人脐带间充质干细胞生物学特性及体内移植促进皮肤创面修复的实验研究

发布时间：2018-04-12 16:50

本文选题：间充质干细胞 + 脐带　；参考：《南昌大学》2008年硕士论文

【摘要】： 目的本研究旨在建立一种分离、培养扩增脐带源间充质干细胞(human umbilical cord derived mesenchymal stem cells,hUC-MSCs)的方法,通过对其基本生物学特性、体外诱导分化能力、绿色荧光蛋白(green fluorescent protein GFP)基因质粒对其标记示踪的可能性及体内移植促进皮肤创面修复的可行性等的研究,以期为组织工程种子细胞提供新的细胞来源,为烧伤、肿瘤等导致的皮肤缺损患者的临床治疗提供新思路。方法1、首先建立hUC-MSCs分离和培养扩增的方法并通过细胞形态学、细胞增殖的研究、细胞免疫表型分析及体外诱导分化能力的检测对其基本生物学特性进行研究并与生物学特性研究较多的脐血间充质干细胞(human umbilicalcord blood hUCB-MSCs)进行比较(骨髓间充质干细胞Bone marrow mesenchymal stem cells BM-MSCs的生物学特性研究最多,但本实验由于骨髓标本来源受限)。2、将已转化的GFP-N2大肠杆菌109菌种,进行质粒提取,脂质体(lipofectamine 2000)介导转染hUC-MSCs,分别于转染24h,96h后计算荧光的转染效率,以未标记的同期细胞作为对照。3、将已标记绿色荧光蛋白基因质粒的hUC-MSCs移植至表皮缺损的裸鼠表皮缺损处,采用组织切片、免疫荧光等方法检测裸鼠表皮愈合及hUC-MSCs在皮肤缺损处的分布。结果1、hUC-MSCs接种后24-48h贴壁,成纤维细胞样克隆形成(Unit-fibroblast like colony-forming,CFU-F)时间为5-7d,首次传代时间为12-16d,对数生长期细胞倍增时间为29h,均较hUCB-MSCs短。免疫表型分析显示hUC-MSCs和hUCB-MSCs均表达粘附分子和基质细胞标记CD13、CD29、CD105、CD166,不表达造血细胞标记CD14、CD34、CD45,不表达内皮细胞标记CD31,两者表型基本一致,无明显区别。此外,实验还证实hUC-MSCs具有向成骨细胞和脂肪细胞诱导分化能力。2、绿色荧光蛋白基因质粒转染的hUC-MSCs 24h后绿色荧光表达率为37%,而且短期内随着培养时间延长,表达率有上升趋势。3、实验组裸鼠大体观察表皮愈合较对照组好,组织切片见实验组皮肤结构层次较清晰而对照组大多数为疤痕愈合,结构层次不清楚,免疫荧光观察见hUC-MSCs在表皮缺损愈合的新生血管中有存活,对照组为阴性。结论1、成功建立了从人足月脐带中分离培养扩增hUC-MSCs的方法,操作简单、易行,分离培养的hUC-MSCs具有MSCs的基本生物学特性和免疫表型,且比hUCB-MSCs含量更丰富,具有更强的增殖能力,可以作为组织工程新的细胞来源。 2、脂质体介导的绿色荧光蛋白基因质粒转染hUC-MSCs标记示踪技术是一种理想的较稳定示踪标记技术,可以应用为组织工程种子细胞示踪的实验研究。 3、hUC-MSCs机体内移植能促进皮肤缺损的愈合,可为皮肤缺损患者的临床治疗提供理论基础。
[Abstract]:Objective to establish a method for isolating and amplifying human umbilical cord derived mesenchymal stem cells (hUC-MSCs) from umbilical cord derived mesenchymal stem cells.The possibility of labeling green fluorescent protein GFP gene plasmid and the feasibility of in vivo transplantation to promote the repair of skin wound were studied in order to provide a new cell source for tissue engineering seed cells and burn injury.The clinical treatment of skin defect caused by tumor provides a new idea.Methods 1.Firstly, the method of hUC-MSCs isolation and culture was established, and the cell proliferation was studied by means of cell morphology and cell proliferation.The basic biological characteristics of human umbilicalcord blood hUCB-MSCs were compared with those of cord blood mesenchymal stem cells (umbilicalcord blood hUCB-MSCs).The biological characteristics of Bone marrow mesenchymal stem cells BM-MSCs were studied most frequently.However, due to the limited origin of bone marrow specimens, the transformed strain of GFP-N2 Escherichia coli 109 was extracted and transfected into hUC-MSCs mediated by liposome lipofectamine 2000. The transfection efficiency was calculated after transfection for 24 h or 96 h, respectively.The hUC-MSCs labeled with green fluorescent protein gene plasmid was transplanted into the epidermal defect of nude mice with unlabeled synchronous cells as control. Tissue sections were used.The epidermal healing and the distribution of hUC-MSCs in skin defect of nude mice were detected by immunofluorescence.Results (1) 24 to 48 h after inoculation of hUC-MSCs, fibroblast-like like colony-forming CFU-F was found to be 5-7 days, the first passage time was 12-16 days, and the logarithmic cell doubling time was 29 h, which was shorter than that of hUCB-MSCs.Immunophenotypic analysis showed that both hUC-MSCs and hUCB-MSCs expressed adhesion molecule and stromal cell marker CD13, CD29, CD105, CD166, hematopoietic marker CD14, CD34, CD45, and endothelial cell labeled CD31. The phenotypes of the two were basically the same, but there was no significant difference between them.In addition, hUC-MSCs had the ability of inducing differentiation into osteoblasts and adipocytes. The green fluorescent expression rate of hUC-MSCs transfected with green fluorescent protein gene plasmid was 37% 24 hours after transfection.The expression rate showed an upward trend. The experimental group showed that the epidermal healing was better than that of the control group, and the skin structure of the experimental group was clear, while the control group was mostly scar healing, and the structure level was not clear.Immunofluorescence observation showed that hUC-MSCs survived in the neovascularization of epidermal defect healing, but negative in the control group.Conclusion 1. The method of isolating and amplifying hUC-MSCs from human umbilical cord has been successfully established. The method is simple and easy to operate. The isolated and cultured hUC-MSCs has the basic biological characteristics and immunophenotype of MSCs, and the content of MSCs is more abundant than that of hUCB-MSCs, and it has stronger proliferative ability.It can be used as a new cell source for tissue engineering.2. Liposome mediated green fluorescent protein gene plasmid transfection with hUC-MSCs labeled tracer technique is an ideal and stable tracer technique, which can be used as a tracer for tissue engineering seed cells.In vivo transplantation of hUC-MSCs can promote the healing of skin defects and provide a theoretical basis for the clinical treatment of skin defects.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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