两种细胞来源的45,XOips细胞系比较及神经分化研究
发布时间：2018-09-07 11:44
【摘要】：目的特纳综合征(Turner Syndrome,TS)是一条X染色体全部或部分缺失所致的性染色体异常综合征,其具体发病机制目前并不清楚。诱导多能干细胞(Induced pluripotent stem cells,iPS cells)是通过人为方法过表达胚胎干细胞主要转录因子基因,重编程成体细胞而获得的一类“类胚胎干细胞”细胞,为人们探讨疾病发病机理提供了新的手段。本研究拟建立两种细胞(绒毛细胞及外周血单个核细胞)来源的45,XO诱导多能干细胞系,比较两种组织来源的细胞建系效率,并同时研究45,XO诱导多能干细胞从全能性细胞向神经细胞诱导分化的过程。为探讨特纳综合征的发病机制及X染色体功能提供较好的细胞模型。方法1.采用仙台病毒载体法将人Oct,Sox2,Klf4,c-Myc 4个基因转入到染色体核型为45,XO的绒毛细胞和外周血单个核细胞中,使其重编程为诱导多能干细胞。2.对诱导多能干细胞进行核型鉴定,用碱性磷酸酶染色、免疫荧光染色、畸胎瘤实验等对两株iPS细胞系的多能性和体内外分化能力进行鉴定。3.通过比较两种细胞重编程后出现干细胞克隆的时间长短以及克隆数目对两种细胞来源的建系效率进行比较。4.对iPSCs进行体外神经干细胞的诱导分化并比较诱导效率,进行免疫荧光染色;获得神经干细胞后进一步诱导其向神经元分化,进行免疫荧光染色鉴定。结果1.45,XO绒毛细胞在感染后第5天形态上发生变化,感染第15天有干细胞样克隆形成。45,XO外周血单个核细胞感染第9天有干细胞样克隆形成。机械法挑选建系,这些细胞能够长期在体外稳定传代并保持核型45,XO,维持自我更新。2.两株45,XO iPS细胞系分别传代进行碱性磷酸酶染色呈强阳性,免疫荧光染色显示SSEA3,SSEA4,Oct4阳性;诱导多能干细胞在体外自然分化可形成三胚层来源细胞;将克隆细胞注射于SCID小鼠皮下形成的畸胎瘤内出现三胚层样结构。3.1X10~5个感染后的两种类型的细胞接种在饲养层细胞上,在感染后25天进行碱性磷酸酶染色,外周血单个核细胞来源的得到300个阳性克隆,效率为0.300%。绒毛细胞得到25个克隆,效率为0.025%。经χ2检验P0.05,差异有统计学意义。4.对两株45,XO-iPSCs与一株正常人46,XX-iPSCs进行神经诱导分化,两株45,XO-iPSCs与一株46,XX-iPSCs体外分化神经干细胞免疫荧光结果显示神经干细胞特异性标记Nestin呈阳性,比较诱导效率无显著差异。继续将神经干细胞诱导分化为神经元细胞系,细胞免疫荧光检测结果显示神经元细胞特异性标记Tuj1、NeuN的表达为阳性。结论1.建立了两株分别来源于绒毛细胞及外周血单个核细胞的ips细胞系,两株ips细胞系在体外长期传代中能维持异常核型及干细胞多能性,可作为特纳综合征研究的细胞模型;2.比较两种细胞建系的效率发现利用外周血单个核细胞建立45,XO-i PSCs效率高于绒毛细胞,外周血单个核细胞可作为制备45,XO-iPSCs的理想细胞来源;3.两株45,XO-iPSCs在神经分化过程中,分化效率与正常的iPSCs相比无显著差异。
[Abstract]:Objective Turner Syndrome (TS) is a sex chromosome abnormality syndrome caused by complete or partial deletion of an X chromosome. The pathogenesis of TS is unclear. Induced pluripotent stem cells (iPS cells) are recombined by artificially overexpressing the major transcription factor genes of embryonic stem cells. A class of "embryonic stem cell-like" cells derived from adult cells provides a new way to explore the pathogenesis of diseases. In this study, we intend to establish 45, XO-induced pluripotent stem cell lines derived from two kinds of cells (villous cells and peripheral blood mononuclear cells), compare the efficiency of cell line-building from two kinds of tissue sources, and study the induction of 45, XO at the same time. Methods 1. Human Oct, Sox2, Klf4, c-Myc genes were transfected into chromosome 45, XO villous cells and peripheral blood mononuclear cells by Sendai virus vector method. Cells were reprogrammed as induced pluripotent stem cells. 4. Induction and differentiation of neural stem cells from iPSCs in vitro were compared and immunofluorescence staining was performed. After obtaining neural stem cells, they were further induced to differentiate into neurons and identified by immunofluorescence staining. On the fifth day of infection, stem cell-like clones were formed on the fifteenth day of infection, and stem cell-like clones were formed on the ninth day of infection of XO peripheral blood mononuclear cells. Phosphatase staining was strongly positive, immunofluorescence staining showed SSEA3, SSEA4, Oct4 positive; induced pluripotent stem cells differentiated spontaneously in vitro to form triembryonic cells; cloned cells were injected into the teratoma of SCID mice subcutaneously to form a triembryonic layer-like structure. After 25 days of infection, 300 positive clones were obtained from peripheral blood mononuclear cells with an efficiency of 0.300%. 25 clones were obtained from chorionic villi with an efficiency of 0.025%. By_2 test, P 0.05, the difference was statistically significant. 4. Two strains of 45, XO-iPSCs and a normal 46, XX-iPSCs were induced to differentiate into neurons, and two strains were 45. XO-iPSCs and a strain of 46,XX-iPSCs differentiated neural stem cells in vitro immunofluorescence results showed that the specific marker Nestin of neural stem cells was positive, and there was no significant difference in induction efficiency. Two iPS cell lines derived from chorionic villi and peripheral blood mononuclear cells were established. two iPS cell lines maintained abnormal karyotype and stem cell pluripotency during long-term passage in vitro, which could be used as cell models for Turner's syndrome. 2. comparing the efficiency of two cell lines, we found that peripheral blood mononuclear cells were used for the study of Turner's syndrome. The efficiency of XO-i PSCs was higher than that of chorionic villus cells. Peripheral blood mononuclear cells could be used as an ideal cell source for the preparation of 45,XO-iPSCs.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596
本文编号：2228169
[Abstract]:Objective Turner Syndrome (TS) is a sex chromosome abnormality syndrome caused by complete or partial deletion of an X chromosome. The pathogenesis of TS is unclear. Induced pluripotent stem cells (iPS cells) are recombined by artificially overexpressing the major transcription factor genes of embryonic stem cells. A class of "embryonic stem cell-like" cells derived from adult cells provides a new way to explore the pathogenesis of diseases. In this study, we intend to establish 45, XO-induced pluripotent stem cell lines derived from two kinds of cells (villous cells and peripheral blood mononuclear cells), compare the efficiency of cell line-building from two kinds of tissue sources, and study the induction of 45, XO at the same time. Methods 1. Human Oct, Sox2, Klf4, c-Myc genes were transfected into chromosome 45, XO villous cells and peripheral blood mononuclear cells by Sendai virus vector method. Cells were reprogrammed as induced pluripotent stem cells. 4. Induction and differentiation of neural stem cells from iPSCs in vitro were compared and immunofluorescence staining was performed. After obtaining neural stem cells, they were further induced to differentiate into neurons and identified by immunofluorescence staining. On the fifth day of infection, stem cell-like clones were formed on the fifteenth day of infection, and stem cell-like clones were formed on the ninth day of infection of XO peripheral blood mononuclear cells. Phosphatase staining was strongly positive, immunofluorescence staining showed SSEA3, SSEA4, Oct4 positive; induced pluripotent stem cells differentiated spontaneously in vitro to form triembryonic cells; cloned cells were injected into the teratoma of SCID mice subcutaneously to form a triembryonic layer-like structure. After 25 days of infection, 300 positive clones were obtained from peripheral blood mononuclear cells with an efficiency of 0.300%. 25 clones were obtained from chorionic villi with an efficiency of 0.025%. By_2 test, P 0.05, the difference was statistically significant. 4. Two strains of 45, XO-iPSCs and a normal 46, XX-iPSCs were induced to differentiate into neurons, and two strains were 45. XO-iPSCs and a strain of 46,XX-iPSCs differentiated neural stem cells in vitro immunofluorescence results showed that the specific marker Nestin of neural stem cells was positive, and there was no significant difference in induction efficiency. Two iPS cell lines derived from chorionic villi and peripheral blood mononuclear cells were established. two iPS cell lines maintained abnormal karyotype and stem cell pluripotency during long-term passage in vitro, which could be used as cell models for Turner's syndrome. 2. comparing the efficiency of two cell lines, we found that peripheral blood mononuclear cells were used for the study of Turner's syndrome. The efficiency of XO-i PSCs was higher than that of chorionic villus cells. Peripheral blood mononuclear cells could be used as an ideal cell source for the preparation of 45,XO-iPSCs.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596
【参考文献】
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1 徐龙勇;陈德桂;;组蛋白去甲基化酶研究进展[J];生命科学;2010年02期
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