一种改良的间歇性低氧细胞模型方法  

An improved method to develop cell culture model of intermittent hypoxia

作　　者：蔡晓红[1] 洪芳芳[1] 陈利亚[1] 梅红芳[1] 林晶[1] 王红霞[2] 李秀翠[1] 梁冬施[1] 温正旺[1] 

机构地区：[1]温州医科大学附属第二医院育英儿童医院儿童呼吸科,浙江温州325027 [2]焦作市人民医院,河南焦作454150

出　　处：《中国病理生理杂志》2016年第1期187-192,共6页Chinese Journal of Pathophysiology

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)

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(P<0.05);6IH和9IH组HIF-1α的mRNA和蛋白分别高于4SH和6SH组(P<0.05);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。AIM： To establish and validate a novel model of cultured cells for imitating intermittent hypoxia.METHODS： In a chamber with experiment cabin and simulated air control cabin,the frequency and duration of the intermittent hypoxia model according to the time of hypoxia and reoxygenation were evaluated. The A549 cells were randomly divided into 7 groups,named as control（ Con） group,6 h intermittent hypoxia（ 6IH） group,9 h intermittent hypoxia（ 9IH） group,6 h simulated air control（ 6AC） group,9 h simulated air control（ 9AC） group,4 h sustained hypoxia（ 4SH） group,6 h sustained hypoxia（ 6SH） group,respectively. When the model was established,the cellular morphology was observed under inverted microscope. The mRNA expression of hypoxia-inducible factor（ HIF）-1α was detected by real-time PCR. The protein expression of HIF-1α was analyzed by immunohistochemistry. RESULTS： The intermittent hypoxia cycle（ 5% O_260 min-20% O_230 min for 6 cycles） was established. The damaged A549 cells were observed in 6IH group,9IH group and 6SH group. Compared with 6IH group,the expression of HIF-1α at mRNA and protein levels was significantly increased in 9IH group（ P 0. 05）. The expression of HIF-1α at mRNA and protein levels in 6IH group and9 IH group was higher than that in 4SH group and 6SH group,respectively（ P 0. 05）. No significant difference among the control group,6AC group and 9AC group was found. CONCLUSION： The model（ 5% O_260 min-20% O_230 min for 6cycles） can simulate the pathological process of obstructive sleep apnea hypopnea syndrome. This model is suitable for studying intermittent hypoxia in adherent cells.

关 键 词：间歇性低氧 人肺腺癌细胞 缺氧诱导因子1Α 

分 类 号：R363[医药卫生—病理学] R563[医药卫生—基础医学]