谷氨酰胺对体外培养肠上皮细胞屏障通透性的影响

Effect of glutamine on intestinal epithelial barrier permeability in vitro

目的:探讨谷氨酰胺保护肠黏膜屏障功能的作用机制.方法:Caco-2细胞(20-30代)应用含有不同浓度谷氨酰胺(0、0.1、1、4 mmol/L)的DMEM培养液在Transwell上培养21 d,检测跨上皮电阻的变化.免疫荧光染色检测紧密连接蛋白occludin的定位、表达.应用不同裂解液制备NP-40可溶性及不溶性蛋白框架,分别进行蛋白印迹杂交检测活性和非活性occludin蛋白的相对含量.结果:与0 mmol/L GLN组比较,补充GLN后,跨上皮电阻明显升高(21.4±0.1,124.5±0.3,173.6±0.2 vs 11.3±0.3,均P<0.05).免疫荧光染色可见0 mmmol/L GLN组occludin呈团状分布于细胞胞质内,随着GLN浓度的增加,occludin阳性荧光染色呈沿细胞膜分布的蜂巢状线性荧光.GLN缺乏不影响65 kDa occludin蛋白的表达,但能降低85 kDa occludin蛋白的表达(1.04±0.03,1.17±0.04,1.29±0.03,1.43±0.06,P<0.05).结论:谷氨酰胺缺乏能够减少活性紧密连接蛋白occludin的表达,增加肠上皮细胞屏障通透性,补充GLN能够阻断这些改变.

AIM：To explore the mechanism of glutamine（GLN）underlying maintenance of intestinal barrier permeability.METHODS：Caco-2 cells were placed on Tran-swell filters and grown in a culture medium composed of DMEM with indicated concentra-tion of GLN（0,0.1,1,4 mmol/L）for 21 days before treatment.Next,transepithelial electrical resistance（TEER）was measured.The localiza-tion and expression of tight junction（TJ）protein occludin were measured under immunofluo-rescence light microscopy.Cells were lysed into detergent-soluble and-insoluble protein frac-tions and the expression of active or non active occludin protein were measured using Western blot.RESULTS：Transepithelial electrical resistancewas significantly raised following GLN supple-mentation compared with 0 mmol/L GLN group（21.4±0.1,124.5±0.3,173.6±0.2 vs 11.3± 0.3,P〈0.05）.Immunofluorescence test showed that occludin protein was localized in cytoplasm,with increasing GLN,positive occludin ap-peared as continuous belt-like structures encircl-ing the cells at the apical cellular junctions in 0 mmol/L GLN group.GLN deprivation did not affect 65-kDa occludin expression,but 85-kDa occludin expression was down-regulated（1.04± 0.03,1.17±0.04,1.29±0.03,1.43±0.06,P〈0.05）.CONCLUSION：Deprivation of glutamine de-creases active occludin protein expression and increases the intestinal barrier permeability,which is partially reversible with GLN supple-mentation.