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0.5 ng/mL KGF培养基、含10.0 ng/mL HIF-1α培养基及同时含0.5 ng/mL KGF和30.0 ng/mL HIF-1α培养基,放入氧气体积分数为21％的细胞培养箱培养24 h.(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24 h;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0.5 ng/mL KGF培养基、含10.0 ng/mL HIF-1α培养基及同时含0.5 ng/mL KGF和30.0 ng/mL HIF-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24 h.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24 h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24 h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24 h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)％,均显著低于常氧对照组的(106.3±12.3)％,P＜0.001.低氧KGF组、低氧HIF-1α组及低氧联合组细胞存活率均明显高于低氧对照组(P=0.023、0.009、＜0.001),低氧联合组细胞存活率明显高于低氧KGF组和低氧HIF-1α组(P=0.017、0.045).(3)培养24 h后,低氧对照组G1期细胞百分比显著高于常氧对照组(P=0.030),低氧对照组、低氧KGF组、低氧HIF-1α组S期细胞百分比显著低于常氧对照组(P =0.020、0.031、0.026),其余各组各期细胞百分比与常氧对照组相近(P =0.516、0.107、0.052、0.985、0.637、0.465、0.314、0.591).培养24 h后,低氧对照组、低氧KGF组及低氧HIF-1α组G1期细胞百分比明显高于低氧联合组(P=0.001、0.030、0.014),且S期细胞百分比显著低于低氧联合组(P=0.001、0.012、0.010).(4)培养24 h后,与常氧对照组比较,低氧对照组细胞凋亡率明显升高(P =0.018),低氧联合组细胞凋亡率明显降低(P=0.008).培养24 h后,与低氧对照组比较,低氧KGF组和低氧联合组细胞凋亡率明显降低(P=0.004、0.001);低氧联合组细胞凋亡率明显低于低氧KGF组和低氧HIF-1α组(P=0.032、0.002).(5)培养24 h后,与常氧对照组比较,低氧联合组细胞ATP含量无明显变化(P=0.209),其余各组细胞ATP含量均明显降低(P=＜0.001、0.001、0.002);与低氧对照组比较,低氧HIF-1α组和低氧联合组细胞ATP含量明显升高(P=0.044、0.001);低氧联合组细胞ATP含量明显高于低氧KGF组和低氧HIF-1α组(P=0.011、0.020).(6)培养24 h后,与常氧对照组比较,低氧对照组、低氧KGF组、低氧HIF-1α组细胞p53蛋白表达量明显升高(P＜0.001),低氧联合组细胞p53蛋白表达量显著低于低氧对照组、低氧KGF组和低氧HIF-1α组(P=0.001、0.001、0.002). 结论 KGF联合HIF-1α对低氧应激大鼠IEC-6细胞具有显著的保护作用,可通过降低其细胞周期阻滞程度及凋亡水平,提高细胞的能量代谢水平,从而促进低氧环境下细胞的存活.%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.Data were processed with one-way analysis of variance and least significant difference test.Results (1) After being cultured for 24 h,cells cultured in normoxic incubator grew well with oval or round shapes and clear cytoplasm,and cells cultured in hypoxic incubator showed irregular shapes such as fusiform or starlike shape,with black particle in cytoplasm.(2) After being cultured for 24 h,cell survival rates of normoxia blank group,normoxia KGF group,normoxia HIF-1α group,and normoxia combine group were (107.4 ± 8.7)％,(109.8 ± 2.9) ％,(115.8 ± 7.4) ％,and (112.8 ± 10.6) ％ respectively.There was no significantly statistical difference in general comparison of cell survival rates among the above groups (F =0.685,P =0.586).After being cultured for 24 h,cell survival rates of hypoxia control group,hypoxia KGF group,hypoxia HIF-1α group,and hypoxia combine group were (35.1 ±4.6)％,(52.9 ±6.8)％,(56.2 ±3.1)％,and (71.2 ±9.6)％ respectively,which were significantly lower than (106.3 ± 12.3)％ of normoxia control group (P ＜0.001).Survival rates of cells in hypoxia KGF group,hypoxia HIF-1α group,and hypoxia combine group were significantly higher than the rate of cells in hypoxia control group (P =0.023,0.009,＜0.001).Survival rate of cells in hypoxia combine group was significantly higher than the rates of cells in hypoxia KGF group and hypoxia HIF-1α group (P =0.017,0.045).(3) After being cultured for 24 h,percentage of cells in G1 phase in hypoxia control group was significantly higher than that of cells in normoxia control group (P =0.030),percentages of cells in S phase in hypoxia control group,hypoxia KGF group,and hypoxia HIF-1α group were obviously lower than the percentage of cells in normoxia control group (P =0.020,0.031,0.026),and percentages of cells in different phases in other groups were close to those of cells in normoxia control group (P =0.516,0.107,0.052,0.985,0.637,0.465,0.314,0.591).After being cultured for 24 h,percentages of cells in G1 phase in hypoxia control group,hypoxia KGF group,and hypoxia HIF-1α group were obviously higher than the percentage of cells in hypoxia combine group (P =0.001,0.030,0.014),and percentages of cells in S phase in the above 3 groups were obviously lower than the percentage of cells in hypoxia combine group (P =0.001,0.012,0.010).(4) After being cultured for 24 h,compared with that of cells in normoxia control group,apoptosis rate of cells in hypoxia control group obviously increased (P =0.01 8),and apoptosis rate of cells in hypoxia combine group obviously decreased (P =0.008).After being cultured for 24 h,compared with that of cells in hypoxia control group,apoptosis rates of cells in hypoxia KGF group and hypoxia combine group obviously decreased (P =0.004,0.001).Apoptosis rate of cells in hypoxia combine group was obviously lower than those of cells in hypoxia KGF group and hypoxia HIF-1 α group (P =0.032,0.002).(5) After being cultured for 24 h,compared with that of cells in normoxia control group,the content of ATP of cells in hypoxia combine group changed unobviously (P =0.209),and content of ATP of cells in the other groups obviously decreased (P =＜0.001,0.001,0.002).Content of ATP of cells in hypoxia HIF-1α group and hypoxia combine group was obviously higher than that of cells in hypoxia control group (P =0.044,0.001).Content of ATP of cells in hypoxia combine group was obviously higher than that of cells in hypoxia KGF group and hypoxia HIF-1 α group (P =0.011,0.020).(6) After being cultured for 24 h,protein expressions of p53 of cells in hypoxia control group,hypoxia KGF group,and hypoxia HIF-1α group were obviously higher than that of cells in normoxia control group (P ＜ 0.001),and protein expression of p53 of cells in hypoxia combine group was obviously lower than those of cells in hypoxia control group,hypoxia KGF group,and hypoxia HIF-1α group (P =0.001,0.001,0.002).Conclusions KGF combined with HIF-1α have significant protective effects on IEC-6 of rats with hypoxia stress,and can improve its survival in hypoxic environment by inhibiting cell cycle arrest,reducing the level of apoptosis,and increasing level of energy metabolism.
