【摘要】：本實(shí)驗(yàn)通過模擬骨髓內(nèi)的低氧張力對人骨髓間充質(zhì)干細(xì)胞(hMSCs)進(jìn)行體外低氧培養(yǎng),探討低氧對hMSCs成管及內(nèi)皮分化傾向的影響及其作用機(jī)制。體內(nèi)實(shí)驗(yàn)中利用pLEGFP-N1逆轉(zhuǎn)錄病毒載體對hMSCs進(jìn)行標(biāo)記,通過EGFP來追蹤hMSCs植入體內(nèi)的變化;以BALB/C裸鼠為實(shí)驗(yàn)動(dòng)物模型,通過Matrigel或腫瘤血管生成實(shí)驗(yàn)研究體內(nèi)低氧環(huán)境對hMSCs內(nèi)皮分化、血管生成作用的影響及其機(jī)制。結(jié)果如下:①成功地建立了一套hMSCs分離培養(yǎng)、純化、鑒定及擴(kuò)增的標(biāo)準(zhǔn)化技術(shù)平臺(tái);P12代內(nèi)hMSCs生物學(xué)性狀穩(wěn)定并保持未分化狀態(tài),可以作為后續(xù)實(shí)驗(yàn)工作的細(xì)胞來源。②體外低氧培養(yǎng)能通過加速細(xì)胞的鋪展、促進(jìn)細(xì)胞外基質(zhì)的降解和細(xì)胞的遷移而促進(jìn)hMSCs成管及內(nèi)皮分化,其機(jī)制為:a.低氧誘導(dǎo)hMSCs表達(dá)HIF-1α,進(jìn)而上調(diào)VEGF與MT1-MMP的表達(dá),從而促進(jìn)hMSCs成管及內(nèi)皮分化;b.低氧可能通過促進(jìn)hMSCs細(xì)胞Ca~(2+)通道開放,而促進(jìn)其向內(nèi)皮細(xì)胞分化。③hMSCs能促進(jìn)雞胚絨毛尿囊膜血管新生。④pLEGFP-N1逆轉(zhuǎn)錄病毒載體能有效地轉(zhuǎn)導(dǎo)hMSCs,轉(zhuǎn)導(dǎo)后的細(xì)胞維持其原有的生物學(xué)特性。⑤Matrigel血管生成實(shí)驗(yàn)證實(shí)體內(nèi)低氧環(huán)境能促進(jìn)hMSCs血管生成及內(nèi)皮分化,但大部分血管為宿主源性的新生血管,只有極少數(shù)血管的內(nèi)皮細(xì)胞是由hMSCs分化而來的。⑥hMSCs能促進(jìn)腫瘤的血管生成,這與腫瘤造成的低氧張力有關(guān),其作用也主要是通過促進(jìn)宿主源性的血管新生實(shí)現(xiàn)的。 綜上所述,本實(shí)驗(yàn)為MSCs的內(nèi)皮誘導(dǎo)分化提供了一個(gè)新的技術(shù)路線和實(shí)驗(yàn)方法,也為今后更好地應(yīng)用MSCs治療臨床缺血性疾病或血管依賴性疾病,以及利用組織工程化MSCs抗腫瘤治療提供了理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。
[Abstract]:The aim of this study was to investigate the effect of hypoxia on the tendency of hMSCs tubule and endothelial differentiation and its mechanism by simulating hypoxia tension in bone marrow to culture human bone marrow mesenchymal stem cell (hMSCs) in vitro. In vivo experiment, pLEGFP-N1 retrovirus vector was used to label hMSCs, and EGFP was used to track the changes of hMSCs implantation in vivo. Using BALB/C nude mice as experimental animal model, the effects of hypoxia on endothelial differentiation and angiogenesis of hMSCs in vivo and its mechanism were studied by Matrigel or tumor angiogeny. The results are as follows: (1) A set of standardized technical platform for isolation, culture, purification, identification and amplification of hMSCs was successfully established. The biological characters of hMSCs in P12 generation are stable and remain undifferentiated, which can be used as the cell source of subsequent experimental work. 2 hypoxia culture in vitro can accelerate the spread of cells. The mechanism of promoting the degradation of extracellular matrix and the migration of cells to promote the tubule and endothelial differentiation of hMSCs is as follows: a. Hypoxia induced the expression of HIF-1 偽 in hMSCs, and then upregulated the expression of VEGF and MT1-MMP, thus promoting the tubule and endothelial differentiation of hMSCs. Hypoxia may promote the differentiation of Ca~ (2) channels into endothelial cells by promoting the opening of Ca~ (2) channels in hMSCs cells. 3hMSCs can promote the neovascularization of chorioallantoic membrane in chicken embryo. 4pLEGFP-N1 retrovirus vector can effectively transfer hMSCs,. The transferred cells maintained their original biological characteristics. 5 Matrigel angiogenesis assay confirmed that hypoxia in vivo can promote hMSCs angiogenesis and endothelial differentiation, but most of the blood vessels are host neovascularization. Only a very small number of vascular endothelial cells are differentiated from hMSCs. 6 h MSCs can promote tumor angiogenesis, which is related to the hypoxia tension caused by tumor, and its effect is mainly achieved by promoting host angiogeny. In summary, this study provides a new technical route and experimental method for endothelial differentiation of MSCs, and also provides a better application of MSCs in the treatment of clinical ischemic diseases or vascular dependent diseases in the future. And the use of tissue engineering MSCs anti-tumor therapy provides a theoretical and experimental basis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 傅文玉,路艷蒙,樸英杰;人骨髓間充質(zhì)干細(xì)胞的分化與端粒酶活性[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2001年11期

2 路艷蒙,傅文玉,樸英杰,喬?hào)|訪,安連兵;人骨髓間充質(zhì)干細(xì)胞的超微結(jié)構(gòu)[J];電子顯微學(xué)報(bào);2002年04期

3 李海生,陳金武,朱玲玲,趙彤,趙惠卿,吳麗穎,丁愛石,范明;持續(xù)低氧增強(qiáng)人骨髓間充質(zhì)干細(xì)胞體外增殖[J];基礎(chǔ)醫(yī)學(xué)與臨床;2005年03期

4 李秀森,郭子寬,楊靖清,劉曉丹,侯春梅,唐佩弦,毛寧;骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性[J];解放軍醫(yī)學(xué)雜志;2000年05期

5 張超紀(jì);組織工程化血管種子細(xì)胞研究現(xiàn)狀[J];生物醫(yī)學(xué)工程與臨床;2003年03期

6 陳福真,楊鈺;組織工程與血管移植材料[J];外科理論與實(shí)踐;1998年04期

7 郭新,李偉,朱桂彬,何旭,張麗紅,李玉林,李一雷;基質(zhì)蛋白與整合素參與人骨髓間充質(zhì)干細(xì)胞體外內(nèi)皮的分化[J];中國臨床康復(fù);2005年14期

8 申麗紅,張均田;干細(xì)胞研究進(jìn)展[J];中國藥理學(xué)通報(bào);2001年05期

9 王煜,肖波;缺氧誘導(dǎo)胚胎大鼠神經(jīng)干細(xì)胞凋亡的初步研究[J];中國病理生理雜志;2003年04期

10 馬麗萍,李偉,李玉林,張麗紅,邵明玉;外周血中血管內(nèi)皮前體細(xì)胞的分離和鑒定[J];中華病理學(xué)雜志;2005年02期

，

本文編號(hào)：2492454