定點整合hFⅧ的人胚胎干細胞定向誘導成間充質干細胞的研究
發(fā)布時間：2019-07-06 14:07
【摘要】：血友病A(hemophilia A, HA)是X染色體連鎖的單基因隱性遺傳病,其致病機理是患者有編碼人凝血Ⅷ因子(factor Ⅷ, FⅧ)基因的功能缺陷,目前該病是基因治療領域的研究熱點之一。本研究組前期已經(jīng)成功利用人核糖體基因區(qū)打靶載體(pHrneo)將人凝血因子VⅧ(hFⅧ)靶入到人胚胎干細胞(human embryonic stem cells, hESCs) rDNA區(qū),統(tǒng)一命名為ET5,再擬將ET5分化為適宜血友病A移植的特異組織細胞,以進行血友病A的基因治療研究。間充質干細胞(mesenchymal stem cells, MSCs)是一種具有多向分化潛能的成體干細胞,在自體、同種異體甚至異種異體體內,能有效修復甚至完全重建功能嚴重受損的組織器官而不被排斥,不易發(fā)生惡性轉化,已經(jīng)成為基因治療及組織修復等領域內的研究熱點。已有大量研究表明MSCS可成為血友病A的理想靶細胞。但是成體MSCs取材較困難,而且難以體外長期擴增培養(yǎng),從而一定程度上限制了MSCs在作為基因治療靶細胞中的應用。因此本研究擬建立hESCs定向誘導成MSCs的平臺,為獲取數(shù)量足夠的MSCs提供新的途徑；本研究將以ET5為對象,定向誘導成MSCs,并檢測分化后細胞中hFⅧ的表達情況,為后續(xù)血友病A基因治療的臨床前研究提供實驗基礎。 目的：初步建立hESCs定向誘導成MSCs的技術平臺,并研究由hESCs定向誘導的MSCs是否與骨髓來源MSCs的特性一致,以及外源基因在ET5誘導的MSCs中的表達情況。 方法：1.對ET5進行堿性磷酸酶染色檢測后,聯(lián)合bFGF,PDGF-BB等細胞因子誘導ET5向MSCs分化,并進行核型檢測；2.鑒定ET5誘導的MSCs在體外向成骨細胞、軟骨細胞和成脂細胞分化的能力；3.流式分析ET5、ET5誘導的MSCs以及骨髓來源MSCs表面標志物的表達情況；4. RT-PCR檢測ET5誘導成MSCs后Oct4、 Sox2和Nanog基因的表達變化；5RT-PCR檢測由ET5誘導的MSCs中hFⅧ在RNA水平的表達情況。 結果：1.ET5堿性磷酸酶染色陽性,表明ET5具備多潛能性,將ET5誘導成MSCs后,具有與骨髓來源的MSCs一致的細胞形態(tài),在體外長期培養(yǎng)傳代后仍保持核型正常；2.ET5誘導成的MSCs,在體外仍具備多向分化潛能,可分化成成骨細胞、軟骨細胞和成脂細胞；3.流式分析ET5誘導的MSCs和骨髓來源的MSCs表面標志物表達情況一致；4RT-PCR檢測定ET5誘導成MSCs后,Oct4、Sox2和Nanog基因表達下調；5.在ET5誘導的MSCs'中可檢測到外源基因hFⅧ在RNA水平的表達。 結論：本研究成功將定點整合hFⅧ的hESCs定向誘導成MSCs,并具有較高的誘導效率；外源基因hFⅧ能在定點整合的hESCs誘導而來的MSCs中表達。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
本文編號：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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
【參考文獻】
相關期刊論文 前1條
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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