Isoflavone Inhibits Experimental Study Of The Mechanisms Of Vascular Smooth Muscle Cell Proliferation

OBJECTIVE The proliferation of vascular smooth muscle cells (VSMC) are the samepathologic character of Arthrosclerosis and restenosis. Puerarin can inhibitproliferation and promote apoptosis of rabbit aorta SMC. Daidzein can inhibitproliferation of many tumor cell lines and rat aortic SMC, meanwhile represssynthesis of DNA in rat aorta SMC. This study intends to observe the inhibitioneffect of puerarin and daidzein on the proliferation of umbilical arterial vascularsmooth muscle cell (HUASMC), then try to find the mechanism in genetranscription and protein expression, thus provide theoretical base for preventingAs and restenosis by puerarin and daidzein.METHODS1. Culture HUASMC in vitro, divide into groups including control group and puerarin, daizein groups in different concentration. Estimate Cellular activity by MTT and detect the state of cellular hyperplasia by growth curve to find the most effective concentration and time of puerarin and daizein.2. Differential display genes relative to the inhibition effect of puerarin and daizein on HUASMC proliferation: culture HUASMC in vitro, stimulate cells with puerarin and daizein, extract RNA, carry out DD-PCR, screen differential fragments.3. Clone differential fragments: Amplify and purify the different fragments and ligate it with pGEMT vector by T4 ligase . Transform the recombinant into JM109. Choose and confirm the positive recombinant. Sequence the recombinant and search the sequence in Genbank.4. Confirm the expression of differential gene in HUASMC: Culture HUASMCs and stimulate them with puerarin and daizein. Extract RNA and carry out competitive RT-PCR.5. Express the differential gene : Design the primer according to the coding region of the differential gene, amplify the gene with the whole cDNA, ligate PCR products with pGEM-T, transform the recombinant into JM109, build up expression recombinant with pET32a,transform the expression recombinant into Origami DE3 , induce the expression of protein with 1mM IPTG, purify the protein with Ni-NTA His Band Resins, cut the fusion protein with enterokinase, confirm it by SDS-PAGE electrophoresis and Coomassie blue staining.6. Assay the effect of protein on HUASMC migration and cellar activity: Stimulate HUASMC with expressed protein , daidzein, and puerarin. EstimateA4 葛根异黄酮抑制血管平滑肌细胞增殖作用机制的研究Cellular activity by MTT. Use Borden loculus to detect the effect of expressedprotein , daidzein, and puerarin on cellar migration.RESULTS1. 2.5 10-5M 1 10-4M daidzein 2.5 10-5M puerarin can inhibit the proliferation, the most effective stimulation time is 24h. The inhibition effect of puerarin 25μM daidzein is stronger than 25μM puerarin .2. There are 11 EST fragments which are strongest in daidzein group, weaker in puerarin group, weakest in control group 4 EST fragments which are strongest in puerarin group, weaker in daidzein group, weakest in. control group, and 4 EST fragments which are strongest in control group, weaker in puerarin group, weakest in daidzein group.3. Compared the genes cloned with Genbank. Exclude the gene coding ribosome and house-keeping genes , and find three luxury genes, one of which is H3A. H3A has a high identity with FSTL1(gi34304366).The mRNA of FSTL1 is 3705bp.4. Competitive PCR shows 2.5 10-5M 1 10-4M daidzein can up-regulate the H3A expression, but puerarin has no effect.5. SDS-PAGE shows there is a 35kD band in samples which have been purified by Ni-His band and Sephadex and enzymed by Enterokinase. We obtain 0.8μg/μl purified protein.6. 10ng/ml 100ng/ml H3A protein can inhibit the migration and proliferation of HUASMC.CONCLUSION1. Daidzein and puerarin at certain concentrations can inhibit the proliferation of HUASMC, the former may be stronger.2. Daidzein may promote expression of H3A in the course of inhibiting the proliferation of HUASMC.3. The protein o...