Experimental Explore Of Intratumoral Injection Treatment For Colorectal Cancer With Bevacizumab And5-FU

[Objective]To explore the antitumoral mechanism of intratumoral injection treatment for nude mouse model of colorectal cancer with bevacizumab combined with5-fluorouracil in terms of cell proliferation, cell cycle and angiogenesis.[Research Methods]1. Determine the cell proliferation by MTT assay:We treated the human colorectal cancer cell line HT-29cultured in vitro with different concentrations of5-FU, bevacizumab or bevacizumabin combined with5-FU, then continued to culture for24hours,48hours,72hours and96hours, respectively. After that the optical densities at490nanometer light were determined by the enzyme-linked immunometric meter, and the inhibition rate of cell proliferation of all groups were compared by the single factor analysis of variance method (LSD).Further we selected the most appropriate concentration of bevacizumab and5-FU for cell cycles investigation.2. Detect the cell cycle distribution using flow cytometry instrument:After culturing human colorectal cancer cell line HT29in vitro for24hours, we respectively added5-FU, bevacizumab or bevacizumabin combined with5-FU for interference. Meanwhile we set up blank control group. The distributions of each phase in cell cycle were detected by flow cytometry instrument48hours later,and the effects were analysed. 3. Detect the expressions of VEGF and growth of microvascula in xenografted colorectal tumors in nude mice:The pathological sections were made from tumor tissues of previous animal experiment. We detected the expressions of VEGF and CD31by immunohistochemistry, and calculated microvascular density. The expressions of VEGF were compared by chi-square test,while the differences of angiogenesis were compared by independent sample t test.[The Results]1.5-FU effectively inhibited the proliferation of human colorectal cancer cell line HT-29and showed dose dependence and time dependence. The time dependence gradually decreased with time going.Among all5-FU groups, the differences were statistically significant (P=0.000). Comparing each of5-FU groups with the control group, the differences were statistically significant (P<0.05). Bevacizumab slightly inhibited the proliferation of HT-29, and only showed time dependence. The time dependence gradually increased with time going. Among all bevacizumab groups, the differences were statistically significant (P=0.000). Comparing0.1ug/ml group with the control group, there was no statistically significant difference (P>0.05). Comparing each of other groups with the control group, the differences were statistically significant (P<0.05). The inhibition of cell proliferation between10ug/ml and80ug/ml groups were relatively significant,and after24hours,48hours,72hours, or96hours the rate of cell inhibition were14.11%vs12.38%、14.34%vs14.21%、17.47%vs18.38%、24.58%vs25.18%. Bevacizumab combined with5-FU had significant inhibitory effect on cell proliferation of HT-29. Among all groups, the differences were statistically significant (P=0.000). Comparing experimental groups with the control group, the difference were statistically significant (P<0.05). Comparing with5-FU groups, combined with bevacizumab could improve the rate of cell inhibition. That was to say, bevacizumab and5-FU had synergistic inhibition of cell proliferation.2. All groups affected the cell cycle distribution:For5-FU group,it mainly showed the proportion of G1phase cells increased and S phase cells decreased. The bevacizumab group was characterized by decreasing G2phase cells,however G1phase and S phase were stable. Bevacizumab combined with5-FU group showed both characteristics at the same time.3. Bevacizumab by intratumoral injection could effectively inhibit the expression of VEGF and microvascular in nude mouse model,and combined with5-FU further improved this inhibition. Comparing bevacizumab group or combination group with the control group there were statistically significant difference in the expression of VEGF and microvascular density (P=0.000).Comparing combination group with bevacizumab group there was statistically significant difference in the expression of VEGF and microvascular density (P<0.05).The expression of VEGF and microvascular density in5-FU group had no significant difference comparing with the control group(P>0.05).[Conclusion]1. Both bevacizumab and5-FU could inhibit the proliferation of colorectal cancer cell line HT29, and the combination group could enhance the inhibitory effect.2. Bevacizumab and5-FU had synergy effects on the cell cycle distribution of human colorectal cancer cell line HT29.5-FU mainly affected G1-S phase,and bevacizumab inhibited G2phase.3. Intratumoral injection with bevacizumab could effectively inhibit the expression of VEGF and decrease microvascular density. When combined with5-FU,the inhibitory effect of bevacizumab was improved.