| Objective:To observe the effect of gentianella acuta on H22 tumor bearing mice and human hepatocellular carcinoma Bel-7402 cells and explore its mechanism.MethodsIn vitro experiment:The preparation of low,medium and high dose gentianella acuta alcohol extract containing serum(EEGA),human hepatocellular carcinoma Bel-7402 cells cultured in vitro.The experiment was divided into blank group,EEGA serum containing low,medium and high dose group and positive drug group.Using MTT method to study the effect of EEGA containing serum on the proliferation of human hepatocellular carcinoma Bel-7402 cells;Effect of EEGA containing serum by flow cytometry and the distribution of adjusted death rate cycle of human hepatocellular carcinoma Bel-7402 cells;Detection of Bax,Bcl-2 and mRNA caspase-3 levels in human hepatocellular carcinoma Bel-7402 cells by RT-PCR;Western blot detection of human hepatoma Bel-7402 cells Bax,caspase-3 protein levels of Bcl-2 and activated;In vivo experiments:Establish H22 tumor-bearing mice.They were randomly divided into six groups:Control group,model group,EEGA low,medium and high dose group and positive group.Control group,model group was given normal saline.EEGA low,medium and high dose groups were fed quantitative pharmaco,positive drug group were injected 5-Fu.View Chahe mice-like living conditions,determination of tumor inhibition rate;Using TNF-a,IL-1?,the content of each group of mouse plasma IL-6 Elisa assay;Flow cytometry in peripheral blood of mice in each group C D 3+?C D 4+?C D 8+lymphocytes;Flow cytometry in each group of mice spleenC D 3+?C D 4+?C D8+lymphocytes;HE staining of tumor tissue embedded sections after treatment,TUNEL staining of tumor cell apoptosis;By immunohistochemical detection of tumor cells Bax,Bcl-2,caspase-3,VEGF levels;RT-PCR in the detection of tumor cells Bax,Bcl-2 and caspase-3mRNA level;Western blot assay of tumor tissue Bax,caspase-3 protein levels of Bcl-2 and activated.Results1In vitro experiment1.1 MTT assay test results show,Compared with the control group,EEGA each group and positive group were effectively inhibited human hepatocellular carcinoma Be1-7402 cell proliferation..EEGA each group with the drug concentration increased,the role of time increases,the inhibition rate increased.1.2 Apoptotic cells by flow cytometry results show,Cultured normal human hepatoma Bel-7402 cells and apoptosis rate of normal rat serum after action is not very different,about 5-10%.EEGA low,medium and high dose group and positive group were able to significantly induce apoptosis in human hepatoma Bel-7402 cells.1.3 Flow cytometry analysis showed that cell cycle distribution results,EEGA each group and positive group were caused by human hepatoma Bel-7402 cell cycle arrest,Performance increase in the proportion of cells G0/G1 phase,reduce the proportion of cells in S phase.1.4 RT-PCR showed,Normal rat serum of human hepatoma Bel-7402 cells Bax,no significant effect on Bcl-2 and caspase-3 mRNA levels,EEGA positive group and each group can make the expression of Bax was significantly increased,at the same time decrease the expression of Bcl-2 gene,the ratio significantly increased Bax/Bcl-2 mRNA of,caspase-3 gene expression was significantly upregulated,EEGA among dose groups show,EEGA intracellular Bax,Bcl-2 and affect caspase-3 mRNA levels showed a dose-effect relationship.1.5 Western blot test results showed that normal rat serum of human hepatoma Bel-7402 cells within Bax,Bcl-2 and activation of caspase-3 protein levels had no significant effect,EEGA groups and positive group were able to significantly induce the expression of Bax protein inhibit the expression of Bcl-2 protein was significantly increased Bax Bcl-2 protein ratio/,increased activation of caspase-3 protein levels.Wherein,EEGA low,medium and high dose groups,with the increase of drug concentration,this effect is growing.2..In vivo experiments2.1 H22 tumor bearing mice modeling success rate of 100%,Late model modeling composition mice tumor rapid growth,all aspects of the state of deterioration,weight loss.Positive drug group compared with model group,slowed tumor growth,but body weight decreased significantly in all aspects poor state.Comparison,slowed tumor growth EEGA each dose group and model group,all aspects of good condition.2.2 Compared with the model group,EEGA each group and positive group mean tumor weight was significantly reduced.EEGA low,medium and high dose groups inhibitory rates were:17.62%,27.75%,38.28%,positive group inhibition rate was 64.18%.2.3 EEGA medium and high dose group,compared with model group increased slightly,was statistically significant;Each dose group and model group compared with 5-FU group and normal group,spleen index had significant difference;Prompt EEGA can enhance the immunity of the mice a tumor-burdened,and 5-FU in inhibiting tumor at the same time,also destroys the body's immune function.2.4 EEGA thymus index of each dose group were greater than a tumor-burdened group,but without statistical significance;And can make the thymus index of 5-FU group was obviously lower,compared with each group have differences.2.5 Elisa method to detect tumor-bearing mice plasma TNF-a,IL-1?,IL-6 content results show,With the blank group,model group,the content of plasma cytokines decreased.With model group,the content in each dose group EEGA elevated plasma cytokines,cytokine content in the plasma of these positive group has not improved.2.6 Flow cytometry results showed that compared with the control group,model group,peripheral blood T lymphocytes reduce the proportion of CD4+cells,CDs+cell ratio increased,CD4+/CD8+ratio decreased.With model group,EEGA each dose group increased the proportion of CD4+cells,CD8+cells was reduced,CD4+/CDs+ratio increased,while positive group no improvement.2.7 Flow cytometry results showed that compared with the control group,model mice spleen T lymphocytes CD4+cell percentage reduced,CD8+cell ratio increased,CD4+/CDs+ratio decreased.With model group,EEGA each dose group increased the proportion of CD4+cells,CDs+cells was reduced,CD4+/CD8+ratio increased,while positive group no improvement.2.8 HE staining showed strong growth of tumor cells in model group,showing abundant microvessels.EEGA groups and positive group of tumor cell growth limitations,fewer microvessels.2.9 TUNEL staining showed that the model of tumor cells apoptosis rate of around 10%,EEGA low,medium and high dose group and positive drug group apoptosis rate of tumor tissue was 25.88±1.96%,respectively,35.38±2.13%,47.38±3.58%,74.63±2.07%.2.10 Immunohistochemistry showed that,compared with the model group,EGGA low,Bax positive cells in the high dose group and positive group increments,Bcl-2 positive cells decreasing,Bax/Bc1-2 ratio increase,activation of caspase-3 increase the proportion of positive cells.The VEGF positive expression was no significant difference between the groups.2.11 RT-PCR test results showed that compared with the model group,EEGA each group and positive group were able to upregulate the expression of Bax gene down-regulated the expression of Bcl-2 gene,increased Bax/Bcl-2mRNA ratio,increased caspase-3 gene expression and showed a dose-effect relationship in EEGA each dose group.2.12 Western blot test results showed that compared with model group,EEGA groups and positive group were able to significantly induce the expression of Bax protein inhibits the expression of Bcl-2 protein was significantly increased Bax/Bcl-2 protein ratio,increased activation of caspase-3 protein level,and showed a dose-effect relationship in EEGA each dose group.Conclusions1.Gentianella acuta extract containing serum can inhibit liver cancer Bel-7402 cell proliferation,it can induce apoptosis and cell cycle distribution.2.Gentianella acuta H22 tumor bearing mice inhibits tumor cell growth and induce apoptosis.3.Gentianella acuta improve immunity in mice bearing H22.4.Gentianella acuta through adjusting the levels of caspase-3 and Bax ratio and activation of Bcl-2 to achieve the anti-liver cancer effects. |
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