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Construction of GPERshRNA Expression Vector and Its Effect on Vascular Endothelial Cell Proliferation

Author: LuAiFen
Tutor: HuangJun
School: Nanchang University
Course: Internal Medicine
Keywords: G protein-coupled estrogen receptor RNA interference Vascular endothelial cells
CLC: R54
Type: Master's thesis
Year: 2011
Downloads: 27
Quote: 0
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Abstract


Objective: To construct a shared human and mouse G-protein-coupled estrogen receptor (G-protein-coupled receptor 30, GPER) short hairpin RNA (short hairpin RNA, shRNA) eukaryotic expression plasmid, was identified in human vascular endothelial Cells (Human vascular endothelial cells, HVECs) and murine endothelial cells (mouse vascular endothelial cells, MVECs) GPER expression, and to observe its effect on human and murine endothelial cell proliferation. Methods: in vitro synthesis and targeting (human and mouse) genes encoding GPER specific short hairpin RNA (short hair RNA, shRNA) oligonucleotide sequences, and synthesize a wrong sequence defined as non-specific negative control , annealed, ligated into the restriction endonuclease Bgl Ⅱ Hind Ⅲ digestion process pSUPER linearized plasmid and transformed into E. coli, positive clones was extracted recombinant plasmid for EcoR Ⅰ gt; Hind Ⅲ restriction enzyme digestion and sequencing electrophoresis identification, construct shRNA plasmid; cultured human and murine endothelial cells (vascular endothelial cells, ECs), and thus will build three groups of recombinant plasmids were transfected liposome human and mouse endothelial cells, proteins blotting (Western blot) to detect the protein expression of GPER by MTT assay recombinant plasmid on human vascular endothelial cells and murine endothelial cell proliferation. Results: GPER shRNA recombinant plasmid by restriction digestion and sequencing analysis showed that: the nucleotide sequence inserted successfully projected sites and sequences were identical; GPER shRNA plasmid transfected human vascular endothelial cells and vascular endothelial cells in mice with control group can be reduced GPER protein expression (P lt; 0.05), which GPER shRNA2 strong inhibition of plasmid transfection after 48 h GPER protein inhibition rate can reach 84.2%; recombinant plasmid human and murine endothelial cells, significantly inhibited (human and mouse) vascular endothelial cell proliferation. Conclusion: We successfully constructed simultaneously targeting human and mouse GPER recombinant vector RNA interference; successfully constructed recombinant plasmid can effectively inhibit human vascular endothelial cells and mouse endothelial cells GPER protein expression; recombinant plasmid transfected with mouse vascular endothelial cells, can significantly inhibit the proliferation of mouse vascular ECs, for the latter lay the foundation for a number of studies provide important tools.

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