Studies On Anti-tumor Activity And Its Mechanism Of Fibrinolyric Protein (EFP) And Induced Apoptosis Of Tumor Form Eupolyphage Sinesis Walker

Eupolyphaga sinensis Walker, Which is in the motherland medicine traditional blood circulation removing extravasted blood class traditional Chinese medicine, is the quality goods raw material for medicine which "the Chinese Pharmacopoeia" records. The Chinese medicine prescription also used it treating tumor, especially the digestive tract tumor to be more common, folk also used for primary liver cancer, stomach cancer, melanin lump, pharyngeal cancer and so on. The laboratory earlier period research took the traditional Chinese medicine Eupolyphaga sinensis Walker as a raw material, used the homogenate, salting-out, ammonium sulfate fractionation, DEAE-52 cellulose column chromatography to obtain fibrinolytic protein (EFP), and had proven EFP has the remarkable cytotoxicity function to the partial tumour cell by in vitro experiment.Based on this study, a novel of enzyme actuation mechanics method has determined the EFP enzyme vigor and the michaelis constant Km value, and the Affinity chromatography analysis method was used to separate EFP in this paper. The experiment has adopted 2 kinds separately for the formula type (directly for medicine and including medicine blood serum), further discussed the different concentrations of EFP and EFP containing serum were acting on the A549, HepG-2, MCF-7, HUVEC cell lines, using MTT assayed at 48h in each group of serum and EFP tumor cell inhibition rate. According to the blood serum Pharmacology's method, affter different concentrations EFP affecting A549 cell by 48h, Annexin V-FITC/PI fluorescence double dyeing examined apoptosis rates; the flow cytometer (FCM) used to determine the Cell cycle; the Immunohistochemistry analyzed apoptosis related gene Bcl-2, the Bax expression level change. Based on the previous experiment, which Li Xingnuan and so on had proven EFP had induced HUVEC apoptosis and cell cycle change, after different concentrations EFP affecting HUVEC cell by 48h, apoptosis related gene Bcl-2, the Bax expression level was detected by immunohistochemistry. The result turned out:The EFP enzyme vigor is 3790.85 BAEE unit/mg; michaelis constant Km is 0.571 mmol/L; the maximum speed of the enzyme is 0.0364. This paper conclude that the chicken ovomucin is not the EFP inhibitor from Affinity chromatography experiments. The EFP direct medication has certain multiplication function to the partial tumour cell, for example EFP does not have the cytotoxicity to HepG-2, But EFP containing serum group, A549, HepG-2, MCF-7, HUVEC cell proliferation were inhibited, that with the increasing concentration of drug-containing serum, inhibition rate gradually increased; The serum pharmacology can reflect pharmacological action actually. EFP can change the A549 cell's cyclical distribution, causes the cell to hinder in the G0/G1 time, and strengthens A549 cell gene bax expression, suppresses bcl-2 expression, thus the induction cell apoptosis. Affter EFP affects HUVEC cell 48h, along with medicine concentrations increase. The Bcl-2 expression is weaken gradually, and the Bax expression strengthens gradually.