摘要
目的观察慢病毒介导的HGF-ShRNA部分干扰大鼠骨髓间充质干细胞(BMSCs)及肝星状细胞(HSCs)来源的肝细胞生长因子(HGF)表达,对BMSCs与HSCs共培养体系中HSCs凋亡的影响,探讨两种细胞来源的HGF对HSCs凋亡的促进作用。方法应用6孔培养板,建立BMSCs及HSCs双层细胞共培养体系,实验分4组:(1)HSCs单独培养组:HSCs单独培养;(2)BMSCs+HSCs共培养组:BMSCs与HSCs共培养;(3)BMSCs转染共培养组:使用慢病毒介导的HGF-ShRNA转染BMSCs后与HSCs共培养;(4)HSCs转染共培养组:使用慢病毒介导的HGF-ShRNA转染HSCs后与BMSCs共培养。分别于培养24、48、72 h后,采用Annexin V-FITC/PI双染法检测各组HSCs凋亡率,Western blot、荧光定量PCR分别检测各组DR5、Caspase-8蛋白及mRNA的表达。结果BMSCs+HSCs共培养组HSCs的凋亡率明显高于HSCs单独培养组和BMSCs转染共培养组,且呈时间依赖性(P<0.01),而与HSCs转染共培养组比较差异无统计学意义(P>0.05);BMSCs+HSCs共培养组HSCs中DR5、Caspase-8蛋白及mRNA表达量均明显高于HSCs单独培养组及BMSCs转染共培养组(P<0.01),而与HSCs转染共培养组比较差异无统计学意义(P>0.05)。结论 BMSCs旁分泌HGF通过上调HSCs的DR5、Caspase-8的表达促进HSCs的凋亡。HSCs自分泌的HGF在共培养体系中促进其凋亡的作用甚少。
Objective To investigate the effects of lentiviral mediated HGF -ShRNA interfere in primary hepatic stellate cells （HSCs） and bone marrow mesenchymal stem cells （BMSCs） on the expression of hepatocyte growth factor （HGF） and the apoptosis of HSCs. To explore the roles of BMSCs and HSCs derived HGF in the apoptosis of HSCs. Methods BMSCs and HSCs were used to establish the upper and lower co - culture system. Experiment was divided into four groups： HSCs blank group （HSCs cultured alone）, co- cultured group （BMSCs co- cultured with HSCs） , BMSCs transfection co - culture group （ BMSCs transfected with lenti - HGF - ShRNA and co - cultured with HSCs）, and HSCs transfection co - culture group （ HSCs transfected with lenti - HGF - ShRNA and co - cultured with BMSCs）. After 24, 48 and 72 h cultured, apoptosis of HSCs was determined by Annexin - V - FITC/PI; while the expression of DR5 and Caspase - 8 protein and mRNA in HSCs was determined by Western blot and FQ - PCR, respectively. Results The apoptosis rate of HSCs in co - culture group was significantly higher than that in HSCs blank group or BMSCs transfection co - culture group in a time - dependent manner （ P 〈 0. 01 ）, but with no significant difference from HSCs transfection co - culture group （P 〉 0.05）. The expression of DR.5 and Caspase - 8 in both protein and mRNA in co - culture group was significantly higher than that of HSCs blank control group and BMSCs transfection co - culture group （P 〈 0. 01 ）, but with no significant difference from HSCs transfection co - cuhure group （ P 〉 O. 05 ）. Conclusion BMSCs co - cultured with HSCs can promote apoptosis of HSCs, which may be associated with the increased expression of HSCs DRA and Caspase -8 by HGF derived from BMSCs. HSCs derived HGF play less role in promoting the apoptosis of HSCs in the co - culture system.
出处
《广东医学》
CAS
CSCD
北大核心
2014年第19期2976-2979,共4页
Guangdong Medical Journal
基金
广西自然科学基金资助项目(编号:2012GXNSFAA053091)
关键词
骨髓间充质干细胞
肝星状细胞
肿瘤坏死因子相关凋亡诱导配体
肝细胞生长因子
死亡受体5
bone marrow mesenehymal stem cells
hepatic stellate cells
tumor necrosis factor -related apoptosis - inducing ligand
hepatocyte growth factor
death receptor - 5
