胰腺癌细胞来源的微囊泡影响糖代谢的初步探讨  

Preliminary study on the effects of pancreatic cancer-derived microvesicles on glycometabolism

作　　者：庞文璟[1] 戴欣[2] 陈熹[3] 袁耀宗[2] 

机构地区：[1]同济大学附属天佑医院消化内科,上海200331 [2]上海交通大学医学院附属瑞金医院消化内科 [3]南京大学生命科学院

出　　处：《中华消化杂志》2015年第12期839-843,共5页Chinese Journal of Digestion

摘　　要：目的探讨胰腺癌细胞来源的微囊泡及其包裹的miRNA在胰腺癌诱导糖尿病发生中的作用。方法收集3种胰腺癌细胞株SW1990、BxPC3及PANCl的培养上清液，使用梯度离心法分离微囊泡，通过蛋白质印迹试验及荧光标记的方法证实胰腺癌来源的微囊泡可进入胰岛细胞株MIN6中。分别测定3种胰腺癌细胞分泌的微囊泡及去除微囊泡的上清液中miRNA19a的含量。以经3种胰腺癌细胞来源的微囊泡分别处理后的MIN6细胞为3个实验组，未处理的MIN6细胞为对照组，测定miRNA19a水平及胰岛素分泌功能变化。通过脂质体转染的方式将miRNA-19a前体及其抑制物分别转入胰岛细胞MIN6中，观察其对胰岛素分泌的影响。结果经蛋白质印迹试验检测微囊泡的标志蛋白CD63及AG02，证实胰腺癌细胞来源的微囊泡可以进入胰岛细胞MIN6中。SW1990、BxPC3、PANC1细胞微囊泡和去除微囊泡的培养上清液中miRNA-19a的含量分别为（132．7±16．0）和（32．8±4．3），（78．4±8．9）和（22．6±3．3），（63．3±12．0）和（23．3±3．3）pmol／L，差异均有统计学意义（t=10．44、10．12、5．56，P均〈O．01）。与对照组MIN6细胞相比，经SWl990、BxPC3和PANCl来源的微囊泡处理后MIN6细胞的miRNA-19a表达量均明显升高，其2-△△ct值分别为2．02±0．50、1．80±0．41和2．11±0．59，差异均有统计学意义（t=2．97、2．77、2．84，P均〈0．05）。在高糖刺激下，经SW1990、BxPC-3、PANCl来源微囊泡处理的3组胰岛细胞均出现了胰岛素分泌量下降，分别为（103．73±16．49）、（141．17±11．26）、（138．24±13．97）ng·mg蛋白-1·h-1对照组胰岛素分泌量为（256．24±33．05）ng·mg蛋白-1·h-1，差异均有统计学意义（t=4．13、3．30、3．29，P均〈0．05）；转染miRNA-19a前体的MIN6细胞的胰岛素分泌量较对照组明显下降，分别为（126．17±62．87）和（316．72±91．87）ngObjective To explore the role of pancreatic cancer-derived microvesicles （MV） and their enclosed microRNAs （miRNA） in the pathogenesis of pancreatic cancer induced diabetes mellitus （DM）. Methods The supernatants of three pancreatic cancer cell lines SW1990, BxPC3 and PANC1 were collected, and MV were isolated with gradient centrifugation. The entrance of MV into pancreatic islet cell line MIN6 was proved by Western blot assay and fluorescence-label method. The miRNA-19a levels were measured in MV and MV-free supernatants of three pancreatic cancer cells lines. The three experimental groups were MIN6 cells separately treated by MV derive from SW1990, BxPC3 and PANC1, and untreated MIN6 cells were assigned to the control group. The miRNA-19a levels as well as changes of glucose stimulated insulin secretion （GSIS） were measured. Afterward, pre miRNA-19a and anti-miRNA-19a were transfected into MIN6 cells by liposome, and the effects of them on GSIS were observed. Results CD63 and AGO2 as the protein markers of MV and the entrance of MV from pancreatic cancer into pancreatic islet cell line MIN6 were detected by Western blotting. The miRNA-19a levels in MV and MV free supernatants of SW1990, BxPC3 and PANC1 were （132.7±16.0）, （32.8±4.3）, （78.4±8.9）, （22.6±3.3）, （63.3±12.0） and （23.3±3.3） pmol/L, respectively, and the differences were statistically significant （t=10.44, 10.12 and 5.56,ali P〈0.01）. Compared to the MIN6 control group, the miRNA- 19a levels of MIN6 treated by MV from SW1990, BxPCS and PANC1 significantly increased, and the 2△△ct value was 2. 02±0. 50, 1. 80±0. 41 and 2. 11±0. 59, respectively, and the differences were statistically significant （t=2.97, 2.77, 2.84; all P〈0.05）. Stimulated with high glucose, the GSIS of pancreatic islet cells treated by SW1990, BxPC3 and PANC1 in three groups decreased, which were （103.73±16.49）, （141.17±11.26）, and （138.24±13.97） ng mg protein-1 . h-1 MV, respectively, and that of control gro

关 键 词：胰腺肿瘤 糖尿病 微RNAS 微囊泡 

分 类 号：R735.9[医药卫生—肿瘤]