Investigate The Effects Of DNA Methyl Transferases 1 On Proliferative Of Human Umbilical Vein Smooth Muscle Cells Induced Byhomocysteic Acid

Objective:To observe the change of the expression of transfection DNMT1 gene , through method of gene recombination eukaryotic expression plasmid pcDNA3.1-DNMT1, to explore whether recombinantion plasmid pcDNA3.1-DNMT1 could be the target which influence the homo sapien umbilical vein smooth muscle cell (HUVSMC) proliferation caused by homocysteic acid, at same time to establish foundation for the clinical treatment of atherosclerosis (AS).Method:1. Construction of recombinate pcDNA3.1-DNMT1 plasmid: Total RNA was extracted from HUVSMC and amplified into the sequence of cDNA encoding human DNMT1 gene by RT-PCR. The pcDNA3.1(+) plasmid and DNMT1 gene were double digested by restriction enzyme EcoRⅠand XbaⅠ, and reclaimed by agarose gel. Connected each other of the reclaimed products after purification, then the conjunction product was converted to E.coli DH5α. The positive recombinate plasmid was analyzed by restriction endonuclease digestion and DNA sequencing techniques.2. The recombinate plasmid of pcDNA3.1-DNMT1 transfected into smooth muscle cell: According to the instruction of positive ion liposome Lipofectamine2000, HUVSMC was divided into three groups:①transfection pcDNA3.1-DNMT1 plasmid group;②transfection plasmid pcDNA3.1(+) group;③control group(group of normal smooth musclecells). Used G418 400μg/mL for two weeks to select positive cells. All the groups were observed under fluorescence microscope.3. Effect of recombinant plasmid of pcDNA3.1-DNMT1 in HUVSMC proliferation caused by homocysteic acid: After succeed reproduce of the multiplicative model; HUVSMC were transfected by pcDNA3.1-DNMT1. HUVSMC were divided into three groups (n=6): group of normal smooth muscle cells (CON group), group of HUVSMC induced by 100μmol/L Hcy (Hcy group), group of HUVSMC induced by 100μmol/L Hcy and transfected recombination plasmid DNMT1 (Hcy+DNMT1 group). Then the proliferations of HUVSMC were observed using growth curve drawed by Cytometry and MTT, and the expression quantity of DNMT1 by PCR.Results:1. Succeed construction of recombinat plasmid of pcDNA3.1-DNMT1: The recombinant plasmid of pcDNA3.1-DNMT1 was analyzed respectively by restriction endonuclease digestion, and the results were correct. The analysis of blast indicated that the sequence of pcDNA3.1-DNMT1 had 100% homology with the DNMT1 sequence in Genbank.2. There was green fluorescence on the VSMCs after transfecting the recombinant plasmid of pcDNA3.1-DNMT1 under fluorescence microscope. However, there was not green fluorescence in pcDNA3.1 (+) group and the control group.3. Recombinat plasmid pcDNA3.1-DNMT inhibited proliferation model of HUVSMC caused by Hcy: MTT test indicated growth velocity of HUVSMC in Hcy group was faster than CON group (P<0.05); growth velocity of HUVSMC in Hcy+DNMT1 group more was slower than Hcy group (P<0.05). It was explained that recombinat plasmid pcDNA3.1-DNMT inhibited proliferation model of HUVSMC caused by Hcy. RT-PCR test indicated the expression contents of DNMT1 mRNA in Hcy group was lower than CON group (P <0.05), but expression contents of DNMT1 mRNA in Hcy+DNMT1 group was higher than Hcy group (P<0.05). It was explained that recombinat plasmid of pcDNA3.1-DNMT increased expression of DNM1 mRNA in HUVSMC.Conclusion:1. The recombinant plasmid of pcDNA3.1-DNMT1 was constructed successfully.2. The HUVSMC was transfected by recombinat plasmid of pcDNA3.1-DNMT1 successfully.3. Recombinat plasmid pcDNA3.1-DNMT may inhibit proliferation of HUVSMC caused by Hcy.