Screening The High Expression Of VEGF And VEGFR In Liver Cancer Cell Lines And The Preliminary Intervention Research Of Matrine

Objective:Hepatocellular carcinoma (HCC) is the enrichment of blood vessels of tumor, whose growth and transfer are closely related with angiogenesis, the vascular endothelial growth factor (VEGF) is a powerful promote angiogenic factors, through combination with VEGF receptor (VEGFR), cause a series of signal transmission, and release of growth factors and cytokines, promote endothelial cell proliferation and migration, directly or indirectly involved in angiogenesis in liver cancer occurrence, its development and prognosis plays important position. This experiment through screening the high expression of VEGF and VEGFR in liver cancer cell lines, observation the matrine and some other effective ingredients of traditional Chinese medicine’s preliminary intervention role, which is aimed at the establish the screening system through targeted VEGF/VEGFR signaling pathways of inhibit angiogenesis liver cancer cells, screening the drugs targeted VEGF/VEGFR signaling pathways inhibit angiogenesis liver cancer, to provide a new train of thought for the modernization of traditional Chinese medicine and liver cancer drug treatments. Methods:(1) screening the high expression of VEGF and VEGFR in liver cancer cell lines:culture human liver cancer cell lines HepG2, SMMC-7721and Bel-7402, and then by immune cells chemical method to detect the expression of VEGF and its receptor Fit-1, KDR; using the ELISA(Enzyme-Linked Immunosorbent Assay, ELISA) method to detect the expression of VEGF in the cell supernatant, take the method of Reverse Transcription Polymerase Chain Reaction(RT-PCR) technology to detect the expression of VEGF mRNA, and in the end to screen VEGF and VEGFR are both with high expression cell lines;(2) The preliminary intervention role matrine and other traditional Chinese medicine:To intervene Bel-7402cell after the above experiment respectively had high expression of VEGF and VEGFR with the traditional Chinese medicine of berberine, quercetin and matrine, Use the CCK-8kit (cell counting kit-8) method to detect cell proliferation, and calculation the half inhibition concentration (IC50), screen the strong effective components of traditional Chinese medicine, and then use the RT-PCR testing the expression of Bel-7402VEGF mRNA after the intervention of the strong effective components. Results:(1) The screening the high expression of VEGF and VEGFR in liver cancer cell lines:the immunohistochemical shows:the HepG2and Bel-7402cells have VEGF, Flt-1and KDR expression, but SMMC-7721cells only have VEGF and Flt-1expression, with no KDR receptor’s expression. ELISA test:the Bel-7402cell has the most expression of VEGF protein in the cell supernatant, SMMC-7721cell has the least. RT-PCR detection:Bel-7402cell has the most expression of VEGF mRNA, SMMC-7721cell has the least(t=6.928, F=0.000). Therefore, this study selected Bel-7402with the VEGF and VEGFR are high expression as experimental use cells.(2) Quercetin, matrine and berberine solvent in DMSO under with antibody in5%o can also have the inhibition of Bel-7402cells, and have the trend with the drug’s concentration enhanced the inhibition rate increased, through the CurveExpert1.3have the berberine, quercetin and matrine’s IC50respectively are:221.12ug/mL,1186.58ug/mL and78.38ug/mL, matrine’s proliferation inhibitory effect is the strongest to the Bel-7402cell. RT-PCR testing results show that the matrine intervened in Bel-7402after48hours, the expression of VEGF mRNA has significantly reduced, and has a good concentration dependence relationship. Conclusion:By immunohistochemical, ELISA and RT-PCR to screen human liver cell lines of HepG2, SMMC-7721and Bel-7402, the Bel-7402cell has highly expression of VEGF and VEGFR, which can be used to deliver targeted VEGF/VEGFR signaling pathways inhibit angiogenesis liver cancer cells screening system. Matrine can significantly inhibited Bel-7402cell proliferation, reduce VEGF mRNA expressions, preliminary point out that matrine may have targeted VEGF/VEGFR signaling pathways inhibit angiogenesis liver function, which needs to be further research.