摘要
目的:观察肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)刺激大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)情况下,共培养体系中BMSCs对大鼠肝星状细胞(hepatic stellate cells,HSCs)凋亡的影响并探讨其机制.方法:全骨髓贴壁法分离、纯化S D大鼠BMSCs,传至第3-4代使用.运用6孔Transwell板建立共培养体系,将TNF-α刺激BMSCs后与HSCs共培养.实验分HSCs空白对照组、BMSCs空白对照组、正常共培养组、刺激共培养组;采用流式细胞术检测HSCs凋亡,RT-PCR、Western blot分别检测RhoA与HGFmRNA及蛋白表达,ELISA测定细胞培养上清液中HGF含量.结果:刺激共培养组24h、48h HSCs RhoA蛋白(24h:0.864±0.006,48h:0.688±0.013)及mRNA(24h:0.809±0.004,48h:0.494±0.010)表达进行性下降,与正常共培养组和HSCs空白对照组比较有显著性差异(P<0.01),BMSCsHGF蛋白(24h:1.032±0.003,48h:1.060±0.003)及mRNA(24h:0.857±0.004,48h:1.195±0.010)表达呈时间依赖性递增,与正常共培养组比较差异有统计学意义(P<0.05);刺激共培养组24h、48h HSCs凋亡率分别为6.583%±0.091%、29.960%±0.223%,与正常共培养组(24h:4.700%±0.168%,48h:23.140%±0.115%)比较差异有统计学意义(P<0.01).结论:TNF-α刺激BMSCs后与HSCs共培养明显促进HSCs凋亡,其机制可能是BMSCs通过旁分泌HGF抑制HSCs RhoA表达实现的.
AIM： To observe the effect of rat bone marrow mesenchymal stem ceils （BMSCs） stimulated by TNF-α on apoptosis of rat hepatic stellate cells （HSCs） in a co-culture system and to explore the possible mechanisms involved. METHODS： BMSCs from SD rats were isolated, cultured, and purified by the whole bone mar- row adherence method. BMSCs at passages 3-4 were stimulated by TNF-α and then co-cultured with HSCs in a Transwell co-culture system. Cells were divided into HSCs blank group, BM- SCs blank group, normal co-culture group, and TNF-α-stimulated co-culture group. Apoptosis of HSCs was detected by flow cytometry; expres- sion of RhoA and HGF mRNAs and proteins was tested by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively; and concentration of hepatocyte growth factor （HGF） in cell supernatants was ＇determined by ELISA. RESULTS： Compared to the normal co-culture group and HSCs blank group, the expression of RhoA protein （24 h： 0.864±0.006, 48 h： 0.688±0.013） and mRNA （24 h： 0.809±0.004, 48 h： 0.494±0.010） in HSCs was significantly lower in the TNF-α-sfirnulated co-culture group （all P 〈 0.01）. The expression of HGF protein （24 h： 1.032±0.003, 48 h： 1.060±0.003） and mRNA （24 h： 0.857±0.004, 48 h： 1.195 ±0.010） in BMSCs was significantly higher in the TNF-α-stimulated co-culture group than in the normal co-culture group （all P 〈 0.05）. The apoptosis rate of HSCs was significantly in- creased in the TNF-α-stimulated co-culture group （24 h： 6.583%±0.091%; 48 h： 29.960%± 0.223%） compared to the normal co-culture group （24 h： 4.700%±0.168%, 48 h： 23.140%±0.115%; both P 〈 0.01）. CONCLUSION： BMSCs stimulated by TNF-α enhance the apoptosis of HSCs possibly via para- crine production of HGF by BMSCs to down- regulate RhoA signaling in HSCs.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第19期1713-1719,共7页
World Chinese Journal of Digestology
基金
广西自然科学基金资助项目
No.0897008~~
