發(fā)布時(shí)間：2018-03-29 21:01

  本文選題：人脂肪間充質(zhì)干細(xì)胞　切入點(diǎn)：TGF-β　出處：《山西醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 第一部分人脂肪間充質(zhì)干細(xì)胞(ADMSCs)的體外分離、培養(yǎng)、鑒定及其生物學(xué)特性的觀察 目的:體外分離、培養(yǎng)、鑒定ADMSCs并對(duì)其生物學(xué)特性進(jìn)行觀察。 方法:采用酶消化法和貼壁培養(yǎng)法分離培養(yǎng)ADMSCs并進(jìn)行傳代擴(kuò)增,采用倒置相差顯微鏡觀察細(xì)胞形態(tài)學(xué)特點(diǎn),用透射電鏡觀察細(xì)胞的超微結(jié)構(gòu),用四氮唑藍(lán)比色法繪制細(xì)胞生長(zhǎng)曲線,通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞表面抗原CD29、CD44、CD31和CD34的表達(dá)及細(xì)胞周期。 結(jié)果:⒈原代培養(yǎng)的ADMSCs光鏡下形態(tài)不規(guī)則,經(jīng)傳代純化后細(xì)胞形態(tài)呈均一梭形生長(zhǎng),透射電鏡示細(xì)胞較為幼稚、核大、核仁較為明顯,常染色質(zhì)多,異染色質(zhì)少,細(xì)胞器少且結(jié)構(gòu)和種類簡(jiǎn)單。 ⒉流式細(xì)胞儀檢測(cè)顯示第1代、第3代和第5代ADMSCs均高表達(dá)CD29和CD44,而第1代、第3代和第5代ADMSCs均不表達(dá)CD31,CD34在第1代和第3代ADMSCs呈弱陽(yáng)性表達(dá),第5代時(shí)轉(zhuǎn)為隱性。 ⒊流式細(xì)胞儀檢測(cè)結(jié)果顯示已純化的ADMSCs中G0/G1、S、G2/M的細(xì)胞分別占90.14%、3.77%和6.09%,提示只有少部分細(xì)胞處于對(duì)數(shù)增殖期,而大多數(shù)細(xì)胞處于靜止期。 ⒋ADMSCs生長(zhǎng)曲線呈“S”形,第1天至第3天為細(xì)胞生長(zhǎng)潛伏期,第4天開(kāi)始進(jìn)入對(duì)數(shù)生長(zhǎng)期,第10天達(dá)頂點(diǎn)。 第二部分細(xì)胞因子體外誘導(dǎo)ADMSCs向平滑肌細(xì)胞的分化 目的:體外利用細(xì)胞因子誘導(dǎo)ADMSCs向平滑肌細(xì)胞的分化。方法:利用不同的細(xì)胞因子誘導(dǎo)ADMSCs向平滑肌細(xì)胞的分化。采用免疫熒光法檢測(cè)胞漿α平滑肌肌動(dòng)蛋白(α-SMA)對(duì)平滑肌細(xì)胞進(jìn)行鑒定并計(jì)算其誘導(dǎo)率,實(shí)驗(yàn)分為三組:⑴轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)誘導(dǎo)組:取已純化的第3代ADMSCs,以2×103/cm2細(xì)胞密度接種于預(yù)先置有蓋玻片的6孔板中,分為三個(gè)不同濃度的TGF-β誘導(dǎo)組和一個(gè)對(duì)照組,誘導(dǎo)組中分別添加TGF-β終濃度為1ng/ml、2.5ng/ml和5ng/ml;對(duì)照組中不添加TGF-β。 ⑵血小板衍生生長(zhǎng)因子-BB(PDGF-BB)誘導(dǎo)組:取已純化的第3代ADMSCs,以2×103/cm2細(xì)胞密度接種于預(yù)先置有蓋玻片的6孔板中,分為三個(gè)不同濃度的PDGF-BB誘導(dǎo)組和一個(gè)對(duì)照組,誘導(dǎo)組分別添加PDGF-BB終濃度為5ng/ml、10ng/ml、20ng/ml;對(duì)照組中不添加PDGF-BB。⑶聯(lián)合細(xì)胞因子誘導(dǎo)組:取已純化的第3代ADMSCs,以2×103/cm2細(xì)胞密度接種于預(yù)先置有蓋玻片的6孔板中,分為一個(gè)誘導(dǎo)組和一個(gè)對(duì)照組,誘導(dǎo)組聯(lián)合添加TGF-β和PDGF-BB,兩種細(xì)胞因子的濃度分別取前兩組實(shí)驗(yàn)的最佳誘導(dǎo)濃度;對(duì)照組中不添加任何細(xì)胞因子。各組均于14d后結(jié)束培養(yǎng),進(jìn)行免疫熒光鑒定并計(jì)算每孔的誘導(dǎo)率[1],選取最佳的誘導(dǎo)方法,用倒置相差顯微鏡觀察誘導(dǎo)后細(xì)胞的形態(tài)學(xué)特點(diǎn),用透射電鏡觀察誘導(dǎo)后細(xì)胞超微結(jié)構(gòu)的改變,用RT-PCR技術(shù)檢測(cè)平滑肌細(xì)胞胞漿蛋白SM calponin、SMMHC、SM 22αmRNA的表達(dá),對(duì)平滑肌細(xì)胞進(jìn)行進(jìn)一步的鑒定。 結(jié)果:⒈光鏡下觀察經(jīng)誘導(dǎo)后的細(xì)胞變得更狹長(zhǎng),由成纖維細(xì)胞狀變?yōu)殚L(zhǎng)梭狀,胞膜清晰,無(wú)空泡,可重疊生長(zhǎng),融合后細(xì)胞形成“峰”和“谷”狀,呈良好的去分化狀態(tài)。免疫熒光法檢測(cè)經(jīng)誘導(dǎo)14d后部分細(xì)胞α-SMA表達(dá)陽(yáng)性,說(shuō)明有部分細(xì)胞分化為平滑肌細(xì)胞,而對(duì)照組中未發(fā)現(xiàn)α-SMA表達(dá)陽(yáng)性的細(xì)胞。 ⒉免疫熒光結(jié)果顯示,在TGF-β誘導(dǎo)組,5ng/ml劑量組誘導(dǎo)效率最高。在PDGF-BB誘導(dǎo)組,20ng/ml劑量組誘導(dǎo)效率最高。聯(lián)合使用5ng/ml TGF-β和20ng/mlPDGF-BB誘導(dǎo)效率高于單獨(dú)使用20ng/mlPDGF-BB劑量組,但低于單獨(dú)使用5ng/ml TGF-β劑量組。在所有實(shí)驗(yàn)組中,誘導(dǎo)效率最高為5ng/ml TGF-β劑量組。 ⒊透射電鏡觀察顯示誘導(dǎo)后的細(xì)胞胞漿內(nèi)可見(jiàn)肌絲結(jié)構(gòu),周圍細(xì)胞器較未誘導(dǎo)組的ADMSCs明顯增多,可見(jiàn)核糖體、線粒體、粗面內(nèi)質(zhì)網(wǎng)等多種細(xì)胞器,細(xì)胞器較未誘導(dǎo)組的ADMSCs更加發(fā)達(dá)。 ⒋RT-PCR結(jié)果顯示誘導(dǎo)后的細(xì)胞可檢測(cè)到平滑肌細(xì)胞胞漿蛋白SM calponin、SMMHC和SM22α的陽(yáng)性條帶,對(duì)照組胞漿蛋白未見(jiàn)陽(yáng)性條帶。 結(jié)論: ⒈成人脂肪組織中可以分離培養(yǎng)出具有多向分化潛能的間充質(zhì)干細(xì)胞。 ⒉細(xì)胞因子TGF-β和PDGF-BB在體外均可誘導(dǎo)人脂肪間充質(zhì)干細(xì)胞分化為平滑肌細(xì)胞,其中以5ng/ml TGF-β劑量誘導(dǎo)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.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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