正交设计优化低温应激诱导IEC-6细胞损伤条件的实验研究

Experimental study on orthogonal design to optimize the damage condition of IEC-6 cells induced by hypothermia stress

目的采用正交实验设计法,优化低温应激诱导大鼠小肠隐窝上皮细胞(intestinal epithelial crypt,IEC-6)损伤的条件。方法以温度、处理时间和接种密度为正交优化的3个因素,每个因素设定3个水平,分别为处理温度(4℃,10℃,22℃),低温处理时间(2 h,4 h,6 h),接种密度(1×105/m L,2. 5×105/m L,5×105/m L),根据三因素三水平设计正交实验表,得到9个实验组,按相应条件处理,细胞增殖/毒性检测试剂法分析细胞毒性,在50%<细胞存活率<90%范围内获得最优组合条件。应用最优组合条件处理实验组IEC-6细胞,同时设置正常对照,镜下观察细胞形态,流式细胞术检测细胞凋亡和线粒体膜电位。结果 (1)在50%<细胞存活率<90%范围内,低温应激诱导IEC-6细胞损伤的最优组合条件为:处理温度4℃,接种密度5×105/m L,低温处理时间4 h。(2)与对照组相比,最优组合条件下实验组细胞失去正常形态,同时线粒体膜电位去极化增强,细胞凋亡显著加重(P<0. 01)。表现为前者以早期凋亡为主,细胞凋亡率为(4. 41±1. 36)%,低强度红色荧光细胞(R2)占比(5. 24±0. 57)%,后者则以晚期凋亡和坏死为主,细胞凋亡率为(23. 70±2. 94)%,低强度红色荧光细胞(R2)占比(31. 12±3. 66)%。结论在50%<细胞存活率<90%范围内,低温应激诱导IEC-6细胞损伤的最优组合条件为处理温度4℃、接种密度5×105/m L、低温处理时间4 h,在该条件下,IEC-6细胞出现明显凋亡。

Objective To optimize the rat small intestinal epithelial crypt （IEC-6） parameters of hypothermia stress-induced damage of （：ells aeeoding to orthogonal experimental design. Methods In our study, the impact of treatment temperature, treatment time and cell inoeulum density were investigated. Three levels of each factor were defined as treatment temperature （4 ℃, 10 ℃, 22 ℃）, treatment time （2 h, 4 h, 6 h）, cell inoeulum density （1×10^5/mL, 2.5×10^5/mL, 5×10^5/mL）. According to the and three-factor three-level orthogonal design, nine experimental groups were obtained. The eytotoxieity was analyzed with cell counting kit-8, and the optimal combination was obtained within the range of cell viability from 50% to 90%. The optimal combination conditions was used to treat the experimental group IEC-6 cells, and the normal control was performed. The cell morphology was observed under microscope. Apoptosis and mitoehondrial membrane potential were detected by flow eytometry. Results ①Within the cell viability range of 50% to 90%, the optimum combination conditions of hypothermia stress-induced IEC-6 cells damage were treatment temperature of 4 ℃ , cell inoeulum density of 5×10^5/mL, and treatment time of 4 h. ②Compared with the control group, the cells in the experimental group lost normal morphology under optimal combination conditions, the mitoehondrial membrane potential depolarization enhanced and the vate of apoptosis significantly increased （ P〈0.01 ）. The former displayed mainly as early apoptosis, with the apoptotie rate was （4.41 ± 1.36）%, and low-intensity red fluorescent cells （R2） accounted for （5.24±0.57 ）%. The latter displayed mainly as late apoptotie and necrosis, with the apoptotie rate was （23.70±2.94）%, and the low intensity red fluorescent cells （R2） accounted for （31.12±3.66） %. Conclusion Within the range of cell viability from 50% to 90%, the optimum combination conditions of hypothermia stress-induced IEC-6 cells damage were at treatment temperature of 4 ℃ , cell inoeulum density of 5×10^5/mL, and treatment time of 4 h. Under these conditions, IEC-6 cells showed significant apoptosis.