骨髓间充质干细胞抗肝纤维化的机制研究

【作者】 覃山羽；

【导师】 姜海行；

【作者基本信息】 广西医科大学 ， 消化内科学， 2009， 博士

【摘要】 第一部分体内实验:骨髓间充质干细胞移植抗CCl4诱导大鼠肝纤维化的研究目的:探讨骨髓间充质干细胞移植对CCl4诱导大鼠肝纤维化的作用与机制。方法:MSCs的分离:从SD雄性大鼠的股骨骨髓中获得,经分离,培养基培养,传代至第4代,动态观察细胞形态及功能变化,作为SD雌性大鼠肝纤维化的移植细胞。采用大鼠背部皮下注射CCl4造模,剂量:40% CC14花生油:0.2m1/100g,每周2次,共6周。将造模型成功SD大鼠60只,随机等量分为3组:①模型对照组:鼠尾静脉注射等量的生理盐水;②MSCs实验组:鼠尾静脉注射含MSCs细胞悬液;③HGF诱导MSCs实验组:鼠尾静脉注射经HGF诱导14天后的MSCs细胞悬液。以造模6周末时鼠尾静脉输注2×105 MSCs细胞悬液1次。每组分别在鼠尾静脉输注2×105 MSCs细胞悬液后,于第1w,第2w,第3w,第4w末不同时点,以戊巴比妥钠麻醉大鼠,眼球取血,分离血清,保存于-20℃备用;迅速称取约50-100 mg肝组织放入液氮中迅速冷冻后,转移放置于-80℃超低温冰箱保存,分别用于抽提肝组织总RNA及蛋白质。于肝左叶同一部位取肝组织约1 cm×1 cm×l cm大小,固定于10%中性福尔马林溶液,做光镜观察和免疫组化用。采用酶联免疫吸附(ELISA)法检测血清肝纤维化指标透明质酸(HA)、层粘蛋白(LN)、Ⅳ胶原(Col-Ⅳ);采用HE染色及Masson染色光镜下观察肝组织纤维化程度;RT-PCR检测骨髓干细胞移植后大鼠肝脏Col-I, RhoA,Cdc42,Rac1 mRNA表达,Western blot检测骨髓干细胞移植后大鼠肝脏Col-I, RhoA,Cdc42,Rac1蛋白质表达。结果:(1)MSCs细胞移植后对肝纤维化大鼠肝组织形态学的影响:HE染色病理结果显示:肝纤维化模型组大鼠肝脏正常肝小叶结构被破坏,由大小不等的圆形或椭圆形肝细胞结节所构成的假小叶取代,结节周围纤维组织增生,少量炎细胞浸润。移植未诱导MSCs组,2w可见新生的卵圆形细胞,也可见少量新生的血管与小胆管, 4w后时肝小叶肝细胞排列较整齐,出现中央静脉、肝窦少量充血。移植经HGF诱导后的MSCs组,与对照组比较,2w可见大量卵圆形细胞增生,并形成较多新生血管与小胆管,向肝小叶迁移, 4w时肝脏基本恢复正常,炎性细胞消失。Masson染色结果显示:诱导MSC组与MSC组的纤维化程度评分逐渐下降,随时间递增而评分递减,且在第4周诱导MSC组与MSC组比较有统计学差异(P<0.05)。(2) ELISA法测定血清HA,LN,IV型胶原含量:移植2周后,诱导MSCs组血清IV型胶原与对照组比较有统计学差异(P<0.05),移植3w,4w后,诱导MSCs组和MSCs组各指标与对照组比较有统计学差异(P<0.05, P<0.01),诱导MSCs组IV型胶原与MSCs组比较有统计学差异(P<0.05);各组HA,LN,IV型胶原量在1w,2w,3w未见差异,但4w与3w比较有统计学差异(P<0.05)。(3) RT-PCR检测MSCs移植后对大鼠肝脏Col-I, RhoA,Cdc42,Rac1 mRNA表达的影响:移植1w后,诱导MSC组Cdc42与对照组比较有统计学差异(P<0.05);移植2w后,诱导MSC组与对照组的RhoA和Cdc42降低比较有统计学差异(P<0.01, P<0.05),且诱导MSCs组比MSCs组比较更明显( P<0.05),MSCs组Cdc42表达与对照组比较有统计学差异(P<0.05);移植3w,4w后,诱导MSC组与对照组各指标比较均有统计学差异(P<0.01, P<0.05),且3w后诱导MSCs组Col-I和Cdc42表达比MSCs组下降更明显(P<0.05),4w后诱导MSCs组比MSCs组Col-I和RhoA, Cdc42下降更明显(P<0.01)。(4) Western blot检测MSCs移植后大鼠肝脏Col-I,RhoA,Cdc42,Rac1蛋白质表达:移植1w后,诱导MSC组RhoA和Cdc42蛋白表达与对照组比较有统计学差异(P<0.05);移植2w后,诱导MSCs各指标与对照组比较有统计学差异(P<0.05), MSCs组与对照组Col-I,RhoA比较有统计学差异(P<0.05)。移植3w,4w后,诱导MSC组和MSC组与对照组各指标比较均有统计学差异(P<0.01, P<0.05),且3w后诱导MSCs组Col-I和RhoA表达比MSCs组下降更明显(P<0.05),4w后诱导MSCs组比MSCs组Col-I下降更明显(P<0.05)。结论:(1)MSCs移植可抑制纤维化大鼠肝组织中胶原纤维增生,加快组织学修复,降低大鼠肝纤维化组织胶原代谢产物HA、LN、IV胶原的分泌。(2)MSCs移植下调肝纤维化大鼠肝组织Rho信号通路相关因子RhoA,Cdc42,Rac1 mRNA及蛋白表达,提示其拮抗CCl4诱发SD大鼠肝损害,可能是MSCs抗肝纤维化的作用机制之一。(3)肝细胞生长因子诱导MSCs使其形态和功能发生改变,用于移植促进肝纤维化转归较单纯MSCs作用更强。第二部分体外实验大鼠骨髓间充质干细胞体外抑制肝星状细胞Rho信号因子与胶原-I表达的研究目的:研究在体外大鼠骨髓间充质干细胞对HSCs增殖和抑制纤维合成的作用,探讨其抗肝纤维化机制。方法:MSCs的分离:从SD雄性大鼠的股骨骨髓中获得,经分离,培养,传代至第4代使用;大鼠肝星状细胞(HSC-T6)和纤维原细胞系复苏后传代使用。应用6孔朔料培养板,在半透膜(transwell insert)上层接种MSCs (2×105cells/ well),在下层接种HSC-T6细胞(2×105cells/ 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阴性对照组:纤维原细胞(Fibroblasts)与HSCs共培养,③MSC组: MSCs与HSCs共培养;④诱导MSC组:HGF诱导14天的MSCs与HSCs共培养。以上体系培养观察72 h。每天于倒置相差显微镜下动态观察活体细胞形态学改变;采用免疫组化法a-SMA表达,用细胞计数板对星状细胞增殖率进行测定;RT-PCR检测MSCs与HSCs共培养后HSCs的Col-I,RhoA,Cdc42,Rac1 mRNA表达;Western blot检测MSCs与HSCs共培养后HSCs的Col-I,RhoA,Cdc42,Rac1蛋白质表达,对以上各个表达参数进行相关性分析。结果:(1) HSCs与MSCs共培养24 h ,48h,72h,HSCs表现明显增殖抑制(P<0.01),且呈现时间依赖性。诱导MSC组和MSC组与阴性对照组比较均有显著性差异(P<0.01),诱导MSC组与MSC组比较有显著性差异(P<0.01)。(2) RT-PCR检测MSCs与HSCs共培养后HSCs的Col-I, RhoA,Cdc42,Rac1 mRNA表达:共培养1d后,诱导MSCs组RhoA,Cdc42,Rac1 mRNA表达与对照组比较有统计学差异(P<0.05或P<0.01),MSCs组Cdc42表达与对照组比较有统计学差异(P<0.05);共培养2d后,诱导MSCs组和MSCs组各指标与对照组比较有统计学差异(P<0.01, P<0.05),且诱导MSCs组的Cdc42与MSCs组比较有统计学差异(P<0.05),共培养3d后,诱导MSCs组和MSCs组各指标与对照组比较均有统计学差异(P<0.01),且诱导MSCs组Col-I,Cdc42和Rac1与MSCs组比较有统计学差异(P<0.05)。(3) Western blot检测MSCs与HSCs共培养后HSCs的Col-I, RhoA,Cdc42,Rac1蛋白质表达:共培养1d后,诱导MSC组RhoA,Cdc42表达与对照组比较有统计学差异(P<0.05);共培养2d后,诱导MSCs组和MSCs组各指标与对照组比较有统计学差异(P<0.01, P<0.05),且诱导MSCs组Cdc42表达与MSCs组比较有统计学差异( P<0.05);共培养3d后,诱导MSCs组和MSCs组各指标与对照组比较均有统计学差异(P<0.01),且诱导MSCs组Col-I,RhoA和Cdc42与MSCs组比较有统计学差异( P<0.05或P<0.01)。(4)相关分析显示:Col-I与RhoA,Cdc42,Rac1m RNA表达的相关性分析:细胞共培养第一天:Col-I与RhoA,Cdc42 mRNA表达有相关性(r=0.711, P<0.05;r=0.812, P<0.05);第二天:Col-I与Rac1有相关性(r=0.751, P<0.01);第三天:Col-I与RhoA,Cdc42有相关性(r=0.826,P<0.05;r=0.646,P<0.05)。Col-I与RhoA,Cdc42,Rac1蛋白表达的相关性分析:共培养第一天:Col-I与RhoA,Cdc42蛋白表达有相关性(r=0.865, P<0.05;r=0.733, P<0.05);第二天:Col-I与RhoA,Cdc42,Rac1有相关性(r=0.825, P<0.01;r=0.744, P<0.05;r=0.586, P<0.05);第三天:Col-I与RhoA,Cdc42有相关性(r=0.841, P<0.01;r=0.665, P<0.05)结论:(1)骨髓间充质干细胞可在体外抑制肝星状细胞的活化及胶原表达。(2)骨髓间充质干细胞抑制星状细胞胶原表达涉及下调Rho信号通路,可能通过旁分泌途径诱导星状细胞凋亡而抑制其增殖。(3)骨髓间充质干细胞与肝星状细胞共培养后首先抑制Rho信号通路相关因子表达,Rho信号通路可能是星状细胞胶原合成的上游通路之一。(4)肝细胞生长因子诱导间充质干细胞后抑制星状细胞活化及表达胶原较单纯间充质干细胞作用显著增强。更多还原

【Abstract】 PART 1 EXPERIMENT in vivo: THE EFFECT OF MSCs TRANSPLANTATION ON THE ANTI-HEPATIC FIBROSIS INDUECED BY CCL4 IN RATSOBJECTIVE: To study the effect of MSCs transplantation against hepatic fibrosis and the effect on expression of Col-I, RhoA,Cdc42,Rac1 in rats with CCl4-induced hepatic fibrosis. fibrosis.METHODS: For MSC isolation, male Sprague-Dawley (SD) rats were killed by cervical dislocation and the limbs removed. MSC were flushed with Dulbecco’s Modified Eagle medium(DMEM)culture medium from the medullary cavities of femurs using a 25-G needle.MSC were separated, grown, and propagated in culture for 4 pasagges and were characterized morphologically. A part of the 4th pasagge cells were treated with HGF for 2 week. The 4th pasagge MSCs and HGF-induced MSCs were then infused into the tail vein of female rats with CCl4–induced liver fibrosis.Hepatic fibrosis model of SD rats were induced by subcutaneous injection (s.c.) of CCl4 on the dorsum at dose of 0.2 m1 of 40% CCl4/100 g (40% CCl4=4ml CCl4 : 6ml peanut oil), twice a week. For rats in the normal control group, normal saline ( NS ) were given, s.c., 0.2 m1 of NS/100g , twice a week. Sixty femail SD hepatic fibrosis rats were divided into 3 groups randomly:①model control group(C, n=20): treated by infusion of normal saline.②MSC group(M, n=20): treated by injection of MSCs suspension via tail vein.③HGF-induced MSC group(HM, n=20): treated by injection of HGF-induced MSCs suspension via tail vein . All fibrosis model rats were treated with infusion of MSC suspension or sham via tail vein once time at the beginning of 7th week, on the different pointed time after treatment(on weeks of 1,2,3 and 4), rats were anesthetized with the sodium pentobarbitol, the blood of the rats was taken from eyeball, and separated serum was finally frozen at -20℃until assay. About 50-100 mg hepatic tissue was treated with the liquid nitrogen rapidly, then being frozen in -80℃freezer for extracting total RNA and protein. Two pieces of hepatic tissue in the identical spot of liver, size 1 cm3×1 cm3×l cm3, were taken, one was soaked in 10% neutral formalin solution for the light microscope observation and immunohistochemistry.The serological indexes, e.g. hyaluronic acid (HA), laminin (LN), collagen IV (Col-IV) were assayed by enzyme linked immunoabsorbent assay (ELISA). The degree of fibrosis in hepatic tissue was observed under light microscope by hematoxylin and eosin(HE). The collagen expression degree was evaluated by Masson staining method. mRNA expression of Col-I, RhoA,Cdc42,Rac1 in hepatic tissues were detected by RT- PCR, whereas protein expression of Col-I,RhoA,Cdc42,Rac1 in hepatic tissues were detected by Western blot.RESULTS:①The influence of MSCs on serological indexes in hepatic fibrosis rats: Compared with the model control group, the serological concentrations of HA, LN, and Col-IV in HGF-induced MSC treated group or MSC treated group were decreased significantly at different pointed time (P<0.01, P<0.05), furthermore, the serological concentrations of HA, LN, and Col-IV in HGF-induced MSC treated group were significantly lower than MSC treated group (P<0.01 , P<0.05).②The influence of MSCs on histomorphology in hepatic fibrosis rats: We analyzed liver histology in rats with MSCs and HGF-induced MSCs in CCL4 modals by HE staining and MT staining.HE staining showed that architecture of liver got better in MSCs and HGF-induced MSCs. Inflammation reduced and pseudolobules were resolved.Collagen accumulation and fatty degeneration were significantly lower in MSCs and HGF- induced MSCs compared to the model control. The necrosis of hepatocyte was replaced by regenerate.The thickened septal fibrosis become thinner or disappeared. Histopathological examination of liver tissue showed that MSCs had a significant antifibrotic effect as evidence by the decrease in liver collagen stained with masson trichrome compared to the model group that exhibited marked periportal fibrosis in hepatic lobules.③The influence of MSCs transplantation on Col-I, RhoA,Cdc42,Rac1 mRNA expression in hepatic fibrosis rats. Compared with the model control group, MSCs group and HGF-induced MSCs group could down regulate significantly Col-I, RhoA,Cdc42,Rac1 mRNA expression (P<0.01, P<0.05) on week 3 and 4 after treatment.④The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 protein expression in hepatic tissues of hepatic fibrosis rats: After treament with MSCs at week 3 and 4, the protein expression of Col-I , RhoA,Cdc42,Rac1 in hepatic tissues reduced significantly as compared with the model control group (P<0.05, P<0.01).Moreover, we observed that the antifibosis effect of MSCs in HGF-induced MSCs group was superior to MSCs group.CONCLUSIONS : MSCs transplantation may suppress collagenous fibre-proliferation in tissues of hepatic fibrosis rats, down-regulate the expression of Col-I, RhoA,Cdc42,Rac1 mRNA and its protein in tissues of hepatic fibrosis rats. The results indicate that MSCs could ameliorate liver fibrosis induced by CCl4 in SD rats, and reduce the collagen synthesis and the deposition, the underlying mechanism may be involvement of inhibition of Rho signaling pathway, which has been implied as an important new target for suppressing the deposition of ECM. PART 2 EXPREMENT in vitro: THE CONCOMITANT INHIBITORY EFFECTS OF MSCs ON THE EXPRESSION OF Col-I and RhoA,Cdc42,Rac1 IN HEPATIC STELLATE CELLSOBJECTIVE: To investigate the effects of mesenchymal stem cells (MSCs) on the mRNA and protein expression of Col-I, RhoA,Cdc42,Rac1 in hepatic stellate cells (HSCs) in vitro, and explore the underlying mechanism of anti- fiborsis of MSCs on the HSCs.METHODS : MSCs were isolated from BM in rats and grown, and propagated in culture flask. HSCs were recoveried and activated morphologically , a-SMA expression in HSCs was evaluated immuno- histochemically. An indirect coculture system between MSC and HSC or fiberblast cells was established using Transwell membranes (24mm diameter, 0.4μm pore size). Approximately 2.0×105 HSCs were placed in the lower chamber with either 2.0×105 MSCs or fiberoblast cells placed on the membrance insert. Cocultures were maintained in HSC medium for three days.Four groups were divided randomly:①HSC control group②Fiberoblast control group.③M SC group.④H GF- induced MSC group.At the different pointed time(on days of 1, 2 and 3), the HSCs in co-culture system were collected and the mRNA expressions of Col-I, RhoA,Cdc42,Rac1 in HSCs were detected by RT- PCR . The protein expressions of Col-I,RhoA,Cdc42,Rac1 in HSCs were evaluated by Western blot.RESULTS:①With the coculture time lasting, the inhibitory rate of HSCs proliferation with MSCs coculture were significant higher than HSC control group and Fiberoblast control group at different pointed time(P < 0.01).②The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 mRNA expression in HSC of coculture system: Compared with the control group, the mRNA expression of RhoA,Cdc42,Rac1 in HGF-induced MSCs group and Cdc42 expression in MSCs group were significantly reduced on day 1(P<0.05, P<0.01), and all of the parameters in HGF-induced MSCs group and MSCs group were significantly decreased on day 2 and day 3(P<0.01, P<0.05, moreover, the expression of Cdc42 in HGF-induced MSC group was significant lower than MSC group on day 2( P<0.05) and the expression of Col-I, Cdc42, Rac1 in HGF-induced MSC group were significant lower than MSC group on day 3( P<0.05).③The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 protein expression in HSC of coculture system: Compared with the control group, the protein expression of RhoA,Cdc42 in HGF-induced MSCs group were significantly reduced on day 1(P<0.05),and all of the parameters in HGF-induced MSCs group and MSCs group were significantly decreased on day 2 and day 3(P<0.01, P<0.05), furthermore, the expression of Cdc42 in HGF-induced MSCs group was significant lower than MSCs group on day 2( P<0.05) and the expression of Col-I, RhoA , Cdc42 in HGF-induced MSCs group were significantly lower than MSCs group on day 3( P<0.05或P<0.01).④The statistical analysis the relationship of the expession of Col-I and RhoA,Cdc42 ,Rac1 in HSC after treatment with MSC:There were positive relationship between the mRNA expression of Col-I and RhoA,Cdc42(r=0.711, P<0.05;r=0.812, P<0.05)on day 1,so as for the mRNA expression of Col-I and Rac1(r=0.751, P<0.01) on day 2, and the mRNA expression of Col-I and RhoA,Cdc42(r=0.826,P<0.05, r=0.646,P<0.05)on day 3.There were positive relationship between the protein expression of Col-I and RhoA,Cdc42 (r=0.865, P<0.05;r=0.733, P<0.05) on day 1;so as for Col-I and RhoA,Cdc42,Rac1(r=0.825, P<0.01;r=0.744, P<0.05;r=0.586, P<0.05) on day 2; as well as Col-I and RhoA,Cdc42(r=0.841, P<0.01;r=0.665, P<0.05) on day 3.CONCLUSIONS:①MSCs could inhibit the proliferation and activation of HSC and induce apoptosis of HSC in vitro, so that reducing the synthesis of ECM and alleviate the hepatic fibrosis.②MSCs could down-regulate the expression of Col I mRNA and its protein in HSC, finally reducing the synthesis of ECM, which may be one of the possible molecular mechanism againist hepatic fibrosis.③HGF-induced MSC may be superior to MSC for anti-fibrosis and inhibitory proliferation in HSCs.更多还原