HGF质粒诱导脐血干细胞向肝系细胞分化的研究

发布时间：2018-03-15 08:01

本文选题：脐带血　切入点：造血干细胞　出处：《南京医科大学》2006年博士论文　论文类型：学位论文

【摘要】：第一部分人脐血CD34~+造血干细胞的分离、纯化及鉴定目的：探讨人脐血CD34~+造血干细胞的分离、纯化及鉴定方法。方法：选择我院足月妊娠产妇，，产前检查HBsAg和抗-HCV均阴性，收集其脐血。密度梯度离心分离单个核细胞，再通过免疫磁式分选法(magnetic activating cell sorting，MACS)收集CD34~+的造血干细胞。纯化得到的干细胞直接用于细胞移植，或加入冻存液后置于液氮中备用。应用流式细胞术分析检测CD34~+造血干细胞的表面标志。结果：经密度梯度离心后，得到的MNC数约为每份脐血WBC的1／10。而每份MNC经纯化后，得到的CD34~+细胞数约为起始细胞数的1／100。流式细胞仪检测MNC和经纯化后CD34~+细胞的含量，每份标本CD34~+细胞的回收率波动于17.8～24.1％，其平均值为20.7％。纯化的人脐血CD34~+细胞经流式细胞仪检测，CD34~+阳性百分率均值为92％，最高为98％。结论：通过免疫磁式分选法可从人脐血中筛选出高纯度的CD34~+造血干细胞。第二部分 HGF质粒尾静脉注射促使体内高表达目的：采用质粒尾静脉快速输注获得HGF小鼠体内高表达。方法：抽提扩增HGF质粒，并酶切鉴定。将不同剂量的HGF质粒通过尾静脉快速注入6-8周的NOD／SCID小鼠体内，ELISA法检测
[Abstract]:The first part: isolation, purification and identification of CD34 ~ ~ hematopoietic stem cells from human umbilical cord blood. Objective: to study the isolation, purification and identification of human umbilical cord blood CD34 ~ hematopoietic stem cells. Methods: HBsAg and anti-HCV were negative in pregnant women in our hospital. The umbilical cord blood was collected and mononuclear cells were isolated by density gradient centrifugation. Then the CD34 ~ hematopoietic stem cells were collected by magnetic activating cell sorting method. The purified stem cells were directly used in cell transplantation. The surface markers of CD34 ~ hematopoietic stem cells were detected by flow cytometry. Results: the number of MNC obtained by density gradient centrifugation was about 1 / 10 of that of WBC in umbilical blood. After purification of MNC, the number of CD34 ~ cells obtained was about 1 / 100 of the number of initial cells. Flow cytometry was used to detect the content of MNC and purified CD34 ~ cells. The recovery rate of CD34~ cells in each specimen fluctuated from 17.8 to 24.1.The average value was 20.7.The percentage of CD34~ + in purified human umbilical cord blood detected by flow cytometry was 922 ~ + and the highest was 98 ~ +. Conclusion: high purity CD34 ~ hematopoietic stem cells can be screened from human umbilical cord blood by immunomagnetic sorting. The second part of HGF plasmid injection into caudal vein to promote high expression in vivo. Objective: to obtain high expression in HGF mice by rapid infusion of plasmid tail vein. Methods: HGF plasmids were extracted and amplified, and identified by enzyme digestion. HGF plasmids of different dosages were injected into NOD/SCID mice for 6-8 weeks by tail vein and detected by Elisa.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2

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