Screening And Evaluation Of Traditional Chinese Medicine Which Up-regulate The Endogenious T-PA Expression By Endothelial Cell

The tissue type plasminogen activator (t-PA) is a primary factor in fibrinolysis system. As a therapeutic medicine for thrombosis, exogenous t-PA is among the hotspots of thrombolytic researching. However, no matter natural or recombinated t-PA, there are disadvantages in their application, such as short-lived half life, narrow-ranged safe dosage, expensive price etc. In this thesis, drug screening was started with Traditional Chinese Medicine (TCM) and t-PA was used as a target. The purpose is to find t-PA up-regulating TCM. This is very helpful for avoiding the above shortages and for the development of new anti-thrombosis agents.A realtime RT-PCR was constructed for detecting t-PA mRNA expression from Human microvascular endothelial cell (HMEC-1). SYBR Green I was used in development of real-time PCR amplification standards curves. The stability, specificity and sensitivity of the method were evaluated. With high specificity the method could be used to detected as low as 103 copies t-PA mRNA with the linear range from 103 to 1010 copies. The standard curves showed high correlations(r2 > 0.990). The intra-assay and inter-assay variation of the method was blow 3.10 % and 10.73 %, respectively.The effect of all-trans ratinoic acid (ATRA) on the expression of t-PA by HMEC-1 cells was examined by constructed realtime PCR and enzyme-linked immunosorbent assay (ELISA). The ATRA up-regulated t-PA mRNA and protein expression. The effect is concentration dependent. Compared with negative control, 10.00μmol /L ATRA could up-regulated t-PA mRNA 2.8 folds higher on HMEC-1 cells.Through twice screening of more than 300 TCMs, 4 TCMs were found have the activity to enhance t-PA expression. They were numbered 1-1,1-3,22-5 and 30-5. Compared with negative control, the drug 1-1 could up-regulated t-PA mRNA 2.4 folds higher. Beside, PAI-1 antigen and activity were tested to evaluate the effect of the 4 drugs on fibrinolysis system. Except 30-5, other 3 TCMs showed no significant influence on PAI-1 expression by HMEC-1 cells.