人端粒酶反转录酶重组绿色荧光表达载体转染椎间盘正常髓核细胞.docx

脊柱组织构
人端粒酶反转录酶重组绿色荧光表达载体转染椎间盘正常髓核细胞
Recombinant green fluorescent protein plasmids expressing human telomerase reverse transcriptase transfect normal nucleus pulposus cells in the intervertebral disc

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摘要 
背景：椎间盘髓核细胞分离培养困难，老化较快，迫切需要一种标准细胞株用于实验研究。
目的：探讨人端粒酶反转录酶重组绿色荧光表达载体的构建及其转染正常髓核细胞构建永生化细胞的可行性研究。
方法：通过目的基因克隆、真核表达质粒中目的基因序列测定、目的基因真核表达质粒的构建、转染人端粒酶反转录酶表达检测等步骤进行实验。
结果与结论：构建出人端粒酶反转录酶重组绿色荧光表达载体，成功转染正常髓核细胞并在细胞中稳定表达。结果表明运用人端粒酶反转录酶转染椎间盘髓核细胞构建永生化细胞是一种可行的方法。
关键词 ： 组织构建； 脊柱组织构建； 髓核细胞； 人端粒酶反转录酶基因； DH5α感受态大肠肝菌； 转染； 永生化； 目的基因； 髓核细胞转染退行性椎间盘病； 省级基金； 组织构建图片文章；   
Abstract：
BACKGROUND: Nucleus pulposus cells are difficult to be isolated and cultured from the intervertebral disc, and the cells age rapidly. An urgent standard cell line is required for experimental research.
OBJECTIVE: To investigate the construction of recombinant green fluorescent protein plasmid expressing human telomerase reverse transcriptase and to assess the feasibility of constructing immortalized human nucleus pulposus cell line.
METHODS: Target gene cloning, target gene sequencing in eukaryotic expression plasmids, construction of eukaryotic expression plasmids expressing target genes, and transfection of human telomerase reverse transcriptase were performed in order.
RESULTS AND CONCLUSION: Nucleus pulposus cells transfected with recombinant green fluorescent protein plasmid expressing human telomerase reverse transcriptase have a stable expression. It is anticipated that human telomerase reverse transcriptase can be useful for creating the immortalized nucleus pulposus cells.
Key words： tissue construction; spinal cord tissue construction; nucleus pulposus cells; human telomerase reverse transcriptase gene;DH5α competent Escherichia coli; transfection; immortalized; target gene; nucleus pulposus cells transfection in degenerative disc diseases;provincial fund; tissue construction photographs-containing paper;
收稿日期: 2012-07-11     
中图分类号: 
R318

基金资助: 广州市医药卫生重点项目(2009-zdi-04)；2010年广东省自然科学基金(10151022001000005)；2011年广东省自然科学基金(S2011010000910)。
研究亮点:
1. 运用人端粒酶反转录酶转染髓核细胞构建永生化髓核细胞，构建的髓核细胞加入重组绿色荧光蛋白质粒，可以随时示踪，检测人端粒酶逆转录酶的表达。
2 .实验将增强型绿色荧光蛋白-人端粒酶反转录酶重组质粒转染髓核细胞，并通过荧光显微镜动态检测转染髓核细胞的增强型绿色荧光蛋白-人端粒酶反转录酶表达
3.建立一种能够长期培养、表型稳定的永生化髓核细胞株，希望能为细胞组织工程研究领域提供标准髓核细胞。