Caco-2细胞缺氧复氧损伤后二肽载体表达及生物学功能的改变

目的：探讨Caco-2细胞缺氧复氧（anoxia/reoxygenation,A/R)损伤后二肽载体表达及生物学功能的变化。方法：采用Caco-2细胞A／R方法，模拟临床缺血再灌注（ischemia/reperfusion injury,I/R)。Caco-2细胞正常培养和A／R后（缺氧90min，复氧30min)后，测定二肽载体对头孢氨苄的摄取功能；采用流式细胞术观察损伤后Caco-2细胞的凋亡情况;员时比较两种培养情况下二肽载体的mRNA水平。结果：A／R后二肽载体对头孢氨苄的摄取能力下降；损伤后Caco-2细胞凋亡水平较正常细胞明显提高；Northern blotting显示损伤后二肽载体mRNA水平下调。结论：A／R后Caco-2细胞二肽载体mRNA下降从基因水平下调了二肽载体对头孢氨苄的摄取功能。提示Caco-2细胞A／R或临床I／R后肠上皮细胞刷状缘二肽载体对二肽的摄取功能下降。

Changes of Expression and Functions of Dipeptide Transporter after Anoxia/reoxygenation in Caco-2 Cells

AIM:To determine the changes of biological functions and expression of dipeptide transporter in Caco 2 cells with anoxia/reoxygenation(A/R) injury. METHOD:The human adenocarcinoma cell line Caco 2 cells were as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco 2 cells grown on multiple well dishes (24 pore) were cultured with or without A/R injury and then uptake of cephalexin was measured. Apoptosis of Caco 2 cells was examined by Flow cytometry, and PepT1 mRNA were also determined by Northern blotting. RESULT:The uptake of cephelaxin across apical membranes of Caco 2 cells after the injury of A/R was significantly decreased compared with that of controls ( P <0 05). Examination of Flow cytometry indicated that the apoptosis index of injuried Caco 2 cells remarkably increased. Northern blotting analysis showed that the level of PepT1 mRNA of injuried Caco 2 cells was greatly decreased compared to controls. CONCLUSION:The present results indicated that the functions of dipeptide transporter in Caco 2 cells were greatly downregulated after A/R injury. The alteration in the gene expression may be a mechanism of regulation of PepT1 In addition, Caco 2 cells take up cephalexin by a Proton dependent dipeptide transporters that closely resemble the transporters present in the intestine. Caco 2 cells represent an ideal cellular model for future studies of the dipeptide transporter.