CAJ | 학술논문

本试验旨在建立家兔空肠上皮细胞的体外分离培养方法，为进一步研究外源物质对家兔小肠上皮结构功能的影响及其作用机制提供体外模型。试验以新生新西兰白兔（1～3日龄）空肠为研究对象，利用由链霉蛋白酶 E、胶原酶Ⅳ和二硫苏糖醇组成的混合消化液消化空肠组织，通过2％山梨醇密度梯度离心来富集隐窝细胞团。运用相差消化法和差速贴壁法纯化家兔空肠上皮细胞，并从形态学观察、碱性磷酸酶染色、细胞角蛋白8免疫荧光染色和肠上皮细胞标志物基因表达分析4个方面鉴定家兔空肠上皮细胞。结果表明：应用链霉蛋白酶 E、胶原酶Ⅳ和二硫苏糖醇混合消化液可获得大量的细胞团，细胞团12 h 贴壁并向周围辐射生长，生长的细胞呈典型的三角形或多角形，5～7 d 融合成片，呈单层生长、互不重叠；原代细胞能够进行传代、纯化；经碱性磷酸酶染色和细胞角蛋白8免疫荧光染色均呈阳性；可表达肠上皮细胞标志蛋白（脂肪酸结合蛋白2、肠肽酶、E-钙黏蛋白、紧密连接蛋白-4）的基因。以上结果表明，本试验成功建立了家兔空肠上皮细胞的体外分离培养方法。
본시험지재건립가토공장상피세포적체외분리배양방법，위진일보연구외원물질대가토소장상피결구공능적영향급기작용궤제제공체외모형。시험이신생신서란백토（1～3일령）공장위연구대상，이용유련매단백매 E、효원매Ⅳ화이류소당순조성적혼합소화액소화공장조직，통과2％산리순밀도제도리심래부집은와세포단。운용상차소화법화차속첩벽법순화가토공장상피세포，병종형태학관찰、감성린산매염색、세포각단백8면역형광염색화장상피세포표지물기인표체분석4개방면감정가토공장상피세포。결과표명：응용련매단백매 E、효원매Ⅳ화이류소당순혼합소화액가획득대량적세포단，세포단12 h 첩벽병향주위복사생장，생장적세포정전형적삼각형혹다각형，5～7 d 융합성편，정단층생장、호불중첩；원대세포능구진행전대、순화；경감성린산매염색화세포각단백8면역형광염색균정양성；가표체장상피세포표지단백（지방산결합단백2、장태매、E-개점단백、긴밀련접단백-4）적기인。이상결과표명，본시험성공건립료가토공장상피세포적체외분리배양방법。
This study was aimed to establish a method to culture jejunal epithelial cells( JEC)isolated from rabbits,and provide an in vitro model for the further study of the effects of exogenous substances on rabbit in-testinal epithelial structure and mechanism. Newborn New Zealand white rabbits(1 to 3 days of age)were used,jejunum was removed and JEC were isolated by the application of digestive juice mixed with pronase E, collagenase Ⅳ and dithiothreitol(DTT),and the crypt cell aggregates were enriched by density gradient cen-trifugation of 2% sorbitol. The rabbit JEC were purified by differential digestion method and differential veloci-ty adherent method. Cells were identified by morphological observation,alkaline phosphatase staining,cytoker-ain-8 immunofluorescence staining and the gene expression analyses of enterocyte markers. The results showed that the application of digestive juice mixed with pronase E,collagenase Ⅳ and DTT could successfully get a lot of rabbit JEC aggregates,which readily adhered to culture dishes within 12 hours and grown to around ra-dially,growing cells showed typical triangular or polygonal,reached confluence within 5 to 7 days,and showed monolayer and non-overlapping;cells could be passaged and purified;cells were stained positive for alkaline phosphatas staining and cytokerain-8 immunofluorescence staining;enterocyte markers,such as fatty acid binding protein 2,enterokinase,E-cadherin,claudin-4,were expressed in JEC. These results show that an ideal method for isolating and culturing epithelial cells of rabbit jejunum is established.