比较不同人脂肪源性间充质干细胞生物学特性差异的研究

发布时间：2018-03-14 05:25

本文选题：脂肪源性间充质干细胞　切入点：分化　出处：《中国人民解放军军医进修学院》2008年硕士论文　论文类型：学位论文

【摘要】： 背景:干细胞治疗急性心肌梗死是近年来研究的热点。脂肪源性间充质干细胞(adipose tissue-derived mesenchymal stem cells,ADMSCs)作为一种新的细胞工程的种子细胞因为取材方便、来源充足、更易被人们所接受等优点逐渐被大家接受。国内、外对于ADMSCs的研究还外于起步阶段,其治疗机制的基础研究还很有限,在临床应用前尚有许多问题有待解决。目前干细胞移植治疗心梗的可能机制主要有两种:1、MSCs可分化为心肌细胞。2、通过旁分泌生长因子(如HGF、VEGF)等来发挥作用。最近科学家们发现干细胞的分化能力存在性别差异,从雌性小鼠肌肉中提取的干细胞比雄性肌肉中提取的更易分化为身体组织。那么人干细胞是否也存在分化的性别差异呢?性别或其它个体因素(如年龄、是否有高血压、糖尿病、冠心病等)对于干细胞增殖、分化、旁分泌、抗凋亡能力等生物学特性是否有影响呢?这些问题的解决对于了解ADMSCs本身的生物学特性,指导ADMSCs基础研究和临床应用具有十分重要的意义。目的:一是要建立从人腹部皮下脂肪组织中分离、培养ADMSCs方法,并从表面标志物及多向分化潜能方面对其进行鉴定,为后续研究做准备。二是观察比较不同人ADMSCs增殖、分化、旁分泌、抗凋亡能力的差别,从细胞水平探索ADMSCs是否存在个体差异,为今后干细胞的基础研究及临床应用提供一种新的视角。内容和方法:根据以上目的,本研究分成为两个部分: 第一部分研究人ADMSCs的分离及体外培养;并通过对获取的细胞进行CD13、CD44、CD45、HLA-DR和VWF免疫组化细胞表面间充质干细胞相关标志的鉴定及成脂、成骨、成心肌多向分化潜能的检验来验证其是否为间充质干细胞。第二部分对第一部分获取的人ADMSCs分别采用MTT比色实验、ELIsA双抗体夹心法、流式细胞计数技术来比较不同人ADMSCs在增殖、旁分泌、分化及抗凋亡能力方面的差别。结果: 从人腹部皮下脂肪组织分离培养出长梭形细胞。这些细胞的细胞表面分子CD13、CD44皆阳性,CD45、HLA-DR、VWF均阴性;具有成脂、成骨、成心肌的多向分化能力。 MTT比色实验示:人ADMSCs由OD值描绘的生长曲线大致形状相似,呈S型。在第5d、7d、9d女性h-ADMSCs的OD值明显高于相应的男性h-ADMSCs(P＜0.01),同时年轻个体ADMSCs的OD值更高。经10umol/L 5-aza诱导24小时后,流式细胞技术示女性h-ADMScs的TNT的表达率普遍高于男性h-ADMSCs(27.3%±2.4%VS 18.9%±2.5%,P＜0.01),而且年轻个体的ADMSCs更容易被诱导并表达TNT。Elisa结果示:女性h-ADMSCs分泌VEGF和HGF的能力要强于男性h-ADMSCs,前者为1582±132 pg/10~6个细胞VS 1265±133 pg/10~6个细胞P＜0.01,后者为13316±1404 pg/10~6个细胞VS 11790±1541 pg/10~6个细胞P＜0.01。用含500umol/L H_2O_2的培养基诱导30分钟后,流式细胞技术示女性h-ADMSCs的凋亡率低于男性h-ADMSCs(18.68%±1.35%VS 25.21%±1.57%,P＜0.01),同时年轻个体的ADMSCs更容易被诱导凋亡。结论: 1.通过酶消化的方法成功地从人皮下脂肪组织分离培养出具有多向分化潜能的ADMSCs。 2.h-ADMSCs在增殖、分化、旁分泌、抗凋亡能力上存在性别差异。年龄也是影响h-ADMSCs增殖、分化和凋亡的一个重要因素。个体本身的冠心病、高血压、糖尿病状况并不会影响其ADMSCs的生物学特性。
[Abstract]:Background: stem cell therapy for acute myocardial infarction is a research hotspot in recent years. Adipose derived mesenchymal stem cells (adipose tissue-derived mesenchymal stem cells, ADMSCs) as a new seed cell for cell engineering because of convenient, abundant and easier to be accepted and other advantages is gradually accepted in China. Outside, for the research of ADMSCs is in the initial stage, the treatment mechanism of basic research is still very limited, which need to be solved before clinical application. At present, there are many problems stem cells transplantation mechanism mainly has two kinds: 1, MSCs can differentiate into myocardial cells.2 by paracrine growth factors (such as HGF VEGF, etc.) to play a role. Recently, scientists found that stem cell differentiation due to gender differences, extracted from female mice in muscle cells than male muscle extracted more easily divided into body tissues. So whether there is a gender difference in stem cell differentiation? Gender or other individual factors (such as age, whether hypertension, diabetes, coronary heart disease etc.) for stem cell proliferation, differentiation, paracrine, whether it will affect the biological characteristics of anti apoptosis ability? To solve these problems for understanding the biological characteristics of ADMSCs itself. Has very important significance in basic research and clinical application of ADMSCs guidance.
Objective: to establish a is isolated from human adipose tissue culture, ADMSCs method, and the surface markers and multilineage differentiation potential of their identification, for the further research. The two is to observe and compare the different ADMSCs proliferation, differentiation, paracrine, anti apoptosis ability difference, to explore whether there was ADMSCs individual differences in the cell level, provide a new perspective for the basic research and clinical application of stem cells in the future.
Content and method: according to the above purpose, this study is divided into two parts:
The first part of the isolation and in vitro culture of human ADMSCs; and through CD13, the cell CD44, CD45, HLA-DR and VWF and adipogenicdifferentiation, immunohistochemistry of cell surface mesenchymal stem cell associated markers identification of osteogenic differentiation potential, inspection to verify whether the myocardial mesenchymal stem cells.
The second part of the first part of the human ADMSCs is MTT colorimetric test, ELIsA double antibody sandwich method, flow cytometry technology to compare ADMSCs proliferation, paracrine, differentiation and anti apoptotic ability of different people.
Result:
The spindle cells from human abdominal subcutaneous adipose tissue were isolated and cultured. All the cell surface molecules CD13 and CD44 were positive, but CD45, HLA-DR and VWF were negative. They had the ability to differentiate into adipocytes, osteoblasts, and into cardiomyocytes.
MTT assay showed the growth curve of ADMSCs by depicting the general shape of OD is similar to that of a S type. In 5D, 7d, 9D was significantly higher than those of the female h-ADMSCs male h-ADMSCs (P < 0.01), while the higher ADMSCs od young individuals. By 10umol/L 5-aza after 24 hours of induction, the expression of flow cytometry shows female h-ADMScs TNT was generally higher than that of male h-ADMSCs (27.3% + 18.9% 2.4%VS + 2.5%, P < 0.01), and younger individuals are more likely to be induced by ADMSCs and expression of TNT.Elisa showed: female h-ADMSCs and HGF secretion of VEGF was better than that of male h-ADMSCs, the former is 1582 + 132 pg/10~6 cells VS 1265 + 133 pg/10~6 cell P < 0.01, the medium was 13316 + 1404 pg/10~6 11790 + 1541 pg/10~6 cell VS cell P < 0.01. with 500umol/L H_2O_2 induced apoptosis after 30 minutes, flow cytometry shows the rate is lower than the female h-ADMSCs Male h-ADMSCs (18.68% + 1.35%VS 25.21% + 1.57%, P < 0.01), while ADMSCs in young individuals was more likely to be induced to apoptosis.
Conclusion:
1. ADMSCs. was successfully isolated and cultured from human subcutaneous adipose tissue by enzymatic digestion.
There are gender differences in proliferation, differentiation, paracrine and anti apoptotic ability of 2.h-ADMSCs. Age is also an important factor affecting h-ADMSCs proliferation, differentiation and apoptosis. The individual's coronary heart disease, hypertension and diabetes do not affect the biological characteristics of ADMSCs.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【共引文献】