The Growth Inhibition Effect On Primary Vascular Smooth Muscle Cells Of Paclitaxel And All-trans-retinoic Acid

Background and objective: Now coronary heart disease(CHD) is one of diseases which jeopardize the man health. The therapy of CHD includes drug therapy,interventional therapy and vascular bypass plantation. But there are high restenosis rate in interventional therapy, especially of diabetes patients even to 30%-50%. Wide use of stent was limited to a certain extent. Restenosis is caused by vascular elastic bound, adverse revascular and neointima proliferation together. While in-stent restenosis is mainly caused by vascular smooth muscle cells (VSMCs) proliferation and transplant. Now the research about VSMCs growth inhibitors is a hotspot. So we pay more attention to drug-eluting stent(DES). Now DES used on clinic are CYPHER stent and TAXUS stent. But both have around 5-10% rate of restenosis in big scale studies. And clinic study showed that CYPHER stent had lower restenosis rate than TAXUS stent. The possible mechanism was that paclitaxel inhibited the proliferation of VSMCs as well as endothelial cells, and this delayed endothelium healing. If it is used together with the drug that inhibits VSMCs proliferation and protects endothelial cells, we could reduce the dose of paclitaxel to avoid delaying endothelium healing. But now there is not the research about this aspect. A research showed: at low doses of paclitaxel (<0.1nmol/l), the growth of haEC was less inhibited than haSMC growth . Whereas high doses of paclitaxel (0.1 to 10nmol/) exerted comparable effects in both haEC and haSMC. At the same time paclitaxel can induce VSMCs to apoptosis even die at higher doses. Some study indicated all-trans-retinoic acid (ATRA) could inhibit VSMCs proliferation, promote endothelium cell growth and accelerate to resume damaged endothelium function. This research will use paclitaxel and ATRA together to inhibit VSMCs growth, and try to find a concentration to inhibit VSMCs proliferation. This will help to popularize drug-eluting stent on CHD therapy. Methods: Rat VSMCs were cultured according to the explant technique. The cells were identified through a-SMC-actin single clone antibody. Passages 3-6 were used on the study. They were divided into Paclitaxel group,ATRA group ,Paclitaxel and ATRA group,ethanol group,DMSO group,ethanol and DMSO group,control group. The cells were analyzed after 48h since the drugs entered. MTT was measured to determine the effect of these drugs on inhibiting vascular smooth muscle cells growth. And the apoptosis and effect on cell cycle will be analyzed through FCM. The test was repeated 3 times. Results: (1)Successfully we culture VSMC which is identified by inverted phase contrast microscope and immunocytochemistry. (2) Paclitaxel inhibited the growth of vascular smooth muscle cells singly in a concentration-dependent manner during 1-100nmol/l. Paclitaxel of 1nmol/l began to inhibit VSMCs proliferation by 17%. The rate of inhibiting VSMCs growth by Paclitaxel of 10nmol/l is 27.5%. The inhibition rate of 100nmol/l is high to 36%. Three different doses of paclitaxel have significant difference (p<0.05). Compared to control group paclitaxel group has significant difference but ethanol has no significant effect on cells growth (p=0.172). (3)Inhibiting effect on VSMC by ATRA became more and more strong with the dose enhancing during 1-100nmol/l. Theinhibition rate of ATRA of 1nmol/l was only 8.6%. That of 100nmol/l is 29.8%。Three different doses of paclitaxel have significant difference (p<0.05). Compared to control group DMSO group has no significant effect on cells growth (p=0.205). VSMCs were occluded in G1 by ATRA and this was enhanced with the ATRA concentration increasing. When ATRA is 100nmol/l, the G1 cells is 91.12%. (4)At 1,100noml/l of concentration pacilitaxel inhibited VSMC more intensely than ATRA. And there was significant difference between ATRA and paclitaxel(p<0.05). But at 10nmol/l there was no significant difference between ATRA and paclitaxel .(5) The inhibition rate of Paclitaxel and ATRA together is higher than that of ATRA of 100nmol/l singly, and also higher than...