摘要
目的探讨间歇性高糖对大鼠胰岛细胞瘤细胞系Ins-1的损伤作用及对第10号染色体缺失的张力同源性磷酸酶（PTEN）表达的影响。方法将Ins．1细胞分为正常组（CG组，11．1mmol／L葡萄糖培养）、持续高糖组（SHG组，33．3mmol／L葡萄糖培养）、间歇性高糖组（IHG组，11．1mmol／L及33-3mmol／L葡萄糖交替培养12h），3组细胞均培养3d。欧文比色法（MTS）~定细胞增殖活性，膜联蛋白v-异硫氰酸荧光素／碘化丙啶（AnnexinV／PI）双染细胞后，以流式细胞仪测定各组细胞的凋亡率，放免法测各组细胞胰岛素分泌，2’，7’．二氯荧光黄双乙酸盐（DCFH—DA）荧光探针测定各组细胞内活性氧簇（ROS）水平，倒置相差显微镜下观察各组细胞形态，钙离子荧光探针法（Fluo3-AM）测定细胞内钙离子浓度，Westernblotting法测定各组细胞PTEN蛋白表达。各组内均数比较采用单因素方差分析。结果SHG组细胞凋亡率、细胞内ROS水平、细胞内钙离子浓度及PTEN表达均较CG组显著增加（t=8．310、10．261、17．890、5．123，均P〈0．05），细胞增殖活性、胰岛素分泌均较CG组显著降低（t=-19．630、-2．053，均P〈0．05）。IHG组细胞凋亡率、细胞内ROS水平以及钙离子浓度及PTEN表达均较CG组及SHG显著增加（t=6．200～18．227，均P〈0．05），细胞增殖活性、胰岛素分泌均较CG组及SHG组均显著降低（t=-27．800、-2．790、-8．167、-1．503，均P〈0．05）。IHG组较SHG组及CG组Ins-1细胞异型性明显、细胞数量显著减少且排列紊乱。结论氧化应激参与高糖对Ins．1细胞的损伤作用，可能与PTEN表达增高有关，间歇性高糖对INS．1细胞的损伤作用较持续性高糖严重。
Objective To study the effect of intermittent hyperglycemia on rat insulinoma cell line Ins- 1 apoptosis and the expression of phosphatase and tensin homologue- deleted chromosome ten gene （PTEN） in those cells. Methods Ins-1 cells were divided into three groups： control group （CG, cultured in 11.1 mmol/L glucose）, sustained high glucose group （SHG, cultured in 33.3 mmol/L glucose） and intermittent high glucose group （IHG, cultured in 33.3 or 11.1 mmol/L glucose each for 12 h） respectively. After three days of incubation, cell viability was determined by Owen＇ s reagent（MTS）, apoptosis rate was detected by flow cytometry after staining with Annexin V/PI, insulin secretion was measured but radioimmunoassay, intracellular reactive oxygen species（ROS） was detected by DCFH-DA fluorescence probe, the morphology of cells of the three groups were observed under inverted phase contrast microscope. The data was compared with one-way ANOVA analysis. Results Compared with CG group, the cellularapoptosis rate, intracellular ROS levels, calcium levels, and the expressions of PTEN protein in cells of SHG group were all significantly increased （t=8.310, 10.261, 17.890, 5.123, all P〈0.05）, while cell viability and insulin secretion decreased significantly（t=-19.630,-2.053, both P〈0.05）. However, compared to both of the CG group and the SHG group, the cellular apoptosis rate, intracellular ROS levels, intracellular calcium levels, and the expression of PTEN protein in ceils of IHG group increased significantly（t=6.20-18.227, all P〈0.05）, while cell viability and insulin secretion decreased significantly（t=-27.800, -2.79, -8.167, -1.503, all P〈0.05）. Compared to CG group and the SHG group, the pleomorphism of the cells, the reduction in cell number and the disarrangement of ceils in the IHG group were all the most significant. Conclusions Oxidative stress may play an important role in the apoptosis of Ins- 1 cells under high glucose environment and it may be related to the high expressions of PTEN. The adverse effect of intermittent high glucose on the Ins-1 cell-line may be greater than that of sustained high glucose.
出处
《中华糖尿病杂志》
CAS
CSCD
2015年第11期689-693,共5页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金青年基金项目（81200653）
辽宁省科学技术计划项目（2012225079、2012225020）
沈阳市科技计划项目（F13-220-9-02）
