定点整合hFⅧ的人胚胎干细胞定向诱导成间充质干细胞的研究
发布时间：2019-07-06 14:07
【摘要】：血友病A(hemophilia A, HA)是X染色体连锁的单基因隐性遗传病,其致病机理是患者有编码人凝血Ⅷ因子(factor Ⅷ, FⅧ)基因的功能缺陷,目前该病是基因治疗领域的研究热点之一。本研究组前期已经成功利用人核糖体基因区打靶载体(pHrneo)将人凝血因子VⅧ(hFⅧ)靶入到人胚胎干细胞(human embryonic stem cells, hESCs) rDNA区,统一命名为ET5,再拟将ET5分化为适宜血友病A移植的特异组织细胞,以进行血友病A的基因治疗研究。间充质干细胞(mesenchymal stem cells, MSCs)是一种具有多向分化潜能的成体干细胞,在自体、同种异体甚至异种异体体内,能有效修复甚至完全重建功能严重受损的组织器官而不被排斥,不易发生恶性转化,已经成为基因治疗及组织修复等领域内的研究热点。已有大量研究表明MSCS可成为血友病A的理想靶细胞。但是成体MSCs取材较困难,而且难以体外长期扩增培养,从而一定程度上限制了MSCs在作为基因治疗靶细胞中的应用。因此本研究拟建立hESCs定向诱导成MSCs的平台,为获取数量足够的MSCs提供新的途径；本研究将以ET5为对象,定向诱导成MSCs,并检测分化后细胞中hFⅧ的表达情况,为后续血友病A基因治疗的临床前研究提供实验基础。 目的：初步建立hESCs定向诱导成MSCs的技术平台,并研究由hESCs定向诱导的MSCs是否与骨髓来源MSCs的特性一致,以及外源基因在ET5诱导的MSCs中的表达情况。 方法：1.对ET5进行碱性磷酸酶染色检测后,联合bFGF,PDGF-BB等细胞因子诱导ET5向MSCs分化,并进行核型检测；2.鉴定ET5诱导的MSCs在体外向成骨细胞、软骨细胞和成脂细胞分化的能力；3.流式分析ET5、ET5诱导的MSCs以及骨髓来源MSCs表面标志物的表达情况；4. RT-PCR检测ET5诱导成MSCs后Oct4、 Sox2和Nanog基因的表达变化；5RT-PCR检测由ET5诱导的MSCs中hFⅧ在RNA水平的表达情况。 结果：1.ET5碱性磷酸酶染色阳性,表明ET5具备多潜能性,将ET5诱导成MSCs后,具有与骨髓来源的MSCs一致的细胞形态,在体外长期培养传代后仍保持核型正常；2.ET5诱导成的MSCs,在体外仍具备多向分化潜能,可分化成成骨细胞、软骨细胞和成脂细胞；3.流式分析ET5诱导的MSCs和骨髓来源的MSCs表面标志物表达情况一致；4RT-PCR检测定ET5诱导成MSCs后,Oct4、Sox2和Nanog基因表达下调；5.在ET5诱导的MSCs'中可检测到外源基因hFⅧ在RNA水平的表达。 结论：本研究成功将定点整合hFⅧ的hESCs定向诱导成MSCs,并具有较高的诱导效率；外源基因hFⅧ能在定点整合的hESCs诱导而来的MSCs中表达。
[Abstract]:Hemophilia A (hemophilia A, HA) is a single gene recessive disease linked to X chromosome. The pathogenesis of hemophilia is that patients have functional defects encoding human coagulation factor VIII (factor VIII, F VIII) gene. At present, the disease is one of the research hotspots in the field of gene therapy. In the early stage, human coagulation factor V VIII (hF VIII) was successfully targeted into the (human embryonic stem cells, hESCs) rDNA region of human embryonic stem cells by using human ribosomal gene region targeting vector (pHrneo), which was named ET5, and differentiated into specific tissue cells suitable for hemophilia A transplantation in order to study the gene therapy of hemophilia A. Mesenchymal stem cell (mesenchymal stem cells, MSCs) is a kind of adult stem cell with multidirectional differentiation potential. In autologous, allogenic and even heterogeneic allogenes, it can effectively repair or even completely reconstruct the severely damaged tissues and organs without rejection, and is not easy to undergo malignant transformation. It has become a hot research topic in the field of gene therapy and tissue repair. A large number of studies have shown that MSCS can be an ideal target cell for hemophilia A. However, it is difficult to obtain adult MSCs, and it is difficult to amplify and culture it for a long time in vitro, which limits the application of MSCs in gene therapy target cells to a certain extent. Therefore, this study intends to establish a platform for directed induction of hESCs into MSCs to provide a new way to obtain a sufficient number of MSCs. In this study, ET5 was directed into MSCs, and the expression of hF VIII in differentiated cells was detected, which provided an experimental basis for the follow-up preclinical study of hemophilia A gene therapy. Aim: to establish a technical platform for directed induction of MSCs by hESCs, and to study whether the MSCs induced by hESCs is consistent with the characteristics of bone marrow-derived MSCs and the expression of foreign genes in ET5-induced MSCs. Method: 1. After alkaline phosphatase staining of ET5, the differentiation of ET5 into MSCs was induced by bFGF,PDGF-BB and other cytokines, and the karyotype was detected. 2. To identify the ability of ET5-induced MSCs to differentiate into osteoblasts, cartilage cells and adipocytes in vitro. The expression of MSCs induced by ET5,ET5 and the surface markers of bone marrow-derived MSCs were analyzed by flow cytometry. 4. The expression of Oct4, Sox2 and Nanog genes induced by ET5 into MSCs was detected by RT-PCR, and the expression of hF VIII in MSCs induced by ET5 was detected by 5RT-PCR. Results: 1.ET5 alkaline phosphatase staining was positive, indicating that ET5 had multipotential. After induction of ET5 into MSCs, it had the same cell morphology as bone marrow-derived MSCs, and remained normal after long-term culture and passage in vitro. MSCs, induced by 2.ET5 still had multidirectional differentiation potential in vitro and could differentiate into osteoblasts, cartilage cells and adipocytes. The expression of MSCs surface markers induced by ET5 was the same as that of MSCs from bone marrow by flow cytometry, and the expression of Oct4,Sox2 and Nanog genes was down-regulated by 4RT-PCR assay after ET5 induction into MSCs. 5. The expression of foreign gene hF VIII at RNA level could be detected in MSCs' induced by ET5. Conclusion: the hESCs of site-directed integration of hF VIII was successfully induced into MSCs, and had high induction efficiency, and the foreign gene hF VIII could be expressed in MSCs induced by site-directed integration of hESCs.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2511075
[Abstract]:Hemophilia A (hemophilia A, HA) is a single gene recessive disease linked to X chromosome. The pathogenesis of hemophilia is that patients have functional defects encoding human coagulation factor VIII (factor VIII, F VIII) gene. At present, the disease is one of the research hotspots in the field of gene therapy. In the early stage, human coagulation factor V VIII (hF VIII) was successfully targeted into the (human embryonic stem cells, hESCs) rDNA region of human embryonic stem cells by using human ribosomal gene region targeting vector (pHrneo), which was named ET5, and differentiated into specific tissue cells suitable for hemophilia A transplantation in order to study the gene therapy of hemophilia A. Mesenchymal stem cell (mesenchymal stem cells, MSCs) is a kind of adult stem cell with multidirectional differentiation potential. In autologous, allogenic and even heterogeneic allogenes, it can effectively repair or even completely reconstruct the severely damaged tissues and organs without rejection, and is not easy to undergo malignant transformation. It has become a hot research topic in the field of gene therapy and tissue repair. A large number of studies have shown that MSCS can be an ideal target cell for hemophilia A. However, it is difficult to obtain adult MSCs, and it is difficult to amplify and culture it for a long time in vitro, which limits the application of MSCs in gene therapy target cells to a certain extent. Therefore, this study intends to establish a platform for directed induction of hESCs into MSCs to provide a new way to obtain a sufficient number of MSCs. In this study, ET5 was directed into MSCs, and the expression of hF VIII in differentiated cells was detected, which provided an experimental basis for the follow-up preclinical study of hemophilia A gene therapy. Aim: to establish a technical platform for directed induction of MSCs by hESCs, and to study whether the MSCs induced by hESCs is consistent with the characteristics of bone marrow-derived MSCs and the expression of foreign genes in ET5-induced MSCs. Method: 1. After alkaline phosphatase staining of ET5, the differentiation of ET5 into MSCs was induced by bFGF,PDGF-BB and other cytokines, and the karyotype was detected. 2. To identify the ability of ET5-induced MSCs to differentiate into osteoblasts, cartilage cells and adipocytes in vitro. The expression of MSCs induced by ET5,ET5 and the surface markers of bone marrow-derived MSCs were analyzed by flow cytometry. 4. The expression of Oct4, Sox2 and Nanog genes induced by ET5 into MSCs was detected by RT-PCR, and the expression of hF VIII in MSCs induced by ET5 was detected by 5RT-PCR. Results: 1.ET5 alkaline phosphatase staining was positive, indicating that ET5 had multipotential. After induction of ET5 into MSCs, it had the same cell morphology as bone marrow-derived MSCs, and remained normal after long-term culture and passage in vitro. MSCs, induced by 2.ET5 still had multidirectional differentiation potential in vitro and could differentiate into osteoblasts, cartilage cells and adipocytes. The expression of MSCs surface markers induced by ET5 was the same as that of MSCs from bone marrow by flow cytometry, and the expression of Oct4,Sox2 and Nanog genes was down-regulated by 4RT-PCR assay after ET5 induction into MSCs. 5. The expression of foreign gene hF VIII at RNA level could be detected in MSCs' induced by ET5. Conclusion: the hESCs of site-directed integration of hF VIII was successfully induced into MSCs, and had high induction efficiency, and the foreign gene hF VIII could be expressed in MSCs induced by site-directed integration of hESCs.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
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1 ;Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro[J];World Journal of Gastroenterology;2005年22期
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