Study On The Effects And Mechanism Of Leech And Eupolyphaga Extract On Invasion And Metastatic Ability Of Human Hepatoma Carcinoma Cell

Objective:To analyse the effectts and mechanism of leech extract (ELC) and eupolyphaga extract (EEP) on invasion and metastatic ability of human hepatoma carcinoma cell.Methods:The ELC and EEP were prepared by methods of rapid freezen-thaw with liquid nitrogen,and following precipitation with acetone. The invasive ability was assessed with a transwell cell culture chamber in HCCLM3treated with these drugs. Cell scratch experiment was used to detecte the cell moving ability. To set up model of orthotopic grafting carcinoma for HCCLM3in nude mouse, and observe its tumor growth and metastasis by intragastric method of ELC and EEP. Using immunocytochemistry methods to detect G-CSFR in different metastatic potential of human hepatoma cell. To detect G-CSFR in nude mouse tumor tissue with Immunohistochemical and real-time quantitative PCR.Result:①The ELC and EEP can promote the migration of HCCLM3. The promotion effect is dose-dependent. The healing rate increased with increasing drug concentration. Using EC50drug interfere with HCCLM3, scratches healing results show that ELC and EEP group healing rate was significantly higher than control group, the difference was statistically significant (P<0.05). Transwell invasion chamber results also showed that ELC and EEP group HCCLM3cells through transwell membrane cell numbers was much more than control group. The difference was notable statistically significant (P<0.01).②The relative size of nude mice liver tumors and tumors weiht in ELC and EEP group are bigger than control group, the difference is statistically significant (P<0.05). The metastasis rate of ELC and EEP group are higher than the control group. The difference was notable statistically significant (P<0.01).③The expression of G-CSFR in different metastatic potentials of three HCC cell lines was determined by immunocytochemistry and the results showed that the the positive expression rate of G-CSFR was all significantly decreased with the decline of metastatic potentials. Difference between HCCLM3and Bel-7402(P<0.01) or Bel-7402and HepG2(P<0.01) or HCCLM3and HepG2(P<0.01) were notable statistically significant.④The expression of G-CSFR in nude mouse tumor tissue was determined by Immunohistochemistry and the results showed that the positive expression rate of G-CSFR was all significantly increased in the ELC group and the EEP group than that in control group (P<0.01). The positive expression rate of the ELC group and the EEP group, the different was no statistically significant (P>0.05).⑤The ELC and EEP group can up-regulate the expression of G-CSFR mRNA, compared with control group, the difference was statistically significant (P<0.05). The expression of G-CSFR mRNA in the ELC and the EEP group, differences was no statistically significant (P>0.05).Conclusion:①ELC、EEP are both promote the motility and invasion of HCCLM3cell in vitro.②ELC、EEP are both promote the metastasis of nude mice liver tumor in vivo.③By detecting the expression of G-CSFR protein, we find that the ELC and the EEP increase the G-CSFR protein expression levels in order to promote hepatoma cells invasion and metastasis. There may be an important molecular mechanism of expelling blood stasis of Chinese medicine for liver metastasis.