Study On The Inhibition Effect And Mechanism Of PI3K Inhibitor On Cd4~+Cd25~+Foxp3 Treg Cells In Pancreatic Cancer Of Tumor Bearing Mice

Objective:To establish pancreatic cancer xenograft model of Kunming mouse; to investigate the effect of CAL-101,the PI3K inhibitor,on the tumor formation and survival of tumor-bearing mice;to investigate the effect of CAL-101,the PI3K inhibitor,on Treg cells in tumor-bearing Kunming mouse;preliminary to explore the inhibition mechanism of the inhibitor on Treg cells.Methods:1.Recovery of MPC-83 cells of Kunming mouse pancreatic cancer,cultured in vitro and Subculture, and to establish pancreatic cancer xenograft model of Kunming mouse with cell suspension.Group of tumor-bearing mice:normal group (A),group of propylene glycol(B),little inhibitor group(C) and more inhibitor mice group(D).2. In the first day,mice of groups B were given 50 μl propylene glycol, groups of C lwere given 50 μl of CAL-101 inhibitors and groups of D were given 100 μl of CAL-101 inhibitors, groups of A were given nothing.Mouse tumor cases and the average survival were observed,tumor growth was measured with calipers.3.Peripheral blood and the spleen of the Kunming mice were analyzed by flow cytometry to detect the level of CD4+CD25+ Foxp3 T cells.4.The expression of Foxp3 mRNA in spleen cells of the Kunming mice were detected with RT-PCR.5. The expression of PI3K in spleen cells of the Kunming mice were observed by Western Blot.6. Statistical analysis:In this study,using SPSS 13.0 statistical software to analyze data,P<0.05 was considered statistically significant.Using mean±tandard deviation,namelyx±s,to indicate the results of measurement data,using Pearson correlation analysis between Foxp3 mRNA and PI3K protein to make it clear whether the two were relevant.Results:1.MPC-83 cells of Pancreatic cancer cultured in vitro were in good conditionand and the construction of pancreatic cancer in mice subcutaneous xenograft animal models were successful, the tumor formation rate was 100%.2. Effect of PI3K inhibitor on pancreatic cancer xenograft tumors and tumor-bearing mice growth conditions:The tumor formation of group A and group B was about 5-7 days, the mice in group C were extended to 7-8 days, and the tumor in group D was the latest, and the mice were mainly concentrated in 9-10 days. The tumor volume of mice treated with the inhibitor was significantly smaller than that of the control group (P<0.05), and the survival time was significantly prolonged, and the difference was statistically significant (P<0.05).3. Effect of PI3K inhibitor on pancreatic cancer CD4+CD25+Foxp3 Treg cells in tumor bearing mice:Flow cytometry was used for 4 groups(group A, B, C and D) of Kunming mice to detect peripheral blood and spleen CD4+CD25+Foxp3 Treg cells, results of CD4+CD25+Foxp3 cell percentage of Treg (%) respectively:peripheral blood (17.94±3.91、17.58±3.82、12.82±2.71、9.97±2.52)% and the spleen (19.23 ±5.56、19.03±5.18、13.01±3.62、11.98±2.66)%. Tumor bearing mice either peripheral blood or spleen cells, CD4+CD25+Foxp3 Tregs were significantly higher than inhibitor treated mice and inhibitor dose increases, the number of Treg decreased more obviously (P< 0.05).4. Expression of Foxp3 mRNA in mouse spleen cells RT-PCR:Foxp3 mRNA expression of 4 groups(group A, B, C and D) in Kunming mice spleen were 1.805± 0.241、1.798±0.237、1.198±0.347、0.832±0.210, the expression of mRNA Foxp3 in tumor bearing mice was significantly higher than that in the inhibitor treatment group, and the expression of mRNA Foxp3 was significantly decreased (P< 0.05).5. PI3K protein levels of spleen cells in Kunming mice were detected by Western blot:Western Blot detected PI3K protein expression of four group(group A, B, C and D) of Kunming mice spleen, the results were as follows:PI3K/Actin (2.22±0.15、 2.13±0.14、1.78±0.10、1.46±0.08). The expression level of PI3K in tumor bearing mice was significantly higher than that in the inhibitor treatment group, and the expression of PI3K protein was significantly decreased (P< 0.05).6.The expression of Foxp3 mRNA and PI3K protein expressionin in the spleen of Kunming mice were analyzed by Pearson correlation analysis and it was shown that Foxp3 mRNA and PI3K protein expression was positively correlated.Conclusion:1. PI3K inhibitor CAL-101 can make pancreatic xenograft tumor grow slow and prolong survival time of the mice.2. PI3K inhibitor CAL-101 can downregulate the expression of CD4+CD25+Foxp3 Treg levels of peripheral blood and spleen in tumor-bearing mouse.3. PI3K inhibitor CAL-101 can downregulate the expression of Foxp3 mRNA and PI3K protein of peripheral blood and spleen in tumor-bearing mouse.4. The expression of Foxp3 mRNA in the mouse spleen was positively correlated with the expression of PI3K protein,it provided a theoretical basis for further research on PI3K/AKT/mTOR signaling pathway in pancreatic cancer development and immune therapy.