The Enhancing Utility Research And Mechanism Of Serum Pharmacology Of LiuweiDiduang Bolus On HSV1-tk/GCV System

Objective:Malignant melanoma is extremely high incidence,prone to metastasis,poor prognosis, high mortality,and clinical treatment is very difficult.With the development of molecular biology,gene therapy especially suicide gene therapy on malignant melanoma brings hope to patients.following the traditional surgical resection,radiotherapy and chemotherapy,suicide gene therapy becomes to the method of a new anti-tumor treatment. But as a result of issues are still not resoled 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.As the gap junction mechanism is an important mechanism for the bystander effect,improving the exchange information of the tumor cells is one of the important ways to improve the efficiency of suicide gene therapy.Preliminary studies showed that:in vivo Liuweidihuang bolus has obvious synergies on the suicide gene therapy of malignant melanoma.And it has relarions with that Liuweidihuang bolus strengthen Cell-mediated immunity and humoral immunity. and taking into account the multiple channels of Chinese medicine herbs and the role of the characteristics of multi-targets,in addition to immune mechanisms,can Liuweidihuang bolus destruction through the side effects of other mechanisms such as gap junctions, apoptosis and other mechanisms can increase efficiency of suicide gene? Be adopted in this experiment in vitro(excluding the role of the immune mechanism) for their demonstration.In this study,Chinese medicine composed of serum pharmacology method was used. Killing effect and its mechanism were discussed through Liuweidihuangwan combined with HSV1-tk/GCV suicide gene system in vitro to B16 melanoma cells.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 medicineMethods:1.Construction and identification of HSV-tk/GCV System on malignant melanoma B16 cells:The PT67/tk cells virus supernatant infected with the logarithmic phase of B16 cells,G418-resistant positive clones named B16/tk,filter and expand culture;extracted B16/tk cells genomic DNA,PCR method to identify genes tk;adding GCV and observing the activity of tk gene under the microscope.2.The work concentration of GCV and the the proportion of tk ~+:Mixing B16/tk and B16 with the proportion of 0,10%,20%,40%,100%,adding the final concentration of GCV, respectively:OuM,6.25μM,12.5μM,25μM,50μM,100μM,200μM,MTT assay was detected3.The preparation of the control serum and serum containing liuweidihuang bolus and the effect of serum on B16 cells:30 SD rats which are SPF-class healthy were randomly divided into 2 groups,control group and Liuweidihuang bolus group,rats marked as Liuweidihuang bolus group fed with Liuwei Dihuang Pill slurry 8ml(containing pharmacognosy 4g),marked as the control group fed with the same volume distilled water for 4 days,one hour after the last gavage,blood from abdominal aortic,prepare serum and preserve-70℃refrigerator to back-up;set 10%newborn calf serum,10%control serum group,2.5%Liuwei serum group(7.5%control serum+2.5%Liuwei serum),5%Liuwei serum group(5%control serum+5%Liuwei serum),10%Liuwei serum group,MTT assay to detect the killing effect on B16.4.The effect of liuweidihuang bolus combined with tk / GCV system on B16:B16tk mixed with B16 cells at the proportion of 20%,setting10%control serum group,2.5% Liuwei serum group,5%Liuwei serum group,10%liuwei serum group,control serum joint GCV group,2.5%Liuwei serum joint GCV group,5%Liuwei serum joint GCV group, 10%Liuwei serum joint GCV group,MTT assay to detect the inhibition of B16;using Q-analysis of Kim Jong-kwan to analysis the effect of Liuweidihuangwan combined with HSV-tk/GCV suicide gene system5.The synergy effect of Liuweidihuang bolus combined with on mouse malignant melanoma and gap junctional mechanisms:setting control serum group,2.5%Liuwei serum group,5%Liuwei serum group,10%Liuwei serum group,Western Blot and indirect immunofluorescence assays detected the impact on Cx26 and Cx43 protein expression on B16 cells,RT-PCR assay detected the impact on Cx26 and Cx43 mRNA expression; RT-RCR detected the efficiency of Cx26-309,Cx26-337,Cx26-367,Cx26-2098 SiRNA interference of Cx26,choose the most efficient interference -Cx26-2098 SiRNA,observed bystander effect after interferenced Cx26 gene;set 10%control serum group,2.5%Liuwei serum,5%Liuwei serum group,10%Liuwei serum group,10%control serum joint GCV group,2.5%Liuwei serum joint GCV group,5%Liuwei serum joint GCV group,10% Liuwei serum Joint GCV group,2.5%Liuwei serum joint GCV + glycyrrhetinic acid group, 5%Liuwei serum joint GCV + glycyrrhetinic acid group,10%serum Liuwei joint GCV + glycyrrhetinic acid group.Liuwei serum were less than 10%of serum complement by control serum,the final concentration of GCV is 20uM,final concentration of glycyrrhetinic acid is 50μM,MTT assay detected inhibitory rate of cells,observation the effects of the GJIC inhibitor on Liuweidihuang bolus combined tk / GCV system on B16 cells.6.The effect of serums on HSV-tk/GCV system and gap junction protein expression: setting Set 10%newborn calf serum,10%rat serum,10%rat administered serum group, 10%newborn calf serum co-GCV group,10%rat serum combined GCV group,10%rat administered serum joint GCV group,MTT assay detected and calculated inhibition of cells in each group;setting 10%newborn calf serum,10%rat serum,10%rat administered serum Group,Western Blot assay detected the expression of Cx26 and Cx43 proteins, RT-PCR assay detected effects of Cx26 and Cx43mRNAResults:1.Construction and identification of HSV-tk/GCV System on malignant melanoma B16 cells:PCR results showed that after infection the virus supernatant,B16 cell genome has been integrated the tk gene.cells dead after adding GCV,suggesting that the B16 cells received tk gene and show activity.2.The work concentration of GCV and the the proportion of tk +:When GCV concentration above 100μM,B16 cells have a certain toxicity,and low concentrations of GCV(6.25μM) also show some B16/tk cell killing effect.We want to choose the GCV concentration that sensitive to B16/tk and free toxicity to B16 cell(6.25μM -100μM),and the minimum GCV concentration that can kill most cells as optimum concentration of GCV. it is the work of the concentration of GCV as 20μM;40%B16/tk and 100%B16/tk were very sensitive to GCV,low concentrations of GCV(6.25μM) almost killed all cells(B16/tk and B16),that is,mixed 40%B16/tk cells demonstrated strong bystander effect.B16 and 10%B16/tk even high concentrations(100μM) of GCV are not sensitive,that is,10% B16/tk is not observed the bystander effect.In order to facilitate in vitro experiments carried out and observe bystander effect,the mixture of 20%B16/tk cells were used. 3.The preparation of the control serum and serum containing liuweidihuang bolus and the effect of serum on B16 cells:MTT results showed that,10%rat control serum group, 2.5%Liuwei serum group,5%Liuwei serum group,10%Liuwei serum group cell survival rates were respectively:100±1.33%,100.63±1.20%,105.30±0.93%,106.59±2.42%, 109.35±2.51%,and compared to 10%bovine serum group,2.5%,5%,10%Liuwei serum Promote B16 cells proliferation(P<0.01).4.The effect of liuweidihuang bolus combined with tk / GCV system on B16:2.5%,5%, 10%liuwei serum Joint 20%tk + / GCV group,the actual inhibition rate(48.75%,59.39%, 69.28%) was significantly higher than the theoretical inhibition rate(38.13%,38.67%, 39.92%),Kim Jung-kwan,Q values were 1.28,1.54,1.74,are greater than 1.15,with synergy synergy.5.The synergy effect of Liuweidihuang bolus combined with on mouse malignant melanoma and gap junctional mechanisms:Liuweidihuang bolus can increase Cx43 protein and mRNA expression on B16,and has a dose-dependent relationship.with two-way adjustment of Cx26 protein and mRNA expression,the role of inhibition with low-dose and high dose is able to increase its expression;SiRNA interferenced Cx26 gene, Liuweidihuang bolus joint suicide gene therapy did not reduce the bystander effect;2.5%, 5%,10%Liuwei serum joint GCV with no glycyrrhetinic acid blocked groups(48.75%, 59.39%,69.28%) compared to groups with glycyrrhetinic acid blocked(29.14%,38.71%, 58.13%),there were a significant reduction in inhibition rate(P <0.01).6.The effect of serums on HSV-tk/GCV system and gap junction protein expression: There was no significant difference on killing effect when newborn bovine serum,rat serum, rat administered serum respectively combined with tk / GCV system.there was no effect on Cx43 protein and mRNA expression when B16cells cultured with serums.It can increase Cx26 protein and mRNA expression levels by gavage mouse serum on B16.Conclusion:This study was successful to build a HSV-tk/GCV suicide gene system on B16.Found in the study,synergy existed in suicide gene therapy combined with Liuweidihuangwan,the synergy maight be relevant with the gap junction mechanism;further study show that Liuweidihuangwan may be through increased expression of Cx26 and Cx43 to achieve synergies by effective role.