The Influence Of Hedyotic Diffusa Upon The Gene Expression Of Transplanted Tumor Apoptosis Of The Hepatoma-lines-H22 Cells

Hepatoma is one of the five most common cancers in the world, which is also called "the king of all the cancers" because it greatly threatens human health and lives . Heat shock protein (HSP) exists generally in various kinds of body cells, whose characters are both generally irritability and highly conservatism. HSP70 family, the main kind of heat shock proteins, are highly expressed in many kinds of tumor, especially in malignant tumor. It participates in tumor cells apoptosis regulation and relates closely to tumor oncogene and anti-oncogene. Hedyotic diffusa is a Chinese herbal medicine, whose effects are heat-clearing and detoxicating and soften hard lumps and dispel nodes The result of previous research has revealed that the hepatoma-lines-H22 cells treated by hedyotic diffusa can raise its active immunity to the homeotransplantation tumor cells .The result of previous research has revealed that hedyotic diffusa can not only raise the expression of HSP₇₀ on the membrane of the hepatoma-lines-H22 cells in mouse's abdominal dropsy to some extent and kill tumor cells but also can raise the mouse's active immunity to the homeotransplantation tumor cells. The mechanism maybe it could induce more expression of HSP70 on the membrane of the hepatoma carcinoma cell and strengthen it's immunogenicity.Objective:The purpose of this research is to discover the mechanism of specific immunity to tumor induced by hedyotic diffusa from apoptosis aspect and to learn the anticancer mechanism of the heat-clearing and detoxicating herbs and nourishing qi to invigorate spleen herbs and the difference of them. It's our aim to provid clinic objective evidence for using herbs reasonable.Methods:First step, ascetic animal model and detection: Hedyotic diffusa (HD )and was chosen as the objects of the research, at the same time, Codonopsis pilosula (CP)was chosen as the control group which has the function of nourishing qi to invigorate the spleen. the experimental animals were tumorgenicitied by injecting the pre-cultured Hepatoma-lines-H22 cells.Second step, solid tumor animal model and detection: Each group ascetic tumor cells having been treated by Chinese medicine were divided into two teams. One was complexed with anti-HSP70 so as to block the expression of HSP₇₀, the other wasn't treated. All mice were divided into five groups: HD group(immunized with HD—treated H22 cells), blocked HD group(immunized with HD—treated H22 cells blocked by HSP₇₀ monoclonal antibody) , CP group (immunized with CP—treated H22 cells) . blocked CP group(immunized with CP—treated H22 cells blocked by HSP70 monoclonal antibody)and blank control group(immunized with RPMI - 1640). We injected the mice subcutaneously by using every group tumor cells twice every week. At the same time, one control group was injected subcutaneously 1640 basic culture fluid. One week after the second injection, all of the mice were vaccinated by injecting the pre-cultured Hepatoma-lines-H22 cells subcutaneously.Third step, The Pathological sections of the tumor,spleen and thymus were made. Then, the tissue pathological morphous was detected by light microscope .In the same time, the transplant tumor cell apoptosis and the expression of apoptosis related gene Bcl-2 and Bax were analyzed by ISEL and immunohistochemistry.Result:1.From the light microscope ,we found more serious necrosis HD group mice's tissue ,thickening cortex of thymus and spleen white pulp, pyknotic tissue and substantial macrophage. 2. Hedyotic diffusa can promote apoptosis obviously and there is not significant difference between HD group and blocked HD group. Compared with Codonopsis pilosula, Hedyotic diffusa can promote apoptosis more effectively although Codonopsis pilosula has the similar character with Hedyotic diffusa .3. Bcl-2 and Bax : Hedyotic diffusa can depress the expression of Bcl-2 and raise the expression of Bax. There is not obvious difference between HD group and blocked HD group. Codonopsis pilosula has the similar character and there is no obvious difference between CP group and blocked CP group . Compared with Codonopsis pilosula , Hedyotic diffusa has more significant effect on raising Bax expression but has no advantage on depressing Bcl-2 expression.Conclusion: The H22 liver cancer cells pretreated by HD can increase mice active immunity on the same kind of transplanted tumor. The mechanism may be related to the induction of the HSP7o expression in H22 liver cancer cells and the reinforcement of its immunogenicity. Hedyotic diffusa can not only raise the expression of oncogene Bax but also depress the expression of oncogene Bcl-2 and Bcl-2 / Bax value to some extent by inducing expression of HSP70.S0, it can promote tumor cells apoptosis and step down the development of liver caner. According to the result, there is this tendency. From the experimental data analysis, however , there is no significant difference between HD group and blocked HD group .The influencing factors may be those as follow: First, the sample size is not enough. Second., maybe the dosage of HSP₇₀ monoclonal antibody is not suitable. Third, maybe there are cell necrosis and apoptosis simultaneously. Fourth, there are so many genes relate to apoptosis that single or several gene can't explain the mechanism of liver cancer nosogenesis .The result also reveals that there is no significant difference between CP group and blocked CP group although Codonopsis pilosula can depress tumor to certain extent and Hedyotic diffusa has more obvious effect than Codonopsis pilosula. Associating with prophase experiments we further discover that there may be different anti-tumor mechanism between heat-clearing and detoxicating herbs and the herbs nourishing qi to invigorate spleen.