| With the aim to pursue a new dosage form for the traditional antitumour drug, mitomycin were prepared with nanometer fiber(PLCL, LA/CL 80:20) as a carrier. The goal is to examine its antitumour activities,so T24 was served as models, at the same time we will investigate the mechanism of anti-tumor activity of mitomycin further.Part1.The preparation of nanofiber as a carrier of mitomycinWe established analytical methods for mitomycin nano-fiber and evaluated its quality. The results of the test showed that the release rate of mitomycin nanofiber is about 10%.Part2.The degradation of mitomycin nano-fiber in vivo and in vitroThe aim of the test want to make sure whether the nano-fiber can be degraded and accepted by the host by using different methods in vivo and in vitro.In vivo the answers of the test displayed that nanofiber can be degraded, and it have not the inflammatory effect on the host;The results of the tset also told us that nano-fiber can be completely degraded in vitro.Part3.The safety evaluation of mitomycin nano-fiberUsing the methods of MTT and the test of micronucleus to investigate the toxicity of mitomycin nanofiber on the L929 and mouse. As shown in results, all dosages mitomycin nano-fiber do not have any toxicity on L929. Even in 8 mg mitomycin nanofiber (the release concentration:80μg/mL) the results of the test showed that it has not any toxicity, compared with the medium toxicity of 80μg/mL mitomycin; The test of micronucleus displayed that mitomycin nano-fiber can not induce the increasion of the red cell micronuclear rates in bone marrow.Part4.Effects of mitomycin nano-fiber on proliferation of human bladder cancer T24 cellsWe testd the effects of mitomycin nano-fiber on human bladder cancer T24 cells with different concentration of mitomycin nanofiber (2mg,4mg and 8mg) treatment for 12h,24h,36h,48h and 72h by the methods of MTT assay. By the methods of flow cytometry.The cell cycle arrest effect of mitomycin nano-fiber on human bladder cancer T24 cells was decided with the indicated concentration of mitomycin nano-fiber treatment for 48h. Cyclin D1 and CDK4 was determined by using the methods of Real time-PCR and western blotting analysis the cell cycle modulating protein WAF-1/p21. The answer displayed:the mitomycin nano-fiber treatment produced obvious cytotoxic effects on T24 cells in a dose-dependent (0-80μg/ml) and time-dependent manner; contrasted with the vehicle-treated controls, the mitomycin nano-fiber treatment caused an appreciable arrest of T24 cells in G1 phase of cell cycle; Using the method of Real time-PCR and immunoblot analysis the answer displayed that mitomycin nanofiber treatment upregulated WAF-1/p21 expression in a obvious dose-dependent manner. Also, The answer displayed that mitomycin nanofiber treatment of T24 cells leads to a dose-dependent decrease in protein expression for cyclin Dl and CDK4.Part5.mitomycin nano-fiber induced apostosis of human bladder cancer T24 cellsBy the methods of flow cytometry we assessed the effects of mitomycin nano-fiber on apoptosis of T24 cell We rationed the extent of apoptosis was by tsst of flow-cytometric analysis of mitomycin nanofiber-treated cells labeled with the method of PI and annexin V. We decided alterations in signaling events in the method of Western blot analysis. As displayed, treatment T24 cell with mitomycin nano-fiber can increased the number of early apoptotic cell in a dose-dependent manner; using the method of western blot, we discovered mitomycin nanofiber can upregulate the protein expression of Bax,caspase-3 and PARP,but it can reduse the protein expressionof p-ser473-Akt, p-ERK1/2Bcl-2,Bcl-xL,procaspase-3,procaspase-9 and Bax/Bcl-2 in a dose dependent manner.Preparation technology of mitomycin nano-fiber is feasible, and perfo rmances of the test are stable.mitomycin nano-fiber can remarkebly increase antitu mour activity, and degrade toxicity and side effect of mitomycin. The research established science foundation for clinic application of nano-fiber in future.
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