骨髓间质干细胞抑制大鼠肝纤维化形成的可能性:受体肝内干细胞存活时间、肝星状细胞活化程度及生存分析的7周观察

Possibility of rat liver fibrogenesis inhibited by bone marrow-derived mesenchymal stem cells:a 7-week observation on survival time of stem cells and activation of hepatic stellate cells in liver of recipients and a survival analysis

目的:观察在体外培养条件下,具有强大的增殖和分化为肝细胞能力的骨髓间质干细胞输注治疗肝纤维化模型大鼠对其肝纤维化形成的影响及肝功能和生存分析,并对治疗组、空白对照组和病理对照组7周内的相关情况予以对照比较。方法:实验于2003-08/2004-10在中山大学基础医学院病理学于病理生理实验室完成。①动物分组:选用雄性6周龄Wistar大鼠5只为供体,供分离骨髓间质干细胞;雌性6周龄Wistar大鼠60只,其中50只被造成肝纤维化模型,在首次用二甲基亚硝胺处理后的第10天,将造模大鼠随机分为2组:骨髓间质干细胞治疗组10只(为受体),于治疗39d后结束实验,取材分析病理对照组40只。另10只作为空白对照组(仅静脉注射生理盐水)。②肝纤维化模型制作:在实验的当天,给予10g/L二甲基亚硝胺溶液(生理盐水配置)1mL/kg,腹腔注射,每周连续3次,共6周。③骨髓间质干细胞治疗组:每只大鼠输注3×106个骨髓间质干细胞;病理对照组:使用等体积生理盐水代替骨髓间质干细胞。④动物的一般生活状态:每天观察饮食、活动、二便、体质量、皮毛、呼吸情况等。⑤肝脏的组织细胞形态结构:将肝组织以苏木精-伊红染色后观察。⑥肝星形细胞及其活化程度检测:前者检测肝脏α-平滑肌肌动蛋白含量,用免疫组织化学染色法,后者以德国IBAS2.5图像分析软件半定量分析。⑦肝脏胶原分布的相对含量检测:采用Masson三色染色法,从而反映肝纤维化程度。⑧肝脏羟脯氨酸含量测定:采用氯胺T法。⑨血清总胆红素和谷丙转氨酶水平检测:采用全自动生化分析仪。⑩血清层粘蛋白和透明质酸水平测定:采用放射免疫法。瑏瑡雄性大鼠骨髓间质干细胞在雌性大鼠肝内的Y染色体性别决定区基因的表达检测:采用聚合酶链式反应。瑏瑢统计学分析:两组样本均数比较采用t检验;多组样本均数比较采用方差分析,两两比较采用q检验;生存分析采用Ka-plan-Meier法。结果:雌性大鼠60只进入结果分析。①实验动物生存状况:造模6周后,骨髓间质干细胞治疗组生存率为80%,而病理对照组生存率为10%,空白对照组生存率为100%,说明骨髓间质干细胞治疗可能通过阻止肝纤维化发展,从改而善大鼠的生存率。②肝脏组织学显示,骨髓间质干细胞治疗组的肝脏结构明显改善,炎症减轻、假小叶结构减少或消散。相反,病理对照组出现广泛的肝脏结构重塑,包括增厚的纤维间隔形成和明显的桥接坏死。半定量分析胶原染色,骨髓间质干细胞治疗组较病理对照组显著减低(8.12±1.98,15.71±2.13,t=6.11,P<0.05)。③肝脏星状细胞活化的标志α-平滑肌激动蛋白的阳性表达:空白对照组仅见血管壁有少量α-平滑肌激动蛋白的阳性表达,而病理对照组和骨髓间质干细胞治疗组可见在纤维间隔、肝窦和汇管区内明显表达。半定量分析α-平滑肌激动蛋白的阳性染色,骨髓间质干细胞治疗组较病理对照组显著减低(11.06±2.03,18.50±3.87,t=4.47,P<0.05)。④Y染色体性别决定区基因的表达:经雄性Wistar大鼠(供体)骨髓间质干细胞治疗39d的雌性大鼠,肝脏DNA经聚合酶链式反应检测结果显示,Y染色体性别决定区基因的表达阳性,而空白对照组和病理对照组则无此阳性信号。说明骨髓间质干细胞移植后,能够在受体肝内存活5周以上。⑤反映肝脏纤维化程度的肝脏羟脯氨酸含量及血清层黏蛋白和透明质酸水平:骨髓间质干细胞治疗组明显高于空白对照组(P<0.05)但明显低于病理对照组(P<0.05),说明骨髓间质干细胞治疗后能够减少肝脏胶原蛋白的含量,从而减少肝纤维化的形成,使肝纤维化得到控制。⑥反映肝功能的血清总胆红素、谷丙转氨酶水平:骨髓间质干细胞治疗组明显低于病理对照组(P<0.05),接近空白对照组。说明了骨髓间质干细胞治疗有助于改善肝功能状态。结论:①二甲基亚硝胺能成功诱导大鼠肝纤维化模型的建立。②骨髓间质干细胞可改善肝纤维化大鼠的生存状况和肝功能。③骨髓间质干细胞能够减少肝脏胶原蛋白的产生,抑制肝纤维化形成。④肝纤维化发生发展过程中活化态的肝星状细胞数量减少可能是骨髓间质干细胞抑制肝纤维化形成的原因之一。⑤雌性大鼠肝脏内存在雄性供体Y染色体性别决定区基因的信号说明骨髓间质干细胞能够在受体肝内存活5周以上。

AIM:To investigate the influence of infusion of bone marrow-derived mesenchymal stem cells (BMSCs) on the liver fibrogenesis in rats according to the strongly proliferative ability of BMSCs and their differential potency into hepatocytes under the condition of culture in vitro,analyze the liver function and survival of BMSCs,and perform a controlled comparison among treatment group,blank control group and pathological control group within 7 weeks. METHODS:The study was conducted in the laboratory of Department of Pathophysiology,College of Basic Medical Sciences,Sun Yat-sen University from August 2003 to December 2004.①Five male Wistar rats aged 6 weeks old as donors and MSCs were isolated from bone marrow of these rats for their culture and proliferation in vitro.Another 60 female Wistar rats aged 6 weeks were studied following the protocol.Fifty random rats were induced to liver fibrogenesis models with dimethylnitrosamine(DMN),and then they were randomly divided into 2 groups on the 10th day:BMSC treatment group(recipient,n=10) which finished experiment after 39-day treatment,and pathological control group(n=40) which was sampled to analyze pathologically.The rest 10 of the 60 rats were designed in blank control group which were intravenously infused with normal saline.②Establishment of liver fibrogenesis models:On the right day of experiment,10 g/L DMN(dispensed with saline) was intraperitoneally injected 1 mL/kg body mass 3 consecutive days of each week for 6 weeks.③Rats in the BMSC treatment group were infused with BMSCs at a dose of 3×106 cells intravenously,and those in the pathological control group were infused with the same volume of normal saline instead of BMSCs.④Common living status was detected including daily diet,activity,urine and stool,body mass,fur and breath.⑤Liver sections were stained with hematoxylin and eosin to observe the structure and form of liver histocytes.⑥The content of smooth muscle α-actin(α-SMA) of liver was detected by immunohistochemistry to observe hepatic stellate cells(HSCs).The activity of HSCs was analyzed semi-quantitatively by IBAS 2.5 imaging analysis software.⑦The relative content of the degree of liver collagen was assessed with Masson Trichrome(MT) staining so as to reflect the degree of liver fibrogenesis.⑧Hydroxyproline(Hyp) content of liver was assayed by chloramines-T method.⑨Levels of serum alanine aminotransferase aspartate(ALT) and serum total bilirub(TB) were assessed by using an automatic biochemistry analyzer.⑩Levels of laminin(LN) and hyaluronic acid(HA) in serum were tested by radioimmunoassay.{11} Sex determination region on the Y chromosome(sry) gene in male rats was explored by polymerase chain reaction(PCR) in the liver of female recipients.{12} Statistical analysis:Significance difference was determined by t test in two groups.Mean of multiple samples were compared by analysis of variance,and the comparison of random two samples assayed by q test.Analysis of survival was conducted based on the Kaplan-Meier method. RESULTS:Sixty female rats were analyzed in the result analysis.①After 6 weeks of modeling,infusion of BMSCs kept a higher survival rate of rats in the BMSC treatment group(80%) than rats in the pathological control group(10%),while rats in blank control group were all alive at the survival rate of 100%.It implied that infusion of BMSCs could improve the survival condition of rats through the inhibition of liver fibrogenesis.②Liver histology showed that structure of liver got obvious recovery,inflammation relieved and pseudolobules were reduced or dissipated in rats in the BMSC treatment group.In contrast,massive rearrangement of the liver structure including septal thick fibrosis and bridging necrosis was obviously shown in rats in the pathological control group.Semi-quantitative analysis of MT staining of liver sections demonstrated a significant decrease of collagen in BMSC treatment group as compared with that in pathological control group(8.12±1.98 vs 15.71±2.13,t=6.11,P< 0.05).③The positive expression of α-SMA(the represen