The Enhancing Effect Of Serum Pharmacology Of LiuweiDiduang Bolus On "Bystander Effect" Of Suicide Gene Therapy Of Liver Cancer

The malignant tumor is the disease of endangering the human health badly. Primary liver cancer is one of them. With the development of molecular biology, the suicide gene therapy of the tumor has represented the good application prospect as a kind of new approach. But the problem in this remedy is insufficient killing ability, low efficiency of transfection and uncertain safety. It's reported that increasing the bystander effect is a good approach to resolve this problem. Considering the most important mechanism of bystander effect conducted by the gap junction intercellular communication (GJIC) of cell, researchers today enhance the bystander effect by method of medical inducement to elevate GJIC. But there were not many medicines especially natural medicine with this effect had been found until today. Although some ingredients of TCM had been proved by the clinic or basic investigations on the effects of inducing apoptosis of tumor cells and increasing immunity of the patients, few articles were released about the investigation on increasing bystander effect and cooperating the tumor gene therapy with TCM or their ingredients. However, it's tested that LiuweiDiduang Bolus had the effect of enhancing cAMP, and the elevation of cAMP could increase the expression of connexin protein, which elevated GJIC to enhance the bystander effect.Therefore, according to the development and trend of the studies in the mechanism of bystander effect and bystander effect-increasing induced by medicine, we presented the following hypothesis, there were some active ingredients in the LiuweiDiduang Bolus which could improve the GJIC, induce the apoptosis of tumor cells, improve the local inflammatory immune environment of tumor or enhance the function of immune system, and exert the effect of anti-tumor by the manner of increasing bystander effect of suicide-gene.Objective: In our research, all above served as the purpose to investigate the possibility improving the bystander effect of suicide gene therapy cooperated with Serum Pharmacology of Liuwei Diduang Bolus. After getting the effective Serum Pharmacology of LiuweiDiduang Bolus on the cell level, we would validate them on the whole level repeatedly to find the real effective ingredients of LiuweiDiduang Bolus.During the research, we hoped to explore the possible mechanism of adjusting cell GJIC and inducing of cell apoptosis with Serum Phamacology of LiuweiDiduang Bolus, then we could get the further comprehension about the mechanism of increasing bystander effect with Serum Phamacology of LiuweiDiduang Bolus.Methods:1 Preparation of Serum Phamacology of LiuweiDiduang Bolus and control serum: SD rat (230～280g), divided into two groups: one was control serum group, the other was Serum Phamacology of Liuwei Diduang Bolus(Administration: 6～8 times of isoeffect dosage). Intragastric administration was also divided into two groups: Four-day Serum and Eight-day Serum. Getting blood from abdominal aorta after the Intragastric administration for one hour, Conservation of serum: -70℃fri ge.2 The construction of HSV-tk/GCV system (Advisor's Topic Group had accomplished).3 The effect observation of herbs serum of Liuwei Diduang Bolus combining with HSV1-tk/GCV system on rat hepatocarcinoma cell CBRH7919 in vitro: Detection of MTT assay: the sensitivity of herbs serum of Liuwei Diduang Bolus and control serum on CBRH7919/tk~+; the GCV sensitivity of CBRH7919/tk~- and CBRH7919/tk~+, with the results to decide the suitable Medication level. The Q-value analysis was used to estimate the synergistic effects of the herbs serum of Liuwei Diduang Bolus on the suicide gene system. The Q-value was equal to the ratio of the actual effect of combined treatment to its theoretical effect. The effect was classified into three categories: antagonistic effect (Q≤0.85), additive effect (0.85＜Q＜1.15), and synergistic effect (Q≥1.15).4 Exploring the mechanism of the synergistic effects of the herbs serum of Liuwei Diduang Bolus: The cell cycle analysis and cell apoptotic index were performed using a FACScan cell analyzer; the effects of herbs serum of Liuwei Diduang Bolus on the GJIC of CBRH7919 were detected by SLDT.Results:1 The results of herbs serum of Liuwei Diduang Bolus combining with HSV1-tk/GCV system on rat hepatocarcinoma cell CBRH7919 in vitro:1.1 The work concentration of GCVThe suitable concentration was 39.2μM for avoiding the inhibiting effect of GCV on CBRH7919 cell. Besides the rat serum could interfere with the effect od GCV.1.2 The preparation of cat serum and the addition of herbs serum of Liuwei Diduang BolusThe control serum and herbs serum of Liuwei Diduang Bolus showed a promotion effect on CBRH7919 when the final concentration was 5%～15% but showed inhibiting effect when higher than 20%(micro. Observation: the cells transformed into vacuoles). There was no statistical difference comparing the influences between the herbs serum of Liuwei Diduang Bolus and control serum on CBRH7919. (P＞0.05) According to the results of MTT and FACS, the effect of Eight-day Serum was better than the Four-day Serum.1.3 Perfect ratio of tk~+: tk~- in the therapy of HSV1-tk/GCV systemThere were synergistic effects when combining with 10%tk~+/GCV system.1.4 The dose-effect relationship of the serum of Liuwei Diduang Bolus to the enhancing impact of bystander effect of HSV1-tk/GCV system on CBRH7919When combining with 10%tk~+/GCV system, herbs serum of Liuwei Diduang Bolus both Eight-day Serum and Four-day Serum showed a markedly synergistic effect at 10% concentration (Q values＞1.15). But the incubation time of herbs serum were different: Four-day Serum for 6h; and Eight-day Serum for 24h.2 The results of the mechanism researchs of the synergistic effects of herbs serum of Liuwei Diduang Bolus combining with HSV1-tk/GCV system2.1 The results of the cell cycle and apoptosis of the effects of herbs serum of Liuwei Diduang Bolus combining with HSV1-tk/GCV system on CBRH7919PI single stain: The herbs serum of Liuwei Diduang Bolus showed an apoptotic rate-elevating effect on CBRH7919 when combining with HSV1-tk/GCV (10%tk~+) system. Futhermore, it showed a S-phase lagging effect On CBRH7919 cell cycle. Roughly observation: the effect of Eight-day serum was better than the Four-day serum.Annexinⅴ/PI double stain: The herbs serum of Liuwei Diduang Bolus showed an apoptotic rate-elevating effect on CBRH7919 when combining with HSV1-tk/GCV (10%tk~+) system. There was no striking difference between Eight-day serum and Four-day serum.2.2 The results of the herbs serum of Liuwei Diduang Bolus to GJIC of CBRH7919 cellThe result of SLDT showed that the ability of GJIC function of CBRH7919 was up-regulated by herbs serum of Liuwei Diduang Bolus. But there was no striking difference between Eight-day serum and Four-day serum.Conclusion:We explore the effects of herbs serum of Liuwei Diduang Bolus on tumor HSV1-tk/GCV suicide gene therapy system. Results show that herbs serum of Liuwei Diduang Bolus exhibits synergistic killing effect on suicide gene system. Mechanisms of the synergistic effect of herbs serum of Liuwei Diduang Bolus is related to the restorationof GJIC, or/and the apoptotic inducement.