本文選題：人臍帶間充質(zhì)干細(xì)胞 + 心肌細(xì)胞�。� 參考：《遵義醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：探討人臍帶間充質(zhì)干細(xì)胞的培養(yǎng)及凍存復(fù)蘇的方法。初步探討人臍帶間充質(zhì)干細(xì)胞向心肌樣細(xì)胞誘導(dǎo)分化的方法及HLA-G蛋白在臍帶間充質(zhì)干細(xì)胞和心肌樣細(xì)胞中的表達(dá)。 方法：第4代人臍帶間充質(zhì)干細(xì)胞進(jìn)行傳代培養(yǎng),并觀察其形態(tài)。取第5代細(xì)胞進(jìn)行凍存3個(gè)月后復(fù)蘇細(xì)胞,顯微鏡下觀察細(xì)胞形態(tài)。取第8代細(xì)胞經(jīng)胰酶(0.125%)消化后接種于T75培養(yǎng)瓶中。待細(xì)胞生長達(dá)到70%-80%融合時(shí),用10μmol/L5-Aza進(jìn)行誘導(dǎo)24h,對(duì)照組用含10%FBS的α-MEM培養(yǎng)液培養(yǎng)。每2d換1次培養(yǎng)液,并在不同誘導(dǎo)階段進(jìn)行細(xì)胞形態(tài)學(xué)觀察。誘導(dǎo)第四周行免疫細(xì)胞化學(xué),RT-PCR檢測。 結(jié)果：第4代人臍帶間充質(zhì)干細(xì)胞在傳代后24小時(shí)貼壁,一周時(shí)觀察到細(xì)胞已長滿瓶底。細(xì)胞呈長梭形或不規(guī)則形,大小不一,類似成纖維細(xì)胞。第5代細(xì)胞凍存后細(xì)胞的貼壁生長能力及形態(tài)與凍存前相同。誘導(dǎo)第2周可見部分細(xì)胞體積逐漸增大,呈球狀和桿狀細(xì)胞。誘導(dǎo)4周時(shí)經(jīng)免疫細(xì)胞化學(xué)可檢測到分化后的細(xì)胞表達(dá)心肌細(xì)胞標(biāo)記cTnT、Cx43和desmin, RT-PCR檢測到有心肌細(xì)胞標(biāo)記desmin的表達(dá)。誘導(dǎo)前后行免疫細(xì)胞化學(xué)法可觀察到臍帶MSCs及心肌樣細(xì)胞均表達(dá)HLA-G。 結(jié)論：人臍帶間充質(zhì)干細(xì)胞在體外具有較強(qiáng)的增殖能力,并能在5-Aza誘導(dǎo)下分化為心肌樣細(xì)胞,表達(dá)心肌細(xì)胞的表面特異性標(biāo)志,人臍帶間充質(zhì)干細(xì)胞可做為作心肌細(xì)胞移植的來源。HLA-G在臍帶MSCs及心肌樣細(xì)胞表達(dá)持續(xù)陽性,并不因?yàn)槠浞只鴨适А?br/>[Abstract]:Objective: To investigate the culture of human umbilical cord mesenchymal stem cells and the methods of cryopreservation and resuscitation. The method of inducing differentiation of human umbilical cord mesenchymal stem cells to cardiac myocytes and the expression of HLA-G protein in umbilical cord mesenchymal stem cells and cardiac myocytes were discussed.
Methods: fourth generations of human umbilical cord mesenchymal stem cells were cultured and observed, and their morphology was observed. Fifth generation cells were recovered after 3 months of cryopreservation. The cell morphology was observed under microscope. Eighth generation cells were inoculated into T75 culture bottle after trypsin (0.125%). When the cell growth reached 70%-80% fusion, 24 cells were induced to induce 24. H, the control group was cultured with 10%FBS containing alpha -MEM culture. 1 cultures were changed per 2D, and the cell morphology was observed at different induction stages. The induction of immunocytochemistry and RT-PCR detection were induced for fourth weeks.
Results: the fourth generations of human umbilical cord mesenchymal stem cells were adhered to the wall for 24 hours after the passage, and the cells were observed at the bottom of the bottle at one week. The cells showed long shuttle or irregular shape, different size, similar to fibroblasts. The cell wall growth ability and shape of the cells after the fifth generation of cells were the same as that before the freezing. The volume of the cells was gradually induced to be visible for second weeks. CTnT, Cx43 and desmin were detected by immunocytochemistry at 4 weeks by immunocytochemistry. The expression of desmin in cardiac myocytes was detected by RT-PCR. The expression of HLA-G. in umbilical cord MSCs and myocardial like cells could be observed before and after induction by immunocytochemical method.
Conclusion: human umbilical cord mesenchymal stem cells have strong proliferation ability in vitro, and can differentiate into myocardial like cells under the induction of 5-Aza, and express the specific markers of the surface of cardiac myocytes. Human umbilical cord mesenchymal stem cells can be used as the source of.HLA-G in the umbilical cord MSCs and cardiac myocyte like cells to be the source of continuous positive expression of the human umbilical cord mesenchymal stem cells. Its differentiation is lost.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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