【摘要】： 目的:構(gòu)建永生化的人骨髓間充質(zhì)干細(xì)胞。 方法:采用陽離子脂質(zhì)體介導(dǎo)法將病毒質(zhì)粒pBabepuro-hTERT導(dǎo)入包裝細(xì)胞內(nèi),嘌呤霉素(Puro)篩選出病毒表達(dá)陽性的細(xì)胞克隆,測定病毒滴度。取培養(yǎng)病毒的上清液感hBMSCs細(xì)胞、篩選出轉(zhuǎn)基因細(xì)胞,我們將其命名為hTERT-hBMSCs細(xì)胞。采用端粒酶重復(fù)序列擴(kuò)增(telomere repe at amplification protocol assay ,TRAP)酶鏈免疫吸附試驗(ELISA)法檢測hBMSCs細(xì)胞轉(zhuǎn)染前后端粒酶活性的變化;采用MTT法(四甲基偶氮唑鹽微量酶反應(yīng)比色法)測繪轉(zhuǎn)染前后細(xì)胞生長曲線,觀察hTERT轉(zhuǎn)入后對細(xì)胞增殖的影響;克隆形成實驗檢測hTERT-hBMSCs細(xì)胞的增殖能力;染色體核型分析觀察hTERT-hBMSCs細(xì)胞染色體是否正常;成骨試劑盒檢測hTERT-hBMSCs細(xì)胞向成骨分化的能力。 結(jié)果:骨髓間充質(zhì)干細(xì)胞為貼壁生長的梭形細(xì)胞,表面抗原CD44表達(dá)陽性,CD34陰性。TRAP-ELISA法檢測轉(zhuǎn)染hTERT的hBMSCs細(xì)胞的端粒酶為陽性,而未轉(zhuǎn)錄的正常人的hBMSCs細(xì)胞則為陰性,說明轉(zhuǎn)入hTERT基因后細(xì)胞端粒酶激活。hTERT-hBMSCs細(xì)胞比hBMSCs細(xì)胞增殖活躍,兩組相比有顯著的差異性,目前已傳了11代,尚未見衰老跡象。染色體核型分析所示:hTERT-hBMSCs細(xì)胞染色體數(shù)目和結(jié)構(gòu)正常,未見缺失、斷裂、異位等畸變。成骨誘導(dǎo)結(jié)果表明hTERT-hBMSCs細(xì)胞仍具有向成骨分化的能力。克隆形成實驗結(jié)果顯示hTERT-hBMSCs細(xì)胞的克隆形成率較低,為0.66%,為正常細(xì)胞。 結(jié)論:導(dǎo)入外源性hTERT可激活細(xì)胞端粒酶的活性。hTERT基因轉(zhuǎn)入細(xì)胞后,不影響其生物學(xué)特性和分化功能,仍然保持向成骨分化的能力。
[Abstract]:Objective: to construct immortalized human bone marrow mesenchymal stem cells. Methods: the viral plasmid pBabepuro-hTERT was introduced into the packaging cells by cationic liposome mediated method. The positive cell clones were screened by purine mycin (Puro) and the viral titer was determined. The supernatant hBMSCs cells of the culture virus were isolated and the transgenic cells were screened. We named them hTERT-hBMSCs cells. Telomerase activity of hBMSCs cells before and after transfection was detected by polymerase chain immunosorbent assay (ELISA) with telomerase repeat amplification (telomere repe at amplification protocol assay trap. The growth curve of hTERT-hBMSCs cells before and after transfection was measured by MTT method, and the effect of hTERT on cell proliferation was observed, and the proliferation ability of hTERT-hBMSCs cells was detected by clone formation assay. Chromosome karyotype analysis was used to observe whether the chromosomes of hTERT-hBMSCs cells were normal, and the ability of hTERT-hBMSCs cells to differentiate into osteoblasts was detected by osteoblast kit. Results: bone marrow mesenchymal stem cells (BMSCs) were spindle cells with adherent growth. The positive expression of surface antigen (CD44) was detected by CD34 negative. TRAP-ELISA was used to detect telomerase activity in hBMSCs cells transfected with hTERT, while hBMSCs cells in untranscribed normal persons were negative. The results showed that telomerase activation of hTERT-hBMSCs cells was more active than that of hBMSCs cells, and there was significant difference between the two groups. Chromosome karyotype analysis showed that the chromosome number and structure of human hTERT-hBMSCs were normal, and no aberrations such as deletion, breakage and ectopic were found. Osteogenic induction showed that hTERT-hBMSCs cells still had the ability to differentiate into osteoblasts. The clone formation rate of hTERT-hBMSCs cells was 0.66, which was normal. Conclusion: the transfection of exogenous hTERT can activate the telomerase activity of HTERT gene into cells without affecting its biological characteristics and differentiation function and maintain the ability of osteogenic differentiation.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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