细胞因子诱导人脂肪间充质干细胞向平滑肌细胞分化的实验研究

发布时间：2018-03-29 21:01

本文选题：人脂肪间充质干细胞　切入点：TGF-β　出处：《山西医科大学》2009年硕士论文

【摘要】： 第一部分人脂肪间充质干细胞(ADMSCs)的体外分离、培养、鉴定及其生物学特性的观察目的:体外分离、培养、鉴定ADMSCs并对其生物学特性进行观察。方法:采用酶消化法和贴壁培养法分离培养ADMSCs并进行传代扩增,采用倒置相差显微镜观察细胞形态学特点,用透射电镜观察细胞的超微结构,用四氮唑蓝比色法绘制细胞生长曲线,通过流式细胞仪检测细胞表面抗原CD29、CD44、CD31和CD34的表达及细胞周期。结果:⒈原代培养的ADMSCs光镜下形态不规则,经传代纯化后细胞形态呈均一梭形生长,透射电镜示细胞较为幼稚、核大、核仁较为明显,常染色质多,异染色质少,细胞器少且结构和种类简单。 ⒉流式细胞仪检测显示第1代、第3代和第5代ADMSCs均高表达CD29和CD44,而第1代、第3代和第5代ADMSCs均不表达CD31,CD34在第1代和第3代ADMSCs呈弱阳性表达,第5代时转为隐性。 ⒊流式细胞仪检测结果显示已纯化的ADMSCs中G0/G1、S、G2/M的细胞分别占90.14%、3.77%和6.09%,提示只有少部分细胞处于对数增殖期,而大多数细胞处于静止期。 ⒋ADMSCs生长曲线呈“S”形,第1天至第3天为细胞生长潜伏期,第4天开始进入对数生长期,第10天达顶点。第二部分细胞因子体外诱导ADMSCs向平滑肌细胞的分化目的:体外利用细胞因子诱导ADMSCs向平滑肌细胞的分化。方法:利用不同的细胞因子诱导ADMSCs向平滑肌细胞的分化。采用免疫荧光法检测胞浆α平滑肌肌动蛋白(α-SMA)对平滑肌细胞进行鉴定并计算其诱导率,实验分为三组:⑴转化生长因子-β(TGF-β)诱导组:取已纯化的第3代ADMSCs,以2×103/cm2细胞密度接种于预先置有盖玻片的6孔板中,分为三个不同浓度的TGF-β诱导组和一个对照组,诱导组中分别添加TGF-β终浓度为1ng/ml、2.5ng/ml和5ng/ml;对照组中不添加TGF-β。 ⑵血小板衍生生长因子-BB(PDGF-BB)诱导组:取已纯化的第3代ADMSCs,以2×103/cm2细胞密度接种于预先置有盖玻片的6孔板中,分为三个不同浓度的PDGF-BB诱导组和一个对照组,诱导组分别添加PDGF-BB终浓度为5ng/ml、10ng/ml、20ng/ml;对照组中不添加PDGF-BB。⑶联合细胞因子诱导组:取已纯化的第3代ADMSCs,以2×103/cm2细胞密度接种于预先置有盖玻片的6孔板中,分为一个诱导组和一个对照组,诱导组联合添加TGF-β和PDGF-BB,两种细胞因子的浓度分别取前两组实验的最佳诱导浓度;对照组中不添加任何细胞因子。各组均于14d后结束培养,进行免疫荧光鉴定并计算每孔的诱导率[1],选取最佳的诱导方法,用倒置相差显微镜观察诱导后细胞的形态学特点,用透射电镜观察诱导后细胞超微结构的改变,用RT-PCR技术检测平滑肌细胞胞浆蛋白SM calponin、SMMHC、SM 22αmRNA的表达,对平滑肌细胞进行进一步的鉴定。结果:⒈光镜下观察经诱导后的细胞变得更狭长,由成纤维细胞状变为长梭状,胞膜清晰,无空泡,可重叠生长,融合后细胞形成“峰”和“谷”状,呈良好的去分化状态。免疫荧光法检测经诱导14d后部分细胞α-SMA表达阳性,说明有部分细胞分化为平滑肌细胞,而对照组中未发现α-SMA表达阳性的细胞。 ⒉免疫荧光结果显示,在TGF-β诱导组,5ng/ml剂量组诱导效率最高。在PDGF-BB诱导组,20ng/ml剂量组诱导效率最高。联合使用5ng/ml TGF-β和20ng/mlPDGF-BB诱导效率高于单独使用20ng/mlPDGF-BB剂量组,但低于单独使用5ng/ml TGF-β剂量组。在所有实验组中,诱导效率最高为5ng/ml TGF-β剂量组。 ⒊透射电镜观察显示诱导后的细胞胞浆内可见肌丝结构,周围细胞器较未诱导组的ADMSCs明显增多,可见核糖体、线粒体、粗面内质网等多种细胞器,细胞器较未诱导组的ADMSCs更加发达。 ⒋RT-PCR结果显示诱导后的细胞可检测到平滑肌细胞胞浆蛋白SM calponin、SMMHC和SM22α的阳性条带,对照组胞浆蛋白未见阳性条带。结论: ⒈成人脂肪组织中可以分离培养出具有多向分化潜能的间充质干细胞。 ⒉细胞因子TGF-β和PDGF-BB在体外均可诱导人脂肪间充质干细胞分化为平滑肌细胞,其中以5ng/ml TGF-β剂量诱导效率最高。
[Abstract]:In vitro isolation, culture, identification and biological characteristics of human adipose mesenchymal stem cells (ADMSCs)
Objective: to isolate, culture, identify and observe the biological characteristics of ADMSCs in vitro.
Methods: using enzyme digestion and adherent culture ADMSCs were cultured and passaged and amplified, the morphological features were observed by inverted phase contrast microscope, the cell ultrastructure was observed by transmission electron microscope, using MTT cell growth curve was drawn through the detection of cell surface antigen CD29, flow cytometry, and the expression of CD44. The cell cycle of CD31 and CD34.
Results: the primary cultured ADMSCs under light microscope, irregular shape, purified by passage after cells were spindle cell growth, TEM showed relatively naive, large nuclei and obvious nucleolus, euchromatin, heterochromatin, organelle structure and species less and simple.
The detection showed that the first generation, flow cytometry, the third and fifth generation ADMSCs were high expression of CD29 and CD44, and the first generation, third generation and fifth generation ADMSCs expressed CD31. The expression of CD34 was weakly positive in the first and third generation ADMSCs, the fifth generation is recessive.
Test results show that G0/G1 has been purified by flow cytometry, ADMSCs S, G2/M cells accounted for 90.14%, 3.77% and 6.09%, suggesting that only a few cells in the logarithmic growth phase, and most of the cells in the stationary phase.
The ADMSCs growth curve was "S", first days to third days for the cell growth incubation period, fourth days into the logarithmic growth phase, the tenth Tianda summit.
Differentiation of ADMSCs into smooth muscle cells induced by the second part of cytokine in vitro
Objective: to induce ADMSCs differentiate into smooth muscle cells by cytokines in vitro. Methods: ADMSCs induced differentiation into smooth muscle cells with different cytokines. The cytoplasm of alpha smooth muscle actin was detected by immunofluorescence (alpha -SMA) for identification of smooth muscle cells and calculate the induction rate, were divided into three groups: the transformation of growth factor beta (TGF- beta) induced group: take the third generation of ADMSCs have been purified, with 2 * 103/cm2 cells were inoculated in 6 well plates pre coverslips, divided into three different concentrations of TGF- beta induced group and a control group, were added to TGF- induced beta mediated group in the final concentration of 1ng/ml TGF-, 2.5ng/ml and 5ng/ml; beta does not add in the control group.
The platelet derived growth factor -BB (PDGF-BB) induced group: take the purified ADMSCs of the third generation, with 2 * 103/cm2 cells were inoculated in 6 well plates pre coverslips, divided into three different concentrations of PDGF-BB induced group and a control group, induction group were added to final concentration of PDGF-BB was 5ng/ml, 10ng/ml 20ng/ml; in the control group, without adding PDGF-BB., combined with cytokine induced group: the purified ADMSCs of the third generation, with 2 * 103/cm2 cells were inoculated in 6 well plates pre coverslips, divided into a treated group and a control group, induction group with the addition of TGF- beta and PDGF-BB, the best induction the concentration of two kinds of cytokines were taken in two groups before the experiment; do not add any cytokines in the control group. Each group was cultured in 14d after the end, identified by immunofluorescence assay and the induction rate of [1] per hole, select the best method of inducing, inverted The morphological characteristics of the cells after induction were observed by phase contrast microscope. The ultrastructural changes of the cells after induction were observed by transmission electron microscope. The expression of SM calponin, SMMHC, SM 22 alpha mRNA in smooth muscle cells was detected by RT-PCR technology, and further identification of smooth muscle cells was carried out.
Results: 1. Under the light microscope after induction the cells become more narrow, shaped by the fibroblasts were long spindle shaped, clear membrane, no vacuoles, overlapping growth and fusion of cells to form a "peak" and "Valley", is good to differentiation was detected by immunofluorescence after induced. 14d after cell alpha -SMA expression, indicating that some cells differentiate into smooth muscle cells, while the control group was not found in -SMA expression positive cells.
The immunofluorescence results showed that in the induction group TGF- beta, 5ng/ml group induced the highest efficiency. In the induction group PDGF-BB, 20ng/ml group induced the highest efficiency. The combined use of 5ng/ml TGF- beta and 20ng/mlPDGF-BB induced efficiency is higher than that of 20ng/mlPDGF-BB group used alone, but less than the use of 5ng/ml TGF- beta dose group alone. In all the experimental groups, induced the highest efficiency for the 5ng/ml TGF- beta dose group.
Observation, transmission electron microscopy showed that the induced cells in the cytoplasm of myofilament structure around the organelle is not induced by group ADMSCs increased significantly, visible ribosomes, mitochondria, endoplasmic reticulum and other organelles, cells than non induced group ADMSCs is more developed.
The RT-PCR results showed that the induced cells were detected in smooth muscle cells of cytoplasmic protein SM calponin positive bands of SMMHC and SM22 alpha protein, the control group no positive bands of cytoplasm.
Conclusion:
The adult adipose tissue can be isolated and cultured multipotent mesenchymal stem cells.
The cytokine TGF- beta and PDGF-BB in vitro can induce human adipose derived mesenchymal stem cells differentiate into smooth muscle cells, of which 5ng/ml TGF- beta dose induced the highest efficiency.

【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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