CAJ | 학술논문

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(PDE5 shRNA1、PDE5 shRNA2、PDE5 shRNA3)并设置阴性对照(NC shRNA),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ADSCs PDE5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-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)、PDE5 shRNA-ADSCs+I/R损伤组(PDE5 sh I/R组).采用CCK-8法及Edu法检测各组细胞24 h、48 h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,I/R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,PDE5 sh I/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.
목적 탐토만병독개도적린산이지매5(PDE5)기인억제대대서지방간세포(ADSCs)증식、조망급분비공능적영향급기가능궤제. 방법 (1)분리순화대서ADSCs,전대배양급감정.구건침묵PDE5기인불동위점적3조PDE5만병독표체재체(PDE5 shRNA1、PDE5 shRNA2、PDE5 shRNA3)병설치음성대조(NC shRNA),채용만병독포장계통은정전염ADSCs,응용RT-PCR검측ADSCs PDE5 mRNA표체,응용Western blotting검측ADSCs PDE5단백표체,이사선출최고효적전염세포주.(2)실험분위4조:정상세포조、결혈-복양(I/R)손상세포조(정상세포+I/R손상)、음성대조조(NC shRNA-ADSCs+I/R손상)、PDE5 shRNA-ADSCs+I/R손상조(PDE5 sh I/R조).채용CCK-8법급Edu법검측각조세포24 h、48 h증식정황,채용류식세포의검측세포조망정황,채용ELISA법검측상청액중혈관내피생장인자(VEGF)、표피생장인자(FGF)급세포내배린조감(cGMP)수평.결과 성공구건료PDE5만병독표체재체,사선병성공건립료은정저표체PDE5기인적ADSCs세포주PDE5 shRNA-ADSCs.여정상세포조상비,I/R손상세포조ADSCs증식솔명현강저,조기、만기급총조망솔균명현증가,상청액중VEGF、FGF수평급세포내cGMP수평명현증고,차이균유통계학의의(P＜0.05);음성대조조여I/R손상세포조비교차이균무통계학의의(P＞0.05);이여음성대조조상비,PDE5 sh I/R조ADSCs증식현저증가,조망솔명현하강,차상청액중VEGF、FGF수평급세포내cGMP수평진일보승고,차이균유통계학의의(P＜0.05). 결론 PDE5기인표체피억제후가능통과상조cGMP수평,유효개선ADSCs재I/R상태하적증식、조망급분비공능,우화기효능.
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 lentivims 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 per;ormed 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 apoptosis rate,and significantly increased VEGF and FGF levels in the liquid supematant and cGMP level in the cells (P＜0.05).Conclusion The proliferation,apoptosis and secretion of ADSCs could be effectively improved and the stem cell efficiency was modified via up-regulating cGMP level after PDE5 gene expression being inhibited in ADSCs.