端粒酶逆轉(zhuǎn)錄酶基因修飾人骨髓間充質(zhì)干細胞的實驗研究
發(fā)布時間：2018-10-13 19:34
【摘要】：人骨髓中除造血干細胞(hematopoietic stem cell,HSC)外還存在另外一類干細胞—骨髓間質(zhì)干細胞(mesenchymal stem cell,MSC),是中胚層發(fā)育的早期細胞。新近研究發(fā)現(xiàn)MSC不僅具有向中胚層來源的細胞(如:成骨細胞、軟骨細胞、脂肪細胞、成肌細胞和心肌細胞)發(fā)育的潛能,而且具有向中胚層外細胞(如神經(jīng)細胞、肝細胞、內(nèi)皮細胞等)分化的潛能。 盡管如此,但由于骨髓有核細胞中平均每10~6個細胞中只有1個MSC,且人MSCs和其它正常體細胞一樣,具有有限的生命,在經(jīng)過有限次的細胞傳代后,就會停止增殖,發(fā)生衰老和死亡,這就限制了MSCs在科研與臨床的進一步應(yīng)用,所以如何實現(xiàn)MSCs在體外既長期擴增又保持多潛能分化狀態(tài)是當(dāng)前亟待解決的問題。 研究表明,端粒與細胞壽命的控制密切相關(guān)。隨著細胞分裂次數(shù)增加,端粒進行性縮短,當(dāng)縮短到一定限度即不能維持染色體的穩(wěn)定時,細胞失去分裂能力,進而衰老死亡。而端粒長度的維持需要端粒酶的激活,端粒酶是一種核糖核蛋白,由RNA和蛋白質(zhì)組成,具有逆轉(zhuǎn)錄的活性,其活性主要由端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase,hTERT)表達水平的高低決定。hTERT是端粒酶的催化亞單位,在端粒酶的激活中起關(guān)鍵作用。 鑒于上述,本實驗旨在將攜帶有EGFP報告基因和hTERT目的基因的質(zhì)粒pEGFP-hTERT通過陽離子脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)入人骨髓MSCs中,設(shè)想通過外源性hTERT基因的異位表達,誘導(dǎo)人骨髓MSCs的端粒酶活性,維持端粒長度的穩(wěn)定,從而延長人骨髓MSCs的生命周期并保持其多潛能分化特性。
[Abstract]:In addition to hematopoietic stem cell (hematopoietic stem cell,HSC), there is another kind of stem cell, bone marrow mesenchymal stem cell (mesenchymal stem cell,MSC), which is the early stage of mesoderm development. Recent studies have found that MSC not only has the potential to develop to mesodermal cells (such as osteoblasts, chondrocytes, adipocytes, myoblasts and cardiomyocytes), but also to mesodermal extracellular cells (such as nerve cells, hepatocytes). The potential for differentiation of endothelial cells, etc. Nevertheless, because of the fact that there is on average only one MSC, per 10 to 6 cells in nucleated bone marrow cells and that human MSCs, like other normal somatic cells, has limited life, it will cease to proliferate after a limited number of cell passages. Aging and death limit the further application of MSCs in scientific research and clinic. Therefore, how to realize the long-term expansion of MSCs in vitro and maintain the state of multipotential differentiation is an urgent problem to be solved. Studies have shown that telomere is closely related to the control of cell life. With the increase of cell division times, telomere gradually shortened. When the telomere was shortened to a certain limit, it could not maintain the stability of chromosome, the cell lost the ability of division and then died of senescence. The maintenance of telomere length requires the activation of telomerase, a ribonucleoprotein that is composed of RNA and proteins and has the activity of reverse transcription. Its activity is mainly determined by the level of telomerase reverse transcriptase (human telomerase reverse transcriptase,hTERT) expression. HTERT is the catalytic subunit of telomerase and plays a key role in the activation of telomerase. In view of the above, the aim of this study was to transfer the plasmid pEGFP-hTERT carrying EGFP reporter gene and hTERT target gene into human bone marrow MSCs by cationic liposome transfection. It was envisaged that the telomerase activity of human bone marrow MSCs could be induced by heterotopic expression of exogenous hTERT gene. To maintain the stability of telomere length, prolong the life cycle of human bone marrow MSCs and maintain its multipotential differentiation characteristics.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329.2
本文編號：2269680
[Abstract]:In addition to hematopoietic stem cell (hematopoietic stem cell,HSC), there is another kind of stem cell, bone marrow mesenchymal stem cell (mesenchymal stem cell,MSC), which is the early stage of mesoderm development. Recent studies have found that MSC not only has the potential to develop to mesodermal cells (such as osteoblasts, chondrocytes, adipocytes, myoblasts and cardiomyocytes), but also to mesodermal extracellular cells (such as nerve cells, hepatocytes). The potential for differentiation of endothelial cells, etc. Nevertheless, because of the fact that there is on average only one MSC, per 10 to 6 cells in nucleated bone marrow cells and that human MSCs, like other normal somatic cells, has limited life, it will cease to proliferate after a limited number of cell passages. Aging and death limit the further application of MSCs in scientific research and clinic. Therefore, how to realize the long-term expansion of MSCs in vitro and maintain the state of multipotential differentiation is an urgent problem to be solved. Studies have shown that telomere is closely related to the control of cell life. With the increase of cell division times, telomere gradually shortened. When the telomere was shortened to a certain limit, it could not maintain the stability of chromosome, the cell lost the ability of division and then died of senescence. The maintenance of telomere length requires the activation of telomerase, a ribonucleoprotein that is composed of RNA and proteins and has the activity of reverse transcription. Its activity is mainly determined by the level of telomerase reverse transcriptase (human telomerase reverse transcriptase,hTERT) expression. HTERT is the catalytic subunit of telomerase and plays a key role in the activation of telomerase. In view of the above, the aim of this study was to transfer the plasmid pEGFP-hTERT carrying EGFP reporter gene and hTERT target gene into human bone marrow MSCs by cationic liposome transfection. It was envisaged that the telomerase activity of human bone marrow MSCs could be induced by heterotopic expression of exogenous hTERT gene. To maintain the stability of telomere length, prolong the life cycle of human bone marrow MSCs and maintain its multipotential differentiation characteristics.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329.2
【參考文獻】
相關(guān)期刊論文 前1條
1 馮凱,裴雪濤;間充質(zhì)干細胞——現(xiàn)代組織工程的新資源[J];國外醫(yī)學(xué).生物醫(yī)學(xué)工程分冊;2000年06期
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