| ObjectiveWe aim to examine SRp20 expression in normal pancreatic cell as well as pancreatic cancer cells and also to examine SRp20 expression in Pancreatic Cancer Cells treated by Triptolide with a dose- and time-dependent manner,meanwhile,to determine the role of SRp20 played in the p53-dependent apoptosis mechanism induced by Triptolide.Methods1.Use the DMEM of 10% fetal bovine serum to culture pancreatic cancer cell of HPDE6-C7,As PC-1, PANC-1 and Bxpc-3, proceeding to next experiment when the goal cell of growing in logarithmic phase and passaging to 10th- 20 th. 2. Study Triptolide- induced anti-proliferation and apoptosis effectof As PC-1 by using MTT and Annexin V-FITC/PI methods. 3.Examine the expressions of SRp20 and p53β in normal or cancel pancreatic cell with Western Blot, simultaneously,analyze proteins mentioned above expression difference by relative quantitative method comparing with undisposed and distinct experimental treatment cells.Results1. We use the cell microscope to observe As PC 1 cells treated by different TPL treating for 48 h, the outcome showed that dosing of 12.5,25and50 n M/Lgroups cells densi-ty become more and more rare and appeared the intracellular swelling as well as forming vacuoles compared with the normal control. 2.As PC-1 Cell were treated with triptolide in different dose and time, MTT assay showed that the proliferation rate of As PC-1 cell were obviously inhibited,in addition,the proliferation value demonstrated a positive correlation effect following the rising of TPL dose(12.5, 25, 50 n M/L) and treat time(24,48,72h). among the result of induc-ed by TPL 48 h,dosing of 12.5,25and50 n M/Lgroups suppression ratio were the25.28±2.18%(12.5n M/L),36.01±1.82%(25 n M/L),60.03±1.67(50 n M/L) [each group compared with control group, p <0.01]. 3.As PC-1 cell treated by different TPL for 48 h,the Flow Cytometry technology showed that apoptosis value of dosing12.5, 25 and 50 n M/Lgroups respectively were 9.45±1.92(12.5n M/L), 30.1± 2.57%(25 n M/L)and 44.23±4.36%(50 n M/L)(each group compared with control group,p<0.01).It means the apoptosis value also increased significantly following the rising of TPL dose. 4.According to the Westrenblot experimental results, we corroborated that SRp20 highly expressed in pancreatic cancer cells than normal pancreatic cells(each group compared with control group,p<0.01),further- more,we also demonstrated that the expression of SRp20 significantly downregulated in Triptolide- induced apoptosis of pancreatic cancer cells compared with non-treated control group,meanwhile, p53、p53β and Bax trended to upregulate. Moreover, In a certain dose range(TPL 6.25~50nmol/L), both expression of SRP20,p53,P53β and Bax were showed in a TPL dose-denpendent manner.(each group compared with control group,p<0.01).Conclusions1. In our study, we showed that SRp20 highly expressed in pancreatic cancer cells(As PC-1,PANC-1,Bx PC-3) than normal pancreatic cells(HDEP6-C7).It suggested that SRp20 may associated with the formation mechanism of pancreatic carcinoma. 2. It were found that the Triptolide were able to downregulate the expression of SRp20 in human pancreatic carcinoma.This result suggested that SRp20 played a potential role associated with the apoptosis mechanism of Triptolide- induced pancreatic cancer cells. 3.We also found that p53β(one of p53 isoforms)which regulated by SRp20 were highly expressed in Triptolide- induced apoptosis of pancreatic cancer cells.It meaned that the the apoptosis mechanism of Triptolide- induced pancreatic cancer cells may correlated with the process of low-level SRp20 regulate up p53β expression. |
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