raf-1基因高表达对鼠肾小球系膜细胞增殖表型的影响  

Effects of raf1 gene overexpression on proliferative phenotype of rat mesangial cells

作　　者：李彪[1] 李晓玫[1] 

机构地区：[1]北京医科大学第一医院肾内科

出　　处：《中华医学杂志》1999年第7期529-532,共4页National Medical Journal of China

基　　金：国家自然科学基金

摘　　要：目的探讨ｒａｆ１基因高表达对肾小球系膜细胞增殖的调控作用。方法应用细胞直接计数、流式细胞仪及特异底物磷酸化法分别检测ｒａｆ１基因转染细胞的增殖率、细胞周期分布及与细胞外信号调节的蛋白酶（ＥＲＫ）和ｃｊｕｎ蛋白酶（ＪＮＫ）的活性。结果ｒａｆ１基因高表达细胞早期生长正常，但于后期生长迟缓，细胞数目（２２．０±２．５）×１０５个／孔与对照组（３３．４±２．７）×１０５个／孔相比较显著减少（Ｐ＜０．０５）；细胞于生长第５天时停滞于Ｇ０／Ｇ１期（８１．３±３．７），（６２．３±２．４）％，（Ｐ＜０．０１），而Ｓ期细胞数量（８．３±０．９）％明显少于对照组（２７．０±１．５）％，（Ｐ＜０．０１）；其ＥＲＫ活性［（２１４±１６）ｐｍｏｌ·ｍｇ－１·ｍｉｎ－１］显著高于对照组［（８２±９）ｐｍｏｌ·ｍｇ－１·ｍｉｎ－１，Ｐ＜０．０５］，但ＪＮＫ活性则较对照组明显降低约５０％。结论在鼠肾小球系膜细胞中，Ｒａｆ１蛋白激酶确可作为ＥＲＫ的上游信号激活细胞增殖信号，但其持续高表达则能导致细胞生长周期停滞，这一作用很可能与ＪＮＫ活性抑制或细胞周期调控蛋白的变化有关。bjective To investigate the regulating effects of raf1 gene overexpression on phenotypic characteristics related proliferation in rat mesangial cells (MC). Methods raf1 gene was stably transfected and overexpressed in rat MC. Growth rate, cell cycle and activity of mitogenactivated protein kinases including ERK and JNK were compared between transfected and normal cells by cell counting, FACS and specific substrate phosphorylation. Results The growth rate of raf1 transfected cells was normal from the 1st to 3rd days, but slower on the 5th day. At the 7th day, the cell number in raf1transfected clones was much less than in controls(22.02.5)105 vs (33.42.7)105,(P<0.05). Most of raf1 transfected cells stayed in the G0/G1 stage in cell cycle (81.33.7)% vs (62.32.4)%, (P<0.01); the percentage of cells in S stage was decreased markedly (8.30.9)% vs (27.01.5)%, (P<0.01). ERK activity was elevated (21416) pmolmg-1min-1 vs (829) pmolmg-1min-1, (P<0.05), but JNK activity was inhibited by about 50% in raf1 overexpressed cells compared to controls. Conclusion Raf1 is a critical intracellular signal for regulating proliferative phenotype in rat MC. Its constitutive overexpression leads these cells to be silent indicating arrest cell cycle, which may be mediated by regulating protein in cell cycle and intracellular JNK activation.

关 键 词：信号转递 蛋白激酶类 基因表达 肾小球疾病 

分 类 号：R692.6[医药卫生—泌尿科学]