HSP70 In Tumor Mediated T Cell Immunity Enhancing Effect After High Intensity Focused Ultrasound Ablation Of SD Rat Original Pancreatic Cancer

BackgroundHigh-intensity focused ultrasound（high-intensity focused ultrasound, HIFU） gets people’s attention as a new local tumor ablation therapy in recent years. HIFU uses good penetration and directional of ultrasound though tissues and focuses energy on the target tumor region so that high temperature makes target region to be coagulation necrosis of instantly, tumor cells are destroyed at the same time without damaging surrounding tissues and organs for tumor therapy.HSP70 is the most important sub-family of heat shock protein（heat shock protein, HSP） family with the ability of molecular chaperone function and involving in antigen synthesis, assembly and presentation as well as the formation of HSP- peptide complexes with antigenic peptides to activate the body specific immune response generated. HSP70 can participate in a variety of immunological processes in vivo: HSP70, as exogenous antigens the immune system, can bind MHC-II receptor molecules on the cell surface directly to activate CD₄⁺ T lymphocytes, then enhance the body immune effector, however, without process of professional APC. After HIFU ablation, local tumor cells has a high expression of HSP result from heat stress, at the same time accompanied by the death of the tumor cells released outside the cell. Whether HSP70 has biological activity after HIFU, nor can present antigen to activate T cell immunity is intend to be further explored.Clinical study found that VEGF and CA125 and other tumor antigen abundantly expressed in cancer tissues; the number of tumor antigen-presenting cells（Antigen presenting cell, APC） increased significantly, the main organization histocompatibility complex MHC-II and co-stimulatory molecules up-regulated after HIFU treatment of breast cancer primary tumor. Further study found that the death of tumor cell in the tumor tissue target significantly increased, the numbers of CD₄⁺T cells, CD₈⁺T and NK cells increased, T lymphocyte function is activated to enhance immune effector after HIFU treatment of breast cancer. These results show that HIFU treatment target tumor after tumor necrotic tissue tumor antigen may activate the body’s way of specific antigen presentation by some mechanism so that specific anti-tumor immune function is enhanced.Our preliminary study found that after HIFU alone or in combination with chemotherapy in pancreatic cancer, pancreatic tumor growth was inhibited or reduced glucose metabolism in a good outcome, the body’s antitumor effect was significantly enhanced. On the basis of above, pancreatic cancer model of SD rats was established, and was chose by high-frequency 12 m Hz B-screened; after HIFU ablation of pancreatic SD rats, local tissues and immune response of the body HSP70 changes was observed, the impact of enhanced immune effector mechanisms HSP70 antigen presentation for HIFU treatment of pancreatic cancer was explored so as to provide a valid scientific basis for clinical application. ObjectBy compared with B-high frequency ultrasound imaging characteristics and common B-ultrasound analysis on SD rat pancreatic combined with histopathological diagnosis, established a diagnostic methods with high-frequency B-imaging of SD rat pancreatic cancer; By observing the change of HSP70, MHC-II and ZAP70 expression level in local tissue after HIFU to explore the effect of HSP70 on local tissue antigen presentation; By observing the change of CD₃+/CD₄⁺T cells, IL-2 and IFN-γ in peripheral blood after HIFU ablation of rat pancreatic SD to explore the effect of HSP70 on differentiation and proliferation of Th cell and the changes of cell factor. Methods1.SD rat orthotopic pancreatic cancer model was made with DMBA-induced, the control group of normal, healthy rats, respectively using high frequency ultrasound 12 m Hz and 5.0 m Hz common ultrasonic cross-sectional scan on SD rats of different position, the contrast analysis of SD rat pancreatic lesions of high frequency ultrasound imaging characteristics and characteristics of ordinary ultrasound the pancreas imaging and joint rat pancreatic cancer histopathological judgment pancreatic tumor characteristics, to establish the rat pancreatic cancer high frequency ultrasound imaging diagnostic methods.2. The use of high frequency ultrasound imaging of pancreatic cancer screening 90 Model SD rats, rats were diagnosed with pancreatic cancer were randomly divided into experimental HIFU ablation group and model control group, 40 healthy rats as normal control group. The high-intensity focused ultrasound（5w, acoustic intensity of 2500W/cm2） applied to the experimental rats, using ablation point the way, each ablation time of 5s, complete ablation; model group and the normal group without ablation process, other processes are identical. Respectively after ablation 2h, 4h, 8h and 12 h from rat pancreatic tissue by immunohistochemistry and Western blot method step HSP70, MHC-II and ZAP70 in local tissue expression; respectively one day after ablation 4 days, 1 week and 2 weeks using the method of heart blood from rat peripheral blood, ELISA peripheral blood Th cells secrete cytokines IL-2 and IFN-γ; were isolated by density gradient centrifugation of peripheral blood lymphocytes, flow cytometric analysis of peripheral blood CD₃+/CD₄⁺T cell ratio subsets. Results1. The rats compared with the normal group, SD rat pancreatic tumor showing hypoechoic, more regular in shape 12 m Hz high-frequency ultrasound scan images, mostly spherical or ellipsoidal, obvious tumor level with the surrounding organs boundaries more obvious; SD rat pancreatic cancer in 5.0m Hz under normal ultrasound scan, failed to find a significant tumor hypoechoic area, the image is more blurred; 12 m Hz ultrasound compared with 5.0m Hz ultrasound, pancreatic tumor frequency B-scan images more for clarity, the tumor is more obvious hypoechoic area; histopathology of pancreatic tumor tissue SD rats show a large number of catheter associated with cell proliferation and invasive growth of pancreatic ductal carcinoma showing pathological features.2.HIFU ablation rat pancreatic SD, peripheral blood IL-2, IFN-γ and CD₄⁺ T cell levels gradually increased, the level of four days ablation of IL-2 group（22.6 ± 1.312） and control group（17.37 ± 0.872） was significantly higher compared with a significant difference（p <0.05）, the level of IFN-γ in one week ablation group（145.37 ± 6.304） was significantly higher compared with the control group（126.87 ± 5.021） phase than having a significant difference（p <0.05）; level of the CD₃+/CD₄⁺ T cells after ablation of one week（45.65 ± 4.384） was significantly higher compared with the control group（28.23 ± 2.991） compared with the significant difference（p < 0.05）; IL-2, IFN-γ and CD₃+/CD₄⁺ T cell levels were significantly increased in 7 to 10 days after HIFU ablation, a slight decrease at 2 weeks but still maintained a high level.3.HIFU ablation rat pancreatic SD, the local tissue HSP70 expression of MHC-II and ZAP70 increased significantly. Compared with the model group（6.570 ± 0.324） and normal control group（6.362 ± 0.650）, HIFU ablation experimental group HSP70 significantly increased ablation 4h expression（32.570 ± 1.173） in having a significant difference（p <0.05）, compared with the control group（6.543 ± 0.547）, model group（7.840 ± 0.547） pancreatic tissue is also a small amount of HSP70 expression, but no significant difference（p> 0.05）; HIFU ablation 4h group of local organizations MHC-II of expression（25.76 ± 1.579） was significantly higher than the model control group（18.760 ± 1.367） and normal group（20.768 ± 1.324）（p <0.05）, the expression of MHC-II normal group was higher than the model control group, but only in 2h and 8h group has significant difference（p <0.05）, after HIFU ablation of MHC-II expression gradually increased, the highest in 8h and then stabilized; the control of the expression of ZAP70 4h group（3.993 ± 0.302） was significantly lower than HIFU ablation experimental group（16.80 ± 0.472） and normal group（12.675 ± 0.856）（p <0.05）, as compared with the control group, HIFU ablation group ZAP70 expression experiments in 2h（11.33 ± 0.529） and 4h（（16.80 ± 0.472）） increased significantly, with a significant difference（p <0.05）, while at 8h and 12 h, no significant difference（p <0.05） groups ZAP70 expression. Conclusions1.DMBA induced rat pancreatic ductal carcinoma formation; image SD rat pancreatic 5.0m Hz normal ultrasound scan is so vague that can’t distinguish pancreatic cancer, 12 m Hz high-frequency ultrasound scanning SD orthotopic pancreatic tumors ultrasound images showed a hypoechoic tumor boundaries more obvious images clearer, high-frequency B-scan 12 m Hz can be used as diagnostic imaging method SD rat pancreatic cancer.2. SD rat pancreatic with HIFU ablation, HSP70 target tumor tissue and involved in antigen presentation of MHC-II expression in a large number in a short time, HSP70 mediated antigen presentation role strengthened, leading to local tissue Th cells is activated, the expression of specific proteins ZAP70 local T cell immune enhancement; HSP70-mediated immune effector T cells persist, HSP70 can stimulate peripheral blood Th cells into CD₄⁺T cells, T lymphocytes secrete IL-2 and elevated levels of IFN-γ, T cells enhance the body’s immune response. In summary, HIFU ablation of pancreatic cancer, local tissue expression of HSP70 by strengthening the role of antigen-presenting cells to stimulate the proliferation and differentiation of T, enhance the body’s immune effector T cells.