多囊蛋白1氨基段融合蛋白对肾小球系膜细胞细胞外基质及其降解基因表达的影响

目的研究多囊蛋白-1氨基段（PC-INF）融合蛋白对大鼠肾小球系膜细胞（RMC）Ⅳ型胶原及其降解调控基因表达的影响。方法用实时荧光定量RT-PCR法检测PC-1NF融合蛋白对RMCIV型胶原和细胞外基质（ECM）降解调控基因基质金属蛋白酶、金属蛋白酶1组织抑制剂（MMP-2、TIMP-1）表达的作用。ELISA方法检测培养的细胞上清液中Ⅳ型胶原含量的变化。免疫细胞化学方法观察融合蛋白对细胞核因子c-jun和c-fos表达的影响。Western印迹检测融合蛋白对蛋白激酶C（PKC）-α信号转导通路的影响。结果4mg／LPC-1NF融合蛋白作用48h后，RMCⅣ型胶原mRNA从（103±16）降至（82±11）拷贝，106GAPDH，与对照组比较差异有统计学意义（P〈0．05），培养上清液中的IV型胶原含量也明显降低；MMP2mRNA从（1150±90）升至（2770±）拷贝，106GAPDH，与对照组比较差异有统计学意义（P〈0．01），而TIMP-1mRNA从（5530±480）降至（3040±370）拷贝，106GAPDH，与对照组差异有统计学意义（P〈0．01）。DAB染色显示，融合蛋白作用后，RMC的c-jun和c-fos表达受到明显抑制。RMC经不同浓度PC-1NF融合蛋白作用48h后，随着融合蛋白浓度升高。PKC-α的表达逐渐减弱。结论PC-1NF融合蛋白可通过上调促进ECM降解的MMP-2和抑制ECM降解的TIMP-1之间的比值而促进Ⅳ型胶原降解；还可能通过PKC-α信号转导通路，抑制c-ju．和c-fos表达，从而抑制RMC的增殖和ECM的合成。

Effect of N-terminal fragment fusion protein of polycystin-1 on collagen Ⅳ synthesis and degradation in rat glomerular mesangial cells

Objective To investigate the effect of N-terminal fragment(LRR-WSC) fusion protein of polycystin-1 (PC-1NF) on collagen Ⅳ synthesis and degradation in rat glomerular mesangial cells and to explore its mechanism. Methods Rat glomerular mesangial cells were treated with different concentrations of PC-1NF in vitro. The mRNA expression of type Ⅳ collagen, metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase 1(TIMP-1) were detected by real-time fluorescence quantitative PCR. The concentration of type Ⅳ collagen was determined by ELISA. The expression level of c-fos and c-jun was examined by immunocytochemistry. The expression of PKC-α was detected by Western blot. Results After treatment with 4 mg/L PC-1NF for 48 hours, the mRNA level of hype IV collagen was down-regulated [（82±11） copies per million GAPDH] compared with that in the control [（103±16）copies per million GAPDH] （P<0.05）, so was the protein level of type Ⅳ collagen. The mRNA level of TIMP-1 was significantly decreased [（3040±370） copies per million GAPDH] compared with that in the control [（5530±480） copies per million GAPDH] （P<0.01）. However, the mRNA level of MMP-2[（2770±170） copies per million GAPDH] was significantly increased compared with that in the control [（1150±90） copies per million GAPDH] （P<0.01）. Meanwhile, PC-1 NF fusion protein treatment resulted in decreased protein levels of c-jun, c-fos and PKC-α. Conclusions PC-1 NF fusion protein may induce collagen Ⅳ degradation through increasing the ratio of MMP-2 to TIMP-1 in rat mesangial cells, inhibit c-jun and c-fos expression by modulating the PKC-α synthesis, and then decrease the production of collagen Ⅳ in rat glomerular mesanglal cells.