| Objective: To explore the effect of Angiotensin-(1-7) [Ang-(1-7)] on the proliferation and extracellular matrix excretion of glomerular mesangial cells(GMCs) induced by transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Methods: Experiments were performed after the mesangial cells had been cultured in the serum-free DMEM for 24 hours. The cells were divided into six groups according to the different intervention factors,final concentration of Ang-(1-7) was 10-6 mol/L,TGF-β1 was 5ng/ml,CTGF was 5ng/ml.①control group:the GMCs were cultured without any treatment;②Ang-(1-7) group: the GMCs were stimulated by Ang-(1-7);③TGF-β1 group: the GMCs were stimulated by TGF-β1;④TGF-β1+Ang- (1-7) group: the GMCs were co-stimulated by TGF-β1 and Ang-(1-7);⑤CTGF group:the GMCs were stimulated by CTGF;⑥CTGF +Ang- (1-7) group:the GMCs were co-stimulated by CTGF and Ang-(1-7).(1) Mesangial cell population was detected by WST-1 assay.The GMCs were prepared 5×104/ml suspension with serum-free DMEM, seeded out into 96 well plates as 6 different groups,with adding 100μl suspension each, and were placed into incubator without serum for 24 hours. These cells were interfered with Ang-(1-7) or/and TGF-β1,CTGF for additional 24 hours. 10μl WST-1 was added into each group. The absorbency at 450 nm of each well plate was measured by the spectrophotometer after incubated 4 hours. (2) LN mRNA,ColⅣmRNA expressions in GMCs were analyzed by RT-PCR: The GMCs were interfered 48 hours with interferation and then the total RNA of each group was extracted with reversed transcription as cDNA for the RT-PCR.After PCR was finished, Reinhoit Zahl of objective gene and intra gene in every group of 15μl production was measured in the 1.5% sepharose,0.5×TBE as electrophoresis buffer,100V voltage electrophoresis for 40min,according to the gelatum imaging and scanning analytical system. (3) The secretion of LN and ColⅣ:The secretion of LN and ColⅣwas measured by radioimmunoassay in culture medium of glomerular mesangial cells after an interference of 24 hours with stimulators. All data was reported as mean±standard deviation. Statistical significance was determined by one-way ANOVA. Significance was defined as a value of P<0.05. The software of SPSS14.0 was applied for statistics analysis. Results: (1) TGF-β1 induced the proliferation of cultured glomerular mesangial cells(P<0.05); Ang-(1-7) significantly inhibited basic and TGF-β1-induced and CTGF-induced proliferation of cultured glomerular mesangial cells; (2) mRNA expression was analyzed by RT-PCR: TGF-β1,CTGF enhanced the expression of LN mRNA and ColⅣmRNA(P<0.05); Ang-(1-7) obviously down-regulated the expressions of LN mRNA and ColⅣmRNA (P<0.05); Ang-(1-7) significantly inhibited the expression of LN mRNA and ColⅣmRNA induced by TGF-β1 or CTGF(P<0.05). (3) Content of LN and ColⅣ:Content of LN and ColⅣin the TGF-β1 group was more obviously advanced than that of control group(P<0.05),but the above index of Ang-(1-7) group was reduced(P<0.05), and Ang-(1-7) significantly inhibited the secretion of LN and ColⅣinduced by TGF-β1 and CTGF(P<0.05). Conclusion:①Ang-(1-7) can inhibit basic and the TGF-β1-induced and CTGF-induced proliferation of cultured glomerular mesangial cells;②Ang-(1-7) can down-regulate the mRNA expression of LN mRNA and ColⅣmRNA basic and the expression of that induced by TGF-β1 , CTGF.③Ang-(1-7) can decrease the secretion of LN and ColⅣbasic and the secretion of that induced by TGF-β1 and CTGF .
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