摘要目的 寻找胰腺癌循环微小核糖核酸(miRNA)的载体.方法 常规培养、传代胰腺癌细胞SW1990、BxPC3,采集6例胰腺癌患者及6例健康体检者(对照组)的血清.应用梯度离心方法获取血清和细胞培养上清中的微囊泡(MV).采用蛋白质印迹法检测RNA诱导沉默复合体的核心元件Ago2蛋白和MV的标志性蛋白CD63,采用荧光定量PCR方法检测MV包裹的和游离的miRNA.结果 胰腺癌细胞株培养上清的MV中含有Ago2、CD63蛋白.胰腺癌组、对照组的血清及MV中均存在miR-20a、miR-21、miR-24、miR-25、miR-191、miR-483-5p,但以MV包裹的量较多,且胰腺癌组MV包裹的miRNA量与对照组不完全一致,其中miR-20a、miR-24、miR-191分别为对照组的(2.93±0.29)、(2.73±0.46)、(2.39±0.51)倍,差异均有统计学意义(F值分别为75.97、25.80、12.94,P＜0.05或＜0.01).结论 MV是胰腺癌循环miRNA的主要载体.

abstractsObjective To investigate the circulating microRNAs carrier in pancreatic cancer.Methods Pancreatic cancer cell lines SW1990 and BxPC3 were routinely cultured and serum of 6 patients with pancreatic cancer and 6 healthy subjects (control group) were collected.Serum and pancreatic cancer cell line supernatant microvesicles (MV) were obtained by gradient centrifugation.The expression of Ago2,CD63 was detected by Western blotting,the expression of microRNA in microvesicle section and microvesicle-free section in serum was detected by using quantitative PCR method.Results The supernatant MV of pancreatic cancer cell lines expressed Ago2,CD63 protein,and these MV carried different microRNAs in different cell lines.In the serum of pancreatic cancer and control group,miR-20a,miR-21,miR-24,miR-25,miR-191,miR-483-5p were detected,but the quantity was relatively higher in MV section,and the expression of microRNAs in pancreatic cancer's MV was inconsistent with that of control group.The expression of miR-20a,miR-24,miR-191 in pancreatic cancer group was (2.93 ± 0.29),(2.73 ± 0.46),(2.39 ± 0.51) times as much as those in control group,and the difference between the two groups was statistically significant (F =75.97,25.80,12.94,P ＜ 0.05 or ＜ 0.01).Conclusions The main circulating microRNAs carrier in pancreatic cancer is microvesicle.