白癜风患者成纤维细胞重编程为诱导多功能干细胞的研究

发布时间：2018-03-25 16:27

本文选题：白癜风　切入点：成纤维细胞　出处：《江苏大学》2016年硕士论文

【摘要】：目的:探讨在体外将白癜风患者白斑患处成纤维细胞重编程为诱导成多功能干细胞(induced pluripotent stem cells,i PSCs)的可能性,并进行形态学及表观遗传学方面的鉴定。方法:(1)获得白癜风患者白斑组织,通过贴壁法取得皮损处成纤维细胞(vitiligo lesional fibroblast,VLF),包装含有Oct4、Sox2、Klf4、c-Myc四种转录因子的逆转录病毒,感染VLF。在显微镜下观察感染病毒后VLF的形态变化。诱导至第12天后挑克隆传代;(2)对重编程出的细胞鉴定:碱性磷酸酶(alkaline phosphatase,AP)染色、细胞免疫荧光检测干性基因蛋白表达情况,实时荧光定量PCR检测内源性干性基因表达及外源性四因子表达,OCT4基因启动子区域去甲基化检测,染色体核型分析以及畸胎瘤形成分析试验。结果:1.重编程白癜风患者诱导多功能干细胞(1)重编程过程中第4天开始出现ES样克隆细胞,并且随培养天数增加长大,有自我生长能力;(2)传代后的VP-i PS(vitiligo patient specific-induced pluripotent stem cell,VP-i PS cell)细胞形状规则,细胞核排列紧密,细胞可见很高的核质比。2.鉴定白癜风患者诱导多功能干细胞(1)AP染色显示VP-i PS细胞为未分化状态;(2)细胞免疫荧光显示干性基因OCT4、NANOG、TRA-1-60、SSEA4蛋白水平表达阳性;(3)q RT-PCR检测内源性干性基因Oct4、Sox2、Nanog、h TERT、Rex1、DNMT3B、GDF3,与阴性对照比较表达水平显著升高(P0.05),检测外源性四因子基因与阳性对照比较表达水平显著下降(P0.05);(4)重亚硫酸盐测序显示VP-i PS细胞OCT4基因启动子区域为去甲基化状态,VLF细胞则为甲基化状态;(5)染色体核型分析显示VP-i PS细胞核型正常,未出现缺失等情况;(6)VP-i PS细胞可以在NOD/SCID小鼠体内形成畸胎瘤,具备向三个胚层分化的能力。结论:可以在体外将白癜风患者白斑患处成纤维细胞重编程为诱导多功能干细胞,并且具备干细胞表观遗传学特征及功能,为进一步研究干细胞治疗和白癜风提供了基础。
[Abstract]:Objective: To investigate the in vitro skin fibroblasts of patients with vitiligo leukoplakia reprogrammed into induced pluripotent stem cells (induced pluripotent stem cells, I PSCs) the possibility, and the morphology and apparent identification of genetics. Methods: (1) vitiligo patients get leukoplakia lesions, made fibroblasts by adherent method (vitiligo lesional fibroblast, VLF), Sox2, packaging containing Oct4, Klf4, c-Myc four transcription factor retrovirus, observe the morphological changes of VLF after infection under the microscope. VLF. infection induced twelfth days to pick cloning passage; (2) the identification of the cell reprogramming: alkaline phosphatase (alkaline phosphatase, AP) staining. The expression of stemness gene protein immunofluorescence detection, real-time PCR to detect the expressions of endogenous and exogenous gene expression of dry four factor, the promoter region of OCT4 gene Methylation detection, karyotype analysis and analysis of the formation of teratoma test. Results: 1. patients with vitiligo reprogramming induced pluripotent stem cells (1) reprogramming process appeared fourth days ES like clone cells, and increased with the days of culture grew up, self growth ability; (2) VP-i PS (after passage vitiligo patient specific-induced pluripotent stem cell, VP-i PS cell) cell shapes and nucleus cells arranged closely, high nucleocytoplasmic ratio.2. identification of patients with vitiligo induced pluripotent stem cells (1) AP VP-i PS staining showed that the cells in an undifferentiated state; (2) immunofluorescence indicated that dry gene OCT4, NANOG, TRA-1-60. The expression level of SSEA4 protein; (3) Q RT-PCR detection of endogenous dry gene Oct4, Sox2, Nanog, h, TERT, Rex1, DNMT3B, GDF3, expression level was significantly increased compared with the negative (P0.05), detection of exogenous factor four Compared with the positive control of gene expression level was significantly decreased (P0.05); (4) bisulfite sequencing analysis showed that VP-i PS cell OCT4 gene promoter region methylation, the methylation status of VLF cells is; (5) the chromosome karyotype analysis showed that VP-i PS cells did not appear to lack normal karyotype, etc.; (6) VP-i PS cells can form teratomas in NOD/SCID mice and have the capability to three differentiation. Conclusion: Patients with vitiligo leukoplakia affected area in vitro reprogramming of fibroblasts into induced pluripotent stem cells, stem cells and have epigenetic features and functions, provide the basis for further study of stem cell therapy and vitiligo.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R758.41

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