多囊蛋白-1氨基段融合蛋白对大鼠肾小球系膜细胞周期及其调控基因表达的影响

Effect of N-terminal Fragment Fusion Protein of Polycystin-1 on Cell Cycle and Its Regulatory Gene in Rat Mesangial Cell

研究多囊蛋白-1氨基段(PC-1NF)融合蛋白对大鼠肾小球系膜细胞(MC)周期及其调控基因表达的影响。应用Brdu-ELISA法检测PC-1NF融合蛋白对MC增殖的作用,流式细胞术观察PC-1NF融合蛋白对MC周期的影响,实时荧光定量RT-PCR方法检测PC-1NF融合蛋白对MC周期调控基因cyclinD1、p21WAF1表达的作用。结果表明PC-1NF融合蛋白能抑制MC增殖,呈现良好的时效与量效关系;PC-1NF融合蛋白能影响MC周期,使G0/G1期细胞增加,S期细胞减少;4μg/mlPC-1NF融合蛋白作用后,cyclinD1mRNA水平比对照组明显下调(P<0.05);而p21WAF1mRNA水平比对照组显著上调(P<0.01)。PC-1NF融合蛋白能抑制MC增殖及周期的进展,其机制可能是通过下调cyclinD1、上调p21WAF1的表达,抑制细胞通过G1-S调控点而介导的。

To investigate the effect of N-terminal fragment （LRR-WSC） fusion protein of polycystin-1 （PC- 1NF） on cell cycle and its regulatory gene in rat mesangial cell and explore its mechanism. Rat mesangial cells were treated with serial concentration of PC-1NF in vitro. The proliferation of rat mesangial cells was measured by Brdu-ELISA. Cell cycle was analyzed by flow cytometry. The mRNA expression of cell cycle regulatory gene cyclin D1 and p21^WAF1 was detected by real-time fluorescence quantitative PCR. Micro-gram per milliliter of PC-1NF could effectively inhibit the proliferation of mesangial cells with dose- and time-dependent correlation to some extent, and cause G0/G1 phase arrest. After treatment with 4 μg/ml PC-1NF, the mRNA level of cyclin D1 was significantly decreased [（2.44±0.27）× 10^4 copies per million GAPDH] compared with that in the control [（4.10±0.32）× 10^4 copies per million GAPDH]. However, the mRNA level of p21^WAF1was significantly increased [（3.73±0.46）× 10^2 copies per million GAPDH] compared with that in the control [（0.85±0.18）×10^2 copies per million GAPDH]. N-terminal fragment fusion protein of polycystin- 1 could effectively inhibit mesangial cell proliferation. The effect could be mediated by down-regulated cyclin D1 and up-regulated p21^WAF1 mRNA expression, subsequently blocking cells passing through Gm-S checkpoint.