Effections Of TIMP-3 Gene Transient Expression Mediated By Cationic Liposome On Proliferation, Migration And Apoptosis Of Rabbit Vascular Smooth Muscle Cells

Objective To observe effections of TIMP-3 on proliferation, migration and apoptosis of rabbit vascular smooth muscle cells(VSMC)transfected by recombinant plasmid vectors mediated by cationic liposome and then discuss the TIMP-3 gene therapy on in-stent restenosis(ISR). Methods Step 1 Construct the expression plasmid vectors of TIMP-3cDNA . Divide 72 holes of VSMC into three groups as normal contrast group, empty plasmid group and recombinant plasmid group. Each group are also divided into three sub-groups according to the special time: 24hours, 48hours and 72hours. Each sub-group have 8 holes. Transfect cells separately by recombinant plasmid and empty plasmid, and no plasmid be added in normal contrast cells. At the time of 24hours, 48hours and 72hours, calculate cells and draw the growth graph. Quantify MMP-2 by ELISA, and analyse TIMP-3 mRNA by RT-PCR. Step 2 Divide 24 holes cells into 3 groups and do gene transfection. After 48hours, measure cell apoptosis rate. Step 3 The migration model is established in the Transwell chamber. Divide 12 chambers into 3 groups. Lay equal volume of diluted ECMgel on each membrane. When it become gel, do gene transfection. Then put equal volume of high- concentration serum medium into lower room, however, add serum-free medium into upper room. After culturing 48hours, take out the chambers, dye with HE, calculate cells migrated into the membrane with microscope, take 5 views randomly. Analyse all datas with SPSS 12.0. Resuls There are no different between normal contrast group and empty plasmid group in cells grow, MMP-2's content , TIMP-3 mRNA expression and apoptosis rate,cells migration.On the contrary, the amount of cells, MMP-2's content and migrating cells of recombinant plasmid group is much less than two groups above, P<0.05. TIMP-3 mRNA express strongly in the recombinant plasmid group, but no or little bar is seen in the gels of normal contrast group or empty plasmid group. Gray level ratio and cell apoptosis rate of recombinant plasmid group are both markedly higher than two groups above, P<0.05. Conclusions It is testified TIMP-3 can strongly inhibit proliferation or migration of VSMC in vitro, but promote apoptosis on the other hand. Cationic liposome is a new medium with efficient, safe advantage which can provide broad application in TIMP-3 gene therapy on ISR.