肠上皮细胞紧密连接模型的建立与验证  

Establishment and verification of tight junction model of intestinal epithelial cells

作　　者：陈婷[1] 张娇 王晓鸽 康楠[4] 王凤云[1] 唐旭东[1] CHEN Ting;ZHANG Jiao;WANG Xiaoge;KANG Nan;WANG Fengyun;TANG Xudong(Institute of Digestive Diseases,Xiyuan Hospital of China Academy of Chinese Medical Sciences,Beijing 100091,China;不详)

机构地区：[1]中国中医科学院西苑医院脾胃病研究所,北京100091 [2]北京市昌平区中医医院脾胃病科 [3]河南中医药大学附属第一医院脾胃肝胆科 [4]济宁医学院附属医院中医科

出　　处：《山东医药》2022年第35期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金面上项目(82074420);中国科协青年人才托举工程专项(CACM-2019-QNRC1-02);中国中医科学院优秀青年科技人才培养专项(ZZ13-YQ-009、ZZ13-YQ-009-C1)。

摘　　要：目的 体外分化Caco-2细胞建立肠上皮细胞紧密连接模型,并对其进行综合评价。方法 将融合至90%的Caco-2细胞以8×104/孔接种于Transwell 24孔培养板上,每孔基底侧加入MEM培养基600μL,顶侧加入细胞悬液100μL;接种后第1周隔日更换培养液,第2周起每日更换培养液,直至培养第21天。细胞培养第3、5、7、15、19、21天,使用细胞电阻仪测定跨膜电阻(TER);细胞培养第5、15、21天,取细胞单层顶侧与基底侧培养基并检测其碱性磷酸酶(ALP)活力,计算顶侧与基底侧ALP活力比;取体外分化培养21天的Caco-2细胞,使用多功能酶标仪检测大分子物质荧光黄(FD-4)通过单层细胞的百分率(即FD-4通透率)及跨膜转运表观渗透系数(Papp),透射电镜下观察细胞形态。结果 Caco-2细胞体外分化培养第3、5、7、15天的TER均明显低于第19、21天(P均<0.05),Caco-2细胞体外分化培养第19和21天TER比较无统计学差异(P>0.05)。Caco-2细胞体外分化培养第5、15、21天顶侧与基底侧ALP活力比分别为1.43±0.16、2.43±0.28、4.85±0.25,Caco-2细胞体外分化培养第5、15天顶侧与基底侧ALP活力比均明显低于第21天(P均<0.05)。体外分化培养第21天的Caco-2细胞FD-4通透率为2.31%±1.12%,Papp为(1.95±0.90)×10^(-7)cm/s。透射电镜观察结果显示,体外分化培养第21天的Caco-2细胞由微绒毛形成的刷状缘排列整齐致密;细胞顶侧面分化出完整的紧密连接结构,表现为连续的融合点,呈“焊接线”样围绕在细胞膜的顶部,相邻细胞膜被“焊接”在一起,形成绳索样结构。结论 Caco-2细胞在Transwell板体外分化21天后可形成肠上皮细胞紧密连接结构,证实成功构建肠上皮细胞紧密连接模型。Objective To differentiate the Caco-2 cells in vitro to construct a tight junction model of intestinal epithelial cells,and to evaluate the model comprehensively.Methods Caco-2 cells with fusion of 90%were inoculated on Transwell 24-well culture plate at 8×10~4/well.MEM medium(600μL)was added to the base side of each well,and 100μL cell suspension was added to the top side of each well.The culture medium was changed every other day for the first week after inoculation,and every day from the second week until the 21st day of culture.On day 3,5,7,15,19 and 21 of cell culture,the transmembrane resistance(TER)was measured by cell resistance meter.On the 5th,15th and 21st days of cell culture,the culture medium of the apical side and the basal side of the cell monolayer was used to detect the ALP activity,and the ALP activity ratio of the apical side and the basal side was calculated.The percentage of FD-4 passing through monolayer cells(FD-4 permeability)and transmembrane apparent permeability coefficient(Papp)were detected by multifunctional enzyme marker.Cell morphology was observed under transmission electron microscope.Results The TER of Caco-2 cells on day 3,5,7 and 15 in vitro was significantly lower than that on day 19 and 21(P<0.05),and there was no statistical difference in vitro differentiation of Caco-2 cells between the 19th and 21st days(P>0.05).The ALP activity ratios of Caco-2 cells on day 5,15 and 21 in vitro were 1.43±0.16,2.43±0.28 and 4.85±0.25,respectively.The ALP activity ratios of Caco-2 cells on day 5 and 15 in vitro were significantly lower than that on day 21(all P<0.05).The FD-4permeability of Caco-2 cells on the 21st day of differentiation and culture in vitro was 2.31%±1.12%,and Papp was(1.95±0.9)×10^(-7).The results of transmission electron microscopy(TEM)showed that the brush margins formed by microvilli of Caco-2 cells on the 21st day of in vitro differentiation and culture were orderly and compact.The cell apical side differentiated into a complete tightly connected structure,

关 键 词：细胞紧密连接 肠上皮细胞 跨膜电阻 碱性磷酸酶 荧光黄通透率 

分 类 号：R574[医药卫生—消化系统]