發(fā)布時(shí)間：2018-02-26 18:31

  本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 永生化 端粒酶逆轉(zhuǎn)錄酶基因(TERT) 恒河猴　出處：《四川大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 背景和目的 骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells，BMSCs)具有多向分化潛能和低免疫原性，是細(xì)胞移植治療和組織工程的良好選擇。然而我們研究發(fā)現(xiàn)，恒河猴骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells from bone marrow of Rhesus monkey，RhBMSCs)體外增殖能力有限，傳至20代細(xì)胞生長(zhǎng)基本停滯。這必然限制了其在細(xì)胞移植和組織工程等領(lǐng)域中的研究和應(yīng)用。建立永生化細(xì)胞系可能會(huì)有效地解決這一問(wèn)題，從而為生物醫(yī)學(xué)研究提供了標(biāo)準(zhǔn)化的工具細(xì)胞。 材料與方法 采用不同的分離方法和不同的培養(yǎng)方案對(duì)3歲齡的恒河猴BMSCs進(jìn)行分離培養(yǎng)，并從形態(tài)學(xué)、表面抗原、分化潛能以及核型等方面進(jìn)行鑒定；將含有人源端粒酶逆轉(zhuǎn)錄酶基因(human telomerase reverse transcriptase gene，hTERT)的質(zhì)粒載體pCI-neo-hTERT穩(wěn)定轉(zhuǎn)入第2代RhBMSCs，并對(duì)轉(zhuǎn)染細(xì)胞進(jìn)行外源基因hTERT表達(dá)的RT-PCR檢測(cè)；通過(guò)特異性抗原(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)檢測(cè)、成骨誘導(dǎo)以及核型分析對(duì)細(xì)胞進(jìn)行鑒定；傳代培養(yǎng)、MTS法和細(xì)胞凋亡的流式檢測(cè)分析細(xì)胞生長(zhǎng)活性；裸鼠致瘤實(shí)驗(yàn)測(cè)定其有無(wú)致瘤性。 結(jié)果 結(jié)果表明hTERT基因可以成功轉(zhuǎn)入RhBMSCs；轉(zhuǎn)基因RhBMSCs表現(xiàn)出旺盛的增殖活性，相對(duì)于第15代未轉(zhuǎn)基因RhBMSCs的33．5％的凋亡率來(lái)說(shuō)，第40代轉(zhuǎn)基因RhBMSCs的凋亡率只有4．5％，RhBMSCs的“壽命”明顯得到延長(zhǎng)；轉(zhuǎn)基因RhBMSCs保持了正常RhBMSCs的細(xì)胞形態(tài)、克隆生長(zhǎng)特性及核型；特異性抗原SH-2、SH-3、SB-10、CD29表達(dá)率均高于95％，CD34、CD45、HLA-DR表達(dá)率均低于4．5％，，表型正常，純度較高；轉(zhuǎn)基因RhBMSCs保持了正常RhBMSCs的分化潛能；裸鼠致瘤實(shí)驗(yàn)結(jié)果呈良性。 結(jié)論 本研究證實(shí)了外源基因hTERT已成功轉(zhuǎn)入RhBMSCs，首次通過(guò)轉(zhuǎn)入hTERT的方式使RhBMSCs的體外傳代“壽命”得以延長(zhǎng)，細(xì)胞除了表現(xiàn)出旺盛的增殖活力外，其維持了與未轉(zhuǎn)染的細(xì)胞相類(lèi)似的表型(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)、核型和分化潛能。這將為應(yīng)用基礎(chǔ)研究提供穩(wěn)定、安全、充足的標(biāo)準(zhǔn)化工具細(xì)胞來(lái)源。另外，在構(gòu)建過(guò)程中涉及到的RhBMSCs分離、擴(kuò)增、轉(zhuǎn)染、鑒定和分化的方法和結(jié)果，不僅有助于我們了解與之近似的人的骨髓間充質(zhì)干細(xì)胞的相關(guān)特性，而且也為以獼猴為動(dòng)物模型的相關(guān)醫(yī)學(xué)實(shí)驗(yàn)和移植臨床前研究提供了基礎(chǔ)數(shù)據(jù)。
[Abstract]:Background and purpose. Bone marrow mesenchymal stem cells (marrow mesenchymal stem cells BMSCs) is a good choice for cell transplantation and tissue engineering because of its multipotential differentiation potential and low immunogenicity. The proliferation ability of mesenchymal stem cells from bone marrow of Rhesus monkey RhBMSCs in vitro was limited. Cell growth is basically stagnant in the 20th generation. This inevitably limits its research and application in areas such as cell transplantation and tissue engineering. The establishment of immortalized cell lines may effectively solve this problem. This provides standardized tool cells for biomedical research. Materials and methods. The BMSCs of 3 years old rhesus monkey was isolated and cultured by different isolation methods and different culture methods, and the morphology, surface antigen, differentiation potential and karyotype were identified. The plasmid vector pCI-neo-hTERT containing human telomerase reverse transcriptase gene (hTERT) was stably transferred into the second generation RhBMSCs, and the exogenous gene hTERT expression was detected by RT-PCR. Osteoblast induction and karyotype analysis were used to identify the cells. MTS method and flow cytometry were used to analyze the cell growth activity. The tumorigenicity of nude mice was determined by tumorigenicity assay. Results. The results showed that hTERT gene could be successfully transferred into RhBMSCs, and transgenic RhBMSCs showed strong proliferative activity. Compared with the apoptosis rate of 33.5% of the 15th generation of non-transgenic RhBMSCs, the apoptosis rate of the 40th generation transgenic RhBMSCs was only 4.5%, and the "life span" of the transgenic RhBMSCs was significantly prolonged. The cell morphology, clone growth characteristics and karyotype of normal RhBMSCs were maintained by transgenic RhBMSCs, and the expression rate of SH-2SH-3HH-3SH-3OSB-10T CD29 was higher than that of 95% CD34 + CD45 + HLA-DR, the phenotype was normal and the purity was high, and the differentiation potential of normal RhBMSCs was maintained by transgenic RhBMSCs. The results of tumorigenesis in nude mice were benign. Conclusion. This study confirmed that the exogenous gene hTERT has been successfully transferred into RhBMSCs, and for the first time, the passage "life span" of RhBMSCs was prolonged by the transfer of hTERT. In addition to the exuberant proliferative activity, the cells showed strong proliferative activity. It maintains similar phenotypic phenotypes as untransfected cells, such as phenotypic phenotypes, SH-2, SH-3, SB-10, CD29, CD34, CD45, and HLA-DRN, karyotype and differentiation potential. This will provide a stable, safe and sufficient source of standardized tool cells for applied basic research. In addition, the RhBMSCs isolation involved in the construction process, The methods and results of amplification, transfection, identification and differentiation not only help us to understand the characteristics of similar human bone marrow mesenchymal stem cells, It also provides basic data for related medical experiments and pre-transplant clinical studies using rhesus monkeys as animal models.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R329

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