| ObjectiveMalignant melanoma usually arise on skin and internal organs. It is difficult to be cured in clinic. In recent years, gene therapy, especially suicide gene therapy has made some progress. But we gradually find many problems during the study, such as the precursor drug of suicide gene function low, has existed toxic side effects and the vector transfection efficiency is not high.These problems lead to suicide gene therapy do not produce the desired therapeutic effect on treating cancer. Due to the existence of bystander effect, to enhance its bystander effect of suicide gene is feasible.Traditional Chinese medicine herbs have multiple targets, the characteristics of multi-channel effects and extensive pharmacological effects, low price, small side effects. Therefore it is a new attempt to combine classical Chinese medicine with suicide gene therapy to treat cancer. The early stage of the study findings:the vivo and vitro experiments showed Liuweidihuang pill jointed with suicide gene can increase the killing effect on tumor. The efficacy of any drug must have material basis. What is the material basis of Liuweidihuang pill improving suicide gene killing effect? What is the mechanism of its effect? On the basis of previous studies, we propose hypotheses:" The major blood transitional ingredient of Liuweidihuang pill is the material basis of it improving tumor suicide gene killing effect ". The assumption was verified by vitro experiments. The results showed that:major five kinds of monomer composition of blood and its mixture had no direct relationship to killing effect of suicide gene therapy efficacy, but major five kinds of monomer composition of blood and its mixture acted in the lymphocytes, lymphocyte culture supernatant of its drug-containing and HSV-tk/GCV system had a synergistic effect on inhibiting B16cells, the synergistic mechanism may be immune-mediated mechanisms, and gap junctions, on this basis, we assumed migrational components in blood of Liuweidihuangwan could enhance suicide gene system to kill melanoma in vivo, and explore their corresponding synergistic mechanism to echo previous experiment in vitro. Above all, we could verify migrational components in blood of Liuweidihuangwan is the material basis of Liuweidihuangwan enhancing tumor suicide gene system to kill melanomaMethods1In order to obtain optimal synergies experimental conditions, we applyed major transitional components in blood of Liuweidihuang pill joint HSV-tk/GCV system in vivo to kill melanoma B16cells.1.1Identification of HSV-tk/GCV system in vivo:the mice were given different doses of GCV after being inoculated100%tk cells in armpit; we identified the activity of tk gene from the efficacy.1.2Determination of the mixing ratio of tk cells and the dose of GCV used for work:we presumed that50mg/kg·d was appropriate dose of GCV. B16/tk cells and B16cells was mixed by the proportion of20%,40%,60%. Then we determined the most suitable mixing ratio from the efficacy. We presumed that40%was the appropriate ratio of B16/tk and B16cells.The mice were given50mg/kg·d,100mg/kg·d,200mg/kg·d doses of GCV. According to efficacy and preliminary experimental results, we determined to the optimal integrated GCV dose1.3Determination of dose that major transitional components in blood from Liuweidihuang pill:We determined to set up the50mg/kg·d dose of GCV and1:1mixed ratio of B16/tk cells and B16cells, considered3:8:1as mass ratio of phenol:(loganic, sweroside, morroniside):5-(hydroxymethyl)-2-furoate acid.We presumed20mg/kg·d was a suitable dose of mixed components, and set up the model group, GCV group, mixed components group, GCV combined with mixed components group. To determine the optimal dose of hybrid components from the efficacy.2Major transitional components in blood of Liuweidihuang pill combined with HSV-tk/GCV system synergies killing melanoma B16tumor cells in vivo experiment:set the GCV dose50mg/kg·d, mixed ratio of B16/tk cells and B16cells was1:1, the dose of transitional component in blood was15mg/kg·d, the dose of Liuweidihuang pill was lOg/kg·d, set up the normal group, model group, GCV group, GCV combined with transitional component group, GCV combined with Liuweidihuang pill group.observed efficacy3The preparation of serum from peripheral blood of the mice which injected by major transitional components in blood of Liuweidihuang pill and study of synergis effect that serum combined with suicide gene killing melanoma3.1The preparation of serum:we inoculated mixed melanoma which the ratio of tk+and tk-was1:1in armpit of mice.divided into model group and transitional components group. The dose of transitional components was15mg/kg·d. After14days we collected the blood of eye, centrifuged to obtain serum at4℃overnight, saved the serum at-20℃. The serum from model group was named for control serum, the serum from drug group was named for medicated serum.3.2The synergism effect of blood components in liuweidinghuangwan on HSV-tk/GCV suicide gene system to melanoma in mice serum was detected by MTT. The culture system concluded10%serum of the total volume. The mixed ratio of B16/tk cells and B16cells was3:7, set up control group (with culture medium),0%medicated serum group (10%control serum), GCV combined with0%medicated serum group (10%control serum), GCV combined2.5%medicated serum group (2.5%medicated serum and7.5%control serum), GCV combined5%medicated serum group (5%medicated serum and5%control serum), GCV combined7.5%medicated serum group (7.5%medicated serum and2.5%control serum), GCV combined10%medicated serum group (10%medicated serum), the final concentration of GCV is30μmol/L, used MTT to detect inhibition rate.3.3The synergism effect of blood components in liuweidinghuangwan on HSV-tk/GCV suicide gene system to melanoma in mice serum was detected by fluorescent traccer technique.The culture system concluded10%serum of the total volume. the mixed ratio of B16/tk cells and B16cells was1:1, set up control group (with culture medium), GCV combined with0%medicated serum group (10%control serum), GCV combined with2.5%medicated serum group (2.5%medicated serum and7.5%control serum), GCV combined with5%medicated serum group (5%medicated serum and5%control serum), GCV combined with7.5%medicated serum group (7.5%medicated serum and2.5%control serum), GCV combined with10%medicated serum group (10%medicated serum), the final concentration of GCV is25 μmol/L, fluorescence microscope photographs was used to observeResults1To explore the optional response of blood component in liuweidinghuangwan to HSV-tk/GCV suicide gene system to kill melanoma.1.1HSV-tk/GCV system in vivo could be successfully constructed, the anti-tumor rate of group given50mg/kg·d dose of GCV was78.3%, the anti-tumor rate of group given100mg/kg·d dose of GCV was85.8%.1.2The optimal dose of GCV was50mg/kg·d. When the dose of GCV was50mg/kg·d, its anti-tumor rate was61.3%; when the dose of GCV was100mg/kg·d, its anti-tumor rate was82.2%;when the dose of GCV was200mg/kg·d, its anti-tumor rate was94.0%. The ratio of tk+cells was50%. Anti-tumor rate of20%tk+group was17.8%, anti-tumor rate of40%tk+group was61.3%anti-tumor rate of60%tk+group was73.4%1.3The dose of five kinds of transitional components was15mg/kg·d Anti-tumor rate of GCV group was5.5%, anti-tumor rate of mixed five kinds of transitional component group was36.6%, anti-tumor rate of GCV combined with five kinds of mixed migration group was30.1%2Major transitional components in blood of Liuweidihuang pill could enhance the HSV-tk/GCV system to kill melanoma B16cells2.1The rate of tumorigenesis and the time of tumor formation:a mouse of model group had not generated tumor, other groups could be touched tumor after eight days; the tumor formation rate of model group was93%, the tumor formation rate of other groups was100%.2.2Tumor volume (unit:mm3) and the growth curve of tumor volume:the difference appeared between three contained GCV groups and model group during the period of drug delivery; at the end of treatment there were statistically significant differences (P<0.01) among every contained GCV group compared with model group on tumor volume2.3Tumor mass (unit:g) and anti-tumor rate:the model group (5.3378±0.3095g), the mixed five kinds of transitional component group (4.6591±0.3572g,12.7%), GCV group (2.8959±0.6147g,45.7%), GCV combined with five kinds of transitional component group (1.7059±0.1001g,68.0%), GCV combined with Liuweidihuangwan group (1.9135±0.1753g,64.2%). There were statistically significant differences (P<0.01) among every contained GCV group compared with model group on tumor mass 2.4Examination of pathology:From the observation of naked eye, tumor volume of every contained GCV group was smaller than model group and transitional component group. From the observation of microscope:the density of tumor cell in two united groups was relatively low, there were more proliferous fibrous connective tissue around the tumor tissue. The results showed that GCV combined with five kinds of transitional components group could inhibit the growth of malignant melanoma, its efficacy was better than GCV treatment and five kinds of mixed migration component. Its efficacy was equivalent of GCV combined with Liuweidihuang pill3Major transitional components in blood of Liuweidihuang pill have a synergistic effect on HSV-tk/GCV system killing melanoma B16cells.3.1Through MTT assay each anti-tumor rate of GCV combined with different serogroup from low to high were showed below:46.4±4.14%,61.9±3.03%,57.1±1.61%,80.1±1.22%. The difference between GCV combined with2.5%medicated serum and GCV combined with the control serum group was statistically significant (P<0.01); The difference between GCV combined with5%medicated serum group and GCV combined with control serum group was statistically significant (P<0.05); the difference between GCV combined with10%medicated serum group and GCV combined with control serum group was statistically significant (P<0.01).3.2Fluorescent tracer method showed that medicated serum could play a certain role on enhancing suicide gene to kill B16melanoma cells Specifically in GCV combined with10%medicated serum group, some red cell debris floated aroud green fluorescent cells, this phenomenon of high medicated serum group was the most obviousConelusionsMajor transitional components in blood of Liuweidihuang pill have a synergistic effect on HSV-tk/GCV system killing melanoma B16cells in vivo, the high medicated serum have a certain amount of effect in vitro. |
PDF Full Text Request