摘要
目的:探讨血小板活化因子(PAF)是否会引起肠黏膜屏障的破坏以及这种破坏机制是否与紧密连接相关,并观察肠三叶因子(ITF)的保护作用.方法:培养人结肠腺癌细胞株Caco-2,分别设正常对照组:不加刺激物及干预因素;实验组:加入PAF,终浓度分别为50 ng/L、100 ng/L和200 ng/L;ITF预防组:先加入ITF0.3 g/L,30 min后加入PAF 100 ng/L;ITF治疗组:先加入PAF 100 ng/L,30 min后加入ITF0.3 g/L,24 h后进行实验.MTT比色法检测细胞活力;测定跨上皮电阻TER和荧光黄的透过量反映肠上皮细胞单层通透性;RT-PCR法检测紧密连接蛋白ZO-1和Occludin mRNA表达的变化;免疫荧光染色观察紧密连接蛋白ZO-1和Occludin的形态学.结果:PAF未影响到细胞的增殖和细胞活力.各浓度PAF作用24 h后,细胞单层通透性增加,跨上皮电阻(TER)下降,荧光黄透过增加.ITF治疗组及预防组TER下降值较模型组明显降低(122.2±14.7,100.3±10.9 vs 210.3±26.4,P<0.05),荧光黄透过量减少(10226.1±556.2,9711.2±364.9 vs 11601.2±693.5,P<0.05),预防组作用更明显.PAF作用后,ZO-1和Occludin mRNA表达均有下降,以100 ng/L组改变最为明显,ITF预防组较之表达增加(1.28±0.06 vs 1.07±0.05,1.13±0.07 vs 0.81±0.06,P<0.05),ITF治疗组改变不明显.免疫荧光染色发现ZO-1和Occludin主要分布在细胞内近胞膜处.PAF作用后,ZO-1及Occludin形态学改变,预防性给予ITF后,可明显减轻这种改变.结论:PAF可引起肠黏膜屏障破坏,其机制可能和紧密连接蛋白的破坏相关;ITF可以通过改变紧密连接蛋白的表达而部分恢复肠黏膜正常通透性,起到保护作用.
AIM: To explore whether platelet-activating factor (PAF) can disrupt the intestinal epithelial barrier anti is associated with tight junction, and to observe the protective effect of intestinal trefoil factor (ITF).
METHODS: Caco-2 cells were cultured withRPMI 1640 containing 15% fetal bovine serum for 7 days. Cells were then treated with different concentrations of PAF (0, 50, 100 and 200 ng/L) for 24 hours until they became fused. The protective effect of ITF was observed, rITF (0.3 g/L) was treated 30 minutes before (prevention group) and after PAF (treat proup) was given and cells were incubated for 24 hours. MTT was used to detect cell vigor. Epithelial monolayer permeability was measured by trans-epithelial electrical resistance (TER) and mucosal to serosal flux of paracellular marker luminal yellow. RT-PCR was used to detect mRNA expression of ZO-1 and Occludin. Indirect immunofluorescence was used to localize ZO-1 and Occludin. RESULTS: PAF had no effect on the Caco-2's vigor and proliferation. Different concentrations of PAF increased the intestinal epithelial paracellular permeability. TER decreased and luminal yellow flux increased. Treatment and prevention group of rITF partially recovered the increased permeability (122.2 ± 14.7, 100.3 ± 10.9 vs 210.3 ± 26.4, P 〈 0.05; 10226.1 ± 556.2, 9711.2 ± 364.9 vs 11601.2 ± 693.5, P 〈 0.05). RT-PCR demonstrated that the mRNA expression of ZO-1 and Occludin in PAF groups treated with different concentrations (especilly 100 ng/L) for 24 hours was significantly different from that in the prevention group (1.07 ± 0.05 vs 1.28 ± 0.06, 0.81 ± 0.06 vs 1.13 ± 0.07, P 〈 0.05). However, treatment of rlTF did not change the expression of mRNA. In the control Caco-2 monolayers, ZO-1 and Occludin proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders. PAF caused a progressive disturbance in the continuity of ZO-1 and Occludin localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact, rITF could partially recover the disrupted distribution of ZO-1 and Occludin proteins.
CONCLUSION: PAF disrupts the intestinal epithelial barrier. Its mechanism may be correlated to the decreased expression of ZO-1 and Occludin proteins, which can be partially recovered after treatment of rITF.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第4期372-378,共7页
World Chinese Journal of Digestology
基金
辽宁省教育厅科研基金
No.20122166~~