人肾小球足细胞表达Nephrin的体外研究

Expression of nephrin by human glomerular podocyte in vitro

目的:观察人足细胞在体外表达Nephrin的分子量大小、细胞内分布。方法:体外培养人肾小球足细胞,采用间接免疫荧光法、免疫蛋白印迹研究原代培养人足细胞表达Nephrin的分布、蛋白质的分子量,逆转录PCR(RT-PCR)法检测Nephrin的mRNA表达等,RT-PCR产物进行序列分析加以确定。结果:体外原代培养的人足细胞可以表达Nephrin,间接免疫荧光显示其沿足细胞的细胞膜、细胞骨架和细胞核分布,并且在细胞内有一聚集中心。免疫蛋白印迹示人足细胞表达2种分子量的Nephrin,其中135kD的分子量与预期分子量一致,180kD的分子量与肾小球蛋白提取物一致,RT-PCR从基因水平确定了该蛋白的表达。结论:人足细胞在体外培养的条件下表达不同分子量的Nephrin,并且在细胞内分布具有一定的规律,为进一步研究其在自身细胞和肾脏病中的作用提供了可靠的实验方法。

Objective: To investigate whether the human glomerular podocytes can express nephrin in vit ro or not, and the distributions of nephrin in podocytes. Methodology:The human glomerular podocytes separated from kidney were cultured in culture me dium (RPMI-1640 with supplements including insulin, transferring, and sodium se lenite), at 37℃ and in 5% CO 2 incubator. The distribution of nephrin in podoc yte was detected by indirect immunofluorescence staining. The molecule size of n ephrin protein was determined by Western Blotting. The mRNA expression of nephri n gene was studied by reverse transcriptional polymerase chain reaction (RT-PCR ) and the RT-PCR products of nephrin were sequenced. Results:Nephrin was distributed along the cell membrane, cytoskeleton, nucleus and also appeared in the mitotic apparatus under immunofluorescence microscopy. Nephrin h ad an organizing center in podocyte plasma. Podocyte in vitro expressed 135 kD a nd 180 kD molecule sizes of nephrin by Western Blotting, the former was similar to expected molecule size, and the later was similar to nephrin from the whole g lomeruli protein lysate. The mRNA expression of nephrin was confirmed by RT-PCR and sequencing its product. Conclusion:The human glomerular podocytes express two molecule sizes of nephrin in vitro. T he nephrin has regular distributive pattern in the cell. Our study supplies one kind method to study the role of podocyte and in renal diseases.