were performed to evaluate the proliferation of cells, flow cytometry was employed to detect the apoptosis of ADSCs, and ELISA was used to quantify the protein expressions of vascular endothelial growth factor （VEGF）, basic fibroblast growth factor （b-FGF） and cyclic guanosine monophosphate （cGMP）. Results Lentiviral expression vectors carried different PDE5 shRNA genes were successfully established and PDE5 sh ADSCs with stable PDE5 expression were established. As compared with cells from normal control group, cells from I/R injury group had significantly decreased ADSCs proliferation rate and increased apoptosis rate at early and late stages, and significantly increased VEGF and FGF levels in the liquid supernatant and cGMP level in the cells （P〈0.05）; these differences were not significant between the I/R injury group and NC group （P〉0.05）; as compared with the NC group, the experimental group had significantly increased ADSCs proliferation rate and decreased a

关 键 词：脂肪干细胞 磷酸二酯酶5 缺血-复氧损伤 细胞增殖 细胞凋亡 

分 类 号：R741[医药卫生—神经病学与精神病学]