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Investigate the Effects of DNA Methyl Transferases 1 on Proliferative of Human Umbilical Vein Smooth Muscle Cells Induced Byhomocysteic Acid

Author: LiuLingJuan
Tutor: LiGuiZhong
School: Ningxia Medical University
Course: Pathology and Pathophysiology
Keywords: Recombinant plasmid pcDNA3.1-DNMT1 Atherosclerosis Homocysteine DNA methylation Gene transfection
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 17
Quote: 0
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Abstract


Objective: The recombinant eukaryotic expression plasmid pcDNA3.1-DNMT1 observed after transfection DNMT1 gene expression changes, and investigate whether the recombinant plasmid pcDNA3.1-DNMT1 can be used as targets of Hcy caused by human umbilical vein smooth muscle cells ( HUVSMC) proliferation, and to lay the foundation for the clinical treatment and prevention of atherosclerosis (AS). Method: 1. Construction of recombinant plasmid pcDNA3.1-DNMT1: HUVSMC total RNA was extracted by the reverse transcriptase polymerase chain reaction (RT-PCR) synthesis DNMT1 gene with restriction endonuclease EcoR Ⅰ and Xba Ⅰ enzyme cut the plasmid pcDNA3.1 () and DNMT1 gene, agarose gel, recovered linearized plasmid pcDNA3.1 (), and the gene product is recovered after purification be connected, and the ligation product is transformed into E. coli DH5α, shake bacteria amplification extract positive recombinant plasmid, the line restriction enzyme digestion and DNA sequencing. Recombinant plasmid pcDNA3.1-DNMT1 transfected smooth muscle cells: instructions cationic liposome Lipofectamine 2000, divided into three groups: (1) transfected with pcDNA3.1-DNMT1 plasmid group; ② transfected with plasmid pcDNA3.1 () group ; (3) control group (normal HUVSMC). The immunofluorescence assay recombinant plasmid expression in smooth muscle cells. In screening After two weeks of G418 (400μg/mL) test. Recombinant plasmid pcDNA3.1-DNMT1 on of Hcy induced HUVSMC proliferation: The recombinant plasmid pcDNA3.1-DNMT1 the transfection to HUVSMC, divided into 3 groups (n = 6): normal smooth muscle cell group (CON) 100μmol / L Hcy stimulation group (Hcy group), 100 μmol / L of Hcy stimulate the recombinant plasmid pcDNA3.1-DNMT1 transfection group (Hcy DNMT1 group). Cell count, MTT assay cell growth curve to detect the proliferation of smooth muscle cells, and DNMT1 gene expression was detected by RT-PCR. Results: 1. Successfully constructed recombinant plasmid pcDNA3.1-expressions of DNMT1: The recombinant plasmid pcDNA3.1 () - DNMT1 digested fragment can be expected to confirm the correctness; published biological sequencing results with GenBank sequence for BLAST comparison of the homology is 100%. Transfection of the recombinant plasmid pcDNA3.1-DNMT1: observed under a fluorescence microscope, the recombinant plasmid transfection HUVSMC visible green fluorescence, while the other two groups. Recombinant plasmid pcDNA3.1-DNMT1 transfected into HUVSMC of Hcy induced HUVSMC, proliferation inhibition: growth experiments showed that the Hcy group than CON the group smooth muscle cell growth increased (P lt; 0.05) of Hcy expressions of DNMT1 group than Hcy group smooth muscle slow down the speed of cell growth (P lt; 0.05). Description transfected with the recombinant plasmid pcDNA3.1-DNMT1 may of Hcy induced HUVSMC, proliferation disincentive. RT-PCR experiments results show the Hcy group than in the CON group DNMT1mRNA expression content is low (P lt; 0.05), while the Hcy DNMT1 group compared with Hcy group DNMT1 mRNA expression levels (P lt; 0.05). Description of the the Hcy role can lead smooth muscle cells DNMT1mRNA of expression decreased recombinant plasmid pcDNA3.1-DNMT1 transfection Hcy induced may DNMT1 mRNA expression levels increased after the model of HUVSMC proliferation. The Conclusion: successfully constructed the recombinant expression plasmid pcDNA3.1-expressions of DNMT1 successfully recombinant plasmid pcDNA3.1-DNMT1 transfected into HUVSMC. 3. Transfected with the recombinant plasmid pcDNA3.1-DNMT1 of Hcy induced HUVSMC, proliferation inhibition.

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