Malignant Melanoma, Hyperthermia, Heat Shock Protein 70 Expression And Immune-related Role

Research background:Tumor local hyperthermia is one kind of tumor treatments which increases the temperature of tumor location in order to kill the tumor cells. As one kind of physical treatments, hyperthermia has little side effects compared with the chemotheraphy and radiotherapy and may be used to treat tumors time after time and is not important to think about side effects accumulating in the body as the chemotheraphy and radiotherapy, so it maybe have applied future in tumor therapies. Tumor local hyperthermia is able to kill tumor cells and is able to activate the body's immune system to enhance the immunological ability to tumors, however the mechanism that hyperthermia enhance the immunological ability is not clarified. Hyperthermia is able to activate tumor cells to express heat shock proteins (HSPs) which may form complexes with tumor antigens, this kind of complexes is considered to be related to tumor immunology. On the other hand, HSPs are also considered to be one kind of protect factors which express in tumor cells after hyperthermia and may enhance the body's resistance ability to harmful factors. So clarifying the action of HSPs in tumor cells after hyperthermia is very important and may offer one new method to treat tumors.Research objects:1. To observe the killing function of different heat dosages to mouse melanoma B16 cells in vitro and to screen the suitable heat dosages killing B16 cells.2. To observe the effects of different heat dosages to the expression of HSP70 mouse melanoma B16 cells in vitro and to screen the suitable heat dosages increasing the expression of HSP70 mouse melanoma B16 cells.3. To heat transplanted tumors in C57 mice by the suitable heat dosages and to investigate the affects of different heat dosages to the survival times of tumor-bearing mice.4. To heat transplanted tumors in C57 mice by the suitable heat dosages and to investigate the affects of different heat dosages to the IL-2 concentration of tumor-bearing mice and the HSP70 expression of transplanted tumors. Research methods:1. There are 18 different dosage experiment groups and one 37℃control group and B16 cells were cultured by DMEM containing 10% FBS which were heated by water, the cells'shape were observed by the inverted phase contrast microscope and the survival rates were detected by the methods of MTT and Trypan blue.2. There 22 different dosage experiment groups and one 37℃control group and B16 cells were cultured by DMEM containing 10% FBS which were heated by water, the HSP70 expression of B16 cells were detected by the methods of immunohistochemistry.3. The transplanted tumors in tumor bearing mice were heated by the electro-thermal light focus heating lamp using the suitable heating dosages screened on the basis of experimental results in vitro, the C57 mice survival times were observed, the IL-2 concentrations of tumor bearing mice in peripheral blood were detected by the method of radioimmunoassay and the HSP70 expressions of transplanted tumors of tumor bearing mice were detected by the method of immunohistochemistry.Research results:1. The results of MTT and Trypan blue showed that survival rates of B16 cells decreased when the heating temperature enhanced and the survival rates of B16 cells decreased when the heating time prolonged on the same heating temperature except the temperature of 40℃. In the light of the survival rate of 37℃being 100%, the survival rates of 40℃were not related to time and it decreased first, then increased and decreased at last. The survival rate of 43℃decreased when the heating time prolonged, the survival rate of 47.5℃*30min group was lowed than the group of 47.5℃*15min and the groups of 45℃*120min and 47.5℃*30min were 17.1% and 18.9%.2. The results of immunohistochemistry showed that the HSP70 expression increased obviously and its'expressions were related to heating temperatures and heating times. On the same heating time, the HSP70 expression quantity increased when the heating temperature enhanced except that tumor cells were too damaged by hyperthermia to express the proteins. On the same heating temperature, the HSP70 expression quantity increased when the heating time prolonged except that tumor cells were too damaged by hyperthermia to express the proteins. 3. Compared with the control group, the survival time of tumor bearing mice prolonged obviously after hyperthermia, the group of 53℃～55℃which was 34.8±6.94 days prolonged most obviously; the results of RIA showed that the IL-2 concentration of peripheral blood in tumor bearing mice increased obviously, but was not related to heating dosages; the results of immunohistochemistry showed that the HSP70 expression increased obviously.Conclusions:1. The survival rates of B16 cells decreased when the heating dosage increased, but the survival rates of 40℃were not related to time and it decreased first, then increased and decreased at last.2. Hyperthermia may activated the HSP70 expression of B16 cells in vitro and the expression quantity increased when the heating dosages increased, hyperthermia may also induced the HSP70 expression of transplanted tumors of tumor bearing mice.3. Hyperthermia was able to prolong survival times of tumor bearing mice and may enhance the IL-2 concentrations of peripheral blood of tumor bearing mice.