【摘要】： 目的:通過構(gòu)建LIM礦化蛋白-1(LMP-1)的真核表達(dá)質(zhì)粒,并使其在分離培養(yǎng)的兔骨髓間充質(zhì)干細(xì)胞(BMSCs)內(nèi)表達(dá),研究LMP-1對(duì)體外培養(yǎng)BMSCs成骨分化的影響。 方法:采用RT-PCR的方法分別從人的胎盤組織和含有人BMP-2的重組質(zhì)粒中,克隆LMP-1和BMP-2,與真核表達(dá)質(zhì)粒pIRES2-EGFP酶切連接,構(gòu)建pIRES2-EGFP-LMP1及pIRES2-EGFP-BMP2重組質(zhì)粒。采用梯度離心方法從兔骨髓中分離培養(yǎng)BMSCs。以第三代細(xì)胞為實(shí)驗(yàn)對(duì)象,脂質(zhì)體介導(dǎo)下分別將pIRES2-EGFP-LMP1、pIRES2-EGFP-BMP2和pIRES2-EGFP質(zhì)粒轉(zhuǎn)染細(xì)胞。熒光顯微鏡下觀察轉(zhuǎn)染效率,7天后收集細(xì)胞檢測(cè)其生物學(xué)性狀的變化。RT-PCR比較LMP-1、BMP-2及Ⅰ型膠原的表達(dá),堿性磷酸酶活性檢測(cè)ALP活性,免疫組化染色比較骨鈣素表達(dá)。 結(jié)果:經(jīng)酶切和測(cè)序鑒定pIRES2-EGFP-LMP1和pIRES2-EGFP-BMP2重組質(zhì)粒構(gòu)建成功。成熟的體外分離培養(yǎng)BMSCs方法方便簡單,收獲細(xì)胞數(shù)量多且增殖能力強(qiáng)。脂質(zhì)體介導(dǎo)重組質(zhì)粒轉(zhuǎn)染BMSCs后,通過熒光顯微鏡觀察轉(zhuǎn)染成功且轉(zhuǎn)染效率15%左右。收集細(xì)胞RT-PCR證實(shí)表達(dá)目的基因,LMP-1和BMP-2轉(zhuǎn)染組可以明顯促進(jìn)細(xì)胞分泌ALP的活性,誘導(dǎo)Ⅰ型膠原和骨鈣素的表達(dá)。
[Abstract]:Aim: to construct eukaryotic expression plasmid of LIM mineralized protein 1 (LMP-1) and express it in (BMSCs) of cultured rabbit bone marrow mesenchymal stem cells (BMSCs), and to study the effect of LMP-1 on osteogenic differentiation of BMSCs cultured in vitro. Methods: LMP-1 and BMP-2 were cloned from human placental tissues and recombinant plasmids containing human BMP-2 by RT-PCR and ligated with eukaryotic expression plasmid pIRES2-EGFP to construct pIRES2-EGFP-LMP1 and pIRES2-EGFP-BMP2 recombinant plasmids. BMSCs were isolated from rabbit bone marrow by gradient centrifugation. The pIRES2-EGFP-LMP1 pIRES2-EGFP-BMP2 and pIRES2-EGFP plasmids were transfected into the cells by liposome-mediated transfection. The expression of LMP-1BMP-2 and collagen 鈪，

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