本文選題：胎盤(pán)絨毛 + 間充質(zhì)干細(xì)胞��； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 間充質(zhì)干細(xì)胞(mesenchymal stem cell, MSCs)是一種具有自我更新和多向分化能力的干細(xì)胞,有著良好的臨床應(yīng)用價(jià)值。以往研究主要集中在人骨髓來(lái)源的間充質(zhì)干細(xì)胞,但由于人骨髓來(lái)源的間充質(zhì)干細(xì)胞的獲得需要通過(guò)骨髓穿刺,而且其數(shù)量隨年齡的增長(zhǎng)而急劇下降,限制了人骨髓源間充質(zhì)干細(xì)胞的應(yīng)用。因此,越來(lái)越多的研究者開(kāi)始尋找間充質(zhì)干細(xì)胞的其他來(lái)源,例如外周血、臍帶血、臍帶、乳牙以及胎盤(pán)。胎盤(pán)作為孕婦妊娠后的廢棄物,易于獲得且對(duì)其研究和應(yīng)用無(wú)倫理道德的爭(zhēng)論,成為間充質(zhì)干細(xì)胞來(lái)源的新的研究熱點(diǎn)。2007年人胎盤(pán)源間充質(zhì)干細(xì)胞研究國(guó)際會(huì)議對(duì)人胎盤(pán)源間充質(zhì)干細(xì)胞的臨床應(yīng)用潛能給予了充分肯定。但是MSCs在胎盤(pán)組織中的分布以及如何有效地在體外獲得和擴(kuò)增是亟待解決的問(wèn)題。 為此,本研究采用常規(guī)方法獲取人胎盤(pán)絨毛層間充質(zhì)干細(xì)胞(Human placenta chorion derived MSCs,hCMSCs),在此基礎(chǔ)上,對(duì)hCMSCs體外擴(kuò)增的條件及生物學(xué)特性作初步分析,從而為實(shí)驗(yàn)研究和臨床應(yīng)用快速有效提供種子細(xì)胞奠定基礎(chǔ)。 第一部分hCMSCs的組織定位及體外分離培養(yǎng)與鑒定 目的:確定hCMSCs的組織定位,從胎盤(pán)絨毛層組織體外分離培養(yǎng)hCMSCs,在此基礎(chǔ)上誘導(dǎo)其向神經(jīng)元樣細(xì)胞和脂肪細(xì)胞分化,為體外優(yōu)化培養(yǎng)hCMSCs提供實(shí)驗(yàn)參數(shù)。方法:采用免疫組織化學(xué)染色確定hCMSCs在胎盤(pán)絨毛層中的組織定位;選擇性剪取胎盤(pán)絨毛層組織,經(jīng)IV型膠原酶消化、貼壁和傳代培養(yǎng)獲得hCMSCs;流式細(xì)胞儀(flow cytometry,FCM)檢測(cè)其表面標(biāo)志;倒置顯微鏡觀察細(xì)胞形態(tài);采用多種誘導(dǎo)條件分別將其向脂肪細(xì)胞、神經(jīng)元樣細(xì)胞方向誘導(dǎo)以觀察細(xì)胞的分化潛能。結(jié)果:(1)免疫組織化學(xué)染色結(jié)果顯示,胎盤(pán)絨毛層中軸血管周?chē)挥蠧D105、CD90陽(yáng)性的間充質(zhì)干細(xì)胞;(2)采用機(jī)械分離和IV型膠原酶消化法體外分離培養(yǎng)能有效獲得hCMSCs;(3)倒置顯微鏡觀察hCMSCs呈典型的成纖維樣貼壁生長(zhǎng),流式細(xì)胞儀分析顯示,hCMSCs高表達(dá)CD73、CD90和CD105,不表達(dá)CD14、CD34、CD45和HLA-DR;(4)hCMSCs向神經(jīng)元樣細(xì)胞誘導(dǎo)后,誘導(dǎo)細(xì)胞呈神經(jīng)元樣細(xì)胞形態(tài),NSE免疫熒光染色呈陽(yáng)性;(5)hCMSCs經(jīng)成脂誘導(dǎo)2周后,胞內(nèi)有明顯的脂滴出現(xiàn),油紅染色呈陽(yáng)性。結(jié)論:從人胎盤(pán)絨毛層組織富集hCMSCs,經(jīng)機(jī)械分離和酶消化法結(jié)合,能有效地獲得hCMSCs,流式細(xì)胞術(shù)及誘導(dǎo)分化結(jié)果提示獲得的hCMSCs具有間充質(zhì)干細(xì)胞標(biāo)志及誘導(dǎo)分化能力,可以成為體外優(yōu)化培養(yǎng)的種子細(xì)胞。 第二部分細(xì)胞因子對(duì)hCMSCs體外促增殖作用及其生物學(xué)特性的影響 目的:優(yōu)化hCMSCs的體外培養(yǎng)條件,并對(duì)優(yōu)化培養(yǎng)條件培養(yǎng)的hCMSCs生物學(xué)特性進(jìn)行初步分析,為實(shí)驗(yàn)研究和臨床應(yīng)用快速有效地提供種子細(xì)胞奠定基礎(chǔ)。方法:選擇性剪取人胎盤(pán)絨毛層組織,通過(guò)0.1%IV型膠原酶消化,按常規(guī)培養(yǎng)方法培養(yǎng)兩代后用于細(xì)胞增殖實(shí)驗(yàn),MTT法檢測(cè)下列不同濃度不同細(xì)胞因子:人白細(xì)胞介素-6(interleukin-6,IL-6)、激發(fā)型鼠抗人IL-6信號(hào)傳導(dǎo)鏈GP130(glycoprotein130 , GP130)、人巨噬細(xì)胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)、人干細(xì)胞生長(zhǎng)因子(stem cell factor,SCF)、人Flt3配體(flt3 ligand,FL)、人堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF)對(duì)hCMSCs的增殖作用;光學(xué)顯微鏡觀察優(yōu)化培養(yǎng)條件下培養(yǎng)的hCMSCs的細(xì)胞形態(tài);流式細(xì)胞儀檢測(cè)優(yōu)化培養(yǎng)條件下培養(yǎng)的hCMSCs的細(xì)胞表型;油紅染色及免疫熒光分別鑒定優(yōu)化培養(yǎng)條件下培養(yǎng)的hCMSCs向脂肪細(xì)胞及神經(jīng)元樣細(xì)胞誘導(dǎo)分化的潛能;RT-PCR分析FL的受體flt3的基因表達(dá)。結(jié)果:(1)MTT法檢測(cè)及細(xì)胞計(jì)數(shù)結(jié)果表明,比較不同濃度的不同細(xì)胞因子對(duì)hCMSCs體外增殖作用的影響,含bFGF、FL的培養(yǎng)條件均能有效地促進(jìn)細(xì)胞的體外增殖,而FL的促增殖作用更強(qiáng);(2)光學(xué)顯微鏡觀察發(fā)現(xiàn)含bFGF、FL的培養(yǎng)條件培養(yǎng)的細(xì)胞呈典型的成纖維樣貼壁生長(zhǎng);(3)流式細(xì)胞儀檢測(cè)結(jié)果顯示含bFGF、FL的培養(yǎng)條件培養(yǎng)的hCMSCs高表達(dá)CD73、CD90、CD105,不表達(dá)CD14、CD34、CD45和HLA-DR;(4)含bFGF、FL的培養(yǎng)條件培養(yǎng)的細(xì)胞經(jīng)神經(jīng)元樣細(xì)胞誘導(dǎo)分化后NSE免疫熒光染色呈陽(yáng)性;向脂肪細(xì)胞誘導(dǎo)后有脂滴的出現(xiàn);(5)RT-PCR結(jié)果顯示hCMSCs表達(dá)flt3的mRNA。結(jié)論:諸多生物因子中bFGF和FL具有較強(qiáng)的促hCMSCs體外增殖作用而FL作用更為明顯,同時(shí)其能保持hCMSCs的細(xì)胞形態(tài)、細(xì)胞表型及多向分化潛能,因此FL可以作為體外培養(yǎng)hCMSCs的良好生長(zhǎng)因子。 本研究明確了人胎盤(pán)源間充質(zhì)干細(xì)胞在胎盤(pán)絨毛層的組織定位,建立了有效分離、體外擴(kuò)增hCMSCs的實(shí)驗(yàn)體系,在此基礎(chǔ)上,證明了FL具有有效地促hCMSCs體外增殖的作用,而且hCMSCs表達(dá)FL的受體flt3,為下一步研究FL對(duì)hCMSCs促增殖作用的作用機(jī)制研究提供實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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