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The Mechanism of Myocardin and Estrogen Receptor α Synergistically Regulating Smooth Muscle Cells Differentiation

Author: ZhangZuo
Tutor: ZhangTongCun
School: Tianjin University of Science and Technology
Course: Microbial and Biochemical Pharmacy
Keywords: Myocardin ERα Transcription factor Vascular smooth muscle cells Differentiation Phenotype conversion
CLC: R543
Type: Master's thesis
Year: 2009
Downloads: 20
Quote: 0
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Abstract


Since Olson et al in 2001 found Myocardin induced cardiac hypertrophy, as well as the mechanism of inhibition of vascular smooth muscle cell proliferation has made great progress. This paper focuses on the smooth muscle cells (vascular smooth muscle cells, VSMCs) study, research transcription factor Myocardin Estrogen receptor a (ERa) in the vascular smooth muscle cell phenotypic mechanism. By the process of the luciferase activity assays (luciferase) in COS-7 cell lines to study the synergistic effect of both functional; functional assay is carried out in the same manner as in the primary cultured rat VSMCs, and smooth muscle cell differentiation marker gene at the RNA levels and protein levels were detected, thereby determining the cell differentiation. Truncated the Myocardin eukaryotic expression plasmid dominant-negative Myocardin (MYCD-dn) with ERa transfected COS-7 cells, initially explored the the missing transcriptional activation domain (TAD domain) build Myocardin and ERa in smooth muscle cell differentiation molecular pathway. Myocardin as Serum Response Factor (SRF)-dependent trans-activation of a variety of smooth muscle cell-specific transcriptional regulation element, plays an important role in the smooth muscle cells. Mainly through a combination of SRF by SRF binding Preparation purpose of the regulation of the gene promoter CArG box (CC (A / T) 6GG) specific sequence, thereby activating the expression of downstream genes. Application of bioinformatics methods, positioning the mouse SM22 gene promoter and determine Myocardin SM22 promoter region -447 to 89 containing two CArG Box binding point. Cloned by PCR method and build Myocardin of ERα eukaryotic expression plasmid and SM22 start luciferase reporter plasmid. Extracted from SD rat aortic smooth muscle cells was detected by RT-PCR method to study the endogenous target genes, including Myocardin, ERα, SM22, α-actin and MHC. Myocardin, ERa and SM22-luc plasmid transfected COS-7 in vitro and in VSMCs detect reporter gene luciferase activity results Myocardin, and ERa able in functional experiments synergistic SM22-luc expression. Simultaneous detection of of Myocardin and ERa the transfection smooth muscle cell differentiation marker gene expression at the RNA and protein levels change, the results show that the the Myocardin ERa co-transfected cells transfected group differentiation marker gene expression has been enhanced. In order to further determine the synergy mechanism Myocardin and ERa in SM22 gene promoter construct eukaryotic expression plasmid of the TAD domain truncated MYCD-dn missing in COS-7 in the co-transfected with ERa. The results showed: MYCD-dn and ERa transfected group, SM22-luc luciferase activity loss. The results of the study to clarify: ERα synergistic effects Myocardin induced smooth muscle cell differentiation. The role is a via Myocardin-CArG box pathway to achieve.

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease
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