XBP1在氯化钴诱导骨肉瘤细胞缺氧环境下抗凋亡作用实验研究

摘要

目的探讨在氯化钴诱导骨肉瘤细胞缺氧情况下，X 盒结合蛋白1（XBP1）在骨肉瘤细胞中表达量的变化、其对细胞凋亡的影响及与低氧诱导因子（HIF）-1α信号转导通路的关系。方法通过氯化钴诱导骨肉瘤 MNNG、MG-63细胞达到模拟缺氧状态，通过实时聚合酶链式反应（PCR）检测不同时间、不同氯化钴浓度下 HIF-1α和 XBP1的表达情况；采用流式细胞仪检测骨肉瘤细胞凋亡率；采用 siRNA 干扰技术，下调 XBP1。实验将骨肉瘤细胞分为干扰组（经 XBP1 siRNA 转染骨肉瘤细胞）和空白对照组（未经 XBP1 siRNA 转染骨肉瘤细胞）。结果XBP1表达具有氯化钴浓度和时间依赖性：一定范围内，随着氯化钴浓度增高和时间延长，XBP1表达升高；敲除 XBP1后，干扰组骨肉瘤细胞凋亡率明显高于对照组（P ＞0．05）；PCR 检测 HIF-1α及下游靶基因 mRNA 表达无明显变化（P ＜0．05）。结论XBP1在氯化钴诱导缺氧的骨肉瘤细胞中表达明显增高，其对骨肉瘤细胞在缺氧环境下抗凋亡有明显作用，其抗凋亡作用与 HIF-1α信号转导通路无直接关联，具体机制有待进一步研究。%Objective To investigate the expression level,anti-apoptotic effect and association with hypoxia inducible factor-1α(HIF-1α) signaling of X-box binding protein 1 (XBP1s)in osteosarcoma cells under CoCl2-induced hypoxic condition.Methods CoCl2 was used to simulate hypoxic environment for MNNG and MG-63 cells.The expression of XBP1s and HIF-1αmRNA of osteosarcoma cells were detected by real-time polymerase chain reaction (PCR)for treatment of different CoCl2 concentration and time span.Flow cytometry was utilized to measure the apoptotic rate of osteosarcoma cells.The siRNA interfering technique was used to reduce the expression of XBP1s.The osteosarcoma cells were divided into the XBP1s-siRNA transfected group and the un-transfected group.Results XBP1s expression was CoCl2-concentration and time-dependent,which increased with the raised CoCl2 concentration and prolonged time in a certain range.After knockdown of XBP1s,the cell apoptotic rate of osteosarcoma cells in the hypoxic group was significantly higher than that in the control group (P >0.05).However,the PCR results showed that there was no significant changes of HIF-1α and its downstream targeted genes after knockdown of XBP1s (P <0.05 ). Conclusion XBP1s could be increased remarkably in osteosarcoma cells after CoCl2-induced hypoxic treatment.It has anti-apoptotic effects on osteosarcoma cells under hypoxic condition.However,there is little relationship between XBP1s and HIF-1αsignaling pathway.The specific mechanism for XBP1s acting as an anti-apoptotic gene in osteosarcoma is needed further deeper investigation.