人脐带间充质干细胞的分离、鉴定以及干细胞特异性转录因子诱导其重编程的研究

发布时间：2018-04-06 23:19

本文选题：脐带间充质干细胞　切入点：分离鉴定　出处：《浙江理工大学》2010年硕士论文

【摘要】： 背景诱导多能干细胞(induced pluripotent stem cell,iPSC)的出现不仅为人体组织工程绕开了胚胎干细胞的伦理障碍,而且为干细胞的研究提供了一种新思路新材料。然而,目前iPSC技术尚不成熟,高额的费用和极低的诱导成功率严重制约了iPSC的发展,因此寻求一种既经济又高效的诱导方法是目前亟待解决的科学难题。间充质干细胞(mesnchymal stem cells,MSCs)是体细胞分化较低的几类细胞之一,因其横向分化能力使其在成体干细胞的研究领域中备受瞩目。相比其他成体细胞,MSCs具有更强的可塑性,因此本课题使用已有的逆转录病毒可能更容易将其诱导成为多能干细胞。腺病毒载体(adenovirus vector)是一种具有高效转染能力的基因载体,因其容量大、产量高、毒副作用小等优点被广泛应用于基因治疗。本课题初步探索利用腺病毒载体携带Oct4、Sox2、Klf4、Nanog四个转录因子,能否诱导脐带间充质干细胞进行重编程,探索一条高效、经济的iPSC诱导方法,为临床应用和细胞治疗奠定基础。实验内容1)由人脐带中分离出间充质干细胞(UCMSCs),于体外培养并鉴定。2)利用已有逆转录病毒载体对其进行iPSC的诱导,并鉴定其相关指标。3)构建嵌合型腺病毒载体Ad5/F11b,且携带Oct4、Sox2、Klf4、Nanog四个转录因子基因。4)利用构建好的携带转录因子基因的嵌合型腺病毒Ad5/F11b感染UCMSCs,并尝试诱导出多能干细胞。实验结果1)从人脐带中分离出间质样细胞,经细胞周期、表面标记、分化潜能等指标确定其为间充质干细胞。2)已有的逆转录病毒可成功将所分离的脐带间充质干细胞诱导为人胚胎干细胞(hESCs)样克隆团细胞,检测发现在mRNA水平上4个转录因子均不同程度的上调表达,且与hESCs相关基因的表达水平也明显提高。可进一步检测其生化指标确定是否为iPSC。3)构建携带Oct4、Sox2、Klf4、Nanog四个转录因子基因的嵌合型腺病骨架质粒,经酶切鉴定确定为正确克隆,并于HEK293细胞中包装出病毒。4)利用携带4个转录因子的嵌合型腺病毒感染UCMSCs,出现类似hESCs的克隆团细胞,还需进一步证实iPSC的诱导情况。结论1)成功于人脐带中分离出间充质干细胞,并通过逆转录病毒诱导成hESCs样克隆团细胞,该细胞与hESCs相关的4个转录因子均不同程度的上调表达,表现出类似hESCs特征。 2)成功构建携带Oct4、Sox2、Klf4、Nanog四个转录因子基因的嵌合型腺病骨架质粒,并于HEK293细胞中包装出病毒。3)利用携带4个转录因子的嵌合型腺病毒感染UCMSCs后,出现类似hESCs的克隆团细胞,还需进一步证实iPSC的诱导情况。
[Abstract]:Background the emergence of induced pluripotent stem cells (pluripotent stem cell sci) has not only bypassed the ethical barriers of embryonic stem cells in human tissue engineering, but also provided a new idea and new material for the research of stem cells.However, at present, the iPSC technology is not mature, the high cost and the extremely low success rate of induction seriously restrict the development of iPSC, so it is an urgent scientific problem to seek an economic and efficient induction method.Mesenchymal stem cells (MSCs) is one of the types of cells with low somatic differentiation, which has attracted much attention in the field of adult stem cells because of its transversal differentiation ability.MSCs have stronger plasticity than other adult cells, so it may be easier to induce them into pluripotent stem cells by using existing retroviruses.Adenovirus (adenovirus) is a kind of gene vector with high efficiency of transfection. It is widely used in gene therapy because of its large capacity, high yield, low toxicity and so on.In this study, we preliminarily explored whether the adenovirus vector carrying four transcription factors of Oct4N Sox2Klf4HG Nanog could induce the reprogramming of umbilical cord mesenchymal stem cells, and explore an efficient and economical method of iPSC induction, which would lay a foundation for clinical application and cell therapy.(1) UCM SCS was isolated from human umbilical cord, cultured and identified in vitro. 2) iPSC was induced by retroviral vector.The chimeric adenovirus vector Ad5 / F11b was constructed and carried four transcription factor genes. 4) the constructed chimeric adenovirus Ad5/F11b containing transcription factor gene was used to infect UCMSCs and induce pluripotent stem cells.Results 1) Leydig cells were isolated from human umbilical cord.The differentiation potential of human embryonic stem cell like human embryonic stem cells can be induced by retrovirus, which has been identified as mesenchymal stem cell (MSCs. 2), and human embryonic stem cells (ESC)-like clone cells can be successfully induced by the isolated umbilical cord mesenchymal stem cells (UMSCs).It was found that the expression of four transcription factors was up-regulated at mRNA level, and the expression level of hESCs related genes was also significantly increased.The cytoskeleton plasmids carrying the four transcription factor genes of Oct4N, Sox2, Klf4 and Nanog could be constructed by further detecting the biochemical index of IPSC.3. The cytoskeleton plasmids of chimeric adenosis were identified by restriction endonuclease digestion and confirmed to be the correct clones.The chimeric adenovirus carrying four transcription factors was used to infect UCMSCs and clone cells similar to hESCs appeared in HEK293 cells. It is necessary to further confirm the induction of iPSC.Conclusion 1) Mesenchymal stem cells were successfully isolated from human umbilical cord and induced into hESCs like colony cells by retrovirus. The four transcription factors associated with hESCs were up-regulated in varying degrees and showed similar hESCs characteristics.2) the chimeric adenosis skeleton plasmids carrying the four transcription factor genes of Oct4G Sox2Klf4nanog were successfully constructed, and the virus was packaged in HEK293 cells. After UCMSCs was infected with the chimeric adenovirus carrying the four transcription factors, hESCs like clone cells appeared.Further confirmation of iPSC induction is needed.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【引证文献】