An Experimental Study On Anti-tumor Cell Immunity Of HSP70-PC Derived From Lung Carcinoma Of Heat Syndrome Of TCM

1 Research Objective:Syndrome is a crucial term uniquely applied in the area of TCM. (clinic curative effect). TCM proposes to make diagnosis first and then conduct treatment based on differentiation of syndromes. Therefore, the correct understanding of the concept and the essential of the Syndrome will cast influence upon clinic curative effect.During a long period, the attention has been focused upon the study of Syndrome. With the rapid development of modern science and technology, the understanding of the essential of Syndrome has been enhanced, which leads the research on Syndrome with TCM up to a new era.With the development of science and technology, besides the surgery, radiotherapy,chemotherapy, and incretion, the tumor with the biological therapy has emerged as a new too. The emergence of immunity RX and game, such as the cell gene, and initiative immunity has brought up a favorable turn for tumor.Heat shock protein 70 is a strongly immunity assistant.It adjust cell's vegetate,growth ,differentiation and doom. Nowadays, it is said that heat shock protein 70 takes part in the reaction process of tumor immunity. Heat shock protein 70-peptide complexes refined from the tumor issues have the specific immunogenicity of tumor, and can produce the specific anti-tumor immunity reaction. As a vaccine of tumor, heat shock protein 70-peptide complexes have a good prospect in clinical use.Researchers have already found HSP molecules derived from tumor including HSP70, HSP90, GP96 are peptide-binding proteins and they are associated with peptides inside of cells to generate heat shock protein-peptide complexes(HSP-PC), which can induce strong anti-tumor protection immunity. But it is unclear that if HSP70 derived from human lungcarcinoma tissue is complexed with T cell antigens and if it has anti-tumor immunity.Recently, some clinical trials on HSP-PC derived from human as tumor vaccine are being carried out at abroad. But the efficiency of HSP-PC immunotherapy is uncertain. There are great difference in the therapeutic effect among cancer patients which refines it widely applied. It has already been proved that the immune effect induced by HSP70 derived from tumor tissue and that from normal tissue is different because they chaperone different antigen peptides. Furthermore, We have already verified from early research that HSR, HSP expression are correlated with the state of different syndrome of the body and HSP70 expresses higher in the heat syndrome in animals with cancers.lt shows that HSP70 expression has the specificity of syndrome of TCM. So, we think that HSP70 purified from tumor with patients of different syndromes may complex with different antigen peptides in quality. We think that the difference in the therapeutic effect among cancer patients with same tumor in clinical application may be related with patients of different syndromes. Considering that heat stress is a primary factor of inducing HSP gene expression and heat shock response (HSR) is similar to heat syndrome of TCM, we carry out some research related HSP family with heat syndrome when we explore the mechanism of syndrome of TCM with biological technology from the level of molecule. So, on the basis of early research results, we purified HSP70-PC from tumor and distal tumor-free lung tissue obtained from patients with different syndrome. On the one hand, we studied whether HSP70-PC purified from human lung carcinoma is able to induce T cell proliferation and tumor cytotoxic reactivity in vitro. On the other hand, we wanted to know if there are different anti-tumor immunity with HSP70-PC derived from patients with different syndrome. It can not only conduce to illuminate the mechanism of tumor immune of HSP70-PC and entend the idea of tumor immune therapy, but also can enlighten the modern research of syndrome of TCM. It is of much significance to use HSP70-PC as tumor vaccine in patients more reasonably and improve the effect of tumor biotherapy.2 Research Methods and Results:Since the mechanism of the anti-tumorefficacy of HSP70-PC may be crrelated with the syndrome of TCM. this research will focus on the study of mechanism through which the anti-tumor efficacy of HSP70-PC may be crrelated with the syndrome of TCM..The research consists of four major parts, literature review, experiment study and study conclusion. Beginning with ancient literature and current reports, the theoretical research focuses on the pathologic mechanism and the treatment development of tumor in TCM and modern medicine. The experiment study includes six parts. In addition to regular pathologic studies, radiate immunity study and immunohistochemical study are performedto observe the effects of HSP70-PC on the level of TNF-a, IL-12 and the expression of gastric mucosal HSPs. The picture analyse of gastric mucosal HSPs (HSP70 and BCL-2) is performed too.The statistical method: All data are expressed in mean plus or minus SD ( X ±SD) . The inter group comparison of means is analyzed by ONE-WAY ANOVA. The comparison of the means between any two groups is conducted by SNK. The correlation of the material is shown by the coefficient of correlation, with Person correlation method. The difference of SD is tested by non-parameter U Test. Duun's method is used in the comparison of the mean of any two groups. All the analysis is performed in the Windows SPSS 11.0 version (0= 0.05).Frist, we chose patients suffering from non-small cell lung carcinoma according to the diagnostic criteria of the disease and the syndrome of TCM and differentiated heat syndrome and non-heat syndrome. Tumor and corresponding non-tumor lung samples were obtained from these patients at the time of open thoracotomy for lung carcinoma.second we purified total proteins from tumor and corresponding non-tumor lung tissue obtained from patients with heat syndrome and non-heat syndrome. The samples were applied to an ADP-Agarose affinity column. Purified protein preparations were resolved on 12% SDS-PAGE. After electrophoresis, the gel was coomassie stained and 70kD molecules were identified. Furthermore, 82kD and 43kD molecules were mixed in the samples. Then the proteins was further purified by DEAE ion exchange chromatography and eluted by a 0-100% 1M Nacl gradient. The proteins of the eluate of 10-20% 1M Nacl were characterized by SDS-PAGE and transferred onto PVDF membrane immunoblotted with anti-HSP70 antibody. It showed that 70kD molecules in these proteins of the eluate are HSP70 which would be quantitated by Bradford assay.Based on the required HSP70-PC, the research will examine how HSP70-PC affects DC to induce and cytokine in different syndromes. The cells showed branching-like and pseudopod-like under inverted light microscope. Significant stimulation was observed with HSP70-PC purified from lung cancer of different syndrome,compared with the control group(P<0.01)It is worthy of being noticed that we could see the trend of increasing in the heat syndrome group.we make sure HSP70-PC availably pulsing DC mature.lt manifests that DC with antigen could be applied into the treatment of tumor as vaccine, which provides a new way to remedy the malignancy.The Methods of Immunofluorescence,Tunel and FCM were adopted to analyze the anti-tumorefficacy of HSP70-PC may be crrelated with the syndrome of TCM. HSP70 and BC1-2 test of lung carcinoma cell of all groups , the positive signal is yellow pellet that located in the cytochylema. Tunel test of lung carcinoma cell of all groups The positivesignal is yellow pellet that located in the nuclei.Apoptosis cell showed typical new moon chromatin margination,condensed nuclei and lots of micronuclei.Significant stimulation was observed with HSP70-PC purified from lung cancer of different syndrome,compared with the control group(P<0.01)It is worthy of being noticed that we could see the trend of increasing in the heat syndrome group.3 Research Conclusion :The research results indicate that the anti-tumor efficacy of HSP70-PC may be crrelated with the syndrome of TCM. Our research suggest that HSP70-PC is valuable forfurther study and development.In conclusion,we obtained very pure HSP70 preparations through the method of chromatographies from human tissue, which is shown to be associated with antigenic peptides,that is HSP70-PC.It was indicated that HSP70-PC purified from tumor tissues of heat syndrome and non-heat syndrome patients chaperoned tumorous peptides which can induce CTL immunity.We can see that the immunological effect elicited by HSP70-PC purified from lung carcinoma may well be correlated with the heat syndrome.There was the trend of increasing in the heat syndrome group and it was expectd to enlarge samples to study.further.Our research resultd allows a subsequent study of anti-tumor immunity of HSP70-PC derived from lung carcinoma of different syndrome. It provides an essential material basis for researers to analyze the antigenic peptides. Furthermore,it also impiled that HSP70-PC derived from lung carcinoma could be a reliable source of tumor-specific antigenic for clinical application and permits an expeditious application of HSP70-PC to the immunotherapy of cancer.We can see that the immunological effect elicite by HSP70-PC purified from lung carcinoma may well be correlated with the heat syndrome .There was the trend of increase in the heat syndrome group and it was expected to enlarged sample to study.further.our researed results allows asubsequent study of anti-tumor immunity of HSP70-PC derived from lung tumor of different syndromes.lt provides an essential material basis for researchers to analyze the antigenic peptides. Furthermore,it also implied that HSP70-PC derived from lung carcinoma and permits an expeditious application of HSP70-PC to theimmunotherapy of cancer.Clinic research in future should focus on big sample size, multi centers, randomized, double blind, and control clinical trials with HSP70-PC, on the establishment of the evaluation system of treatment effect recognized by Chinese and western medicine, on conducting objective and reliable test of HSP70-PC, and finally on providing reliable clinical data for the further development of HSP70-PC.4 Points of creation:First,on the aspect of methodology,we purify HSP70-PC from tumorous tissues and corresponding distal tumor-free lung tissues and establish the method of HSP70-PC purified from human lung tissue.Second, the research on the anti-tumor efficacy of HSP70-PC was focused on the animal models and the kind of cancers being studied were lmited. It is unclear whether HSP70 purified from lung carcinoma tissue has anti-tumor immunity and the mechanism of HSP70 in tumor immune is unclear either.We observe the anti-tumor immunity of HSP70-PC obtained from cancers and the mechanism of HSP70-PC in tumor immune.Third.we first consider that the anti-tumor efficacy of HSP70-PC may be correlated with the syndrome of TCM. By observing the anti-tumor efficacy of HSP70-PC from patients of different syndrome of TCM, which will be an important exploration on the modern research of the syndrome of TCM.These findings will offer some theoretical and experimental evidences for HSP70-PC as canaer vaccine being applied to cancer patients preferably.