摘要
目的探讨骨髓间充质干细胞条件培养基(BMSC-CM)对促炎因子诱导大鼠胰岛素瘤细胞(INS-1)凋亡的影响及其作用机制。方法培养大鼠骨髓间充质干细胞(BMSCs)至第3代,收集、浓缩其上清液,获得富含BMSCs分泌因子的条件培养基。用含促炎因子TNF-α、IL-1β、IFN-γ的培养基处理INS-1细胞48h,造成炎症损伤β细胞模型,加入不同浓度的BMSC-CM继续培养12h,所获细胞分为以下5组:正常对照组,促炎因子组,1×BMSC-CM组,2×BMSC-CM组,4×BMSC-CM组。采用CCK-8法检测INS-1细胞存活率,膜连蛋白V/碘化丙啶(Annexin V/PI)检测细胞凋亡率,二氯荧光素双醋酸盐(DCFH-DA)检测细胞内活性氧(ROS)的生成量。结果与正常对照组相比,促炎因子组INS-1细胞存活率下降至正常对照组的75.84%±1.53%,细胞凋亡率增加(17.23%±1.77%),细胞内ROS水平明显升高(34.3%±0.80%),差异均具有统计学意义(P<0.01)。而BMSC-CM有效对抗了促炎因子导致的INS-1细胞损伤,其作用呈剂量依赖性,4×BMSC-CM组效果最佳,该组INS-1细胞存活率较促炎因子组升高了11.86%,达到87.7%±2.08%,细胞凋亡率下降至8.67%±1.59%,ROS生成量减少至17.1%±2.14%,差异均具有统计学意义(P<0.05)。结论 BMSC-CM可抑制促炎因子诱导的INS-1细胞凋亡,其作用机制可能与BMSC-CM降低细胞内ROS水平有关。
Objective To investigate the effect and mechanism of bone marrow mesenchymal stem cell-conditioned medium（BMSC-CM） on pro-inflammatory cytokines-induced apoptosis of insulinoma cells（INS-1）. Methods The conditioned medium containing various cytokines and growth factors was collected and condensed from the supernatants of culture medium of BMSC of rats at passage three. After exposed to the medium with a pro-inflammatory cytokines cocktail containing TNF-α, IL-1β and IFN-γ for 48 hours, INS-1 cells were cultured with BMSC-CM in different concentrations for another 12 hours. INS-1 cells were divided into the following five groups： control group, pro-inflammatory cytokines-treated group, 1× BMSC-CM-treated group, 2× BMSC-CM-treated group and 4× BMSC-CM-treated group. The survival rate of INS-1 cells was assessed by CCK8 assay. The incidence of cell apoptosis was measured using Annexin V/PI staining. Intracellular reactive oxygen species（ROS） production was determined with dichlorofluorescein diacetate（DCFH-DA）. Results Compared with the control group, prolonged exposure to pro-inflammatory cytokines induced significant decrease in the viability of INS-1 cells（75.84%±1.53%） and a great increase in the incidence of cell apoptosis（17.23%±1.77%）, and also in intracellular ROS level（34.3%±0.80%） with statistically significant difference（P〈0.01）. However, BMSC-CM treatment protected INS-1 cells against pro-inflammatory cytokines-induced injury in a dose-dependent manner. Among the BMSC-CM of three different concentrations, 4× BMSC-CM exhibited best protective effects on pro-inflammatory cytokines-treated INS-1 cells, as reflected by increased cell viability（87.7%±2.08%） and significant reduction in the incidence of cell apoptosis（8.67%±1.59%）, and also in intracellular ROS production（17.1%±2.14%） with statistically significant difference（P〈0.05）. Conclusion BMSC-CM protected INS-1 cells against pro-inflammator y cytokines-induced apoptosis, the possible mechanism underlying this effect might be associated with the reduction of intracellular ROS level.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2015年第5期339-343,共5页
Medical Journal of Chinese People's Liberation Army
基金
国家高技术研究发展计划(863计划)(2013AA020105)~~
