【作者】 陈国忠；

【导师】 姜海行；

【作者基本信息】 广西医科大学 ， 内科学， 2010， 博士

【摘要】 目的骨髓间充质干细胞(BMSCs)移植对多种原因引起的肝损伤具有显著修复作用,可改善甚至逆转肝纤维化,但逆转肝纤维化的机制未明。肝星状细胞(HSCs)的激活在肝纤维化的发生发展中起着关键作用。因此,深入探讨骨髓间充质干细胞对肝星状细胞增殖、凋亡的影响及作用机制,具有重要意义。通过观察体外大鼠骨髓间充质干细胞对肝星状细胞增殖、凋亡和RhoA表达的影响,探讨BMSCs旁分泌肝细胞生长因子(HGF)在其中的作用机制。方法贴壁筛选法培养、纯化SD大鼠BMSCs,传代至第4代使用；大鼠肝星状细胞(HSC-T6)系及纤维原细胞系冻融后传代使用。应用6孔塑料细胞培养盒,在半透膜(transwell insert)上层接种MSCs(1×105)cells／well,在下层接种HSC-T6细胞(1×105)cells/well,建立上下双层细胞共培养体系,常规培养。实验分组：①空白对照组：HSCs单独培养；②实验对照组：纤维原细胞(Fiberoblasts)与HSCs共培养；③MSC组：’BMSCs与HSCs共培养；④C-met抗体预处理组：兔抗C-met多克隆抗体500ng／ml预先封闭HSCs表面C-met受体6h后,BMSCs与HSCs共培养。以上体系培养观察24h和48 h,于倒置相差显微镜下动态观察HSCs细胞形态；免疫组化法检测HSCs a-SMA表达。四甲基偶氮唑蓝(MTT)法检测共培养体系0h、24h、48h HSCs增殖能力；流式细胞术Annexin-V-FITC/PI双染法检测共培养体系24h、48h HSCs凋亡；RT-PCR、Western blot检测共培养体系0h、24h、48h HSCs内RhoA mRNA和RhoA蛋白的表达。收集BMSCs与HSCs共培养、单独BMSCs、单独HSCs培养体系24h、48h时间段上清液,-80℃冰箱保存,酶联免疫吸附法(ELISA)检测共培养上清液中肝细胞生长因子浓度。结果1.BMSCs对HSC增殖具有抑制作用,BMSCs与HSCs共培养后24h、48h的HSCs增殖抑制率分别为(12.21±2.55)%与(35.43±6.17)%。BMSCs在24h已抑制大鼠肝星状细胞的增殖,48h明显抑制大鼠肝星状细胞的增殖并呈现时间依赖性。MSCs组与空白对照组、实验对照组和C-met抗体预处理组比较有统计学差异(P<0.01)。2.BMSCs与HSCs共培养24h、48h后HSCs的凋亡率分别为(7.20±1.70)%与(25.80±3.60)%,BMSCs明显促进24h、48h时间段大鼠肝星状细胞的凋亡,与空白对照组、实验对照组与C-met抗体预处理组比较有统计学差异(P<0.01)。3.BMSCs与HSCs共培养后,RT-PCR法检测共培养24h、48h后HSCs中RhoA mRNA表达分别为(0.68±0.08)与(0.40±0.03)。BMSC组共培养24h后,RhoA mRNA的表达受到抑制；48h时抑制则更为明显,且显著低于空白对照组、实验对照组与C-met抗体预处理组,与上述三组比较有统计学差异(F<0.01)。4. Western blotting检测BMSCs与HSCs共培养24h、48h后HSCs中RhoA蛋白的表达分别为(0.76±0.06)与0.43±0.05)。BMSC组共培养,24h后,RhoA蛋白表达即受到抑制；48h时抑制则更为明显,且显著低于空白对照组、实验对照组与C-met抗体预处理组,与上述三组比较有统计学差异(P<0.01)。5.ELISA检测BMSCs与HSCs共培养24h、48h上清液中HGF浓度分别为(250±35)pg／m1与(570±80)pg／ml,明显高于单独BMSCs培养和单独HSC培养,与上述2组比较有统计学差异(P<0.01)。结论BMSCs与HSCs共培养能抑制HSCs的增殖,促进凋亡,抑制RohA表达,其机制是通过BMSCs旁分泌HGF发挥抑制大鼠肝星状细胞增殖,促进凋亡和抑制RhoA表达的作用。可能是BMSCs抗肝纤维化作用的分子机制之一。更多还原

【Abstract】 Objective To observe the bone marrow mesenchymal stem cells(BMSCs) regulate the proliferation and apoptosis and the expression of RohA in rat hepatic stellate cells(HSCs).The research explore the underlying mechanism of rat BMSCs paracrine HGF to modulate HSCs.Methods BMSCs were isolated from bone marrow in SD rats and cultured and purified in vitro. HSCs and fiberoblast cells were recoveried and activated morphologically, a-SMA expression in HSCs was evaluated with immuno-histochemically. An indirect co-culture system was established using a Transwell membrane system (diameter:24 mm; pore size:0.4μm). The HSCs were seeded in the lower chamber with MSCs or fiberoblast cells (1×105) cells /ml were seeded onto the Transwell membrane of the inner chamber with the ratio of 1:1.Cultures were maintained in HSCs in medium for 24 hour and 48 hour. Rat normal fibroblast cell lines and HSCs (HSC-T6) were respectively cultured as a control group. HSCs were randomly divided into four groups: blank control group (HSCs alone), negative control group (HSCs co-cultrue with fibroblasts), and experimental group (HSCs co-culture with BMSCs). At some conditions, rabbit polyclonal antibody of C-met was pre-administrated 6h before co-cultrue as pre-treatment group according to experiment need. Cell proliferation was determined by MTT and cell apoptosis was determined by flow cytometry. The expression of RohA mRNA in HSCs was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of RohA protein expression by Western blotting respectively. Supernatants were harvested and stored at-70℃until they were analyzed. The HGF concentration in the supernatants of culture was determined by ELISA method.Results 1. BMSCs inhibited the HSCs proliferation. After 24hour,48hour co-clutrue, the inhibition rates of HSCs were (12.21±2.55)%, (35.43±6.17) % respectively. The inhibited rate of HSCs in the experimental group was significantly higher than those in the other three groups (P< 0.01).2.The apoptosis of HSCs was detected by Annexin-V-FITC/PI, the rate of apoptosis of the BMSCs with HSCs group were significantly increased at time of 24 hour and 48 hour,which were significantly higher than those in the other three groups (P<0.01). Flow cytometry found that compared with the other three groups, BMSCs could promote the apoptosis of HSCs.3. The expression of HSCs RohA mRNA in each group after co-cultured was determined by RT-PCR. At time of 48 hour, the expression of RohA mRNA in HSCs was significantly lower on the MSCs Co-cultured with HSCs group than those in the other three groups (P<0.01).BMSCs inhibited the expression of HSCs mRNA.4. By Western blotting detection system and digital image analysis software, we found that BMSCs inhibited the expression of HSCs RohA protein after BMSCs co-cultrued with HSCs at time of 48 hour.Compared with the other three groups,the difference had statistical significance (P<0.01).5. The HGF concentration in the supernatants of BMSCs co-culture with HSCs was higher than MSCs culture and HSCs culture, the difference had statistical significance(P<0.01).Conclusions The rat bone marrow mesenchymal stem cells inhibit the proliferation and promote the apoptosis and decrease the expression of RohA in rat activated hepatic stellate cells in vitro. The underlying mechanism of rat bone marrow mesenchymal stme cells may be by way of paracrine HGF pathway.更多还原