腺病毒介导的突变型人胰岛素原基因转染人脐带间充质干细胞

发布时间：2018-06-14 00:13

本文选题：间充质干细胞 + 脐带　；参考：《中国协和医科大学》2008年博士论文

【摘要】： 研究背景:目前糖尿病已成为仅次于心脑血管病症和癌症的第三大死亡疾病,它是一种影响体内胰岛素和糖含量的疾病,共有两种主要类型,分别为Ⅰ型和Ⅱ型,其共同特征是胰岛β细胞损伤或不能产生足够的胰岛素或机体不能有效的利用胰岛素,所以胰岛素就被作为治疗糖尿病的特效药应用于临床。由于随时需要注射胰岛素,给患者带来了极大的不便,目前国际上对糖尿病患者进行胰岛移植和基因治疗研究成为了热点。但面临的主要问题是供体不足,免疫排斥,胰岛素基因的转染效率和表达调控等。人脐带来源的间充质干细胞可以较易获得和纯化,并且易于基因修饰,可以将外源性基因转染间充质干细胞,建立一种安全的基因治疗的“细胞载体”。研究表明间充质干细胞还有免疫抑制功能,它不但在体外可以抑制T淋巴细胞增殖反应,在体内实验中也表现出了类似免疫抑制剂的作用,所以利用它作为细胞载体可以避免免疫排斥问题。并且我们利用基因重组的方法将胰岛素原基因进行改造,使其可以在非β细胞(间充质干细胞)中高效表达。相信我们的研究可以为Ⅰ型糖尿病的治疗提供一些有力的实验依据。目的:将人胰岛素原基因进行两个部位的突变,通过腺病毒介导将其转染到人脐带间充质干细胞中,研究其表达分泌人胰岛素的能力。方法:应用RT-PCR的方法从健康流产胎儿胰腺组织中扩增pINS的cDNA序列,利用重叠延伸PCR方法对pINS基因在A链-C肽和C肽-B链的连接处进行两个部位的突变,产生Furin蛋白酶酶切位点。采用AdEasy~(TM)系统构建重组腺病毒pAdINS、pAdINS-M2和pAdGFP。分离培养人脐带间充质干细胞,并对其生长特性,免疫表型,分化能力及Furin蛋白酶的表达进行鉴定。pAdINS、pAdINS-M2和pAdGFP感染人脐带间充质干细胞后,利用荧光显微镜和流式细胞仪检测它们的感染效率,并确定最佳的感染复数(multiplicities of infection,MOI)。以感染复数为100的pAdINS、pAdINS-M2或pAdGFP感染人脐带间充质干细胞1、3、5、7、10天后,RT-PCR检测人胰岛素原基因在人脐带间充质干细胞中表达;Western blot检测成熟人胰岛素和人C-肽在人脐带间充质干细胞中的表达;免疫荧光实验检测感染后48小时人胰岛素在人脐带间充质干细胞中的表达:ELISA实验检测感染后1、3、5、7、10天人胰岛素和人C-肽的分泌。结果:自健康流产胎儿胰腺组织中扩增出364 bp的pINS cDNA,连接入穿梭质粒中,构建重组腺病毒。pAdINS-M2经扩增纯化后滴度达到2.57×10~(10) PFU/mL,pAdINS的滴度为1.25×10~(10)PFU/mL,对照病毒AdGFP为4.85×10~9 PFU/mL。成功的从脐带组织中分离培养出间充质干细胞,绘制生长曲线,约3天扩增一代。细胞周期检测,大部分细胞都处于G0-G1期(占94.39%),小部分处于G2-M期(4.15%)和S期(1.45%)。在合适的诱导培养条件下,分离培养的人脐带间充质干细胞能够分化为脂肪细胞和成骨细胞。应用RT-PCR方法在人脐带间充质干细胞中检测到了Furin蛋白酶的存在,表明我们构建的含Furin蛋白酶酶切位点的人胰岛素原基因在此细胞中可以被切割产生成熟的人胰岛素。通过重组腺病毒pAdINS、pAdINS-M2和pAdGFP以不同感染复数感染人脐带间充质干细胞,利用荧光显微镜和流式细胞仪检测感染效率,确定MOI 100为最佳的感染复数。以MOI=100的pAdINS、pAdINS-M2或pAdGFP感染人脐带间充质干细胞1、3、5、7、10天后,RT-PCR方法检测到在pAdINS和pAdINS-M2感染的UC-MSCs中人胰岛素原基因都有表达;Western blot的结果显示pAdINS感染的UC-MSCs表达人胰岛素原,而pAdINS-M2感染的UC-MSCs中检测到了人胰岛素和C.肽,pAdGFP感染的UC-MSCs中两者都没有检测到表达。免疫荧光也检测了pAdINS-M2感染后48小时人胰岛素和C-肽在人脐带间充质干细胞胞浆中表达:ELISA检测感染后1、3、5、7、10天培养上清中人胰岛素和C-肽的浓度。结果显示,自感染后24小时目的基因就开始表达,第3-5天表达量达高峰,一周后表达有所下降。与RT-PCR和Western blot结果一致。结论:成功构建含有Furin蛋白酶切位点的人胰岛素原基因腺病毒重组体(pAdINS-M2);分离培养了人脐带源间充质干细胞,并对其进行了鉴定;重组腺病毒pAdINS-M2感染人脐带间充质干细胞后,表达分泌成熟人胰岛素和C-肽。可以为Ⅰ型糖尿病的治疗提供一些有力的实验依据。
[Abstract]:Background: diabetes has become the third most fatal disease after cardio cerebrovascular disease and cancer. It is a disease that affects insulin and sugar content in the body. There are two main types, type I and type II, which are characterized by islet beta cell damage or failure to produce sufficient insulin or the body is not effective. Insulin is used as a special drug for the treatment of diabetes. Because of the need for insulin injection at any time, it has brought great inconvenience to the patients. Now, the international study of islet transplantation and gene therapy for diabetic patients has become a hot spot. But the main problem is the deficiency of donor, immune rejection, and pancreas. The transfection efficiency and expression regulation of islin gene. Mesenchymal stem cells from human umbilical cord derived mesenchymal stem cells can be easily obtained and purified, and are easily genetically modified. Exogenous genes can be transfected into mesenchymal stem cells to establish a "cell carrier" for a safe gene therapy. It is also shown that mesenchymal stem cells have immunosuppressive function, and it also shows that mesenchymal stem cells also have immunosuppressive function. It can not only inhibit the proliferation of T lymphocyte in vitro, but also show the effect of immunosuppressant in the experiment in vivo, so using it as a cell carrier can avoid the problem of immune rejection. And we use gene recombination method to modify the proinsulin gene, so that it can be used in non beta cells (mesenchymal stem cells). We believe our study can provide some powerful experimental basis for the treatment of type I diabetes. Objective: to transfect the human proinsulin gene in two sites and transfect it into human umbilical cord mesenchymal stem cells via adenovirus to study the ability to express human insulin. Methods: the application of RT-PCR. The cDNA sequence of pINS was amplified from the fetal pancreas of healthy abortion, and the pINS gene was mutated at the junction of A chain -C peptide and C peptide -B chain by overlapping extension PCR method, and the Furin protease tangent site was produced. The recombinant adenovirus pAdINS was constructed by AdEasy~ (TM) system, and the human umbilical cord mesenchyme was isolated and cultured. Mesenchymal stem cells, and identification of their growth characteristics, immunophenotype, differentiation ability and expression of Furin protease,.PAdINS, pAdINS-M2 and pAdGFP infected human umbilical cord mesenchymal stem cells, using fluorescence microscopy and flow cytometry to detect their infection efficiency, and determine the optimal number of multiplicities of infection (MOI). PAdINS, pAdINS-M2 or pAdGFP infected human umbilical cord mesenchymal stem cells for 1,3,5,7,10 days, RT-PCR detected human proinsulin gene expression in human umbilical cord mesenchymal stem cells; Western blot was used to detect the expression of mature human insulin and human C- peptide in human umbilical cord mesenchymal stem cells; immunofluorescence test detected the infection for 48 hours after infection. The expression of human insulin in human umbilical cord mesenchymal stem cells: ELISA test was used to detect the secretion of insulin and human C- peptide in 1,3,5,7,10 days after infection. Results: 364 BP pINS cDNA was amplified from the fetal pancreas tissue from healthy abortion and joined into the shuttle plasmid, and the recombinant adenovirus.PAdINS-M2 was constructed and purified to reach 2.57 x 10~ (10) P after amplification and purification. The titer of FU/mL, pAdINS was 1.25 x 10~ (10) PFU/mL. Compared with the virus AdGFP of 4.85 * 10~9 PFU/mL., mesenchymal stem cells were isolated and cultured from the umbilical cord tissue successfully. The growth curve was plotted for about 3 days. Cell cycle detection, most of the cells were in G0-G1 period (94.39%), and the small part was in G2-M phase (4.15%) and S period (1.45%). The cultured human umbilical cord mesenchymal stem cells can differentiate into adipocytes and osteoblasts under the induced culture conditions. The presence of Furin protease in human umbilical cord mesenchymal stem cells is detected by RT-PCR method, indicating that the human proinsulin gene, which is constructed with the Furin protease cutting site, can be cut in this cell. Human umbilical cord mesenchymal stem cells were infected by recombinant adenovirus pAdINS, pAdINS-M2 and pAdGFP with different infection complex numbers. The infection efficiency was detected by fluorescence microscopy and flow cytometry, and MOI 100 was the best complex number of infection. Human umbilical cord mesenchymal stem cells were infected with MOI=100 pAdINS, pAdINS-M2 or pAdGFP. 1,3,5,7,10 days later, the RT-PCR method detected the expression of human proinsulin gene in both pAdINS and pAdINS-M2 infected UC-MSCs; Western blot results showed that UC-MSCs expressed human proinsulin in pAdINS infection, while both human insulin and C. peptides were detected in UC-MSCs pAdINS-M2 infection, both of which were not detected. Immunofluorescence also detected the expression of insulin and C- peptide in human umbilical cord mesenchymal stem cell cytoplasm 48 hours after pAdINS-M2 infection: ELISA was used to detect the concentration of human insulin and C- peptide in the culture supernatant of 1,3,5,7,10 days after infection. The results showed that the target gene was expressed at the beginning of the infection 24 hours after infection, the expression level reached the peak at day 3-5, and a week later The results were in accordance with the results of RT-PCR and Western blot. Conclusion: the recombinant human proinsulin gene adenovirus recombinant (pAdINS-M2) containing Furin protease site was successfully constructed, and the human umbilical cord derived mesenchymal stem cells were isolated and cultured, and the recombinant adenovirus pAdINS-M2 infected human umbilical cord mesenchymal stem cells and expressed and secreted. Mature human insulin and C- peptide can provide some powerful experimental evidence for the treatment of type I diabetes.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R587.1;R346

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