| BackgroundBladder cancer is one of the most common malignant tumors in the clinical work of urinary surgery in our country.In most countries around the world, bladder urothelial carcinoma (BUC) accounts for over90%of bladder cancers. At present, bladder cancers treatment should be based on surgery and assisted by chemotherapy and radiation therapy.However, due to the further study on the pathogenesis of tumors, the newly emerging molecule targeting therapy in recent years enables us to expect non-surgical treatment of tumors. Belonging to the papaya protein family, cathepsin B (CB) is the representative of cysteine protease and is the most important sulfhydryl protease in lysosome. Recent studies at home and abroad have found that CB is highly expressed in a variety of tumor tissues, and is associated with the malignant phenotypes of tumors such as invasion and metastasis. But no one has yet studied the expression and functions of CB in T24cells of human bladder urothelial carcinoma in vitro.ObjectivesTo discuss the expression of cathepsin B in T24cells of human bladder urothelial carcinoma; and to reduce the expression and activity of cathepsin B by specific inhibitors CA-074so as to observe its influence on the metastasis and invasion of T24cells of human bladder urothelial carcinoma.MethodsA. Preparation: purchase and subculture the T24cells of human bladder urothelial carcinoma for experiment. Dissolve CA-074with dimethyl sulfoxide (DMSO) into appropriate concentration.B. Grouping: the blank control group (normally cultured T24cells), four experimental groups where T24cells are cultured in different final concentrations of CA-074culture medium with concentrations of0.5umol/L,1umol/L,5umol/L and10umol/L respectively; and the experimental control group where T24cells are cultured in a medium containing solvent DMSO. Test the changes of related indicators after culturing for24h,48h and72h.C. Testing indicators and approachesa) Test the mRNA expression of cathepsin B inT24cells of human bladder urothelial carcinoma cells by reverse transcription polymerase chain reaction(RT-PCR).b) Test the protein activity of cathepsin B in T24cells of human bladder urothelial carcinoma cells by cathepsin B cracking substrate experiment.c) Test the protein expression level of cathepsin B in T24cells of human bladder urothelial carcinoma cells by immunocytochemical method.d) Test the protein expression level of cathepsin B in T24cells of human bladder urothelial carcinoma cells by Western blot.e) Test the metastasis and invasion of T24cells of human bladder urothelial carcinoma cells by transwell chamber.D. Statistical analysis: All the experiment data are processed by SPSS17.0software, the differences between groups are dealt with through single factor analysis of variance. the test standard is a=0.05.ResultsA. There were no statistically significant differences in the mRNA expression of cathepsin B between groups of T24cells of human bladder urothelial carcinoma (P>0.05).B. Both of the protein expression level and the activity of cathepsin B in T24cells of human bladder urothelial carcinoma decreased with the increase of the concentration of CA-074and the extension of culturing time. It had statistical significance comparing with the blank control group and the experimental control group (P<0.05). C. The number of transmembrane cells in T24cells of human bladder urothelial carcinoma was statistically significantly reduced with the increase of concentration of CA-074and the extension of culturing time (P<0.05).D. Tests of the blank control group and experimental control group were not statistically significant (P>0.05).ConclusionsA. CA-074, the specific inhibitors of cathepsin B, could significantly inhibit the metastasis and invasion of T24cells of human bladder urothelial carcinoma.B. Cathepsin B is involved and plays an important role in the metastasis and invasion of T24cells of human bladder urothelial carcinoma. |
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