Abstract:Aim To observe the role of puerarin on the proliferation of vascular smooth muscle cells(VSMC) induced by thrombin and the effect of puerarin on the Bcl-2 protein and thrombin receptor(TR) mRNA expression. Methods Cell number and cell cycle analysis using flow cytometry were adopted as two different indicators of effects on proliferation of VSMC.Western blot was used to indicate the changes of Bcl-2 protein,and reverse transcription-polymerase chain reaction(RT-PCR) was used to evaluate TR mRNA expression,with treatment of thrombin and puerarin after 24 h. Results Thrombin can significantly increase the cell numbers of VSMC,the peak of proliferation curve is at 24 h,and the effect of T is in a dose dependent manner(0.1～1.0 u/L).1.5×10~(-5)～1.5×10~(-3) mol/L puerarin can significantly suppress this stimulation of VSMC proliferation and DNA synthesis.Western blot demonstrated that treated with thrombin and puerarin after 24 h,thrombin can significantly increase Bcl-2 protein.1.5×10~(-5)～1.5×10~(-3) mol/L puerarin can significantly suppress this increase.RT-PCR demonstrated that thrombin can increase TR mRNA expression significantly,and 1.5×10~(-3) mol/L puerarin can suppress this increase. Conclusions Puerarin can suppress the proliferation and DNA synthesis of VSMC promoted by thrombin.This inhibitory effects of puerarin are closely related with the suppression of Bcl-2 protein,and partly related with the suppression on TR mRNA.

Abstract:Aim To investigate the process of neointimal formation, level of platelet activation and thrombin receptor mRNA by means of a rat aortic balloon injury model in order to study the mechanism of acute and late restenosis after percutaneous transluminal coronary angioplasty(PTCA). Methods Forty eight male Wistar rats were divided into two groups randomly. Group 1 (n=24) served as controls, group 2 (n=24) were given aortic balloon injury by self-made 2F balloon catheters. The process of intimal thickening, number of platelet GMP-140 and level of thrombin receptor mRNA were investigated at day 3, 7, 14 and 28 after balloon injury by histological method, radioimmunological method and reverse transcription polymerase chain reaction (RT-PCR) technique, respectively. Six rats were examined at each group. Results The expression of thrombin receptor mRNA was at a low level in endothelium and smooth muscle cell of normal arteries, but increased significantly at day 3 after balloon injury, reached its peak at day 14 and decreased at day 28. The number of platelet GMP-140 was higher at day 3 and began to decrease at day 7 after injury. The migration and proliferation of vascular smooth muscle cell had existed at day 3 after balloon injury. The intimal thickening began at day 7 after injury, and it was more significant at day 14. The proliferation of VSMC decreased at day 28, but extracellular matrix increased and the intimal thickening continued. Conclusions The expression of TR mRNA and the number of platelet GMP-140 increase in the process of intimal thickening after balloon injury.

Abstract:Aim To study the influence of thrombin receptor on the proliferation of rat vascular smooth muscle cells,probe into mechanism of antisense sequence of thrombin receptor restrained the proliferation of rat vascular smooth cells. Methods Take the incoporation of 3H-thymidine as the target to evaluate vascular smooth muscle cells proliferation; Antisense sequence of thrombin receptor restrained the expression of mRNA of thrombin receptorin vascular smooth cells was detected by RT-PCR; Antisense sequence of thrombin receptor restrained the expression of protein of thrombin receptor was detected by Western-blot; The influence of antisense sequence of thrombin receptor on phosphate inositol of vascular smooth cells was detected by incoporation of 3H-inositol. Results Antisense sequence of thrombin receptor restrained vascular smooth muscle cells proliferation marketedly (compared with control p<0.05),restrained expression of mRNA and protein of thrombin receptor marketedly;Also restrained metabolism of phosphate inositol in vascular smooth cells. Conclusion Antisense sequence of thrombin receptor restrained SD rat vascular smooth muscle cells proliferation marketedly; Antisense sequence of thrombin receptor restrained vascular smooth muscle cells proliferation which could arrested gene expression of thrombin receptor,restrained signal trasduction of vascular smooth muscle cells.

Abstract:Aim To establish a immunocytochemistry method of detection to thrombin receptor(TR).Methods TR activated by thrombin can release a peptide that contain 41 amino acids(TR1-41)from N-terminal of TR.The TR1-41 was synthesized accord-ing to the amino acids sequence of TR,then mice were immunized utilizing the TR1-41,4 cell strains produc-ing TR-McAb were gotten.Preparations of atheroscle-rotic human left ventricular myocardium of four died patients were cut out.The preparations were made in-to paraffin sections.Results A immunocytochemistry method-alkaline phosphatase antialkaline phosphatase(APAAP)to de-tect the thrombin receptor was established and the TR of myocardium were detected successfully.Conclusion The method established can detect TR expression of tissue and organ.