Methylene Blue Enhances BCNU Therapy Of Rat C6 Glioma

Methylene Blue (MB) is thiazine dye chemical compound,and is the water dissolving quality pigment. Hirschberg and Longatti PL utilize MB as the colour mark of brain tumour, in order to make certain of tumour border, cut down the operation time and decrease the injury to the brain of operation.The study of SK - N - MC and U - 373 MG glioma cell discover that MB may directly restrain proliferation. MB did not significantly alter cellular levels of cGMP in both cells. 8 - Br cGMP, a membrane - permeable analogue of cGMP, did not decrease MB - induced cytotoxicity, indicating that cGMP may not be a major target of the cytotoxic action of MB. However, hydroxyl radical scavengers or intracellular Ca2 + modulators effectively blocked the MB - induced cytotoxicity. These results suggest that hydroxyl radical and intracellular Ca2 + may have an important involvement in the cytotoxic action of MB. These results further suggest that the treatment with MB may be useful for the therapeutic applications of human brain tumors.Apoptosis is a form of cell death characterized by active cellular suicide during T -cell clonal deletion, embryogenesis, and DNA damage. Apoptotic cell death is often associated with distinctive characteristics, such as nuclear fragmentation, cytoplasmic blebbing, and internucleosomal fragmentation of DNA. The Bcl - 2 family plays a central role in the control of apoptosis. The family includes a number of proteins which have amino acid sequence homology, including anti - apoptotic members such as Bcl - 2 and Bcl - xL, as well as pro - apoptotic members including Bax and Bad. Overpression of Bax has been shown to accelerate the cell death. Conversely, Overpression of antiapoptotic proteins such as Bcl - 2 represses the death function of Bax. Thus, the ratio of Bcl - 2 to Bax appears to be a critical determinant of a cell's threshold for under-going apoptosis.In the study, to adopt Wistar rat C6 cell is worthwhile to research the work of the MB. In order to investigate whether methylene blue (MB) could restrain proliferation and induce apoptosis in C6 cell cultures and MB enhances BCNU therapy of experimental rat C6 glioma. It could confirm the resisted glioma action of MB and provide us with theory and the test foundation that the addition of MB to BCNU therapy increases the antineoplastic effect of the BCNU therapy.Material and method1 Material: MB and modified tertrozalium salt ( MTT) were from Sigma (Buchs, Switzerland). The Wistar rat were from China Medical University experimental animal ministry. Monoclonal anti - Bax Bcl - xl antibody were obtained from Boster Co. (Wu Han, CHINA). The RNA collects the TRIzol Reagent that the reagent is the American GIBCOBRL corporation , PCR Kit act as Shanghai student workman's produce ,The BcaBest Kit that the Japan treasure living beings ( Dalian ) corporatioies were fastend to the system is writeed down in the inversion invert.2 Cell line: The cell line C6 was used in our study (Institute of Cytobiology of Chinese Academy of Sciences, Shanghai, China). Cells were grown in culture flasks containing RPMI 1640 culture medium supplemented with 10% FCS at 37℃ in a humidified atmosphere with 5% CO2.3 Method:(1) MTT assay 1 X 105 cells/well in a 96 - well plate after 24 hours incubation were treated with increasing concentrations of MB (0. 04 ~ 0. 20 |xg. ml-1) for 24 hours. 10 ul of 5g. L-1 of MTT was added to the cells in every well and incubated for 4 hours at 37 ℃,. Culture media were discarded followed by addition of 0. 2 ml of DMSO and vibration for 10 minutes. The absorbance ( OD) was measured at 570 nm using a microplate reader. The cell growth inhibitory rate was calculated as follows: ( OD of control group - OD of experimental group )/OD of control group x 100 %.(2) Immune histochemistry staining To observe the expression of PCNAprotein and analysis the significance.(3) Transmission electron microscopy The C6 cells treated with 0. 1 ug/ml MB for 6 12 24 hours were trypsinized and harvested a...