Methylene BlueTrial Research For Apoptosis Of Rat C6 Glioma

IntroductionMethylene Blue (MB) is thiazine dye chemical compound, and is the water dissolving quality pigment. It has some what nerve block effect. The study of SK -N - MC and U - 373 MG gloma cell discover that MB may directly restrain proliferation. It has the same effect to human lung cancer cell (LTEP -23).Tumor is relevent to abnormal apoptosis of cell. Cell apoptosis mostly depend on the activation of the cysteinyl asparate - specific protease ( caspase). Caspase - 3 is the most important key - protease in mamal apoptosis, it has not found the report about that MB induces apoptosis of C6 Glioma and affect the level of caspase -3 mRNA.We detect the cell apoptosis rate of C6 Glioma by flow cytometric; we detect the level of caspase - 3 mRNA by RT - PCR. It could confirm the resisted glioma action of MB and provide us with theory and the test foundation that MB could be the additional therapy to tumor.Material and Method1. MaterialThe cell line used in our study was bought from Institute of Cytobiology of Chinese Academy of Sciences, Shanghai, China.2. ReagentRPMI - 1640 and 10% cow serum were bought from American GIBCOBRL corporation;Pancreatic protease was bought from Americal Sigma Corporation.Caspase - 3 primer was bought from Huamei corporation.100bp DNA Ladder was bought from Huamei corporation.The RNA Collects the TRIzol Reagent that the regent is bought from the A-merica GIBCOBRL corporation. PCR Kit was bought from the Japan treasure living beings ( Dalian) corporation.The BcaBest Kit that the Japan treasure living beings (DaLian) corporation were fastend to the system is writed down in the inversion invert. 3. Method(1) The cells cycle analysis was determined by flow cytometric.(2) Assessing the expression of the caspase -3 mRNA by RT-PCR.Results1. We find that the typical apoptosis peak appears after the C6 gliomal cell affected for 24 hours by 0. ljxg/ml MB from flow cytometric.2. It is certified by RT - PCR that caspase -3 mRNA ascend along with the time and the concentration of MB, and there was statistically significant difference (p <0. 01).DiscussionMethylene blue (MB) , a known inhibator of guanylate cyclase, its appetency and interpenetration are different from the normal cell. It can make tumor cell blue - dyed but the normal epidermis can not be dyed. The test results from the killed tumor cells by MB suggest that it has apparent dependency on the time and concentration.Modern point of view is that tumor is relevent with cell apoptosis. We want to certified that MB can accelerate apoptosis of C6 glioma by flow cytometric.Cell apoptosis is the programmed cell death which is controned by many different genes. The cysteinyl asparate -specific protease (caspase) has the initial and decisive effect in the cell apoptosis. Caspase -3 is the most importand Key -protease in the caspase family. In normal instance, caspase -3 exists as unactive enzyme. Different proteases cut caspase -3 discretely, which induce the autivation of caspase - 3. Contrarily, the active caspase - 3 can also cut dof-ferent subance, which lead to the enlaryement of the reaction and induce cell death. In the other hard, it is reported that caspase - 3 has important effect in cell apotosis by destroyiny the cell anti - apoptosis factors ( eg: bcl - 2 ) , cell framework (eg: frame protein actin, fodrin, lamin).In this test, C6 glioma cell were affeced for 0, 6, 12, 24 hours by 0.ml MB and affected by 0, 0. 05, 0. 1, 0. 15 g/ml MB for 24 hours. Caspase mRNA ascends along with the time and concentration of MB and there was statistically significant difference among the teams ( p < 0. 01). It suggest that MB could induce the apoptosis of C6 gloma through the way of caspase - 3 and the expression of cospase - 3 mRNA concide with the time and concentration of MB.ConclusionsMB has the function of inducing apoptosis on C6 glioma. The regulation pathway of caspase - 3 may be a mechanism of MB - induced apoptosis on C6 glioma.