论文部分内容阅读
目的通过在真核细胞中表达突变基因,探讨NPHS2基因突变对podocin分子表达和分布的影响。方法构建分别含有野生型NPHS2编码区cDNA、467-468insT和503G>A突变NPHS2编码区cDNA的表达载体,分别转染人胚肾细胞(HEK293)后用抗podocin氨基端多克隆抗体(P21)和抗podocin羧基端多克隆抗体(P35)进行免疫荧光染色,在共聚焦显微镜下观察podocin在细胞内的分布。结果与野生型NPHS2编码的podocin分子的表达和分布比较,抗podocin氨基端抗体染色显示,两个突变NPHS2表达的podocin与野生型的一样均为阳性;抗podocin羧基端抗体染色显示503G>A突变NPHS2表达的podocin与野生型的一样为阳性,而467-468insT突变NPHS2表达的podocin呈阴性。野生型NPHS2表达的podocin不但分布在胞核的周围,而且主要在细胞膜上呈连续性的线状分布,而两个突变NPHS2表达的podocin主要分布在胞核周围。结论NPHS2的467-468insT突变将严重影响podocin的分子结构和在细胞内的正常分布。NPHS2的503G>A突变即使没有严重影响podocin的结构,但仍然导致podocin不能正常到达细胞膜,因而不能行使正常功能。podocin的正常功能不但有赖于分子结构也有赖于分子分布。
Objective To investigate the effect of NPHS2 gene mutation on the expression and distribution of podocin by expressing mutant genes in eukaryotic cells. Methods The expression vector of cDNA encoding NPHS2 coding region of wild type NPHS2, 467-468insT and 503G> A respectively was constructed and transfected into human embryonic kidney (HEK293) cells with anti-podocin polyclonal antibody (P21) and Anti-podocin carboxyl terminal polyclonal antibody (P35) immunofluorescence staining observed under confocal microscope podocin intracellular distribution. Results Compared with the expression and distribution of podocin in wild-type NPHS2, the anti-podocin amino-terminal antibody staining showed that the podocin expression of both mutant NPHS2 was the same as that of the wild type. The anti-podocin carboxyl terminal antibody showed 503G> A mutation Podocin expressed by NPHS2 was positive as wild type, while podocin expressed by 46HSHS mutation 467-468insT was negative. The expression of podocin in wild-type NPHS2 was not only distributed around the nucleus, but also mainly in the linear distribution of the cell membrane, while the expression of podocin in NPHS2 was mainly distributed around the nucleus. Conclusion The 467-468insT mutation of NPHS2 will seriously affect the molecular structure of podocin and its normal distribution in the cell. The 503G> A mutation of NPHS2, even though it did not significantly affect the structure of podocin, still causes podocin not to reach the cell membrane normally and therefore can not function properly. The normal function of podocin depends not only on the molecular structure but also on the molecular distribution.