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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 observe the molecular size and intracellular distribution of human Nephrin in vitro. Methods: Human glomerular podocytes were cultured in vitro. The distribution of Nephrin in primary cultured human podocyte was studied by indirect immunofluorescence and Western blotting. The expression of Nephrin was detected by reverse transcription polymerase chain reaction (RT-PCR) Etc., RT-PCR products were identified by sequence analysis. RESULTS: Nephrin was expressed in human primary cultured cells in vitro. Indirect immunofluorescence revealed the distribution of Nephrin along the cell membrane, cytoskeleton and nucleus along the podocyte, with an aggregation center in the cell. Immunoblotting showed that human podocytes expressed two kinds of molecular weight Nephrin, of which the molecular weight of 135kD was consistent with the expected molecular weight. The molecular weight of 180kD was consistent with that of glomerular protein extract. The expression of this protein was confirmed by RT-PCR from the gene level. CONCLUSION: Human podocytes express Nephrin with different molecular weights under in vitro culture conditions and have certain regularity in the cell distribution, which provides a reliable experimental method for further studying its role in autologous cells and kidney disease.