体外诱导人脂肪干细胞向内皮细胞分化的实验研究

发布时间：2018-01-11 06:12

本文关键词：体外诱导人脂肪干细胞向内皮细胞分化的实验研究　出处：《山西医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 第一部分人脂肪来源的间充质干细胞的生物学特性观察目的观察人脂肪间充质干细胞(human adipose-derived stem cells , hADSCs)的形态学,生长动力学及细胞表面标志抗原。方法采用酶消化法分离培养脂肪间充质干细胞,倒置相差显微镜下观察其生长和形态学变化;MTT比色法观察细胞增殖活性;流式细胞技术分别检测细胞周期和原代、第2代、第4代脂肪间充质干细胞表面抗原CD31、CD34、CD44、CD29的表达。结果1.在脂肪组织中存在能不断增殖的未成熟细胞,经多次传代后仍保留很强的增殖能力,倒置相差显微镜下观察呈成纤维细胞样。 2.MTT比色法证实其有很强的增殖活性,随传代降低不明显。 3.流式细胞周期检测显示G1期细胞占绝大多数,达到86.19%,S期为6.09%,表明大多数细胞处于静止期,是一群处于未分化状态的非定向细胞。 4.其形态学及表面标记物与骨髓间充质干细胞类似,间充质干细胞相关的表面抗原CD29、CD44随着传代表达水平逐渐增高,与原代细胞比较没有显著差异(p0.05),内皮细胞标志CD31阴性表达。造血干细胞标志CD34随细胞培养时间延长表达水平明显降低,与原代细胞比较有显著差异(p0.05)。第二部分体外诱导人脂肪干细胞向内皮细胞的分化目的体外诱导人脂肪间充质干细胞向内皮细胞的分化,探索其诱导条件,为组织工程学种子细胞的来源及其临床应用提供实验基础。方法酶消化法分离培养脂肪干细胞,当传至第3代时用内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(b-FGF)诱导其向内皮细胞分化。实验分为VEGF 50ng/ml加b-FGF 10ng/ml和VEGF 20ng/ml加b-FGF 10ng/ml两组,用流式细胞仪检测其表面标记物CD34、CD31的表达;在荧光显微镜下观察其吞噬乙酰化低密度脂蛋白(DiI-ac-LDL)和结合植物凝集素(FITC-Lectin)的能力;用细胞免疫荧光法检测Ⅷ因子的表达;用RT-PCR技术测定细胞中CD133mRNA和CD144mRNA的表达。结果1.流式细胞仪检测结果显示CD31、CD34表达水平随诱导时间的延长而逐渐增加,同一诱导时间的不同浓度组之间比较没有显著差异(p0.05),同一诱导浓度不同诱导时间组之间比较有显著差异(p0.05)。 2.荧光显微镜下观察到细胞吞噬DiI-ac-LDL显红色荧光,阳性率达94%以上;与Lectin结合显绿色荧光,阳性率达95%;双染色为橙色荧光,阳性率达90%以上。 3.荧光显微镜下可见大部分细胞胞浆内呈现绿色荧光,即胞浆内表达Ⅷ因子。 4.CD144mRNA表达水平随诱导时间的延长而逐渐增加,与空白对照组比较有显著差异(p0.05),CD133mRNA在诱导第4天有一定表达,第8天时表达降低,与空白对照组比较均有显著差异(p0.05)。结论 1.通过酶消化法可从人脂肪组织中获取间充质干细胞,其与骨髓间充质干细胞相似,具有很强的增殖分化能力,通过流式细胞技术检测到其表达间充质干细胞相关表面标志。 2. VEGF、b-FGF可诱导脂肪干细胞成内皮样细胞,且对VEGF浓度有很大的依赖性,随着诱导时间的延长其表面标志物由内皮祖细胞向成熟内皮细胞转变。其较内皮祖细胞容易获得,成为组织工程学种子细胞的选择之一。
[Abstract]:The biological characteristics of mesenchymal stem cells from the first part of human fat
Objective To observe the morphology, growth kinetics and surface marker antigen of human adipose-derived stem cells (hADSCs).
Methods by using enzyme digestion method cultured adipose derived mesenchymal stem cells, inverted to observe the growth and morphological changes under the microscope to observe cell proliferation; MTT colorimetric assay; flow cytometry was used to detect the cell cycle and the original generation, second generation, fourth generation of adipose derived mesenchymal stem cell surface antigen CD31, CD34. CD44, the expression of CD29.
Results 1., there were immature cells that could proliferate in adipose tissue. After repeated passage, they still had strong proliferative ability.
2.MTT colorimetric assay showed that it had strong proliferative activity and was not obvious with the decrease of passage.
3. flow cytometry showed that the majority of G1 cells were 86.19% and S 6.09%, indicating that most cells were at a quiescent stage, and they were a group of undirected cells.
4. the morphology and surface markers of bone marrow mesenchymal stem cells, mesenchymal stem cell associated surface antigens CD29, CD44 increased gradually with the passage of the level, and the primary cells showed no significant difference (P0.05), endothelial cell marker CD31 negative expression cells CD34 with cell culture. The extension of time. Significantly lower levels of stem, there was significant difference compared with the primary cells (P0.05).
Differentiation of human adipose stem cells into endothelial cells in vitro in the second part
Objective to induce the differentiation of human adipose derived mesenchymal stem cells into endothelial cells in vitro, and explore the induction conditions, so as to provide experimental basis for the source and clinical application of tissue engineering seed cells.
Isolation and culture of adipose derived stem cells by enzyme digestion method, when passed third generations with endothelial cell growth factor (VEGF) and basic fibroblast growth factor (b-FGF) induced to differentiate into endothelial cells. The experiment was divided into VEGF 50ng/ml and b-FGF 10ng/ml and VEGF 20ng/ml and b-FGF 10ng/ml in the two groups, with the detection of surface markers CD34 flow cytometry, the expression of CD31; observe the phagocytosis of acetylated low density lipoprotein under fluorescent microscope (DiI-ac-LDL) and binding lectin (FITC-Lectin) expression ability; immunofluorescence assay of factor VIII; Determination of the expression of CD133mRNA and CD144mRNA cells by using RT-PCR technology.
The test results of 1. flow cytometry showed that CD31 expression level of CD34, with the prolonged induction time gradually increased, there was no significant difference between different concentration groups at the same time (P0.05), induced by the same concentration induced by different induction time had significant difference between groups (P0.05).
2. under fluorescence microscope, phagocytosis of DiI-ac-LDL showed red fluorescence. The positive rate was over 94%. The positive rate of green fluorescence with Lectin was 95%, and the double staining was orange fluorescence, the positive rate was over 90%.
3. under the fluorescence microscope in the cytoplasm of most cells showed green fluorescence expression of factor VIII in the cytoplasm.
The expression level of 4.CD144mRNA increased with the prolongation of induction time. There was a significant difference between the expression level of P0.05 and the blank control group (CD133mRNA). There was a certain expression of CD133mRNA on the fourth day of induction, and the expression decreased on the eighth day, which was significantly different from that in the blank control group (P0.05).
conclusion
1., through enzyme digestion, mesenchymal stem cells can be obtained from human adipose tissue. It is similar to bone marrow mesenchymal stem cells, and has strong proliferation and differentiation ability. The related surface markers of mesenchymal stem cells can be detected by flow cytometry.
2. VEGF, b-FGF can induce adipose derived stem cells into endothelial like cells, and have a great dependence on the concentration of VEGF, with the extension of time the transformation induced by surface markers by endothelial progenitor cells into mature endothelial cells. The endothelial progenitor cells are easy to obtain, become one of the seed cells for tissue engineering.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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