本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 定向分化 內(nèi)皮細(xì)胞 骨髓間充質(zhì)干細(xì)胞 定向分化 平滑肌細(xì)胞 骨髓間充質(zhì)干細(xì)胞 定向分化 成纖維細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 第一部分骨髓間充質(zhì)干細(xì)胞定向分化內(nèi)皮細(xì)胞的實(shí)驗(yàn)研究 目的:建立可以在體外穩(wěn)定地獲得大量高純度內(nèi)皮細(xì)胞的簡便方法,并對獲得細(xì)胞進(jìn)行形態(tài)觀察及表面標(biāo)志鑒定。 材料和方法:1.取4周齡SD大鼠,雌雄不拘,無菌條件下提取原代骨髓間充質(zhì)干細(xì)胞,體外通過全髓貼壁法培養(yǎng)、純化、擴(kuò)增,將獲得的P3-BMSCs采用流式細(xì)胞術(shù)進(jìn)行細(xì)胞表面抗原鑒定,應(yīng)用的抗體為CD29和CD34。2.取P3-BMSCs分為2組:實(shí)驗(yàn)組和對照組;實(shí)驗(yàn)組細(xì)胞在L-DMEM(20%FBS+10ng/ml VEGF)條件下培養(yǎng)14d;對照組細(xì)胞以未加特殊誘導(dǎo)劑的L-DMEM+10%FBS的培養(yǎng)液培養(yǎng)14d;每日于倒置顯微鏡下觀察細(xì)胞生長情況,培養(yǎng)到7d和14d時取實(shí)驗(yàn)組和對照組細(xì)胞爬片,用Anti-FactorⅧ進(jìn)行免疫細(xì)胞化學(xué)鑒定。 結(jié)果:1.通過全髓貼壁法可獲得均質(zhì)性較好的細(xì)胞,獲得的細(xì)胞培養(yǎng)到第3代時經(jīng)流式細(xì)胞術(shù)鑒定發(fā)現(xiàn),細(xì)胞表達(dá)CD29的陽性率超過96%,而不表達(dá)CD34;BMSCs為均一的長梭形,漩渦狀、團(tuán)簇狀生長,在體外增殖能力強(qiáng)。2.實(shí)驗(yàn)組P3-BMSCs經(jīng)L-DMEM(20%FBS+10ng/ml VEGF)誘導(dǎo)培養(yǎng)后,細(xì)胞逐漸變的粗大, 10d左右倒置顯微鏡下出現(xiàn)明顯的“鵝卵石”樣形態(tài),而對照組仍為均一的長梭形,漩渦狀、團(tuán)簇狀生長,細(xì)胞形態(tài)無明顯變化;用免疫細(xì)胞化學(xué)技術(shù)鑒定發(fā)現(xiàn),誘導(dǎo)7d和14d的細(xì)胞表達(dá)Anti-FactorⅧ的陽性率分別為82%和93%,而對照組細(xì)胞陰性表達(dá)Anti-FactorⅧ。 結(jié)論:通過全髓貼壁法可獲得數(shù)量足夠的、均質(zhì)的骨髓間充質(zhì)干細(xì)胞;BMSCs在L-DMEM(20%FBS+10ng/ml VEGF)誘導(dǎo)條件下可以定向分化為內(nèi)皮細(xì)胞,通過此法可在體外獲得大量高純度的內(nèi)皮細(xì)胞。 第二部分骨髓間充質(zhì)干細(xì)胞定向分化平滑肌細(xì)胞的實(shí)驗(yàn)研究 目的:建立可以在體外穩(wěn)定地獲得大量高純度平滑肌細(xì)胞的簡便方法,并對獲得細(xì)胞進(jìn)行形態(tài)觀察及表面標(biāo)志鑒定。 材料和方法:實(shí)驗(yàn)組細(xì)胞P3-BMSCs在L-DMEM(20%FBS+2ng/ml PDGF-BB)條件下誘導(dǎo)培養(yǎng)14d;對照組P3-BMSCs以未加特殊誘導(dǎo)劑的L-DMEM+10%FBS培養(yǎng)液培養(yǎng)14d;每日于倒置顯微鏡下觀察細(xì)胞生長情況,培養(yǎng)7d和14d時取實(shí)驗(yàn)組和對照組細(xì)胞爬片,分別用Anti-Actin alpha、Anti-Vimentin進(jìn)行免疫細(xì)胞化學(xué)鑒定。 結(jié)果: P3-BMSCs經(jīng)L-DMEM(20%FBS+2ng/ml PDGF-BB)誘導(dǎo)培養(yǎng)后,細(xì)胞體積逐漸變大,呈不規(guī)則梭形,密集與稀疏相交錯,形成“峰-谷”狀形態(tài),而對照組仍為均一的長梭形,漩渦狀、團(tuán)簇狀生長,細(xì)胞形態(tài)無明顯變化;分別應(yīng)用Anti-Actin alpha、Anti-Vimentin進(jìn)行免疫細(xì)胞化學(xué)檢測,結(jié)果顯示:7d和14d時實(shí)驗(yàn)組細(xì)胞表達(dá)Anti-Actin alpha的陽性率分別為78%和87%,而Anti-Vimentin表達(dá)為陰性;對照組細(xì)胞對Anti-Actin alpha和Anti-Vimentin均呈陰性表達(dá)。 結(jié)論: BMSCs在L-DMEM(20%FBS+2ng/ml PDGF-BB)誘導(dǎo)條件下可以定向分化為平滑肌細(xì)胞,通過此法可在體外獲得大量高純度的平滑肌細(xì)胞。 第三部分骨髓間充質(zhì)干細(xì)胞定向分化成纖維細(xì)胞的實(shí)驗(yàn)研究 目的:建立可以在體外穩(wěn)定地獲得大量高純度成纖維細(xì)胞的簡便方法,并對獲得細(xì)胞進(jìn)行形態(tài)觀察及表面標(biāo)志鑒定。 材料和方法:實(shí)驗(yàn)組細(xì)胞P3-BMSCs在L-DMEM(20%FBS+20ng/ml b-FGF)條件下進(jìn)行誘導(dǎo)培養(yǎng)14d,對照組P3-BMSCs以未加特殊誘導(dǎo)劑的L-DMEM+10%FBS的培養(yǎng)液培養(yǎng)14d;每日于倒置顯微鏡下觀察細(xì)胞生長情況,培養(yǎng)7d和14d時取實(shí)驗(yàn)組和對照組細(xì)胞爬片,分別用Anti-Actin alpha、Anti-Vimentin進(jìn)行免疫細(xì)胞化學(xué)鑒定。 結(jié)果: P3-BMSCs經(jīng)L-DMEM(20%FBS+20ng/ml b-FGF)誘導(dǎo)培養(yǎng)后,細(xì)胞逐漸變得細(xì)長,紡錘形,呈束狀、網(wǎng)狀排列生長,而對照組仍為均一的長梭形,漩渦狀、團(tuán)簇狀生長,細(xì)胞形態(tài)無明顯變化;分別應(yīng)用Anti-Actin alpha、Anti-Vimentin進(jìn)行免疫細(xì)胞化學(xué)鑒定,結(jié)果顯示:實(shí)驗(yàn)組細(xì)胞誘導(dǎo)培養(yǎng)7d和14d時,細(xì)胞表達(dá)Anti-Actin alpha的陽性率分別為72%和82%,表達(dá)Anti-Vimentin的陽性率分別為75%和85%;而對照組細(xì)胞對Anti-Vimentin和對Anti-Actin alpha均呈陰性表達(dá)。 結(jié)論: BMSCs在L-DMEM(20%FBS+20ng/ml b-FGF)誘導(dǎo)條件下可以定向分化為成纖維細(xì)胞,通過此法可在體外獲得大量高純度的成纖維細(xì)胞。
[Abstract]:Experimental study on directional differentiation of endothelial cells by bone marrow mesenchymal stem cells
Objective: to establish a simple and convenient method for obtaining a large number of highly purified endothelial cells in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: 1. 4 week old SD rats of both sexes under sterile conditions to extract primary bone marrow mesenchymal stem cells in vitro by whole marrow adherent culture, purification, amplification, the P3-BMSCs obtained by cell surface antigens by flow cytometry, using antibodies to CD29 and CD34.2. P3-BMSCs were divided into 2 groups: experimental group and control group; experimental group of cells in the L-DMEM (20%FBS+10ng/ml VEGF) under the condition of 14d culture medium group; control cells without special inducer in the culture of L-DMEM+10%FBS 14d; daily on cell growth was observed under inverted microscope, the experimental group and control group cell culturing to 7d and 14d, identified by immunocytochemistry with Anti-Factor VIII.
Results: 1. through the whole marrow adherence method can obtain better homogeneity of cells, the cells were cultured to the third generation by flow cytometry showed that the cells, the positive expression rate of CD29 is more than 96%, while the expression of CD34; BMSCs long spindle shape, uniform swirling, cluster growth by L-DMEM in the in vitro proliferation of.2. experimental group P3-BMSCs (20%FBS+10ng/ml VEGF) after induction, the cells gradually become coarse and the apparent "cobblestone" form about 10d under the microscope, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly; found immunocytochemistry identification, induction of 7D and 14d positive expression rate of Anti-Factor VIII respectively 82% and 93%, while the control group were negative expression of Anti-Factor VIII.
Conclusion: sufficient quantity of homogeneous bone marrow mesenchymal stem cells can be obtained by full pulp adherence method. BMSCs can be differentiated into endothelial cells under L-DMEM (20%FBS+10ng/ml VEGF) induction condition, and a large number of high-purity endothelial cells can be obtained in vitro.
Experimental study on directional differentiation of smooth muscle cells by bone marrow mesenchymal stem cells in the second part
Objective: to establish a simple and convenient method for obtaining a large number of high purity smooth muscle cells in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: the experimental group of P3-BMSCs cells in L-DMEM (20%FBS+2ng/ml PDGF-BB) cultured under the condition of 14d; control group P3-BMSCs without special inducer L-DMEM+10%FBS medium 14d; daily on cell growth was observed under inverted microscope, cultured 7d and 14d from the experimental group and the control group were treated with Anti-Actin cell climbing slices alpha, identified by immunocytochemistry Anti-Vimentin.
Results: P3-BMSCs by L-DMEM (20%FBS+2ng/ml PDGF-BB) after induction, cell volume became larger, irregular fusiform, dense and sparse staggered, the formation of "peak valley" shape, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly the application of Anti-Actin alpha Anti-Vimentin; respectively, were detected by immunocytochemistry. The results showed that: 7d and 14d positive cells in the experimental group the expression of Anti-Actin alpha were 78% and 87%, while the expression of Anti-Vimentin was negative; the control group of Anti-Actin alpha and Anti-Vimentin cells were negative.
Conclusion: BMSCs can be differentiated into smooth muscle cells under the induction of L-DMEM (20%FBS+2ng/ml PDGF-BB). By this method, a lot of high purity smooth muscle cells can be obtained in vitro.
Experimental study on directional differentiation of bone marrow mesenchymal stem cells into fibroblasts in the third part
Objective: to establish a simple and convenient method for obtaining a large number of highly purified fibroblasts in vitro, and to observe the morphology of the obtained cells and identify the surface markers.
Materials and methods: the experimental group of P3-BMSCs cells in L-DMEM (20%FBS+20ng/ml b-FGF) were induced and cultured under the condition of 14d, 14d P3-BMSCs in cultured control group without special inducer of L-DMEM+10%FBS; daily on cell growth was observed under inverted microscope, cultured 7d and 14d from the experimental group and the control group cell climbing films, respectively with Anti-Actin alpha, identified by immunocytochemistry Anti-Vimentin.
Results: P3-BMSCs by L-DMEM (20%FBS+20ng/ml b-FGF) after induction, cells became elongated, spindle, a bundle, mesh arrangement growth, while the control group is still long spindle shape, uniform swirling, cluster growth, cell morphology did not change significantly; respectively using Anti-Actin alpha, identified by immunocytochemistry. The results showed that: the experimental group of Anti-Vimentin cells induced by 7d and 14d when cultured cells, the positive expression rate of Anti-Actin alpha were 72% and 82%, the expression of Anti-Vimentin positive rates were 75% and 85%; while the control group of Anti-Actin cells on the expression of Anti-Vimentin and alpha were negative.
Conclusion: BMSCs can be differentiated into fibroblasts under L-DMEM (20%FBS+20ng/ml b-FGF) induction. By this method, a large number of high purity fibroblasts can be obtained in vitro.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊立信 ,徐志云 ,張寶仁 ,黃盛東 ,葉福林;骨髓基質(zhì)干細(xì)胞構(gòu)建組織工程心臟瓣膜的初步研究[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年12期

2 劉幸平;劉清;鄭燕珊;黃展勤;;大鼠血管平滑肌細(xì)胞的培養(yǎng)與鑒定[J];汕頭大學(xué)醫(yī)學(xué)院學(xué)報(bào);2008年03期

3 孟乘飛;張春禮;范宏斌;徐虎;白建萍;;EGF體外誘導(dǎo)骨髓基質(zhì)干細(xì)胞增殖分化為成纖維細(xì)胞的實(shí)驗(yàn)研究[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2007年03期

4 杜俊鵬;張凱倫;傅平;;人骨髓基質(zhì)干細(xì)胞作為心血管瓣膜組織工程種子細(xì)胞的實(shí)驗(yàn)研究[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2006年04期

5 馮濱,劉迎龍,馮凱,龔茹,陳虎;Tie-2單克隆抗體鑒定人骨髓間充質(zhì)干細(xì)胞體外定向誘導(dǎo)分化的內(nèi)皮細(xì)胞[J];中國應(yīng)用生理學(xué)雜志;2005年03期

6 董念國,葉曉峰,史嘉瑋,宋強(qiáng),孫宗全;組織工程瓣膜天然支架去細(xì)胞方法的比較[J];中華實(shí)驗(yàn)外科雜志;2005年03期

7 衛(wèi)向陽,劉維永,孫國成,歐陽輝,顧春虎,劉興光;犬骨髓間充質(zhì)干細(xì)胞體外分離、誘導(dǎo)成纖維細(xì)胞的實(shí)驗(yàn)研究[J];中華外科雜志;2005年18期

8 傅文玉,路艷蒙,樸英杰;人骨髓間充質(zhì)干細(xì)胞的培養(yǎng)及多能性研究[J];中華血液學(xué)雜志;2002年04期

9 張舒;周楊;關(guān)思宇;梁霄玉;歐陽紅生;;血小板源性生長因子(PDGF-BB)促進(jìn)山羊骨髓間充質(zhì)干細(xì)胞向平滑肌細(xì)胞分化[J];中國獸醫(yī)學(xué)報(bào);2008年08期

10 葉福林,徐志云,張寶仁,韓林,黃盛東,王曉偉;去細(xì)胞豬主動脈瓣葉的獲取和內(nèi)皮細(xì)胞的種植[J];中國修復(fù)重建外科雜志;2003年06期

，

本文編號：1481633