【作者】 王波；

【导师】 唐晓鹏；

【作者基本信息】 中南大学 ， 内科学， 2010， 硕士

【摘要】 背景及目的：我国急性肝功能衰竭发生率高,其病理特点是大块或亚大块肝细胞坏死,而残存肝细胞的增殖受到各种因素的抑制难以再生。原位肝移植是治疗终末期肝病的有效方法,但因供肝不足,价格昂贵,而且需要终身服用免疫抑制剂等诸多因素限制了其临床应用。干细胞移植作为辅助治疗方法是肝移植的重要补充。目前已经有大量实验研究证实干细胞在一定的条件下能够向肝细胞定向分化。本文主要观察脐血干细胞和自体骨髓干细胞共同移植治疗D-gal所致大鼠急性肝衰竭疗效,为临床干细胞共移植治疗肝衰竭提供实验依据。方法：1.采集,培养干细胞：采集大鼠脐血和骨髓后,在培养体系中加入特定的细胞因子：促肝细胞生长因子(hepatocyte growth factor, HGF),干细胞因子(Stem cell factor,SCF),观察细胞生长情况,培养21天后对细胞进行检测,采用免疫细胞化学检测培养后的细胞是否表达肝细胞特征产物白蛋白(ALB)和甲胎蛋白(AFP)。2.动物实验：D-gal 1.4g/kg腹腔注射建立大鼠急性肝衰竭模型。将建模后24小时存活的大鼠随机分成4组。每日(总共7天)分别经鼠尾静脉注入单独的脐血干细胞或骨髓干细胞以及混合后的脐血干细胞及骨髓干细胞或生理盐水0.8ml。观察4组大鼠存活率,肝功能及肝组织病理变化程度。结果：1.在SCF,HGF的诱导下,脐血干细胞和骨髓干细胞可以在体外扩增并分化为肝细胞。2.1.4g/kg D-gal所致肝衰竭鼠3天病死率达80%,组织学见大面积肝坏死,血清转氨酶,胆红素明显升高。而干细胞治疗组72小时存活率明显升高,肝组织病变明显减轻,血清转氨酶,胆红素明显降低。3.脐血干细胞和骨髓干细胞共同移植治疗组存活率(77%)高于脐血干细胞治疗组(57%)和骨髓干细胞治疗组(50%),统计学比较,肝功能及组织学改善也优于脐血干细胞治疗组和骨髓干细胞治疗组。结论：1.在SCF,HGF的诱导下,脐血干细胞和骨髓干细胞可以在体外扩增并分化为肝细胞。2.1.4g/kg D-gal可造成大鼠肝组织弥漫性损伤,不经治疗死亡率达80%以上。3.尾静脉分别注射骨髓干细胞,脐血干细胞,骨髓干细胞和脐血干细胞对D-gal致大鼠肝损伤均有一定的治疗作用。4.在肝功能和肝组织病理改变方面,骨髓干细胞和脐血干细胞联合治疗组优于单用骨髓干细胞治疗组和脐血干细胞治疗组。更多还原

【Abstract】 Background and Objective:At present the incidence rate of acute or chronic liver failure is still very high in our country, the pathological feature is mass necrosis of hepatocytes,liver regeneration is inhabited by many factors.OLT is an available effective therapy for terminal liver failure. However, the extensive application is restricted due to some factors, such as increasing shortage of donor livers, high cost, immunosuppresant using all life, and so on.Transplantation of hepatocytes is an important supplement for OLT as an auxiliary therapy. Many researches have proved that stem cell can differentiate into hepatocyte in certain condition. In the hope of providing new treatment clues, we observed the effects of bone marrow stem cell and umbilical cord blood stem cell on the restoration of acute liver injured by D-gal in rats.Methods:1.Selecting, Separating and cultivating stem cells:hepatocyte growth factor (HGF) and stem cell factor (SCF) were added into the culture medium. The growth of the cells were observed and human AFP, ALB were detected by immunohistochemistry. 2.Acute hepatic failure model were produced by injection of D-gal 1.4g/kg into the mice. Divided the mice which were still alive after 24h into four groups.Bone marrow stem cell, umbilical cord blood stem cell, NS,umbilical cord blood stem cell and bone marrow stem cell were injected into the animals with 0.5-0.8ml every day. All the rats were sampled at the appointed times and various target markers were determined as follows:hematoxylin and eosin (HE) staining, as well as the index changes of liver enzymology. The data were applied for evaluating the improvement of hepatic histological damage.Result:1.Bone marrow stem cell and umbilical cord blood stem cell which were added HGF and SCF can be passaged and expanded in vitro.2.The acute liver failure model induced by D-gal exhibited a lethal hepatic insult mimicking fulminant hepatitis, among which 15 mice were died with 3 days(with a survival rate of 20%):massive necrosis of hepatocellular parenchyma were observed histologically, the animal serum levels of aminotranferases were increased remarkably. Treatment with stem cell markedly improved the survival rate of 3 days, lessened the histological damage, significantly lowered the serum levels of aminotranferases.3.The survival rate of 3 days of the groups which were injected bone marrow stem cell and umbilical cord blood stem cell(77%) are higher than the groups which were injected umbilical cord blood stem cell(57%) and the groups which were injected Bone marrow stem cell(50%);The serum levels of aminotranferases were the lowest among three groups.Conclusions:1.Bone marrow stem cell and umbilical cord blood stem cell which were added HGF and SCF can be passaged and expanded in vitro.2.The survival rate of 3 days of the mice which were injected D-gal 1.4g/kg were less than 20% without any treatment, massive necrosis of hepatocellular parenchyma were observed histologically.3.All the stem cell can protect mice, repair the injured liver function and histological structure induced by D-gal to some extent.4.The effect of the group of injected bone marrow stem cell combined with umbilical cord blood stem cell was the best in all groups, which could repair the injured liver function and histological structure significantly更多还原