本文選題：間充質(zhì)干細(xì)胞 + 羊水��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的體外分離培養(yǎng)人羊水間充質(zhì)干細(xì)胞(HAFMSCs, human amniotic fluid mesenchymal stem cells),觀察其生物學(xué)特性。低溫凍存HAFMSCs三個(gè)月,觀察凍存后細(xì)胞的生物學(xué)特性,找出最佳的凍存方案。體外誘導(dǎo)HAFMSCs向內(nèi)皮細(xì)胞分化,分別在常氧及低氧環(huán)境下觀察其分化為內(nèi)皮細(xì)胞的能力。 方法采用四種方法分離培養(yǎng)HAFMSCs,用MTT法觀察各組細(xì)胞的增殖能力。流式細(xì)胞技術(shù)(FACS)檢測(cè)各組細(xì)胞表面抗原CD29, CD34, CD44, CD45, CD73, CD90, HLA-ABC, HLA-DR等的表達(dá)。比較各組細(xì)胞成脂、成骨的分化能力,及檢測(cè)各組細(xì)胞中OCT-4, Nanog等mRNA表達(dá)水平。用不同成分的凍存液凍存HAFMSCs,凍存三個(gè)月后復(fù)蘇細(xì)胞,并分析其存活率、生物學(xué)特性及分化能力。體外采用VEGF和bFGF聯(lián)合誘導(dǎo)HAFMSCs向內(nèi)皮細(xì)胞分化,FACS分析細(xì)胞vWF、CD31及CD133mRNA的表達(dá)水平,采用細(xì)胞免疫熒光的方法鑒定誘導(dǎo)分化后細(xì)胞vWF的表達(dá)情況,觀察誘導(dǎo)后細(xì)胞在Matrigel上形成毛細(xì)血管樣結(jié)構(gòu)的能力。比較低氧環(huán)境與常氧環(huán)境下上述指標(biāo)的差異。 結(jié)果四種不同的培養(yǎng)方法均培養(yǎng)出HAFMSCs。其中Chang培養(yǎng)基兩步培養(yǎng)法培養(yǎng)的細(xì)胞,其貼壁時(shí)間、細(xì)胞集落形成時(shí)間及細(xì)胞傳代時(shí)間較其余三組更短,培養(yǎng)1.5天即有細(xì)胞貼壁,4-5天可見細(xì)胞集落形成,細(xì)胞增殖快。四組細(xì)胞均表達(dá)間充質(zhì)干細(xì)胞標(biāo)記物CD29、CD44、CD73、CD90,而不表達(dá)造血干細(xì)胞標(biāo)記物CD34、CD45,符合間充質(zhì)干細(xì)胞的特征,各組之間無明顯差異。四組細(xì)胞成骨、成脂誘導(dǎo)成功,并分別通過油紅O染色及von Kossa染色證實(shí)。四組細(xì)胞中均檢測(cè)到OCT-4, Nanog mRNA的表達(dá)。凍存三個(gè)月后的HAFMSCs,以凍存液方案為DMEM:FBS:DMSO=5:4:1勺細(xì)胞存活率最高,細(xì)胞增殖能力最強(qiáng)。而復(fù)蘇后細(xì)胞的生長(zhǎng)曲線、細(xì)胞表型、分化能力及OCT-4, Nanog6勺表達(dá)與凍存前細(xì)胞相比無統(tǒng)計(jì)學(xué)差異。HAFMSCs體外誘導(dǎo)分化為內(nèi)皮細(xì)胞,流式細(xì)胞技術(shù)分析比較各組誘導(dǎo)后細(xì)胞中vWF, CD31,CD133等內(nèi)皮相關(guān)標(biāo)記物抗原的水平,以VEGF和bFGF聯(lián)合誘導(dǎo)組誘導(dǎo)效率最高,Mtrigel上形成毛細(xì)血管結(jié)構(gòu)的能力也最強(qiáng)。低氧環(huán)境下經(jīng)VEGF和bFGF聯(lián)合誘組HAFMSCs內(nèi)VEGF, v WF和CD31mRNA水平高于其余組，，毛細(xì)血結(jié)構(gòu)能力也明顯強(qiáng)于其余組。結(jié)論HAFMSCs具有易獲、體外增殖快、多向分化能力等優(yōu)勢(shì)。Chang培基兩步培法能夠在較短時(shí)間內(nèi)培大量HAFMSCs。短期凍存HAFMSCs對(duì)于其生物學(xué)特?zé)o明顯影，最佳凍存配方為DMEM:FBS:DMSO=5:4:1。VEGF和bFGF聯(lián)合誘較為理體外誘HAFMSCs分化為內(nèi)皮細(xì)胞誘方案。低氧能夠促進(jìn)這過程，在短期低氧環(huán)境下，HAFMSCs誘分化為內(nèi)皮細(xì)胞能力與暴于低氧環(huán)境時(shí)間關(guān)。
[Abstract]:Objective to isolate and culture human amniotic fluid mesenchymal stem cells from human amniotic fluid mesenchymal stem cells in vitro and observe their biological characteristics.After cryopreservation of HAFMSCs for three months, the biological characteristics of cryopreserved cells were observed and the best cryopreservation scheme was found out.HAFMSCs was induced to differentiate into endothelial cells in vitro and its ability to differentiate into endothelial cells was observed under normoxic and hypoxic conditions.Methods Haf MSCs were isolated and cultured by four methods. The proliferative ability of each group was observed by MTT method.The expression of CD29, CD34, CD44, CD45, CD73, CD90, HLA-ABC and HLA-DR were detected by flow cytometry.The ability of adipogenic and osteogenic differentiation and the expression of OCT-4 and Nanog were measured.HAF MSCs were frozen with different components of cryopreservation solution for 3 months, and the survival rate, biological characteristics and differentiation ability of Hafs cells were analyzed.In vitro, VEGF and bFGF were used to induce the differentiation of HAFMSCs into endothelial cells. The expression levels of vWF-1 CD31 and CD133mRNA were analyzed in vitro, and the expression of vWF in differentiated cells was identified by cell immunofluorescence.The ability of induced cells to form capillary-like structure on Matrigel was observed.To compare the difference of above indexes between hypoxic environment and normoxic environment.Results HAF MSCs were obtained from four different culture methods.The adherent time, colony forming time and cell passage time of Chang culture medium were shorter than those of the other three groups.All the cells in the four groups expressed mesenchymal stem cell marker CD29, CD44, CD73, CD90, but not hematopoietic stem cell marker CD34, CD45, which was consistent with the characteristics of mesenchymal stem cells, and there was no significant difference among the four groups.Osteogenesis and adipogenesis were successfully induced in the four groups, and were confirmed by oil red O staining and von Kossa staining, respectively.OCT-4 and Nanog mRNA were detected in all four groups.After three months of cryopreservation, the cryopreservation solution was DMEM:FBS:DMSO=5:4:1 with the highest cell survival rate and the strongest cell proliferation.However, there was no significant difference in growth curve, phenotype, differentiation ability and expression of OCT-4 and Nanog6 between resuscitated cells and pre-cryopreserved cells. HAFMSCs were induced to differentiate into endothelial cells in vitro.The levels of endothelium-related markers such as vWF, CD31 and CD133 were analyzed by flow cytometry, and the ability to form capillary structure on Mtrigel was the highest in the combination of VEGF and bFGF.In hypoxic environment, the levels of VEGF, vWF and CD31mRNA in HAFMSCs induced by VEGF and bFGF were higher than those in other groups, and the capillary blood structure ability was significantly stronger than that in other groups.Conclusion HAFMSCs has the advantages of easy to obtain, rapid proliferation in vitro and multidirectional differentiation ability. Chang Peiji two-step culture method can cultivate a large number of HAF MSCs in a short time.Short-term cryopreservation of HAFMSCs had no obvious effect on its biology. The best formula of cryopreservation was the combination of DMEM:FBS:DMSO=5:4:1.VEGF and bFGF to induce HAFMSCs to differentiate into endothelial cells in vitro.Hypoxia can promote this process. The ability of HAFMSCs to differentiate into endothelial cells under short-term hypoxia is related to the time of exposure to hypoxia.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(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

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 秦茂林,蔡文琴,姚忠祥;昆明種系小鼠胚胎干細(xì)胞的分離培養(yǎng)及特性鑒定[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2001年09期

2 王國(guó)云,李棟,張輝,白增亮;小鼠胚胎成纖維細(xì)胞飼養(yǎng)層制備條件的研究[J];山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年05期

3 鄧春艷;李富榮;王新根;齊暉;文錦麗;周雅瑩;;小鼠骨髓間充質(zhì)干細(xì)胞對(duì)同種異體骨髓來源的樹突狀細(xì)胞分化和功能的影響[J];細(xì)胞與分子免疫學(xué)雜志;2009年12期

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