摘要
目的探讨角质细胞生长因子(KGF)联合低氧诱导因子1α(HIF-1α)对低氧应激大鼠小肠隐窝上皮细胞(IEC-6)的保护作用及其机制。方法(1)取常规培养大鼠IEC-6细胞,根据随机数字表法分为常氧空白组、常氧KGF组、常氧HIF-1α组和常氧联合组,分别更换DMEM培养基、含0.5ng/mLKGF培养基、含10.0ng/mLHIF-1α培养基及同时含0.5ng/mLKGF和30.0ng/mLHIF-1α培养基,放入氧气体积分数为21%的细胞培养箱培养24h。(2)另取常规培养大鼠IEC-6细胞,根据随机数字表法分为常氧对照组、低氧对照组、低氧KGF组、低氧HIF-1α组及低氧联合组。常氧对照组细胞更换DMEM培养基,并常规培养24h;低氧对照组、低氧KGF组、低氧HIF-1α组及低氧联合组细胞分别更换DMEM培养基、含0.5ng/mLKGF培养基、含10.0ng/mLHIF-1α培养基及同时含0.5ng/mLKGF和30.0ng/mLHIF-1α培养基,并置于氧气体积分数为5%的三气培养箱中低氧培养24h。取常氧及低氧处理各组细胞,样本数为3,光学显微镜下观察细胞形态学变化。取常氧及低氧处理各组细胞,样本数为3,细胞计数试剂盒8检测细胞存活率。取常氧对照及低氧处理各组细胞,样本数为3,流式细胞仪检测细胞周期变化及凋亡水平,紫外分光光度计和蛋白质印迹法分别检测细胞ATP含量和p53蛋白表达水平。对数据行单因素方差分析及LSD检验。结果(1)培养24h后,常氧处理各组细胞均生长良好,细胞呈圆形或椭圆形,胞质清晰;低氧处理各组细胞均出现梭形、星形等不规则形态,胞质有黑色颗粒沉着。(2)培养24h后,常氧空白组、常氧KGF组、常氧HIF-1α组及常氧联合组细胞存活率分别为(107.4±8.7)%、(109.8±2.9)%、(115.8±7.4)%、(112.8±10.6)%,组间总体比较,差异无统计学意义(F=0.685,P=0.586)。培养24h后,低氧对照组、低氧KGF组、低氧HIF-1α组及低氧联合组细胞存活率分别为(35.1±4.6)%、(52.9±6.8)%、(56.2±3.1)%、(71.2±9.6)%,均显著低�
Objective To investigate the protective effects and mechanism of keratinocyte growth factor (KGF) combined with hypoxia inducible factor-1α (HIF-1α) on intestinal crypt epithelial cells (IEC-6) of rats with hypoxia stress. Methods (1) The routinely cultured IEC-6 of rats were collected and divided into normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group, according to the random number table, and then the previous mediums were respectively replaced with dulbecco′s modified eagle medium (DMEM), medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α. And the cells were cultured in cell incubator with oxygen volume fraction of 21% for 24 hours. (2) Another batch of routinely cultured IEC-6 were collected and divided into normoxia control group, hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group, according to the random number table. The previous mediums were replaced with DMEM, DMEM, medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α respectively. And then, the cells in normoxia control group were cultured routinely for 24 hours, and cells in the other 4 groups were cultured in cells incubator of 3 gases, with oxygen volume fraction of 5% for 24 hours. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and morphological changes of cells were observed with optical microscope. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and survival rates of cells were detected by cell count kit 8. Cells in normoxia control group and cells cultured in hypoxic incubator were collected, with 3 samples in each group. The cell cycle changes and apoptosis rates were detected by flow cytometer, the content of adenosine triphosphate (ATP) was detected by ultraviolet spectrophotometer, and protein expression of p53 was detected by Western blotting.
作者
徐倩
白燕青
曾通旭
杨波
蔡小玲
哈小琴
Xu Qian;Bai Yanqing;Zeng Tongxu;Yang Bo;Cai Xiaoling;Ha Xiaoqin(Department of Clinical Laboratory, the People′s Liberation Army Joint Service Support Unit 940 Hospital, Lanzhou 730050, China;Key Laboratory of Stem Cells and Gene Drugs of Gansu Province, Lanzhou 730050, China)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2019年第1期54-61,共8页
Chinese Journal of Burns
基金
兰州市创新人才项目(2016-RC-61).
关键词
大鼠
缺氧
缺氧诱导因子1
Α亚基
角质细胞生长因子
肠隐窝上皮细胞
Rats
Anoxia
Hypoxia-inducible factor 1, alpha subunit
Keratinocyte growth factor
Intestinal crypt epithelial cells
