| Part 1:Primary Culture and Identification of Rat PodocyteOBJECTIVE:To establish the stable primary culture system of rat podocyte and to study the biology its characteristics.METHODS:Sterile kidneys were taken out of health SD rats which weighted from 100g to 150g.The glomeruli separated by sieving (100mesh,150mesh,200mesh),and then were cultured in culture medium(DMEM/F12 with 10%FBS and supplements including insulin, transferring,and sodium selenite),at 37℃and in 5%CO2 incubator. After one week,the podocytes growing from glomeruli were digested and passaged to coverslip.The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.Results:The glomeruli were integrated capsules,after five days culture, podocytes grew and appeared cobblestone,one week later,podocytes were digested and passaged to coverslip,the podocytes grew foot processes.The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.Nephrin was distributed in cell membrane,cytoplasm and nuclear.WT-1 was distributed in cell nuclear. PI staining is located in nuclear.Both WT-1 and nephrin are positive in podocytes.CONCLUSION:Glomeruli are seperated and podocytes grow from glomeruli and appear cobblestone.Arfter disgestion,podocytes differentiates foot processes.Both WT-1 and nephrin are detected positive in podocytes by indirect immunofluorescence staining.The stable rat podocytes culture system.has been establishedPart 2:The establishment of podocyte injury model by angiotensinⅡand the alteration of nephrin mRNA in podocytesAIM:To study mechanism of renal diseases,The podocyte injury model by angiotensinⅡwhich alterates expression of nephrin mRNA was established.METHODS:The podocyte culture in pore plate and grow well.After being handled to the equal pace,the podocytes were stimulated by angiotensinⅡ.AngiotensinⅡwas given on different time course(24h, 48h,and 72h)and with different dose(10-8mol/L,10-6mol/L,10-4mol/L). The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR).RESULTS:AngiotensinⅡreduced expression of nephrin mRNA at the time of 24h,48h and 72h.The downregulation of nephrin expression by AngⅡwas displayed time-dependent.AngiotensinⅡreduced expression of nephrin mRNA at the dose of 10-8mol/L,10-6mol/L and 10-4mol/L, which were dose-dependent.CONCLUSIONS:AngiotensinⅡcan reduce expression of nephrin mRNA of primary culture podocyte and appear time- and dosedependent.Part 3:Active metabolite A771726 of leflunomide on the expression of nephrin mRNA of injury podocyte by angiotensinⅡAIM:To exam the expression of nephrin mRNA of podocyte which are handled by A771726 after injuried by angiotensinⅡ.METHODS:The podocytes were cultured in pore plate and grew well, then the podocytes were handled to the equal pace.After podocytes were injured by angiotensinⅡ(10-6mol/L),different dose of A771726 (0.3μg/ml,3μg/ml,30μg/ml)was added to medium.The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR)and the RT-PCR products of nephrin were sequenced.RESULTS:A771726(0.3μg/ml,3μg/ml,30μg/ml)partly corrected the down regulation of nephrin mRNA induced by angiotensinⅡ. CONCLUSIONS:AngiotensinⅡdown regulates expression of nephrin mRNA,active metabolite A771726 of leflunomide can partly correct the effects of angiotensinⅡon it.
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