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Effects of Endothelin-1 and Calcitonin Gene-related Peptide on the Transformation of Phenotype and Proliferation of Vascular Smooth Muscle Cells in Rat

Author: XuHongTao
Tutor: WangChen
School: Suzhou University
Course: Human Anatomy and Embryology
Keywords: Endothelin-1 Hypertension-related gene 1 Smooth muscle 22α Calcitonin gene-related peptide phenotypic changes
CLC: R543
Type: Master's thesis
Year: 2007
Downloads: 86
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Abstract

The effects and mechanism of the purpose of the first part of endothelin-1 on the transformation and proliferation of rat vascular smooth muscle cell phenotype: role of ET-1 in rat VSMC and ET-1 receptor signal blocking explore the ET-1 the VSMC phenotype transformation and proliferation of and mechanisms. METHODS: Cultured rat aortic VSMC, experiments take the first 4 to 5 cells. Experiment, the experiment is divided into: A1: control group, B1: ET-1 group C1: of ET-1 Group Effect of BQ123. Synchronized with serum-free DMEM medium for 24h, the cells in each group join the appropriate drugs (ET-1 ,10-7mol / L; Effect of BQ123 ,10 mol / L), after 48h the cells were collected with a 5-BrDU tag cells proliferation; RT-PCR detection VSMC proliferation negative regulatory genes HRG-1 and Phenotypic SM22α expression. The second experiment, the experiment is divided into: A2: serum for 72h; B2: serum control group; C2: ET-1 group; D2: ET-1 BQ123 group. Groups cells, containing 10% NCS DMEM continue for 72h after synchronization with serum-free DMEM medium for 24h, this time A2 terminate cultured cells were collected, the rest of the group replaced with serum-free medium and were added to the corresponding drugs The role of (ET-1 ,10-7mol / L; BQ123 ,10-6mol / L) for 48 hours, the cells were collected, RT-PCR was used to detect the expression of HRG-1 and SM22αmRNA. Results: experiment: immunocytochemistry, ET-1 action group visible proliferation of VSMC while adding marked proliferative cells were significantly reduced after the ET-1 receptor antagonist BQ123. RT-PCR analysis of ET-1 group HRG-1, and SM22αmRNA express significantly down compared with the control group join blocker BQ123 antagonize this role. Experiment II: RT-PCR detection further confirmed to the BQ123 have blocked the ET-1 down VSMC HRG-1 and the SM22α two gene expression. Conclusion: 1.ET-1 on the contractile phenotype VSMC significant effect on the proliferation and enables VSMC phenotype from contractile to synthetic conversion; 2.ET-1 synthetic VSMC also promote phenotypic transformation and proliferation effect, indicating that the role of ET-1 on VSMC factor is an irreversible. 3 receptor signaling pathway is one of the mechanisms of ET-1 to promote the transformation and proliferation of VSMC phenotype. The purpose of the second part of endothelin-1 and CGRP total effect of the transformation and proliferation of vascular smooth muscle cell phenotype: ET-1 and calcitonin gene-related peptide (CGRP) acting on VSMC, research CGRP inhibit ET-1 promote The role and mechanism of the transformation and proliferation of VSMC phenotype. METHODS: Cultured rat aortic VSMC, experiments take the first 4 to 5 cells. First experiment, the experiment was divided into: a1: control group; b1: ET-1 group; c1: ET-CGRP group 1 (ET-1 for 24h, plus CGRP) in; d1: CGRP (CGRP for 24h, ET-1 , together with ET-1). Serum-free DMEM medium synchronization for 24h, the cells in each group join the appropriate drugs (ET-1 10-7mol / L; of CGRP, 2 x 10-7mol / L), cells were collected after 48h, cells with 5-BrDU mark proliferation, RT-PCR was used to detect the expression of HRG-1 and SM22αmRNA. Second experiment, the test is divided into: a2: serum for 72h; b2: serum-free control group; c2: ET-1 group; d2: ET-1 CGRP group (ET-1 role after 24h, plus CGRP) in; e2: CGRP ET-1 (CGRP after 24h, coupled with ET-1). Serum-free DMEM medium synchronization for 24h, the cells in each group switching to continue for 72h with 10% NCS DMEM At this point a2 terminate cultured cells were collected, the rest of the groups replaced with serum-free medium and were added to the corresponding drug ( ET-1 10-7mol / L; CGRP, 2 × 10-7mol / L) role 48h, cells were collected, RT-PCR to detect the expression of HRG-1 and SM22αmRNA. Results: experiment: immunocytochemistry, ET-1 group there are a large number of markers of smooth muscle cell proliferation, adding CGRP proliferating cells decreased slightly, while the number of proliferating cells greatly reduced the first plus CGRP role group. RT-PCR analysis, ET-1 group compared with the control group VSMC HRG-1 and SM22α gene expression was significantly lowered; Join CGRP gene expression has been raised, but not obvious; now add two genes were up-regulated in the the CGRP role group significantly . Experiment II: RT-PCR detection of further evidence, CGRP blocking ET-1 down the VSMC HRG-1 expression and SM22αmRNA, but to join the CGRP effect is more pronounced. Conclusion: 1.CGRP of ET-1 promote VSMC proliferation inhibition as prophylactic administration of effect is more pronounced. 2.CGRP ET promote VSMC phenotype transformation (from contractile to synthetic) reversal effect, and the same as the preventive administration effect more pronounced.

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