人胎盘绒毛层间充质干细胞体外培养的优化及生物学特性的初步分析

发布时间：2018-07-03 02:15

本文选题：胎盘绒毛 + 间充质干细胞　；参考：《苏州大学》2009年硕士论文

【摘要】： 间充质干细胞(mesenchymal stem cell, MSCs)是一种具有自我更新和多向分化能力的干细胞,有着良好的临床应用价值。以往研究主要集中在人骨髓来源的间充质干细胞,但由于人骨髓来源的间充质干细胞的获得需要通过骨髓穿刺,而且其数量随年龄的增长而急剧下降,限制了人骨髓源间充质干细胞的应用。因此,越来越多的研究者开始寻找间充质干细胞的其他来源,例如外周血、脐带血、脐带、乳牙以及胎盘。胎盘作为孕妇妊娠后的废弃物,易于获得且对其研究和应用无伦理道德的争论,成为间充质干细胞来源的新的研究热点。2007年人胎盘源间充质干细胞研究国际会议对人胎盘源间充质干细胞的临床应用潜能给予了充分肯定。但是MSCs在胎盘组织中的分布以及如何有效地在体外获得和扩增是亟待解决的问题。为此,本研究采用常规方法获取人胎盘绒毛层间充质干细胞(Human placenta chorion derived MSCs,hCMSCs),在此基础上,对hCMSCs体外扩增的条件及生物学特性作初步分析,从而为实验研究和临床应用快速有效提供种子细胞奠定基础。第一部分hCMSCs的组织定位及体外分离培养与鉴定目的:确定hCMSCs的组织定位,从胎盘绒毛层组织体外分离培养hCMSCs,在此基础上诱导其向神经元样细胞和脂肪细胞分化,为体外优化培养hCMSCs提供实验参数。方法:采用免疫组织化学染色确定hCMSCs在胎盘绒毛层中的组织定位;选择性剪取胎盘绒毛层组织,经IV型胶原酶消化、贴壁和传代培养获得hCMSCs;流式细胞仪(flow cytometry,FCM)检测其表面标志;倒置显微镜观察细胞形态;采用多种诱导条件分别将其向脂肪细胞、神经元样细胞方向诱导以观察细胞的分化潜能。结果:(1)免疫组织化学染色结果显示,胎盘绒毛层中轴血管周围富有CD105、CD90阳性的间充质干细胞;(2)采用机械分离和IV型胶原酶消化法体外分离培养能有效获得hCMSCs;(3)倒置显微镜观察hCMSCs呈典型的成纤维样贴壁生长,流式细胞仪分析显示,hCMSCs高表达CD73、CD90和CD105,不表达CD14、CD34、CD45和HLA-DR;(4)hCMSCs向神经元样细胞诱导后,诱导细胞呈神经元样细胞形态,NSE免疫荧光染色呈阳性;(5)hCMSCs经成脂诱导2周后,胞内有明显的脂滴出现,油红染色呈阳性。结论:从人胎盘绒毛层组织富集hCMSCs,经机械分离和酶消化法结合,能有效地获得hCMSCs,流式细胞术及诱导分化结果提示获得的hCMSCs具有间充质干细胞标志及诱导分化能力,可以成为体外优化培养的种子细胞。第二部分细胞因子对hCMSCs体外促增殖作用及其生物学特性的影响目的:优化hCMSCs的体外培养条件,并对优化培养条件培养的hCMSCs生物学特性进行初步分析,为实验研究和临床应用快速有效地提供种子细胞奠定基础。方法:选择性剪取人胎盘绒毛层组织,通过0.1%IV型胶原酶消化,按常规培养方法培养两代后用于细胞增殖实验,MTT法检测下列不同浓度不同细胞因子:人白细胞介素-6(interleukin-6,IL-6)、激发型鼠抗人IL-6信号传导链GP130(glycoprotein130 , GP130)、人巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)、人干细胞生长因子(stem cell factor,SCF)、人Flt3配体(flt3 ligand,FL)、人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对hCMSCs的增殖作用;光学显微镜观察优化培养条件下培养的hCMSCs的细胞形态;流式细胞仪检测优化培养条件下培养的hCMSCs的细胞表型;油红染色及免疫荧光分别鉴定优化培养条件下培养的hCMSCs向脂肪细胞及神经元样细胞诱导分化的潜能;RT-PCR分析FL的受体flt3的基因表达。结果:(1)MTT法检测及细胞计数结果表明,比较不同浓度的不同细胞因子对hCMSCs体外增殖作用的影响,含bFGF、FL的培养条件均能有效地促进细胞的体外增殖,而FL的促增殖作用更强;(2)光学显微镜观察发现含bFGF、FL的培养条件培养的细胞呈典型的成纤维样贴壁生长;(3)流式细胞仪检测结果显示含bFGF、FL的培养条件培养的hCMSCs高表达CD73、CD90、CD105,不表达CD14、CD34、CD45和HLA-DR;(4)含bFGF、FL的培养条件培养的细胞经神经元样细胞诱导分化后NSE免疫荧光染色呈阳性;向脂肪细胞诱导后有脂滴的出现;(5)RT-PCR结果显示hCMSCs表达flt3的mRNA。结论:诸多生物因子中bFGF和FL具有较强的促hCMSCs体外增殖作用而FL作用更为明显,同时其能保持hCMSCs的细胞形态、细胞表型及多向分化潜能,因此FL可以作为体外培养hCMSCs的良好生长因子。本研究明确了人胎盘源间充质干细胞在胎盘绒毛层的组织定位,建立了有效分离、体外扩增hCMSCs的实验体系,在此基础上,证明了FL具有有效地促hCMSCs体外增殖的作用,而且hCMSCs表达FL的受体flt3,为下一步研究FL对hCMSCs促增殖作用的作用机制研究提供实验依据。
[Abstract]:Mesenchymal stem cell (MSCs) is a kind of stem cells with self-renewal and multidirectional differentiation, which has a good clinical value. Previous studies focused on mesenchymal stem cells derived from human bone marrow. However, the number of mesenchymal stem cells derived from human bone marrow needs to be punctured by bone marrow, and the number of mesenchymal stem cells from bone marrow is required. As the growth of age increases rapidly, it restricts the application of mesenchymal stem cells in human bone marrow. Therefore, more and more researchers have begun to find other sources of mesenchymal stem cells, such as peripheral blood, umbilical cord blood, umbilical cord, milk teeth, and placenta. Placenta as a pregnant waste of pregnant women, easy to obtain and the research and application of it. The International Conference on human placental mesenchymal stem cells (.2007) International Conference on human placental mesenchymal stem cells has been fully affirmed in the International Conference on human placental mesenchymal stem cells. However, the distribution of MSCs in the placental tissue and how to effectively obtain and amplify in vitro are urgent to be solved. The problem.
In this study, Human placenta chorion derived MSCs (hCMSCs) of human placental villi was obtained by conventional methods. On this basis, the conditions and biological characteristics of hCMSCs amplification in vitro were preliminarily analyzed, thus laying the foundation for the rapid and effective supply of seed cells in experimental research and clinical application.
Part one is tissue localization and isolation, culture and identification of hCMSCs.
Objective: to determine the tissue location of hCMSCs, to isolate and culture hCMSCs from the placental villus tissue in vitro, and to induce it to differentiate into neuron like cells and adipocytes on this basis, and provide experimental parameters for the optimization of hCMSCs in vitro. Methods: immunohistochemical staining was used to determine the location of hCMSCs in the placental villi; HCMSCs was obtained by IV type collagenase digestion, adherent and passages culture. Flow cytometry (flow cytometry, FCM) was used to detect the surface markers; inverted microscope was used to observe the cell morphology; the differentiation potential was observed by a variety of induction conditions to adipocytes and neuron like cells. Results: (1) The results of histochemical staining showed that CD105 and CD90 positive mesenchymal stem cells were rich around the villi in the placental villi; (2) hCMSCs was obtained by mechanical separation and IV collagenase digestion in vitro, and (3) inverted microscope observation showed that hCMSCs was a typical fibroid adherent growth, and the flow cytometry analysis showed that hCMSCs High expression of CD73, CD90 and CD105, did not express CD14, CD34, CD45 and HLA-DR; (4) after the induction of hCMSCs to neuron like cells, the cells were induced to be neuron like cells, and the NSE immunofluorescence staining was positive. (5) hCMSCs after 2 weeks of lipid induction, there were obvious lipid droplets in the cell and positive oil red staining. Conclusion: hCM from the human placental villi tissue is enriched hCM. SCs, through the combination of mechanical separation and enzyme digestion, can effectively obtain hCMSCs, flow cytometry and induced differentiation results suggest that the obtained hCMSCs has the markers of mesenchymal stem cells and the ability to induce differentiation, and can be the optimal culture of seed cells in vitro.
The second part is the effect of cytokines on proliferation and biological characteristics of hCMSCs in vitro.
Objective: to optimize the culture conditions of hCMSCs in vitro, and to make a preliminary analysis of the biological characteristics of hCMSCs in the optimized culture conditions, and lay the foundation for the rapid and effective supply of seed cells for experimental research and clinical application. Method: selectively cut the human placental villi tissue, digest the 0.1%IV collagenase, and cultivate two by the conventional culture method. After the cell proliferation test, the MTT method was used to detect the following different concentrations of cytokines: human interleukin -6 (interleukin-6, IL-6), the IL-6 signal conduction chain GP130 (glycoprotein130, GP130), the macrophage colony stimulating factor (macrophage colony-stimulating factor, M-CSF), and human stem cell growth factor (human stem cell growth factor) Ll factor, SCF), human Flt3 ligand (Flt3 ligand, FL), the proliferation of human basic fibroblast growth factor (basic fibroblast growth factor, bFGF); optical microscopy observation of cultured cell morphology under optimized culture conditions; flow cytometry to detect the phenotype of cultured cells under optimal culture conditions; oil red dye Color and immunofluorescence identified the potential of hCMSCs to induce differentiation of adipocytes and neuron like cells under optimized culture; RT-PCR analysis of FL receptor FLT3 gene expression. Results: (1) the results of MTT assay and cell count showed that the effects of different cytokines on the proliferation of hCMSCs in vitro were compared, including bFG The culture conditions of F, FL can effectively promote the proliferation of cells in vitro, and the proliferation promoting effect of FL is stronger. (2) optical microscope observation found that the cells containing bFGF, FL culture conditions were typical fibroid adherent growth; (3) flow cytometry results showed bFGF, FL culture conditions of hCMSCs high expression CD73, CD90, CD105. CD14, CD34, CD45 and HLA-DR were not expressed; (4) the cells cultured in bFGF, FL culture conditions were positive for NSE immunofluorescence staining after differentiation of neuron like cells, and the presence of lipid droplets after induction of adipocytes; (5) RT-PCR results showed that hCMSCs expressed FLT3 mRNA. conclusion: many biological factors have stronger promoter in vitro. The effect of FL is more obvious, and it can maintain the cell morphology, cell phenotype and pluripotent differentiation potential of hCMSCs, so FL can be used as a good growth factor for the culture of hCMSCs in vitro.
In this study, the tissue location of human placental mesenchymal stem cells in the placental villi was defined. An experimental system for effective isolation and amplification of hCMSCs in vitro was established. On this basis, it was proved that FL could effectively promote the proliferation of hCMSCs in vitro, and hCMSCs expressed FL receptor FLT3, which was the next step to study the effect of FL on the proliferation of hCMSCs. The mechanism research provides the experimental basis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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