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体外瞬时转染TIMP-1基因促进成纤维细胞增殖

ISSN号：1009-587X
期刊名称：《中国中西医结合肾病杂志》
时间：0
分类：R619.6[医药卫生—临床医学;医药卫生—外科学]
作者机构：[1]中国人民解放军总医院,全军肾脏病研究所暨肾脏病重点实验室,北京 100853
相关基金：本课题为国家自然科学基金创新研究群体科学基金资助项目（No.30121005）,国家自然科学基金面上项目（No.30570856）

作者：刘书馨[1], 吕杨[1], 陈香美[1], 谢院生[1], 傅博[1], 洪权[1], 刘维萍[1]

关键词：组织型金属蛋白酶抑制剂1, 细胞增殖, 成纤维细胞, 细胞周期, Tissue inhibitor of metalloproteinase- 1 Cell proliferation Fibroblast Cell cycle

中文摘要：

目的：探讨体外瞬时转染组织型金属蛋白酶抑制剂1（TIMP-1）质粒对小鼠成纤维细胞细胞周期和增殖的影响。方法：构建表达小鼠TIMP-1的质粒，体外转染小鼠成纤维细胞NIH3T3,以转染空质粒的NIH3T3细胞作对照，采用RT—PCR和westem印迹分析检测TIMP—1在基因和蛋白质水平的表达；采用BrdU法和MTT法检测细胞增殖能力和活力；流式细胞仪检测细胞周期的变化。结果：转染TIMP-1质粒的NIH3T3细胞在24h即有明显的TIMP-1mRNA表达和蛋白质表达上调；与转染空质粒的NIH3T3细胞相比，转染TIMP-1质粒的NI心L细胞48h的BrdU摄取率显著升高（P〈0．01），72h的m摄取率显著升高（P〈0．05）。流式细胞仪检测结果显示与转染空质粒的细胞相比，72h时TIMP-1质粒转染组G0／G1期细胞显著减少（P〈0．01），s期细胞比例显著升高（P〈0．01）。结论：体外瞬时转染TIMP-1质粒可以有效地使TIMP-1的基因和蛋白质水平在NIH3T3细胞高表达，TIMP-1高表达显著提高细胞BrdU和MTT摄取率，减少G0／（31期细胞比例，提高s期细胞比例，提示TIMP—1高表达能够促进戍纤维细胞增殖，从而加速间质纤维化的进程。

英文摘要：

Objective：To investigate the possible effect of tissue inhibitor of metalloproteinase- 1 （TIMP- 1 ） gene on the cell cycle and proliferation of fibmblast （NIH3T3）. Methods：Plenti6v5dest- TIMP- 1 plasmid was constructed. NIH3T3 cells were transient transfection of Plenti6v5dest - TIMP- 1 plasmid and Plenti6v5dest plasmid as control. The transfection effects of TIMP - 1 on the mRNA and protein levels were examined by reverse transcription PCR （RT- PCR） and Western blot analysis respectively. Cell proliferation rate and viability were measured by BrdU and MTT. The changes of cell cycles were detected by FCM. Results：TIMP- 1 mRNA and protein expression were obvious upregulated at 24hr in TIMP- 1 plasmid transfected NIH3T3 cells than control plasmid transfected cells. BrdU and MTT incorporation rate were increased significantly at 48hr（P 〈 0.01 ）and 72 hr respectively（P〈 0.05）. Compared to control plasmid transfected cells, cell cycle of G0/G1 stage were much lower（P 〈 0.01 ）and S stage much higher（P〈0.01 ） in TIMP - 1 transfected cells at 48 hr. Conclusion：High expression of TIMP - 1 gene markly increases BrdU and MTT incorporation rate, decreases G0/G1 stage ceils numbers and increases S stage cell numbers in NIH3T3 cells. These resuits suggests that TIMP- 1 stimulates NIH3T3 cell proliferation and ultimately accelerates interstitial fibrosis.

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