The Study Of Apoptosis Induced By TNF-α And IL-2 In Salivary Pleomorphic Adenoma

Objective: With the conception of tumor turning from excessive cell growth to no cell death,apoptosis of tumor cells has been new direction and hot spot of tumor research．Salivary pleomorphic adenoma is a kind of boundary tumor with first morbidity in the salivary gland．It is obviously latent malignant,and easy to implant and to relapse,relapse many times of tumor may lead to carcinomatous change．In this study, inducing apoptosis by TNF-α and IL-2 in salivary pleomorphic adenoma was done,and the mechanism of apoptosis was discussed．The new idea and experimental data will be provided to the clinical therapy of salivary pleomorphic adenoma．Materials and Methods: 1．Material:(1) Cell line:The cells of 12th passage of human salivary pleomorphic adenoma were cultured. (2) Medicine:Recombined human tumor necrosis factor (rhTNF) and interleukin-2 (IL-2)．2．Methods:The cells of salivary pleomorphic adenoma were cultured in RPMI1640 medium (supplemented with 10% fetal bovine serum) at 37℃．The study included two parts:At first,these cells were divided into four experimental groups:group A (treated with 1500U/ml rhTNF-α for24h)；group B (treated with 1500U/ml rhTNF-αfor 48h)；group C (treated with 1500U/ml rhTNF-α for 72h) and group D (treated with 3000U/ml rhTNF-α for 72h) ．Simultaneous,normal saline was added to every corresponding control group．Secondly,these cells were divided into two experimental groups:group E (treated with 1500U/ml IL-2 for 72h),normal saline was added to the corresponding control group as above-mentioned；group F1 (treated with 1500U/ml rhTNF-α and 500U/ml IL-2 for 72h),group F2 (treated with 1500U/ml rhTNF-α and 1500U/ml IL-2 for 72h),group F3 (treated with 1500U/ml rhTNF-α and 3000U/ml IL-2 for 72h),the corresponding control group (treated with 1500U/ml rhTNF-α for 72h)．The cells of salivary pleomorphic adenoma in each group were counted and the percentage of apoptotic cells was detected by the flow cytometer．The cells of salivary pleomorphic adenoma treated with 1500U/ml rhTNF-α and 1500U/ml IL-2 were observed under light microscope (LM)．Results:1. The total cell number of salivary pleomorphic adenoma in group A is 130.33±4.73×104 ,its control group is 154.67±4.04×104 (P<0.01)；group B is 167.33± 5.69×104 ,its control group is 232.67±4.04×104 (P<0.01)；group C is 262.33±15.50×104 ,its control group is 391.67±12.10×104 (P<0.01)；group D is 184.33±6.66×104 ,its control group is 391.67±12.10×104 (P<0.01)；group E is 386.67±12.86×104 ,its control groupis 391.67±12.10×104 (P>0.05)；groups F:the control group is 262.33±15.50×104,while group F1 is 228.33±3.21×104 (P<0.01)；group F2 is 193±5.57×104 (P<0.01)；group F3 is 193±8.19×104 (P<0.01) respectively．2．The result of flow cytometer showed that the typical 'apoptotic peak' was formed in A,B,C experimental groups．The percentage of apoptotic cells in each group: group A is 10.30±0.46%,its control group is 1.77±1.01% (P<0.01)；group B is 12.37±0.80%,its control group is 3.20±0.40% (P<0.01)；group C is 16.57±0.61%,its control group is 5.20±0.50% (P<0.01)．There was significant difference of the percentage of apoptotic cells among A,B and C experimental groups (P<0.01)．The percentage of apoptotic cells is much different between any two groups(A-B:P<0.01；A-C:P<0.01；B-C:P<0.01)．In addition,the cell cycle was changed with different treated time．The percentage of apoptotic cells in group D is 9.20±0.36%,the control group is 5.20±0.50%(P<0.01)．Simultaneous,in group D the membranes of some cells are more permeable to Propidium Iodide (PI) which showed stronger PI staining,it is suggested that some cells in group D underwent necrosis．The percentage of apoptotic cells in group E is 5.03±0.75%,the control group is 5.20±0.50% (P>0.05)．The typical 'apoptotic peak' was formed in groups F also．The percentage of apoptotic cells in each group:Their control group is 16.57±0.61%,group F1 is 18.33±0.56% (P<0.01)；group F...