In Vitro Study On Human Meningioma Proliferation Suppressed And Apoptosis Induced By MFH Combination With HSR1 Antisense Oligodeoxy-nucleotide

Objective: About 90% of human meningioma belongs to WHO I grade, which is benign tumor and grow slowly. Effects of Radiotherapy and Chemotherapy are not effective and immunotherapy is not identified. At present, the popular therapy of human meningioma is neurosurgical operation. However, the recurrence rate after operation just reaches 7% to 32%.Although effect of tumor hyperthermia has been proved in many human tumor therapy, systematic study on hyperthermia of human meningioma is not clearly reported so far. The obtain of thermotolerance of tumor is related with high expression of variety of HSPs in tumor cells. Under heat stimulation, HSF1 protein activation is the key in transcription of HSPs, while HSR1 is the essential factor for HSF1 trimerization. Transfection of HSR1 ASODN suppressed HSF1 protein activation and expression of various HSPs, it has the possibility to change heat sensitivity of human meningioma.With the combination of HSR1 ODN transfection, two other techniques of warm water hyperthermia and ferric oxide MFH are also applied in the study. The aim of the research is to find the possibility and superiority of MFH in treatment of human meningioma by means of studying heat shock response, cell proliferation and cell apoptosis.Methods: Establishing the short-term primary cell culture method of human meningioma first, and then detects the expressions of HSF1 protein, HSP27, HSP70, HSP90 protein by immunohistochemistry (IHC) respectively.To detect the expression of HSF1, HSP27, HSP70 and HSP90 protein under different heat temperature and time by IHC or western blot,and the inhibition rate of tumor cell proliferation by MTT method.To transfect HSR1 ASODN and HSR1 SODN in human meningioma by liposome and FuGENE HD transfection system.The experiments of warm heat hyperthermia and ferric oxide magnetic fluid hyperthermia are conducted respectively right after cell transfection, so as to detect proliferation inhibition rate, apoptosis rate and the change of HSF1,HSP27,HSP70 and HSP90 protein by IHC,western blot,MTT method, various apoptosis detection methods, AO staining method, TUNNEL method and FCM double staining method.Result: Successfully established the short-term primary cell culture of 15 human meningioma cases, including the condition of short-term cell culture, and the methods of passage, Cryopreservation, cell revitalization, identification of tumor cell. It is also found that there exists expression of HSF1 protein in human meningioma, and the constructive expression in HSP27, HSP70 and HSP90 is low. The vitro experiment has proved that the expression of HSF1 protein in human meningioma did not increased, but HSF1 protein trimerization was activated under warm heat hyperthermia(42-44℃, 30-60min) or ferric oxide magnetic fluid hyperthermia(42-43℃,30min), which induced permanent and high expression of HSP27,HSP70 and HSP90 in 24 hours, while tumor cell proliferation was not effected by hyperthermia, which indicates that the heat sensitivity of human meningioma is low under this condition. When heated for 30min under 44℃, the cell proliferation inhibition rate of ferric oxide magnetic fluid in human meningioma was 12.62±0.78%. The inhibitory action could not be increased by this kind of hyperthermia in 3 times. The main channel for the proliferation inhibition was not in the form of increased cell apoptosis, but the cellular necrosis.Tumor cell proliferation and the expression of HSF1 protein and HSPs in human meningioma could not be affected by applying the method of FuGENE HD transfection in human meningioma, but heat sensitivity of tumor cell was changed. In addition, the effect of FuGENE HD transfection was higher in transfection and the cytotoxicity was much lower.In vitro test of hyperthermia after HSR1 ASODN transfection, ferric oxide MFH (42.5℃,30min) and warm water hyperthermia (43℃,30min) could inhibit activity of HSF1 protein, induce expression of HSP27,HSP70,HSP90, inhibit the ability of cell proliferation in human meningioma, and lead to cell apoptosis.Conclusion: Low heat sensitivity of human meningioma is mainly related to permanent and high expression of induced HSP27,HSP70 and HSP90 under heat stimulation.HSR1 ASODN transfection inhibits trimerization activation of HSF1 protein, and suppresses induced expression of HSPs, which leads to the change of heat sensitivity of human meningioma.In vitro test, ferric oxide MFH(42.5℃,30min) with the combination of HSR1 ASODN transfection could suppresse cell proliferation obviously, and induces cell apoptosis through inhabitation activity of HSF1 protein and induced expression of HSP27,HSP70,HSP90.MFH combined with HSR1 antisense transfection may become a brand-new treatment of human meningioma. Innovation:1.Low heat sensitivity of human meningioma is closely related to high expression of induced HSP27, HSP70 and HSP90 under heat stimulation. No reports concerned have been released so far both at home and abroad.2. Under heat stimulation, HSR1 ASODN transfection inhibits trimerization activation of HSF1 protein, and suppresses induced expression of HSPs, which leads to the change of heat sensitivity of human meningioma. No reports concerned have been released so far both at home and abroad.3. Ferric oxide magnetic fluid (44℃,30min) could inhibit the ability of cell proliferation in human meningioma. No reports concerned have been released so far both at home and abroad.4. In vitro test, ferric oxide MFH (42.5℃,30min) with the combination of HSR1 ASODN transfection could suppresse cell proliferation obviously, and induces cell apoptosis through inhibitation activity of HSF1 protein and induced expression of HSP27,HSP70,HSP90. No reports concerned have been released so far both at home and abroad.