鳕鱼皮胶原蛋白肽在Caco-2细胞单层模型中的吸收机制

Absorption Mechanism of Cod Skin Collagen Peptide in Caco-2 Cell Monolayer Model

目的:鳕鱼皮胶原蛋白肽(cod skin collagen peptide,CSCP)对肝损伤具有较好的保护作用,但其吸收机制尚不明确,本实验拟采用人结肠腺癌Caco-2细胞单层模型对CSCP在肠道中的吸收机制进行研究,为CSCP在动物肠道内的吸收机制提供依据。方法:在进行转运实验前,先对CSCP在人工胃、肠液、不同pH值条件及在Caco-2细胞单层中的稳定性进行评价;采用CCK-8(cell counting kit-8)法确定CSCP在转运实验中的最高质量浓度,然后建立Caco-2细胞单层模型并测定跨膜电阻值和碱性磷酸酶活力以检验细胞单层的致密性、完整性和极化现象;考察转运时间、CSCP质量浓度、维拉帕米、MK-571、氧化苯砷和去氧胆酸钠对转运的影响,利用高效液相色谱法检测CSCP质量浓度并计算累积转运量和表观渗透系数。结果:在3h内,CSCP在人工胃、肠液及接近胃肠道pH值环境中基本保持稳定,转运过程中未见多肽发生水解。CSCP的转运具有时间和浓度依赖性,不受维拉帕米和氧化苯砷的影响,去氧胆酸钠和MK-571对CSCP的转运具有非常显著的促进作用(P<0.05)。结论:CSCP跨Caco-2细胞单层转运的机制为细胞旁路途径,其外排受到多药耐药蛋白的介导。

Objective： Cod skin collagen peptide （CSCP） has a good protective effect on liver injury, but the absorption mechanism of CSCP is still not clear. In this study, the Caco-2 cell monolayer model was used to investigate the absorption mechanism of CSCP in order to provide an experimental basis for the study of the absorption mechanism of CSCP in the animal intestine. Methods： The stability of CSCP in artificial gastric juice, artificial intestinal juice, different pH conditions, and Caco-2 cell monolayers were evaluated. Subsequently, the highest concentration of CSCP in the transport experiment was determined by using cell counting kit-8 （CCK-8） assay. The Caco-2 cell monolayer model was established, and its compactness, integrity and polarization were evaluated by measuring transepithelial electrical resistance and the activity of alkaline phosphatase. The effects of transport time, CSCP concentration, vrapamil, MK-571, phenylarsine oxide and odium deoxycholate on the transport efficiency were investigated by using reversed-phase high performance liquid chromatography to determine CSCP concentration and calculating apparent permeability coefficients and accumulated transport of CSCP. Results： CSCP was relatively stable in artificial gastric juice, artificial intestinal juice, different pH conditions and Caco-2 cell monolayers for 3 h. No polypeptides were found to be hydrolyzed during the transport process. The transport of CSCP was positively correlated to the transport time and CSCP concentration and was not affected by addition of verapamil or phenylarsine oxide. In the presence of sodium deoxycholate and MK-571, the transport of CSCP was significantly promoted （P 〈 0.05）. Conclusion： The mechanism of CSCP transport across Caco-2 cell monolayer was related to cell bypass and CSCP efflux was mediated by multidrug resistance proteins.