恒河猴骨髓间充质干细胞永生细胞系构建

发布时间：2018-02-26 18:31

本文关键词： 骨髓间充质干细胞永生化端粒酶逆转录酶基因(TERT) 恒河猴　出处：《四川大学》2007年硕士论文　论文类型：学位论文

【摘要】： 背景和目的骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)具有多向分化潜能和低免疫原性，是细胞移植治疗和组织工程的良好选择。然而我们研究发现，恒河猴骨髓间充质干细胞(mesenchymal stem cells from bone marrow of Rhesus monkey，RhBMSCs)体外增殖能力有限，传至20代细胞生长基本停滞。这必然限制了其在细胞移植和组织工程等领域中的研究和应用。建立永生化细胞系可能会有效地解决这一问题，从而为生物医学研究提供了标准化的工具细胞。材料与方法采用不同的分离方法和不同的培养方案对3岁龄的恒河猴BMSCs进行分离培养，并从形态学、表面抗原、分化潜能以及核型等方面进行鉴定；将含有人源端粒酶逆转录酶基因(human telomerase reverse transcriptase gene，hTERT)的质粒载体pCI-neo-hTERT稳定转入第2代RhBMSCs，并对转染细胞进行外源基因hTERT表达的RT-PCR检测；通过特异性抗原(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)检测、成骨诱导以及核型分析对细胞进行鉴定；传代培养、MTS法和细胞凋亡的流式检测分析细胞生长活性；裸鼠致瘤实验测定其有无致瘤性。结果结果表明hTERT基因可以成功转入RhBMSCs；转基因RhBMSCs表现出旺盛的增殖活性，相对于第15代未转基因RhBMSCs的33．5％的凋亡率来说，第40代转基因RhBMSCs的凋亡率只有4．5％，RhBMSCs的“寿命”明显得到延长；转基因RhBMSCs保持了正常RhBMSCs的细胞形态、克隆生长特性及核型；特异性抗原SH-2、SH-3、SB-10、CD29表达率均高于95％，CD34、CD45、HLA-DR表达率均低于4．5％，，表型正常，纯度较高；转基因RhBMSCs保持了正常RhBMSCs的分化潜能；裸鼠致瘤实验结果呈良性。结论本研究证实了外源基因hTERT已成功转入RhBMSCs，首次通过转入hTERT的方式使RhBMSCs的体外传代“寿命”得以延长，细胞除了表现出旺盛的增殖活力外，其维持了与未转染的细胞相类似的表型(SH-2、SH-3、SB-10、CD29、CD34、CD45和HLA-DR)、核型和分化潜能。这将为应用基础研究提供稳定、安全、充足的标准化工具细胞来源。另外，在构建过程中涉及到的RhBMSCs分离、扩增、转染、鉴定和分化的方法和结果，不仅有助于我们了解与之近似的人的骨髓间充质干细胞的相关特性，而且也为以猕猴为动物模型的相关医学实验和移植临床前研究提供了基础数据。
[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.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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