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Western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NIH3T3细胞48h的BrdU摄取率显著升高(P<0.01),72h的MTT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/G1期细胞比例,提高S期细胞比例,提示TIMP-1高表达能够促进成纤维细胞增殖,从而加速间质纤维化的进程。
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.
出处
《中国中西医结合肾病杂志》
2007年第2期73-76,I0001,共5页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
国家自然科学基金创新研究群体科学基金资助项目(No30121005)
国家自然科学基金面上项目(No30570856)
