脐血干细胞移植体内诱导分化为肝细胞的实验研究

发布时间：2019-02-15 01:26

【摘要】：目的通过脐血干细胞移植在体内诱导分化为肝细胞的实验研究,进一步为脐血干细胞向肝细胞的分化提供证据,证实脐血干细胞的可塑性,为将脐血干细胞移植应用于治疗终末期肝病的研究奠定基础。方法 ① 使用采血袋法采集脐血标本,分别采用改良HES(羟乙基淀粉)沉降分离法和Ficoll分离法两种方法分离脐血干细胞,比较分离前后有核细胞(nuclear cell,NC)数、分离后CD34~+细胞数、分离后有核细胞回收率及台盼蓝拒染率,了解分离后有核细胞的数量和质量,为脐血移植创造条件。 ② 选用BALB/C裸鼠,将其随机分成实验组和对照组,实验组再根据输注有核细胞数的不同分成A、B、C三组。采用Co~(60)治疗仪γ-射线对实验组和对照组进行亚致死剂量350cGy照射,照射后3小时内在无菌条件下由裸鼠尾静脉输入分离的人新鲜脐血有核细胞。实验A、B、C三组分别输入NC1.0×10~7个/只、2.0×10~7个/只、3.0×10~7个/只,对照组经尾静脉注入等体积无菌生理盐水。移植后第1、2、3、4和8周分别检测实验组和对照组外周血白细胞,移植后第4、6和8周通过流式细胞仪检测外周血人源性CD34~+和CD45~+细胞水平,了解人鼠细胞嵌合情况。移植后一个月对实验组A、B和对照组裸鼠分别按0.4ml/kg注射用玉米油稀释到100μl的四氯化碳(carbon tetrachloride,CCl_4),实验组C注射等体积生理盐水。通过CCl_4造成肝脏损伤,诱导脐血干细胞归巢到
[Abstract]:Objective to provide evidence for the differentiation of umbilical cord blood stem cells into hepatocytes by transplantation of umbilical cord blood stem cells in vivo, and to confirm the plasticity of cord blood stem cells. To lay a foundation for the application of umbilical cord blood stem cell transplantation in the treatment of end-stage liver disease. Methods (1) Cord blood samples were collected by blood bag method. Two methods, modified HES (hydroxyethyl starch) sedimentation method and Ficoll method, were used to isolate umbilical cord blood stem cells. The number of nucleated cells (nuclear cell,NC) before and after isolation was compared. The number of CD34~ cells after separation, the recovery rate of nucleated cells and trypan blue rejection rate after separation, the quantity and quality of nucleated cells after separation were understood to create conditions for cord blood transplantation. 2 BALB/C nude mice were randomly divided into experimental group and control group. The experimental group was divided into three groups according to the number of nucleated cells. The experimental group and control group were irradiated with sublethal dose of 350cGy with Co~ _ (60) therapeutic instrument. Within 3 hours after irradiation, human fresh cord blood nucleated cells were injected into nude mice 'tail vein in aseptic condition. NC1.0 脳 10 ~ 7 / mouse, 2.0 脳 10 ~ 7 / mouse and 3.0 脳 10 ~ 7 / mouse were injected into the control group respectively. The control group was injected with the same volume of aseptic saline through the caudal vein. Peripheral white blood cells (PBWs) in the experimental group and the control group were detected at 4 and 8 weeks after transplantation. The levels of human derived CD34~ and CD45~ cells were detected by flow cytometry at the 6th and 8th weeks after transplantation. The chimerism of human mouse cells was investigated. One month after transplantation, the experimental group and the control group were diluted to 100 渭 l carbon tetrachloride (carbon tetrachloride,CCl_4) with corn oil for 0.4ml/kg injection, while the experimental group C was injected with the same volume of normal saline. Liver injury induced by CCl_4 induces umbilical cord blood stem cells to homing
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329

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