| Objective:Liver cancer is extremely high incidence, poor prognosis, high relapse incidence and mortality,low qualityoflife. With the development of molecular biology, gene therapy especially suicide gene therapy on liver cancer brings hope to patients. But as a result of issues are still not resolved such as suicide gene therapy vector transfection efficiency is currently low and targeting not enough, there are still some residual tumor cells leading to tumor recurrence, which is the treatment of major defects exist. Bystander effect,that gene transfection-positive cells can kill gene transfection-negative cells, is a notable feature of suicide gene therapy. Enhancing the bystander effect has become an important strategy on improving efficacy of tumor suicide gene therapy. Preliminary studies showed that:Liuweidihuang bolus has obvious synergies on the suicide gene therapy of malignant tumors in vivo and in vitro,and it related to immune mechanisms and gap junctions. What is the the substance basis of it's efficacy and it's mechanisms? Be adopted in this experiment in vitro for their demonstration.And be based on preliminary studies,we continue to filtrate pharmacological activities which have obvious synergies on the suicide gene therapy of malignant tumors. Through research we want to provide experimental data and scientific evidence for establishment the programs of suicide gene therapy for clinical compound combined with traditional Chinese medicine and western medicine.Methods:1. The influence of composition of LiuweiDihuang Bolus entering the blood combined with tk/GCV system on CBRH7919:CBRH7919/tk mixed with CBRH7919 cells at the proportion of 10%,the mixed cells were treated seperately by composition of LiuweiDihuang Bolus entering the blood with suitable final concentration and GCV with final concentration at 15.7μM, or treated by composition of LiuweiDihuang Bolus entering the blood plus GCV with the same concentration.The final concentration of each composition of LiuweiDihuang Bolus entering the blood were:paeonol/loganin/mixed drugs:25,50,100μM;morroniside/5-(Methoxymethyl)-2-furoic acid/sweroside: 37.5,75,150μM. MTT assay to detect the inhibition of each group,using Q-analysis of Kim Jong-kwan to analysis the effect of composition of LiuweiDihuang Bolus entering the blood combined with HSV-tk/GCV suicide gene system.2. The preparation of lymphocyte supernatants with drug (drug supernatants) and lymphocyte supernatants without drug (blank supernatants) and the effect of drug supernatants combined with tk/GCV system on CBRH7919:SD rats which are SPF-class healthy, taking out spleen and making into lymphocyte cell suspension.Then mixed it with drug (drug supernatants) and without drug (blank supernatants).CBRH7919/tk mixed with CBRH7919 cells at the proportion of 10%,the mixed cells were treated seperately by drug supernatants and blank supernatants with final concentration at 7.5%,15%,30% and GCV with final concentration at 15.7μM, or treated by drug supernatants and blank supernatants plus GCV with the same concentration. MTT assay to detect the inhibition of each group,using Q-analysis of Kim Jong-kwan to analysis the effect of composition of drug supernatants and blank supernatants combined with HSV-tk/GCV suicide gene system.3. The synergy effect of drug supernatants combined with tk/GCV system on CBRH7919 and gap junctional mechanisms:Setting control group,7.5% drug supernatants,15% drug supernatants,7.5% blank supernatants,15% blank supernatants, Western Blot assay detected the impact on Cx43 protein expression on CBRH7919 cells; Setting control group,7.5% drug supernatants,15% drug supernatants,7.5% drug supernatants joint GCV group,15% drug supernatants Joint GCV group,7.5% drug supernatants joint GCV group+30μM glycyrrhetinic acid group,7.5% drug supernatants joint GCV group+60μM glycyrrhetinic acid group,15% drug supernatants joint GCV group+30μM glycyrrhetinic acid group,15% drug supernatants joint GCV group+ 60μM glycyrrhetinic acid group,GCV group,GCV group+30μM glycyrrhetinic acid group,GCV group+60μM glycyrrhetinic acid group, the final concentration of GCV is 15.7μM, MTT assay detected inhibitory rate of cells, observation the effects of the GJIC inhibitor on drug supernatants combined tk/GCV system on CBRH7919 cells.4. The effect of kaempferol combined with tk/GCV system on CBRH7919: CBRH7919/tk mixed with CBRH7919 cells at the proportion of 10%, Setting control group,15μM kaempferol group,30μM kaempferol group,60μM kaempferol group, GCV group,15μM kaempferol joint GCV group,30μM kaempferol Joint GCV group,60μM kaempferol Joint GCV group, the final concentration of GCV is 15.7μM, MTT assay to detect the inhibition of each group,using Q-analysis of Kim Jong-kwan to analysis the effect of kaempferol combined with HSV-tk/GCV suicide gene system.5. The synergy effect of kaempferol combined with tk/GCV system on CBRH7919 and gap junctional mechanisms:Setting control group,15μM kaempferol group,30μM kaempferol group,60μM kaempferol group,parachute assay detected the impact of GJ with kaempferol combined with tk/GCV system on CBRH7919; RT-PCR assay detected the impact on Cx43 mRNA expression; Western Blot assay detected the impact on Cx43 protein expression on CBRH7919 cells.Setting control group,20μM kaempferol group, 40μM kaempferol group,20μM kaempferol group joint GCV group,40μM kaempferol group joint GCV group,20μM kaempferol group joint GCV group+30 u M glycyrrhetinic acid group,20μM kaempferol group joint GCV group+60 u M glycyrrhetinic acid group,40μM kaempferol group joint GCV group+30 u M glycyrrhetinic acid group,40μM kaempferol group joint GCV group+60μM glycyrrhetinic acid group,GCV group,GCV group+30μM glycyrrhetinic acid group,GCV group+60 u M glycyrrhetinic acid group, the final concentration of GCV is 15.7μM, MTT assay detected inhibitory rate of cells, observation the effects of the GJIC inhibitor on kaempferol combined tk/GCV system on CBRH7919 cells.6. Used flow cytometry and comet assay to detect the effect of curcumin on cancer cell cycle and to analyze its impact on the apoptotic index:CBRH7919/tk mixed with CBRH7919 cells at the proportion of 10%, Setting control group,15μM kaempferol group,30μM kaempferol group,60μM kaempferol group, GCV group,15μM kaempferol joint GCV group,30uM kaempferol Joint GCV group,60μM kaempferol Joint GCV group, the final concentration of GCV is 15.7p.M, used flow cytometry to detect the effect of curcumin on cancer cell cycle and to analyze its impact on the apoptotic index; used comet assay to detect the impact of kaempferol combined with tk/GCV system on CBRH7919 through determining average light density and smearing.Results:1. The influence of composition of LiuweiDihuang Bolus entering the blood combined with tk/GCV system on CBRH7919:25μM,50μM,100μM paeonol joint 10% tk+/GCV group,the actual inhibition rate (17.23%,17.89%,18.46%) was lower than the theoretical inhibition rate (19.73%,21.10%,22.75%), Kim Jung-kwan, Q values were 0.87,0.85,0.81, are less than 1.15, was additive effect; 25μM,50μM,100μM loganin joint 10% tk+/GCV group, the actual inhibition rate were 13.17%,13.86%,12.22%, the theoretical inhibition rate were 14.01%,12.15%,12.84%, Kim Jung-kwan, Q values were 0.94,1.14,0.95,are less than 1.15, was additive effect;The actual inhibition rate of 75μM morroniside joint 10% tk+/GCV group approached 75μM morroniside group, the actual inhibition rate of 37.5μM,150μM morroniside joint 10% tk +/GCV group were lower than 37.5μM,150μM morroniside;37.5μM,75μM,150μM5-(Methoxymethyl)-2-furoic acid joint 10% tk+/GCV group, the actual inhibition rate (3.00%,2.71%,1.01%) was lower than the theoretical inhibition rate (5.45%,5.86%,2.48%), Kim Jung-kwan, Q values were 0.55,0.46,0.41, are less than 0.85, was rivalry; The actual inhibition rate of 37.5μM sweroside was-3.22%,was effect of proliferation of cells,75μM,150 u M sweroside joint 10% tk + / GCV group,the actual inhibition rate (7.47%,2.88%) was higher than the theoretical inhibition rate (7.25%,2.72%), Kim Jung-kwan, Q values were 1.03,1.06, are less than 1.15, was additive effect; 25 u M,50 u M,100μM mixed drugs joint 10% tk+ / GCV group,the actual inhibition rate (4.06%,5.44%,7.37%) was lower than the theoretical inhibition rate (14.65%,14.07%,11.13%), Kim Jung-kwan, Q values were 0.28,0.39,0.66, are less than 0.85, was rivalry.2. The preparation of lymphocyte supernatants with drug (drug supernatants) and lymphocyte supernatants without drug (blank supernatants) and the effect of drug supernatants combined with tk/GCV system on CBRH7919:7.5%,15%,30% blank supernatants joint 10% tk+/GCV group,the actual inhibition rate (10.49%,13.16%,19.94%) was lower than the theoretical inhibition rate (12.43%,15.33%,21.42%), Kim Jung-kwan, Q values were 0.85,0.86,0.93,are less than 1.15, was additive effect; 7.5%,15%,30% drug supernatants joint 10% tk+/GCV group,the actual inhibition rate (22.72%,26.68%,30.50%) was significantly higher than the theoretical inhibition rate (15.18%,15.27%,13.64%), Kim Jung-kwan, Q values were 1.50,1.75,2.24, are greater than 1.15, with synergy.3. The synergy effect of drug supernatants combined with tk/GCV system on CBRH7919 and gap junctional mechanisms:Blank supernatants cann't increase Cx43 protein expression on CBRH7919,with two-way adjustment of Cx43 protein expression of drug supernatants, the role of inhibition with low-dose and high dose is able to increase its expression.7.5% drug supernatants joint GCV with 30μM glycyrrhetinic acid blocked groups (27.32%) compared to groups with no glycyrrhetinic acid blocked (27.83%), there were no significant reduction in inhibition rate (P>0.05);7.5% drug supernatants joint GCV with 60μM glycyrrhetinic acid blocked groups (29.56%) compared to groups with no glycyrrhetinic acid blocked (27.83%), there were no significant reduction in inhibition rate (P>0.05); 15% drug supernatants joint GCV with 30μM and 60μM glycyrrhetinic acid blocked groups (31.40%,24.79%) compared to groups with no glycyrrhetinic acid blocked (34.09%), there were a significant reduction in inhibition rate when using 60μM glycyrrhetinic acid (P<0.01).4. The effect of kaempferol combined with tk/GCV system on CBRH7919:15μM,30μM,60μM kaempferol Joint 10% tk+/GCV group,the actual inhibition rate (23.44%,28.75%,53.81%) was significantly higher than the theoretical inhibition rate (15.34%,18.52%,43.58%), Kim Jung-kwan, Q values were 1.53,1.55,1.24, are greater than 1.15, with synergy.5. The synergy effect of kaempferol combined with tk/GCV system on CBRH7919 and gap junctional mechanisms:kaempferol can advance GJ of CBRH7919; kaempferol can increase Cx43 protein and mRNA expression on CBRH7919,and has a dose-dependent relationship; 20μM,40μM kaempferol joint GCV with 30μM,60μM glycyrrhetinic acid blocked groups(26.74%,39.55%; 24.62%,32.93%) compared to groups with no glycyrrhetinic acid blocked (45.06%; 55.11%), there were a significant reduction in inhibition rate (P<0.01).6. Used flow cytometry and comet assay to detect the effect of curcumin on cancer cell cycle and to analyze its impact on the apoptotic index:The S phase of CBRH7919 when kaempferol combined with tk/GCV system significantly increased, the apoptotic index of 15μM,30μM,60μM kaempferol Joint 10% tk+/GCV group(13.88%,24.06%,38.23%) was significantly higher than 15μM,30μM,60μM kaempferol group(8.94%,13.53%,17.80%)and GCV group(15.44%);15μM,30μM,60μM kaempferol Joint 10% tk+/GCV group, average light density significantly declined versus 15μM,30μM,60μM kaempferol group and GCV group,while smearing evidently increased.Conclusion:This study preliminary approved there were no direct action of composition of LiuweiDihuang Bolus entering the blood and mixd drugs on the synergy effect of LiuweiDihuang Bolus combined with tk/GCV system on malignant tumors.Found in the study, synergy existed in suicide gene therapy combined with drug supernatants, the synergy maight be relevant with the gap junction mechanism; Preliminary found kaempferol which have obvious synergies on suicide gene therapy of CBRH7919 combined with tk/GCV system, the synergy maight be relevant with the gap junction mechanism and apoptosis mechanism,further study showed that kaempferol may be through increasing expression of Cx43,inducing apoptosis, regulating cell cycle to achieve synergies by effective role.
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