胰腺星状细胞通过白细胞介素-22影响胰腺癌侵袭转移及化疗耐药的研究

Effects of pancreatic stellate cells on malignant progression and chemoresistance of pancreatic cancer by interleukin-22

目的探讨具有不同白细胞介素-22(IL-22)表达水平的胰腺星状细胞(PSC)对胰腺癌细胞侵袭转移及化疗耐药的影响。方法将人原代胰腺癌间质PSC细胞与胰腺癌PANC-1细胞共培养后检测IL-22的表达及胰腺癌细胞的生长增殖能力、迁移和侵袭能力变化;构建稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养,检测PANC-1细胞的生长增殖、迁移和侵袭能力;利用稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养后检测E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表达情况;将含有不同表达水平的胰腺癌间质PSC细胞与胰腺癌PANC-1细胞体外共培养并加入不同浓度梯度的吉他西滨培养72 h后检测细胞存活率,计算细胞耐受吉他西滨的半数抑制浓度(IC_(50));将稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行混合后种植于裸鼠皮下,40 d后处理裸鼠取出肿瘤并绘制肿瘤生长曲线,比较两组细胞的成瘤能力。采用t检验分析各组间差异的显著性。结果单纯PANC-1培养组IL-22表达水平(0.907±0.025)明显低于PANC-1与PSC共培养组(4.167±0.306),差异有统计学意义(t=18.420,P<0.05)。细胞增殖实验中过表达组细胞吸光度值(0.727±0.040)明显高于空载对照组(0.563±0.055),差异有统计学意义(t=4.141,P<0.05);敲低组细胞吸光度值(0.327±0.038)明显低于空载对照组(0.530±0.020),差异有统计学意义(t=8.225,P<0.05)。体内成瘤实验中过表达组细胞在裸鼠内成瘤体积[(2711.000±24.880)mm^(3)]明显高于空载对照组[(2037.00±25.514)mm^(3)],差异有统计学意义(t=32.758,P<0.05);敲低组细胞成瘤体积[(636.667±33.501)mm^(3)]明显低于空载对照组[(1959.667±50.053)mm^(3)],差异有统计学意义(t=38.046,P<0.05)。Transwell迁移实验中过表达组细胞计数[(375.667±9.451)个/视野]明显高于空载对照组[(283.667±9.073)个/视野],差异有统计学意义(t=12.162,P<0.05);敲低组细胞计数[(71±6)个/视野]明显低于空载对照组[(263.333±13.051)个/视野],差异有统计学意义(t=23.191,P<0.05)。Transwell侵袭实验中,过表达组细胞计数[(459.330±15.370)个/视野]明显高于空载对照组[(210.330±9.012)个/视野],差异有统计学意义(t=24.198,P<0.05);敲低组细胞计数[(84.330±4.160)个/视野]明显低于空载对照组[(211.330±8.960)个/视野],差异有统计学意义(t=22.258,P<0.05)。PT-PCR检测过表达组Vimentin表达水平(3.080±0.085)明显高于空载对照组(1.230±0.045),差异有统计学意义(t=33.167,P<0.05);敲低组Vimentin表达水平(0.413±0.025)明显低于空载对照组(1.123±0.042),差异有统计学意义(t=25.278,P<0.05);过表达组E-cadherin表达水平(0.603±0.047)明显低于空载对照组(0.830±0.046),差异有统计学意义(t=5.964,P<0.05);敲低组E-cadherin表达水平(2.910±0.030)明显高于空载对照组(0.917±0.031),差异有统计学意义(t=80.634,P<0.05)。耐药性实验中过表达组IC_(50)值[(91.130±1.850)μmol/L]明显高于空载对照组[(52.727±2.475)μmol/L],差异有统计学意义(t=21.529,P<0.05);敲低组Vimentin IC_(50)值[(22.897±2.069)μmol/L]明显低于空载对照组(49.957±1.734)μmol/L,差异有统计学意义(t=17.363,P<0.05)。结论本研究提示PSC通过IL-22影响胰腺癌的侵袭转移、EMT和化疗耐药,这有望为胰腺癌寻找新的治疗模式和治疗靶点,切实提高胰腺癌治疗的临床效果。

Objective Interleukin-22(IL-22)is highly expressed in human primary pancreatic cancer mesenchymal pancreatic stellate cells(PSCs).This study aims to investigate the effects of different expression levels of IL-22 on the invasion and metastasis of pancreatic cancer cells and chemotherapy resistance.Methods The interstitial PSCs of primary pancreatic cancer were co-cultured with PANC-1 cells to detect the expression of IL-22 and the changes of growth,proliferation,migration and invasion ability of pancreatic cancer cells.Human primary pancreatic cancer mesenchymal PSCs with stable knockdown or overexpression of IL-22 were constructed and co-cultured with pancreatic cancer PANC-1 cells in vitro to detect the growth,proliferation,migration and invasion of PANC-1 cells.The expression of E-cadherin and Vimentin in human primary pancreatic cancer mesenchymal PSCs with stable knockdown or overexpression of IL-22 was detected by in vitro co-culture with pancreatic cancer PANC-1 cells.Pancreatic cancer mesenchymal PSCs with different expression levels were co-cultured with pancreatic cancer PANC-1 cells and cultured with guitar sibin of different concentration gradients for 72 h.The survival rate of the cells was detected,and the half maximal inhibitory concentration(IC_(50))of the cells tolerant to guitar sibin was calculated.Human primary pancreatic cancer mesenchymal PSCs with stable knockout or overexpression of IL-22 were mixed with pancreatic cancer PANC-1 cells and implanted subcutaneously in nude mice.After 40 days of treatment,tumors were removed from nude mice and tumor growth curves were drawn to compare the tumor-forming ability of the two groups of cells.T test was used to analyze the significance of differences among the groups.P<0.05 was considered statistically significant.Results The expression level of IL-22 in PANC-1 culture group(0.907±0.025)was significantly lower than that in PANC-1 and PSCs co-culture group(4.167±0.306),and the difference was statistically significant(t=18.420,P<0.05).In cell proliferation experiment,the absorbance value of cells in overexpression group(0.727±0.040)was significantly higher than that in no-load control group(0.563±0.055),and the difference was statistically significant(t=4.141,P<0.05).The cell absorbance value in knockdown group(0.327±0.038)was significantly lower than that in no-load control group(0.530±0.020),and the difference was statistically significant(t=8.225,P<0.05).In vivo tumor formation experiment showed the tumor formation volume of overexpression group[(2711.000±24.880)mm^(3)]in nude mice was significantly greater than that of no-load control group[(2037.000±25.514)mm^(3),t=32.758,P<0.05].The tumor volume of the knockdown group[(636.667±33.501)mm^(3)]was significantly less than that of the no-load control group[(1959.667±50.053)mm^(3),t=38.046,P<0.05].In Transwell migration experiment,the cell count of overexpression group[(375.667±9.451)cells/field]was significantly greater than that of no-load control group[(283.667±9.073)cells/field,t=12.162,P<0.05].Cell count[(71±6)cells/field]in the knock down group was significantly less than that in the no-load control group[(263.333±13.051)cells/field,t=23.191,P<0.05].In Transwell invasion experiment,the number of cells in overexpression group[(459.330±15.370)/field]was significantly greater than that in no-load control group[(210.330±9.012)/field,t=24.198,P<0.05].Cell count[(84.330±4.160)cells/field]in the knockdown group was significantly less than that in the no-load control group[(211.330±8.960)cells/field,t=22.258,P<0.05].The expression level of Vimentin in overexpression group(3.080±0.085)was significantly higher than that in no-load control group(1.230±0.045,t=33.167,P<0.05).The expression level of Vimentin in knockdown group(0.413±0.025)was significantly lower than that in no-load control group(1.123±0.042,t=25.278,P<0.05).The expression level of E-cadherin in overexpression group(0.603±0.047)was significantly lower than that in no-load control group(0.830±0.046,t=5.964,P<0.05).The expression level of E-cadherin in knockdown group(2.910±0.030)was significantly higher than that in no-load control group(0.917±0.031,t=80.634,P<0.05).The IC_(50) value of overexpression group[(91.130±1.850)μmol/L]was significantly higher than that of no-load control group[(52.727±2.475)μmol/L,t=21.529,P<0.05].The IC_(50) value[(22.897±2.069)μmol/L]in the low-load group was significantly lower than that in the no-load control group[(49.957±1.734)μmol/L,t=17.363,P<0.05].Conclusion This study suggests that PSCs affect the invasion and metastasis,EMT and chemotherapy resistance of pancreatic cancer through IL-22,which is expected to find a new treatment model and therapeutic target for pancreatic cancer,and effectively improve the clinical effect of pancreatic cancer treatment.