| Objective:To investigate the effects and mechanism of endothelin-1(ET-1) and its receptor antagonist BQ123 influence the transformation of phenotype and proliferation of rat vascular smooth muscle cells (VSMC) in vitro.Methods:The VSMC were cultured with rat aorta in vitro through the explant- attached method. The 4th～5th passage cells were cultured in DMEM culture medium without blood serum. The first experiment: The cells were kept pace with each other, and then divided into: control group (A1), ET-1 group(B1), ET-1and BQ123 group(C1). The final concentration of ET-1 is 10-7mol/L and BQ123 is 10-6mol/L. Terminated the reaction 48h later, 5-BrDU (8×10-4mol/L) was added into the culture medium before 24h of accomplishing samples. The proliferation of cells was labeled by 5-bromodeoxyuridine (5-BrDU) with immunocytochemical method, and the mRNA expression of hypertension related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22α) were determined by detected by reverse transcription- polymerase chain reaction method (RT-PCR). The second experiment: The cells were kept pace with each other, and then continued to incubate for 72h in culture flask containing 10% new-born calf serum(NCS) DMEM culture medium. A quarter of those cells were culminated as serum induced group (A2). Removed the serum in cultures the rest of the cells were divided into three groups: no serum group (B2), ET-1 group (C2), ET-1and BQ123 group (D2). The final concentration of ET-1 is 10-7mol/L and BQ123 is 10-6mol/L. To keep the experiment for 48h, and then the changes of the mRNA expression of HRG-1 and SM22αin the four groups was detected by RT-PCR.Results:In the first experiment, the VSMC had some degree of proliferation in all the groups. We can see that most of proliferous cells were labeled by 5-BrDU in the ET-1 group under the microscope. There were lest of positive nucleuses labeled by 5-BrDU in the ET-1 and BQ123 group. The mRNA expression of HRG-1 and SM22αwas decreased obviously in the ET-1 group, and those effects can be blocked when the cultures added into BQ123。The second experiment: The result of RT-PCR indicated that ET-1 can decrease significantly the mRNA expression of HRG-1 and SM22α, and its receptor antagonist BQ123 blocked those effects.Conclusions:1. ET-1 could stimulate notably proliferation of VSMC that were contractile type and change the phenotype of VSMC from contractile to synthesize type. 2. ET-1 affected remarkably phenotype and proliferation of VSMC that were synthesize type , that told us the effect on the VSMC of ET-1 can not reversion. 3. the receptor signal transduction pathway may be one of the mechanisms of the influence on the phenotype and proliferation of vascular smooth muscle cells of endothelin-1PartⅡInfluence on the phenotype and proliferation of vascular smooth muscle cells in rat by antistatic action of endothelin-1 and calcitonin gene-related peptideObjective:To study whether endothelin-1 and calcitonin gene- related peptide can influence the phenotypic modulation and proliferation of vascular smooth muscle cells (VSMC), and what is the relationship of the two factors.Methods:To establish the cellular models of VSMC from rat aorta in vitro. The 4th～5th passage cells were cultured in DMEM culture medium without blood serum The first experiment: To kept cellular pace with each other, and then divided into: control group(a1), ET-1 group(b1), ET-1 and CGRP group(c1).CGRP and ET-1 group(d1).In c1 group, deal the VSMC with ET-1+CGRP for 24h after ET-1; In d1 group, treated the VSMC by CGRP for 24h before the mixture of CGRP and ET-1. The concentration of ET-1 and CGRP is 10-7mol/L and 2×10-7mol/L, respectively.After 48h, terminated the reaction. In theexperi-ment the proliferation of the cells were labeled by 5-bromodeoxyuridine (5-BrDU) with immunocytochemical method, and the mRNA expression of hypertension related gene-1 (HRG-1) and smooth muscle 22 alpha (SM22α) were determined by detected by reverse transcription- polymerase chain reaction method (RT-PCR). The second experiment: The cells were kept pace with each other, and then continued to incubate for 72h in culture flask containing 10% new-born calf serum(NCS) DMEM culture medium. 1/5 of those cells were culminated as serum induced group (a2). Removed the serum in cultures the rest of the cells were divided into four groups: no serum group(b2), ET-1group(c2), ET-1+CGRP group(d2). CGRP+ET-1 group(e2). In d2 group, deal the VSMC with ET-1+CGRP for 24h after ET-1; In e2 group, treated the VSMC by CGRP for 24h before the mixture of CGRP and ET-1. The concentration of ET-1 and CGRP is 10-7mol/L and 2×10-7mol/L, respectively. To maintain the experiment for 48h, and then the changes of the mRNA expression of HRG-1 and SM22αin the five groups were examined by RT-PCR.Results:It was found that, in the first experiment, there were lots of proliferous cells were labeled by 5-BrDU in the b1 group and c1 group. The positive nucleuses in the d2 group reduced extremely. The mRNA expression of HRG-1 and SM22αwas decreased significantly in the b1 group, and in c1 group the mRNA expression of the two genes were increased, but it was slightly. The mRNA expressions of the two genes were increased in the d1 group notably. The second experiment: The result of RT-PCR showed that in d2 group the mRNA expression of HRG-1 and SM22αwas increased lightly compared with the c2 group, but the expression of the genes increased obviously in the e2 group.Conclusions:1. CGRP could inhabit the proliferation of VSMC induced by ET-1, and the effects were obvious as the precaution. 2. CGRP could reverse the phenotype of VSMC from contractile synthesize to contractile type induced by ET-1, and also the effects were manifest at the prevention stage . |
PDF Full Text Request