| Objective: The nude mice are one of the kind animals that have no hairand no thymus. The function of thymus is to produce T cells. The reason ofnude mice for lack of T cells is the result of one gene mutation, which wecalled nu, Belongs to the recessive genetic, its phenotype can appear onlywhen the nu/nu comBined. The immune function of T cell can assist the Bodyto inhiBit and kill exogenous suBstances that have invaded into the Body. Theimmune function of the nude mice is very bad, Because no thymus to produceT cells. When exogenous substances invaded into the Body, they can’t betimely suppressed or eliminated. Based on the characteristics, the nude miceBecome one of the ideal kind of transplantation tumor model. Usually, thenude mice are often used to study the treatment of malignant tumor. Duringthe experiment, according to the characteristics of the nude mice, the nudemice transplantation tumor model has Become the ideal model, By which theSulforaphane can inhiBit the Bcl-2family in human salivary gland adenoidcystic carcinoma transplanted tumor in this experiment.Salivary gland adenoid cystic carcinoma is a kind of oral andmaxillofacial tumors which are more malignant. Its clinical characteristics isthat the metastatic rate is very high, and the growth is surrounding the nerve,etc. Cytological characteristics:it is a kind of basal cell tumor, composed bythe epithelial cells and myoepithelial cells which arranged in a tubular, screen,and different structures such as solid nest.According to our preliminary experiment, Sulforaphane (SFN) can inhibitthe cell proliferation and induce cell apoptosis of salivary adenoid cysticcarcinoma cell line ACC-M in vitro. The apoptosis process of salivary glandadenoid cystic carcinoma induced By SFN is mediated by Caspases. In addition, during the vitro process, the Bcl-2family play an important role:Sulforaphane could increase the expression of Bax and Bak, which we callapoptosis-promoting protein, and can inhiBit the expression of the Bcl-2andBcl-xl, which we call apoptosis-inhiBiting protein. Our primary experimentshowed that sulforaphane could inhiBit the incremental growth of adenoidcystic carcinoma ACC-M nude mice xenografts in vivo, and induce theadenoid cystic carcinoma ACC-M nude mice xenografts apoptosis. In thatprocess, whether the Bcl-2protein family play the same important role as invitro experiments, to our Best knowledge, has not Been reported yet. In thisexperiment, we use the tumor model, which we inject the ACC-M into thenude mice to construct, to determine whether the expression of Bcl-2family invivo experiment is the same as in the vitro or not By using the method ofimmunohistochemical staining.Methods:1Cell culture: The frozen human salivary gland adenoid cystic cancercells(ACC-M) were recovered and cultured in the constant temperatureincuBator under37℃,5%CO2. When the cell density reaches a certainconcentration, with0.25%of pancreatic enzyme digestion Batches, weharvested the cells and adjust density of cell suspension to2×107/ml.2Set up xenografts model: cell suspension containing106cells wasinjected suBcutaneously on Both left and right flank of each mouse. Micewere randomized into two groups of5mice/group (2tumors/mouse).Experimental animals were treated orally with SFN (6.0mmol SFN in0.1mlPBS)3times/week (Monday, Wednesday and Friday) Beginning the day oftumor cell implantation. Control mice received an equal volume of the vehicle.3Harvest the tumors: Three weeks later, the nude mice were euthanizedwith the method of Broken neck, dissect the tumors with the surgical methodand remove the necrotic tissue and the small Blood vessels on the surface ofthe tumors. The disscted tumor tissues were emBedded in paraffin.4Immunohistochemical staining： For immunohistochemistry, theparaffin Blocks were sectioned toXμM and were stained with the four kind apoptosis factors from Bcl-2protein family, including Bcl-2,Bax,Bcl-xl,Bak.Then the biopsies were observed and scored under the microscope.5Statistical analysis: Rank test for grade data were used to statisticalanalysis and draw the conclusions.Results: Compared with the control group, the experession of anti-apoptotic protein Bcl-2and Bcl-xl decreased，there are statistically significant(P <0.05). the experession of the pro-apoptotic proteins Bax and Bakincreased, but there are not statistically significant (P>0.05).Conclusion: The Bcl-2family play an important role during apoptosisprocess of salivary gland adenoid cystic carcinoma cell line ACC-M inducedby sulforaphane in vivo. |
PDF Full Text Request