转染Megsin基因对体外系膜细胞增殖和Ⅳ型胶原分泌的影响及机制

Effects and mechanism of Megsin gene transfection on mesangial cell proliferation and type IV collagen excretion

目的了解Megsin基因高表达对体外系膜细胞增殖和Ⅳ型胶原分泌的影响,并探讨其机制。方法构建大鼠Megsin基因真核表达载体,体外转染系膜细胞。3H-胸腺嘧啶(3H- TdR)掺入法检测系膜细胞增殖情况。RT-PCR方法检测系膜细胞血小板源生长因子(PDGF)- BB、IL-1β、IL-6、IL—10、TGF-β1和TNF-αmRNA表达变化。ELISA法检测细胞培养上清PDGF- BB、TGF-β1和Ⅳ型胶原浓度。观察PDGF-BB中和抗体对大鼠系膜细胞转染Megsin基因后细胞增殖和TGF-β1 mRNA表达的影响。结果大鼠系膜细胞转染Megsin基因后,细胞内3H-TdR掺入量显著增加,PDGF-BB和TGF-β1 mRNA表达显著上调,细胞培养上清PDGF-BB、TGF-β1和Ⅳ型胶原的浓度显著升高,3者与转染Megsin基因呈明显时间依赖性关系。PDGF-BB中和抗体显著抑制系膜细胞转染Megsin基因后细胞内3H-TdR的掺入,并下调稳定表达Megsin基因的大鼠系膜细胞TGF-β1 mRNA表达。结论Megsin基因高表达在体外可促进系膜细胞PDGF-BB和TGF-β1的表达和分泌,进而促进系膜细胞增生和Ⅳ型胶原分泌。

Objective To investigate the effects and mechanism of Megsin gene transfection on mesangial cell proliferation and type Ⅳ collagen excretion. Methods Rat Megsin cDNA eukaryotic expressing vector was constructed and transfected to cultured rat mesangial cells. Cell proliferation was measured by determining [3^H]-thymidine （3^H-TdR） incorporation. The mRNA expression of PDGF-BB, TGF-β1, IL-1β, IL-6, IL-10 and TNF-α in mesangial cells was detected by RT-PCR. Excretion of PDGF-BB, TGF-β1 and type Ⅳ collagen in supematant of cultured mesangial cells was measured by ELISA. Impacts of PDGF-BB neutralizing antibody in cell proliferation and expression of TGF-β1 mRNA was also detected. Results A time-dependent up-regulation of mRNA expression of PDGF-BB and TGF-β1 was detected in cultured mesangial cells when transfected with Megsin gene. The concentration of PDGF-BB, TGF-β1 and type Ⅳ collagen in the cell supematant rose accordingly. PDGF-BB neutralizing antibody suppressed the incorporation of 3^H-TdR and the mRNA expression of TGF-β1 in rat mesangial cells transfected with Megsin gene significantly. Conclusion Over expression of Megsin gene enhances the mRNA expression, extracellular excretion of PDGF-BB and TGF-β1 in mesangial cells, which further leads to mesangial cell proliferation and type Ⅳ collagen excretion.