衰老的脐带间充质干细胞生物学特性及基因表达谱的研究

发布时间：2018-03-17 15:00

本文选题：脐带间充质干细胞　切入点：衰老　出处：《山东大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的： 1.观察并检测人脐带间充质干细胞体外培养衰老过程中细胞形态学、表型、生长动力学及诱导分化能力等生物学特性的改变； 2.检测人脐带间充质干细胞体外培养衰老过程中基因表达谱的改变并探讨其可能的机制。方法 1.细胞培养：用组织块贴壁法培养人脐带间充质干细胞,取第3代细胞作为对照组,连续培养到第15代的细胞为衰老组,倒置显微镜下观察细胞形态,并分别制成细胞爬片扫描电镜观察并拍照。 2.生长增殖：分别取对照组和衰老组细胞,接种于96孔培养板中,各设8复孔,培养6天。每日检测在450nm波长处测吸光值(OD值),绘制细胞生长曲线。 3.表型检测：对照组和衰老组细胞消化后充分吹打成单细胞悬液,使用流式细胞仪检测并分析两组细胞CD34、CD45、CD44和CD105等表型变化。 4.诱导分化能力鉴定：将对照组和衰老组细胞置于不同的诱导培养液中向成骨细胞及成脂细胞诱导分化,诱导分化14天后,进行固定,并分别进行相应的染色观察诱导分化差异。 5.基因表达谱分析：细胞洗涤后加入’Trizol试剂,提取总RNA,进行基因表达谱芯片杂交、检测和数据分析,并用DNA芯片扫描仪行微阵列扫描。将扫描结果进行标准化分析,与对照组相比log2Ratio|2(4-fold change)为差异表达,并进行GO及KEGG pathway分析。 6.PCR验证：根据基因表达谱的结果,选取了多个典型的差异基因进行实时定量PCR验证。检测对照组和衰老组细胞BAX, FADD, JAK2, IGF3BP5, PDK1, Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2等基因的差异表达。结果 1.细胞形态：对照组细胞呈典型的成纤维状,每平方毫米可达500个细胞；衰老组细胞个体差异较大,细胞变得宽大,每平方毫米仅有约150个细胞,衰老细胞核质比减小。扫描电镜下可见年轻组细胞形态饱满,表面微绒毛分布均匀,而衰老组细胞伪足增多,整体扁平。 2.增殖情况：对照组和衰老组生长曲线在接种后都有一个停滞期(12-24小时),然后进入增殖期,对照组细胞能更快的进入增殖期,并在第5天达到更高的细胞数量后进入平台期。而衰老组停滞期长,进入增殖期后第3天进入平台期,最终的细胞数量也少。 3.表型检测：根据流式细胞仪检测结果,对照组和衰老组细胞CD34和CD45均为阴性表达,CD44和CD105均为高表达,组间阳性率表达并无显著差异。 4.分化能力：在适当的分化条件诱导下,对照组和衰老组细胞均能向成骨和脂肪细胞分化：向成骨细胞诱导14天后大部分细胞出现钙化胞外基质,茜素红S染色阳性碱性磷酸酶染色阳性；向脂肪细胞诱导分化14天后约50%的细胞内出现油红O阳性脂肪颗粒。这些结果证明所获得的贴壁细胞符合间充质干细胞的特征,与对照组细胞相比,衰老组细胞诱导分化能力显著降低。 5.差异基因信号通路及基因聚类分析：将衰老组细胞与对照组差异表达的基因进行KEGG pathway分析(p0.05),可见衰老组显著上调通路有20条,其中包括自身免疫病、退行性疾病相关通路,下调通路49条,涉及通路包括合成代谢、细胞外基质、物质代谢和细胞增殖等。对41000+条基因进行检测,差异基因多达8000个,对显著差异的2000个基因进行聚类分析,显示差异基因涉及转录、翻译、生物合成、物质代谢、信号转导、细胞增殖、细胞迁移、细胞间连接等多方面。 6.PCR验证：荧光定量PCR示衰老组细胞与对照组细胞相比,BAX, FADD, JAK2, IGF3BP5, PDK1和MGMT等基因在衰老细胞中表达上调,而Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2和PAK2等基因表达下调。结论 1.第3代对照组细胞及第15代衰老组细胞具有增殖潜能、相同的细胞表型,并能向成骨、脂肪细胞诱导分化,但衰老组细胞在形态学、增殖速度和诱导分化的能力均较对照组降低。 2.体外培养人脐带间充质干细胞的过程中,细胞随传代次数的增加形态发生改变、增殖减慢、分化及合成代谢能力均减退,细胞趋于老化。 3.衰老组细胞与对照组细胞比较,下调表达的基因和信号通路涉及细胞增殖、细胞迁移、代谢、生物合成、细胞外基质、细胞连接等方面,而免疫紊乱及退行性相关基因表达明显上调。
[Abstract]:Objective:
1. to observe and detect the morphological changes, phenotypes, growth kinetics and differentiation ability of human umbilical cord mesenchymal stem cells in vitro.
2. the changes of gene expression profiles in human umbilical cord mesenchymal stem cells during the process of senescence in vitro were detected and the possible mechanisms were discussed.
Method
1. cell culture: human umbilical cord mesenchymal stem cells were cultured by tissue block adherence method. The third generation cells were taken as control group, and the fifteenth generation cells were continuously cultured into the aging group. The cell morphology was observed under inverted microscope, and then the cell slides were made and observed by SEM.
2. growth and proliferation: cells from control group and aging group were inoculated in 96 hole culture plate, and 8 holes were set up for 6 days. The daily absorbance (OD) value was measured at 450nm wavelength, and cell growth curve was drawn.
3. phenotypes: control group and aging group were digested fully into monoplast suspension, using flow cytometry and analysis of two groups of cells CD34, CD45, CD44 and CD105 phenotypes.
4. identification of differentiation ability: the control group and the aging group were induced to differentiate into osteoblasts and adipocytes in different induction medium, and then differentiated after induced differentiation for 14 days.
Expression analysis of 5. genes: after washing the cells' adding Trizol reagent, extraction of total RNA by microarray hybridization, detection and data analysis, and use the DNA chip scanner for microarray scanning. The scan results were standardized analysis, compared with the control group log2Ratio|2 (4-fold change) for differential expression, and analysis of GO the KEGG and pathway.
6.PCR validation: according to the results of gene expression profiles, a number of typical differentially expressed genes were selected for real-time quantitative PCR validation. The differential expression of BAX, FADD, JAK2, IGF3BP5, PDK1, Col- III, Col-, IV, SMAD2, SAMD4, SMAD2, etc. in control group and senescence group were detected.
Result
1. cell morphology: the control group cells were fibrous into typical, per square millimeter of up to 500 cells; cell aging group individual differences, cells become large, per square millimeter of only about 150 cells, aging cytoplasmic ratio decreases. Under a scanning electron microscope group young cells were plump, microvilli on the surface of uniform distribution, and aging group cell pseudopodia increased, the overall flat.
2. proliferation: control group and aging group growth curve have a period of stagnation after inoculation (12-24 hours), and then enter the proliferating cells in the control group, can quickly enter the proliferation period, and in fifth days to reach a higher number of cells into the platform. After the long period of stagnation and aging group, plateau in third days the proliferation after the final cell number is less.
3. phenotypic detection: according to the results of flow cytometry, CD34 and CD45 were negative in the control group and aging group. CD44 and CD105 were all highly expressed. There was no significant difference in the positive rate between the two groups.
4.: in the induction of differentiation ability of appropriate differentiated condition, control group and aging group cells were able to differentiate into osteoblasts and adipocytes: after 14 days of osteogenic induction. Most of the cells have calcified extracellular matrix, alizarin red S staining positive alkaline phosphatase staining; oil red O positive fat particle induced differentiation after 14 days about 50% to fat cell cells. These results demonstrated that the adherent cells with mesenchymal stem cell characteristics, compared with the control cells, aging group decreased significantly induced cell differentiation.
Analysis of 5. different gene signal pathway and gene clustering: a group of differentially expressed genes will be aging group with control cells by KEGG pathway analysis (P0.05), aging group was significantly up-regulated in 20 pathways were, including autoimmune diseases, degenerative disease pathway, down 49 pathways, including anabolic pathways involved in extracellular. The matrix, substance metabolism and cell proliferation. Detection of 41000+ gene, genetic differences of up to 8000, clustering analysis of 2000 genes showed significant differences, differences in genes involved in transcription, translation, biosynthesis, metabolism, signal transduction, cell proliferation, cell migration, cell junctions and other aspects.
6.PCR validation: fluorescent quantitative PCR showed that the expression of BAX, FADD, JAK2, IGF3BP5, PDK1 and MGMT increased in senescent cells compared with the control group, while Col-, III, Col-, IV, SMAD2, Col-, mRNA, and mRNA were down regulated.
conclusion
1., the third generation of the control group and the 15 generation of aging group had proliferative potential, the same cell phenotype, and could differentiate into osteoblasts and adipocytes, but the morphology, proliferation rate and differentiation ability of the aging group were all lower than those of the control group.
2. in the process of culturing human umbilical cord mesenchymal stem cells in vitro, cell morphology changes, proliferating and slowing down, the ability of differentiation and anabolism decrease, and cells tend to be aging as the number of passages increases.
3., compared with the control group, the down regulated genes and signaling pathways involved in cell proliferation, cell migration, metabolism, biosynthesis, extracellular matrix and cell connection.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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