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

作　　者：赵东长[1] 雷俊霞[2] 陈蕊[1] 张秀明[1] 许瑞云[3] 李树浓[1] 项鹏[1] 

机构地区：[1]中山大学干细胞与组织工程研究中心,广东省广州市510080 [2]中山大学基础医学院病理生理学教研室,广东省广州市510080 [3]中山大学附属第三医院肝胆外科,广东省广州市510630

出　　处：《中国临床康复》2005年第22期52-55,i002,共5页Chinese Journal of Clinical Rehabilitation

基　　金：国家重点基础研究发展规划项目(2001CB509904);广东省科技攻关重大专项(2003A3020103);广州市科技攻关重大专项(2002U13E0011);国家自然科学基金(30100188)~~

摘　　要：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�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 ste

关 键 词：干细胞 骨髓细胞 肝纤维化/治疗 大鼠 二甲基亚硝胺 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]