一种改良的间歇性低氧细胞模型方法

发布时间：2018-10-25 08:44

【摘要】：目的:研制细胞氧舱,建立间歇性低氧(intermittent hypoxia,IH)细胞模型并进行验证。方法:定制细胞实验舱和空气模拟对照舱,根据氧分压-时间曲线设计间歇低氧模式。将人肺腺癌细胞A549随机分为正常对照(Con)组、间歇低氧6 h(6IH)组、间歇低氧9 h(9IH)组、空气模拟对照6 h(6AC)组、空气模拟对照9 h(9AC)组、持续低氧4 h(4SH)组、持续低氧6 h(6SH)组。暴露结束后光镜下观察细胞形态改变,real-time PCR、免疫组化法检测缺氧诱导因子1α(HIF-1α)的mRNA和蛋白表达变化。结果:该模型间歇低氧模式为5%O_260 min-20%O_230 min,6个循环。与Con组比较,6AC、9AC组为原本细胞形态,6IH、9IH和6SH组部分细胞出现突起、变圆,胞质中出现较多黑色颗粒,细胞边界模糊,而4SH组未见明显异常。与6IH组比较,9IH组HIF-1α的mRNA和蛋白表达量明显增加(P0.05);6IH和9IH组HIF-1α的mRNA和蛋白分别高于4SH和6SH组(P0.05);6AC、9AC组与Con组比较差异不显著。结论:5%O_260 min-20%O_230 min的间歇性低氧-复氧细胞模式能模拟阻塞性睡眠呼吸暂停低通气综合征病理生理过程,是研究该疾病较理想的细胞模型。
[Abstract]:Objective: to develop cell oxygen chamber and establish intermittent hypoxia (intermittent hypoxia,IH) cell model. Methods: the intermittent hypoxia model was designed according to the oxygen partial pressure-time curve. Human lung adenocarcinoma cell A549 was randomly divided into normal control (Con) group, intermittent hypoxia group for 6 h (6IH), intermittent hypoxia group for 9 h (9IH), air simulated control group for 6 h (6AC), air simulated control group for 9 h (9AC), continuous hypoxia group for 4 h (4SH) and continuous hypoxia group for 6 h (6SH). The changes of cell morphology were observed under light microscope and the expression of mRNA and protein of hypoxia inducible factor-1 伪 (HIF-1 伪) were detected by real-time PCR, immunohistochemical method. Results: the intermittent hypoxia model consisted of 6 cycles of 5%O_260 min-20%O_230 min,. Compared with the Con group, 6AC9AC group had the original cell morphology, and some cells in 6IHH9IH and 6SH group had processes, became round, more black granules appeared in the cytoplasm, and the boundary of the cells was blurred, but no obvious abnormality was found in the 4SH group. Compared with 6IH group, the expression of mRNA and protein of HIF-1 伪 in 9IH group was significantly increased (P0.05); mRNA and protein of HIF-1 伪 in 6IH and 9IH group were higher than those in 4SH and 6SH group (P0.05); 6AC9AC group had no significant difference with Con group. Conclusion: the intermittent hypoxia / reoxygenation cell model of 5%O_260 min-20%O_230 min can simulate the pathophysiological process of obstructive sleep apnea hypopnea syndrome and is an ideal cell model to study the disease.
【作者单位】：温州医科大学附属第二医院育英儿童医院儿童呼吸科;焦作市人民医院;
【基金】：浙江省科技厅项目(No.2013C33174) 浙江省自然科学基金资助项目(No.Y2110277) 温州市科技合作项目(No.H20130001) 浙江省医药卫生科技计划(No.2014ZDA014) 国家卫计委国家重点临床专科开放课题(No.20130201)
【分类号】：R766

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