摘要
目的研究铜对Caco2细胞单层细胞间通透性、P糖蛋白(Pgp)活性的影响。方法应用Caco2细胞单层模型,通过测定铜暴露后细胞单层的跨上皮细胞电阻(TEER)来反映通透性的改变;通过免疫荧光和荧光染色观察铜对紧密连接蛋白ZO1和F微丝的影响;通过测定细胞单层对罗丹明123的跨膜转运和细胞内罗丹明123的蓄积来反映P糖蛋白活性的改变。结果细胞单层顶面铜暴露(30～100μmol/L,Hanks缓冲溶液,0～3h)可导致TEER值呈浓度和时间依赖性降低,同时伴随着F微丝的解聚,但对紧密连接蛋白ZO1无显著影响;在不影响细胞活性和细胞单层通透性的剂量下,顶面铜暴露(300μmol/L,完全培养基,24h)后细胞单层对罗丹明123的表观通透系数(Papp)BL→AP从对照组的(737±020)10-6cm/s降低为(643±027)×10-6cm/s,PappAP→BL从对照组(123±005)×10-7cm/s增加为(341±008)×10-7cm/s,同时细胞内罗丹明-123的蓄积量从对照组的每膜(031±001)nmol增加为每膜(050±003)nmol。结论铜可显著增加Caco2细胞单层的通透性和抑制Pgp的活性,从而推测可能影响肠道上皮细胞的正常屏障功能。
Objective To investigate the effects of copper on permeability and P glycoprotein(P gp) of Caco 2 cell monolayers Methods The differentiated Caco 2 cell model was used in this study Permeability of cell monolayers was reflected by monitoring transepithelial electrical resistance (TEER); distribution of tight junctional protein ZO 1 was measured by immunofluorescent staining;F actin was measured by fluorescence staining; and Activity of P gp was reflected by changes of transcellular transport and accumulation of Rho 123 in Caco 2 cells Results Apical treatment with copper (30 100 μmol/L, Hanks’buffered salt solution,up to 3 hours) induced a time and concentration dependent increase in permeability reflected by progressive decrease of TEER of Caco 2 cell monolayers, accompanied by deorganization of F actin,but without significant effects on tight junctional protein ZO 1; at a dose without any adverse effects on viability and permeability of Caco 2 monolayers, copper treatment (300 μmol/L,complete medium,24 hours) decreased Papp BL→AP from 7 37±0 20×10 -6 cm/s (controls) to (6 43±0 27)×10 -6 cm/s, the increased Papp AP→BL from (1 23±0 05)×10 -7 cm/s (controls)to (3 41±0 08)×10 -7 cm/s,and enhanced the intracellular Rho 123 from (0 31±0 01) nmol/filter (controls)to (0 50±0 03) nmol/filter Conclusion Copper might alter the barrier functions of Caco 2 cells through increasing the permeability and inhibiting P gp of Caco 2 cells
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2004年第6期406-410,共5页
Chinese Journal of Preventive Medicine
基金
国际铜业协会资助项目(TPT0542E)
