脐带间充质干细胞转染HGF基因对肝损伤再生的研究

发布时间：2018-04-24 07:31

本文选题：间充质干细胞 + 脐带　；参考：《昆明医学院》2008年博士论文

【摘要】： 目的: 肝脏损伤后的再生和修复始终是肝脏疾病研究中重要的课题,应用干细胞对终末期肝病进行细胞移植治疗或是作为转基因治疗的细胞载体是目前研究的热点之一。但干细胞的来源始终是困扰进一步应用的原因。间充质干细胞是成体干细胞中的一种,分布广泛,具有成体干细胞的一切共有特征,并且目前研究证实其在体外具有很强的免疫抑制能力,其作用对象涉及了NK细胞、T淋巴细胞、B淋巴细胞和树突状细胞等几乎所有的免疫细胞。是目前首选的转基因移植的干细胞。最近几年发现从脐带基质中也可以培育出间充质干细胞(脐带间充质干细胞,UC-MSCs),这为今后间充质干细胞广泛的临床应用提供了一个极为简便充足的来源。但目前关于脐带间充质干细胞的特性的研究还较少,至今为止尚未见到UC-MSCs分化为肝细胞的报道。因此,本研究以UC-MSCs为载体细胞,携带HGF基因到受损的肝脏模型,观察其对肝损伤修复再生的作用。材料与方法: 1.人脐带间充质干细胞(UC-MSC)的分离和培养(Isolation and culture of humanUC-MSC) 脐带取自经父母授权同意的医院产科的足月产儿,均在取材后24小时内进行处理。首先,在无菌条件下用PBS冲洗去除样本血液,采用组织块培养法和酶消化法提取wharton's jelly的间充质干细胞。试验比较不同浓度的血清,不同的取材方法来取得的干细胞,并进行流式细胞仪检测和染色体核型分析。 2.用慢病毒载体的构建和肝细胞生长因子(HGF)和绿色荧光蛋白(GFP)基因的转染含人肝细胞生长因子cDNA的构建复合物PUC-SRct/HGF的NotⅠ片段被纯化并被插入到pcDNA3.1(invitrogen)中,继而从pcDNA3.1-HGF构建体中切除肝细胞生长因子的PMEⅠ片段并将其克隆入复制缺陷慢病毒载体pWPI载体,即pWPI-HGF。载体序列的方向和完整性通过测序证实。重组体慢病毒通过磷酸钙转染法瞬时转染入293T细胞中,pWPI和pAPI-HGF的滴定度通过转导的293T细胞的GFP表达量来评估。传代至第三代的MSC(间充质干细胞)在含4μg/mL硫酸鱼精蛋白(Sigma)的MOI 20内进行转导。间充质干细胞的转导效率通过在荧光显微镜(Nikon,Japan)下检测GFP的表达量来评估。用转染后的第三代细胞进行移植。 3.动物模型的建立及间充质干细胞移植治疗制作SD雄性大鼠CCL4急性肝损伤加部分肝切除模型,进行细胞移植试验。大鼠染毒24小时后行部分肝切除术,切除范围占全肝比例约35%,术前眼底采血以备测肝功。SHAM组施予除肝叶切除以外的类似手术操作作为阴性对照。移植治疗后血清酶学检测肝功能以及免疫组化检测绿色荧光蛋白阳性细胞的数目、形态和分布。western blot检测肝组织中肝细胞生长因子的表达情况。RT-PCR方法检测肝中肝细胞生长因子及相关基因表达。结论: 1,脐带基质中能够较容易的获得丰富的间充质干细胞,其细胞表面标志物与其他组织来源的间充质干细胞大致相同。该种间充质干细胞能为今后临床间充质干细胞的应用提供一条简便充足的来源; 2,慢病毒作为载体能够简便有效的转染肝细胞生长因子基因到脐带间充质干细胞并获得稳定的表达; 3,用部分肝切除术加腹腔单次注射CCL4的方法能够成功的建立大鼠肝损伤再生的复合型模型。并且该模型建立方法更简单,与临床情况吻合性更好; 4,将转染了肝细胞生长因子基因的脐带间充质干细胞移植到大鼠肝损伤再生复合型模型中,脐带间充质干细胞能够到达受损肝脏并表达肝细胞生长因子基因参与到受体肝损伤的修复和再生过程中,对肝脏的修复和再生过程产生有益的影响,其机理尚需要进一步的研究。
[Abstract]:Objective:
Regeneration and repair of liver injury has always been an important issue in the study of liver diseases. Cell transplantation for end-stage liver disease by stem cells is one of the hotspots of current research, but the source of stem cells is always the reason for the further application of stem cells. Mesenchymal stem cells are adult cells. A species of stem cells, widely distributed, has all the common characteristics of adult stem cells and has been proved to have strong immunosuppressive ability in vitro. The targets involved in almost all immune cells, such as NK cells, T lymphocytes, B lymphocytes and dendritic cells, are the first preferred transplants. Cell. Mesenchymal stem cells (umbilical cord mesenchymal stem cells, UC-MSCs) have been found in the umbilical cord matrix in recent years. This provides a very simple and sufficient source for the extensive clinical application of mesenchymal stem cells in the future. However, there are few studies on the characteristics of umbilical cord mesenchymal stem cells. So far, UC- has not yet been seen. MSCs is a report of hepatocyte differentiation. Therefore, this study uses UC-MSCs as the carrier cell, carrying the HGF gene into the damaged liver model, and observing its effect on the repair and regeneration of liver injury.
Materials and methods:
Isolation and culture of 1. human umbilical cord mesenchymal stem cells (UC-MSC) (Isolation and culture of humanUC-MSC)
The full-term birth of the umbilical cord in the obstetrics and Gynecology of the obstetrics and Gynecology which was authorized by their parents by their parents has been processed within 24 hours after taking the material. First, the sample blood was removed by PBS washing under the aseptic condition. The mesenchymal stem cells of wharton's jelly were extracted by tissue mass culture and enzyme digestion. The tests were made to compare the different concentrations of serum and different methods of sampling. Stem cells were obtained and analyzed by flow cytometry and karyotype analysis.
2. construction of lentiviral vector and transfection of hepatocyte growth factor (HGF) and green fluorescent protein (GFP) gene.
The Not I fragment of the complex PUC-SRct/HGF containing human hepatocyte growth factor cDNA was purified and inserted into the pcDNA3.1 (Invitrogen), and then the PME I fragment of the hepatocyte growth factor was removed from the pcDNA3.1-HGF construction and cloned into the replication defective lentivirus vector pWPI vector, that is, the direction and integrity of the pWPI-HGF. vector sequence. It was confirmed by sequencing that the recombinant lentivirus was transiently transfected into 293T cells through calcium phosphate transfection, and the titrations of pWPI and pAPI-HGF were evaluated by the GFP expression of the transduced 293T cells. The generation of MSC (mesenchymal stem cells) to third generations was transduced in MOI 20 of the 4 mu g/mL protamine (Sigma). Mesenchymal stem cells were transduced. The transduction efficiency was assessed by detecting the expression of GFP under Nikon (Japan). The third generation of transfected cells was transplanted.
3. establishment of animal models and transplantation of mesenchymal stem cells
A model of CCL4 acute liver injury and partial hepatectomy in SD male rats was made to carry out a cell transplantation test. The rats were treated with partial hepatectomy after 24 hours of exposure. The resection range accounted for about 35% of the whole liver, and the preoperative fundus blood sampling was used to measure the liver function in group.SHAM. Enzyme detection of liver function and immunohistochemistry detection of the number of green fluorescent protein positive cells, morphology and distribution of.Western blot to detect the expression of hepatocyte growth factor in liver tissue..RT-PCR method was used to detect the expression of hepatocyte growth factor and related genes in liver.
Conclusion:
1, abundant mesenchymal stem cells can be obtained easily in the umbilical cord matrix, and the cell surface markers are roughly the same as those of other tissue derived mesenchymal stem cells. This kind of mesenchymal stem cells can provide a simple and sufficient source for the application of mesenchymal stem cells in the future.
2, lentivirus can be used as a vector to transfect hepatocyte growth factor gene into umbilical cord mesenchymal stem cells effectively and obtain stable expression.
3, the method of partial hepatectomy and single intraperitoneal injection of CCL4 can successfully establish a compound model of rat liver injury regeneration, and the method is more simple and better anastomosed with the clinical situation.
4, the umbilical cord mesenchymal stem cells transfected with the hepatocyte growth factor gene are transplanted into the rat liver regeneration complex model. The umbilical cord mesenchymal stem cells can reach the damaged liver and express the hepatocyte growth factor gene in the repair and regeneration process of the recipient liver injury, which is beneficial to the repair and regeneration of the liver. The mechanism needs further study.

【学位授予单位】：昆明医学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

【引证文献】