| Renal fibrosis is the result of various chronic kidney diseases(CKD).The major pathological features of renal fibrosis are glomerulosclerosis and tubularinterstitial fibrosis. The mechanism of renal fibrosis is unknown, and the study for quesing the reasonable way to block or prevent and even reverse the renal fibrosis is still in difficult groping stage. The occurrence of renal fibrosis is the result of multiple factor coaction. In recent years, several lines of studies have demonstrated that TGF-βwas the important fibrogenic cytokines and has critical role in the occurrence and development of renal fibrosis.On the basis of the reasons mentioned above, and to investigate the regulative mechanism of TGF- β 1 protein synthesis, the aims of this study were: 1. to construct recombinant plasmids phTGF contain the different length of TGF-β1 promoter region which named phTGFO. 585, phTGF1. 12, phTGF1. 423, phTGF1.68, phTGF2. 14, to evaluate the activity of each construct phTGF plasmids and to analyze the region of regulation sequence located; 2. to investigate the influence of HGF on TGF-β 1 gene promoter activity and the interaction among them; 3. to study the influence of drugs, lovastatin and verapamil, on the activity of TGF-β1 gene promoter activityPart I Construction of phTGF recombinant plasmids and analysis of their activityUsing human blood genomic DNA as template, five fragments of TGF- β 1 gene upstream sequence with the same 3' end were obtained by PCR method. The five promoter fragments, which length were 0. 585kb, 1. 12kb, 1. 423kb, 1. 68kb, 2. 14kb, were ligated to the chloramphenicol acetyltransferase(CAT) reporter gene-pCAT-Enhancer (vector), respectively. The five deletion constructsobtained were named as phTGFO. 585, phTGFl. 12, phTGFl. 423, phTGFl. 68, phTGF2.14, these constructs were analyzed to be corrected by digested weth restriction enzymes Mlu I and HindUJ, these constructs use the same vector pCAT-enhancer, the length of promoter sequence inserted into it were 0.585kb, 1.12kb, 1.423kb, 1.68kb, 2.14kb respectively. They were correspondent to the sequence of +227~+812bp , -308~+812bp, -611~+812bp, -867~+812bp, -1328~+812bp respectively in the upstream of the human TGF-31 gene. The sequence of these fragments were consistent to the sequence obtained from GenBank(Accession No. J04431). These constructs were transiently transfected into normal rat mesangial cells(RMC) with FuGene transfection reagent. The CAT activity of the reporter gene was assessed by ELISA method and compared their promoter activity. The results showed that the plasmid which contains the -308~+812bp sequence of TGF- P 1 gene had highest activity, the plasmed with the -611~+812bp sequence took the second place, the list of the activity from higher to lower of these constructs were phTGFl.12, phTGFl. 423, phTGF2.14, phTGFl. 68, phTGFO. 585 in turn.Part II Study of transcriptional regulatory of TGF-P 1 gene by HGFObjective: To study the effect of HGF on the proliferation of RMC and expressive activity of TGF-P 1 gene promoter in the RMC. Method: Effect of HGF on the proliferation of RMC were detected by MTT method. The constructs, phTGF2. 14 and phTGFl. 12 , were transfected into rat mesangial cells(RMC) using the method of transient transfection. The cells were interfered with appropriat concentration of HGF(lng/ml, lOng/ml). The CAT activity were assessed by ELISA. Result: The proliferation of RMC were interfered with HGF of 0, 0.1, 1, 10, lOOng/ml. The result of MTT were 0. 70 + 0. 04, 0. 72 + 0. 03, 0. 73 + 0. 04, 0. 75 + 0. 02, 0. 74 + 0. 06, respectively. There were no significant difference between cells treated with differentconcentration of HGF and control(P>0.05). The RMC transfected with phTGF2. 14 were treated with lng/ml and lOng/ml. The CAT activity of control group(phTHG2. 14) was 1. The CAT activity of test groups(HGF: lng/ml and lOng/ml) were 0.76 + 0.1 and 0.78 + 0.15 respectively. There were no significant difference between test groups and control (P>0. 05). The RMC transfected with phTGFl. 12 were treated with lng/ml and lOng/ml. The CAT activity of control group(phTHGl.12) was 1. The CAT activity of test groups (HGF: lng/ml and lOng/ml) were 0. 73 + 0. 06 > 0.89 + 0.21 respectively. There were no significant difference between test groups and control(P>0.05). Conclusion: 1. HGF could not effect the proliferation of RMC. 2. HGF could not inhibit the activity of TGF-pi gene promoter.PartUI Effect of drugs on transcriptional regulatory of TGF-01 gene Objective: To study the effect of lovastatin and verapamil on the proliferation of RMC and expressive activity of TGF-P 1 gene promoter in the RMC. Method: Effect of lovastatin and verapamil on the proliferation of RMC were detected by MTT method. The constructs, phTGF2. 14 and phTGFl. 12 , were transfected into rat mesangial cells (RMC) using the method of transient transfection. The cells were interfered withappropriat concentration of lovastatin(107M and 105M) and verapamil(lmg/U lOmg/L) . The CAT activity were assessed by ELISA. Result: Theproliferation of RMC were interfered with lovastatin of 0, 10 M. 10 M.10"5M, 10"4M. The result of MTT were 1.40 + 0. 07. 0.95 + 0. 05. 0.73 + 0.03. 0.70 + 0.03. 0. 43 + 0. 02 respectively. There were significant difference between cells treated with different concentration of lovastatin and control (P<0. 01). The proliferation of RMC were interfered with verapamil of 0, lmg/L. lOmg/L. 50mg/L. The result of MTT were 1.40 + 0.07. 1.18...
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