不同接种密度对Caco-2细胞吸收模型分化及去极化评价指标的影响  

Effects of Cell Density on Differentiation and Depolarization of Caco-2 Cell Monolayers

作　　者：唐家明[1] 吕林[2] 张丽阳[2] 邹亚学[1] 王秋悦[1] 罗绪刚[2] 李素芬[1] 

机构地区：[1]河北科技师范学院,河北秦皇岛066600 [2]中国农业科学院北京畜牧兽医研究所

出　　处：《河北科技师范学院学报》2016年第3期66-70,共5页Journal of Hebei Normal University of Science & Technology

基　　金：国家自然科学基金项目(项目编号:31272465)

摘　　要：为确定不同接种密度对Caco-2细胞吸收模型分化及去极化评价指标的影响,以及细胞模型适用于吸收和转运试验的时间,采用Caco-2细胞高密度(1×105cells·cm-2)、中密度(5×104cells·cm-2)和低密度(3×104cells·cm-2)等3个接种密度,分别接种于带聚碳酸酯微孔滤膜插槽的Transwell 6孔板上,培养29 d,通过测定跨膜电阻值(TEER)、碱性磷酸酶活性(ALP)和酚红透过率,评价细胞单层的完整性、极化和透过性。结果表明,高、中、低密度接种分别于第12天、第15天和第18天TEER值超过600Ω·cm2,第27天、第29天和第29天TEER值开始下降。高、中、低密度接种分别于第15天、第18天和第21天上下侧碱性磷酸酶活性的比值都能达到10倍以上,第29天开始降低。高、中、低密度接种分别于第9天、第12天和第15天酚红透过率小于0.5%。综合以上3项指标,高密度接种细胞在第15天、中密度接种细胞在第18天、低密度接种细胞在第21天开始,可以用于吸收和转运试验,27 d以后细胞活力就开始下降,不适合做细胞模型。In order to investigate the effects of cell density on differentiation and depolarization of Caeo-2 cell monolayer and determine the optimal time for absorption and transport experiment, the Caeo-2 cells were inoculated at a density of 1 x 105（high, H）, 5 × 10^4（medium, M） or 3 × 10^4（low, L） cells · cm-2 on six-well transwell plates and cultured for 29 days. The transepithelial electrical resistance （TEER） , alkaline phosphatase activities of apical side and basolateral side, and diffusion of phenol red were measured to evaluate the integrity and permeability of cell monolayers. The results showed that the TEER value exceeded 600Ω·cm2 at 12 d, 15 d and 18 d, and decreased at 27 d, 29 d and 29 d when the cells were inoculated at a density of H, M or L, respectively. The ratios of alkaline phosphatase activities of apical side to basolateral side were more than 10 at 15 d, 18 d and 21 d when the cells were inoculated at a density of H, M or L, respectively. The diffusions of phenol red from the apical side to the basolateral side were lower than 0.5% at 9 d, 12 d and 15 d when the cells were inoculated at a density of H, M or L, respectively. According to the above three indexes, the Caco-2 cell monolayers were satisfactory from 15 d, 18 d and 21 d for absorption and transport experiment when the ceils were inoculated at a density of H, M or L, respectively. The Caco-2 cell monolayers more than 27 d were no longer suitable for absorption and transport experiment because of their lower vitality.

关 键 词：CACO-2细胞 跨膜电阻(TEER) 碱性磷酸酶(ALP) 酚红 

分 类 号：R329.27[医药卫生—人体解剖和组织胚胎学]