本文關(guān)鍵詞：大鼠骨髓間充質(zhì)干細(xì)胞永生化的實(shí)驗(yàn)研究　出處：《天津醫(yī)科大學(xué)》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 分化 永生化 端粒酶逆轉(zhuǎn)錄酶催化亞單位 液壓沖擊腦損傷 移植 大鼠

【摘要】：干細(xì)胞具有自我更新能力和多向分化潛能。骨髓間充質(zhì)干細(xì)胞(BMSCs)在體內(nèi)或體外誘導(dǎo)條件下可向神經(jīng)細(xì)胞分化,還能分泌多種神經(jīng)營(yíng)養(yǎng)因子,可作為中樞神經(jīng)系統(tǒng)移植治療的種子細(xì)胞。有研究表明,BMSCs長(zhǎng)期體外培養(yǎng)后,增殖、分化能力逐漸喪失。這會(huì)影響移植細(xì)胞的數(shù)量和質(zhì)量,制約移植治療的開展。 本研究擬通過陽離子脂質(zhì)體介導(dǎo)的人端粒酶逆轉(zhuǎn)錄酶催化亞單位(hTERT)表達(dá)載體轉(zhuǎn)染,建立大鼠BMSCs永生化細(xì)胞系,并對(duì)體外培養(yǎng)條件下永生化BMSCs的增殖及神經(jīng)細(xì)胞方向分化能力進(jìn)行檢測(cè),同時(shí)對(duì)BMSCs的生長(zhǎng)變異及其應(yīng)用安全性進(jìn)行初步評(píng)估。在此基礎(chǔ)上,借助大鼠液壓沖擊腦損傷模型,檢測(cè)創(chuàng)傷性腦損傷(TBI)后永生化BMSCs同種異體腦內(nèi)移植的長(zhǎng)期存活、分化情況,為永生化干細(xì)胞走向臨床提供實(shí)驗(yàn)依據(jù)。 第一部分 大鼠BMSCs的分離培養(yǎng)與鑒定 目的:分離、培養(yǎng)大鼠BMSCs,檢測(cè)其表面標(biāo)記及Nestin表達(dá)。 方法:SD大鼠處:死后,分離雙側(cè)股骨和脛骨,剪開長(zhǎng)骨兩端骨皮質(zhì),露出骨髓腔,用含10%FBS的低糖DMEM培養(yǎng)基反復(fù)沖洗骨髓腔,收集細(xì)胞懸液,接種到10cm塑料培養(yǎng)皿,37℃、5%CO_2、飽和濕度條件下細(xì)胞培養(yǎng)箱中培養(yǎng)。至10～14天單層貼壁細(xì)胞接近90%匯合時(shí),0.25%胰蛋白酶消化,1:2傳代。以后隨細(xì)胞擴(kuò)增均于80～90%匯合時(shí)按1:2持續(xù)體外傳代培養(yǎng)。 體外培養(yǎng)細(xì)胞至第5代,接近90%匯合時(shí),消化收集細(xì)胞,取約1×10~6個(gè)細(xì)胞懸于100μl PBS中,分別加入適量FITC標(biāo)記CD29、CD31、CD44、CD45、
[Abstract]:Stem cells have self - renewal ability and multi - directional differentiation potential . Bone marrow mesenchymal stem cells ( MSCs ) can differentiate into neural cells under the condition of in vivo or in vitro induction , and can secrete various neurotrophic factors , and can be used as seed cells for central nervous system transplantation . In this study , we studied the expression vector of human telomerase reverse transcriptase ( hTERT ) , which was mediated by cationic liposome , and established the immortal cell line in rats . Isolation , Culture and Identification of Bone Marrow in the First Part of Rats Objective : To isolate , culture and detect the surface markers and Nestin expression in rats . Methods : SD rats were divided into two sides of femur and tibia after death . The bone cortex at both ends of the long bone was cut , the marrow cavity was exposed , the cells were cultured in a 10 cm plastic culture dish , incubated in a cell incubator at 37 鈩，

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