| Objective: In order to provide significant data for the mechanism of activated anti-tumor immunity after high intensity focused ultrasound (HIFU) treatment, this study aimed to observe the expression of heat shock protein (HSP) in treated mice bearing H22 hepatic carcinoma and to explore effect of HIFU on tumor antigen presenting. HSP70-peptide complex purified from HIFU-treated tumor was subcutaneously injected as a tumor vaccine for the purpose of immune activation, and specific anti-tumor immune activities were subsequently measured in the mice.Methods: This study consists of three parts as follows: ⑴ Twenty C57BL/6J mice bearing H22 hepatic carcinoma were randomized to HIFU group (n=10) and control group (n=10). The tumor specimens were harvested from either mice immediately after ultrasound exposure in HIFU group, or mice without any treatment in control group. Using HE staining and transmission electron microscopy, histological changes were observed, and SP immunohistochemical technique was performed to assess the expression of HSPs including HSP70 and GP96. Positive expression rates of HSPs in both groups were calculated using x2 test. ⑵Using DEAE52-cellulose and ConA-Sepharose column chromatography, HSP70 prepared from the HIFU-treated tumors was purified. SDS-PAGE and Western bolt were performed to identify. ⑶One hundred C57BL/6J normal mice were randomized to five groups: group A (n=20), in which 10μl saline was injected; group B (n=20), in which H22 tumor supernatant was injected; group C (n=20), in which HSP70-peptide complex prepared from HIFU-treated B16 tumor was inoculated; group D (n=20), in which H22 tumor supernatant from HIFU-treated tumor was injected; group E (n=20), in which HSP70-peptide complex prepared from HIFU-treated H22 tumor was inoculated. They were subcutaneously injected the introduced materials as inoculation on the first and eighth days. Twenty mice bearing H22 tumor were classified as group F after a successful HIFU ablation. Spleen lymphocytes were extracted from ten mice in each group on the 15th days, and MTT assay was performed to assess the cytotoxic activity of lymphocytes against H22 tumor cells. The remaining 10 mice in each group received tumor attack from a subcutaneous injection of 2×106 H22 cells, and then followed up for the evaluation of tumor growth rate, tumor volume, metastasis, survival curve and survival rate.Results: ⑴HIFU induced coagulative necrosis of H22 hepatic carcinoma, and cellular membrane was severely ruptured. The positive rates of HSP70 were 100% in HIFU group and 30% in control group respectively. A significant difference was observed between two groups (P<0.05). But there was no statistical difference in the GP96 expression between two groups. ⑵Using SDS-PAGE and Western blot assay the elution was identified as pure HSP70, and 50μg HSP70 was elicited from 0.7~1.0cm tumor. ⑶ CTL cytotoxicity rates against H22 tumor cells in group A, B, C, D, E and F were 15.9%, 30.7%, 16.2%, 37.5%, 65.3% and 62.7% respectively. The rates were significant higher in group E and group F than those in the remaining four groups (P<0.05). After receiving a subcutaneous injection of 2×106 H22 cells, no tumor growth was identified in group E and F. But during the first week, tumor growth was observed in 50% of mice in group A, 40% in group B, 60% in group C, and 50% in group D, respectively. Five-week survival rates were 40% in group A, 30% in group B, 20% in group C, 30% in group D, 100% in group E, and 100% in group F respectively. Conclusions: ⑴ Cellular membrane is severely destroyed by HIFU exposure, and an increased expression of HSP70 can be detected in the treated-tumor. ⑵ 337μg HSP70 can be obtained from 1g HIFU-treated tumor. This amount of HSP70 may provide tumor antigen enough to stimulate host anti-tumor immunity. ⑶ HSP70-peptide complex prepared from the HIFU-treated H22 tumor has an obvious biological activity. As a tumor vaccine, it can activate specific anti-tumor immune reaction in mice. T...
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