本文關鍵詞： 骨髓間充質干細胞 永生化 端粒酶逆轉錄酶基因(TERT) 恒河猴　出處：《四川大學》2007年碩士論文　論文類型：學位論文

【摘要】： 背景和目的 骨髓間充質干細胞(bone marrow mesenchymal stem cells，BMSCs)具有多向分化潛能和低免疫原性，是細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)，恒河猴骨髓間充質干細胞(mesenchymal stem cells from bone marrow of Rhesus monkey，RhBMSCs)體外增殖能力有限，傳至20代細胞生長基本停滯。這必然限制了其在細胞移植和組織工程等領域中的研究和應用。建立永生化細胞系可能會有效地解決這一問題，從而為生物醫(yī)學研究提供了標準化的工具細胞。 材料與方法 采用不同的分離方法和不同的培養(yǎng)方案對3歲齡的恒河猴BMSCs進行分離培養(yǎng)，并從形態(tài)學、表面抗原、分化潛能以及核型等方面進行鑒定；將含有人源端粒酶逆轉錄酶基因(human telomerase reverse transcriptase gene，hTERT)的質粒載體pCI-neo-hTERT穩(wěn)定轉入第2代RhBMSCs，并對轉染細胞進行外源基因hTERT表達的RT-PCR檢測；通過特異性抗原(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)檢測、成骨誘導以及核型分析對細胞進行鑒定；傳代培養(yǎng)、MTS法和細胞凋亡的流式檢測分析細胞生長活性；裸鼠致瘤實驗測定其有無致瘤性。 結果 結果表明hTERT基因可以成功轉入RhBMSCs；轉基因RhBMSCs表現(xiàn)出旺盛的增殖活性，相對于第15代未轉基因RhBMSCs的33．5％的凋亡率來說，第40代轉基因RhBMSCs的凋亡率只有4．5％，RhBMSCs的“壽命”明顯得到延長；轉基因RhBMSCs保持了正常RhBMSCs的細胞形態(tài)、克隆生長特性及核型；特異性抗原SH-2、SH-3、SB-10、CD29表達率均高于95％，CD34、CD45、HLA-DR表達率均低于4．5％，，表型正常，純度較高；轉基因RhBMSCs保持了正常RhBMSCs的分化潛能；裸鼠致瘤實驗結果呈良性。 結論 本研究證實了外源基因hTERT已成功轉入RhBMSCs，首次通過轉入hTERT的方式使RhBMSCs的體外傳代“壽命”得以延長，細胞除了表現(xiàn)出旺盛的增殖活力外，其維持了與未轉染的細胞相類似的表型(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)、核型和分化潛能。這將為應用基礎研究提供穩(wěn)定、安全、充足的標準化工具細胞來源。另外，在構建過程中涉及到的RhBMSCs分離、擴增、轉染、鑒定和分化的方法和結果，不僅有助于我們了解與之近似的人的骨髓間充質干細胞的相關特性，而且也為以獼猴為動物模型的相關醫(yī)學實驗和移植臨床前研究提供了基礎數(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.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

【相似文獻】

相關期刊論文 前10條

1 ;骨髓間充質干細胞與肝細胞修復及組織重構[J];中國組織工程研究與臨床康復;2011年23期

2 林治平;曾榮;胡資兵;林顥;;Wnt信號通路與骨髓間充質干細胞神經分化[J];廣東醫(yī)學;2011年08期

3 王長軍;殷國勇;李翔;沈國強;;骨髓間充質干細胞與成骨細胞復合培養(yǎng)對成骨細胞增殖的影響[J];江蘇醫(yī)藥;2011年11期

4 張英博;楊大平;;干細胞用于創(chuàng)傷修復的新進展[J];中國美容醫(yī)學;2011年08期

5 石洋;袁偉;;中藥誘導骨髓間充質干細胞分化為成骨細胞的研究進展[J];內蒙古中醫(yī)藥;2010年05期

6 羅俊;顧文娟;徐禮;李江;;骨髓間充質干細胞移植治療對心肌梗死后心功能的影響[J];醫(yī)學與哲學(臨床決策論壇版);2011年06期

7 劉嘉祺;林峰;李孟全;;骨髓間充質干細胞的培養(yǎng)及生物學特性[J];牡丹江醫(yī)學院學報;2011年03期

8 劉偉;趙勁民;蘇偉;李曉峰;秦義武;;柚皮苷對兔骨髓間充質干細胞分化為成骨細胞的影響[J];江蘇醫(yī)藥;2011年14期

9 程書榜;王成友;洪建琴;范子冰;胡迎春;;同種骨髓間充質干細胞靜脈輸注對大鼠CD4~+、CD8~+T淋巴細胞和IL-10、IL-2、TNF-α的影響[J];消化腫瘤雜志(電子版);2010年01期

10 李軍;杜金榮;戴艷萍;;骨髓間充質干細胞移植治療大鼠腦梗死的研究[J];黑龍江醫(yī)學;2011年09期

相關會議論文 前10條

1 黃煒;呂安林;劉博武;候婧;李W

本文編號：1539171