摘要目的 观察肠三叶因子(ITF)和黏蛋白对烧伤血清所致肠上皮细胞免疫功能变化的影响.方法 (1)体外培养大鼠小肠上皮细胞株IEC-6,根据培养液添加物质不同,按照随机数字表法将细胞分为5组:①正常对照组,培养液中含10％(指体积分数,下同)小牛血清；②烧伤对照组,培养液含10％烧伤血清；③ITF+烧伤血清组,培养液含10％烧伤血清及终浓度25μg/mL ITF;④黏蛋白+烧伤血清组,培养液含10％烧伤血清及终浓度250 μg/mL黏蛋白；⑤ITF+黏蛋白+烧伤血清组,培养液含10％烧伤血清及终浓度25 μg/mL ITF、250 μg/mL黏蛋白.向各组细胞加入上述培养液的同时,加入大肠杆菌菌液(1×108 CFU/mL,200 μL).继续培养15 min、30 min、1h、2h、3h后,行瑞氏-吉姆萨染色,于显微镜下观察并统计黏附在细胞上的细菌数；采用锥虫蓝染色法观察并统计细胞存活率.每组每时相点样本数均为20.(2)将IEC-6细胞按照随机数字表法分为4组:烧伤对照组、ITF+烧伤血清组、黏蛋白+烧伤血清组、ITF+黏蛋白+烧伤血清组,分别同前加入相应的培养液(不加菌液)培养3、6、12、24、48 h.采用放射免疫分析法测定各时相点培养上清液中TNF-α、IL-6和IL-8 含量,每组每时相点样本数均为6.对实验数据行t检验.结果 (1)烧伤对照组细胞各时相点细菌黏附数量较正常对照组明显增多(t值为2.947 ～8.149,P值均小于0.01).与烧伤对照组比较,其余3个烧伤血清组在加菌后多数时相点细菌黏附的数量明显偏少(t值为-4.733～-2.180,P＜0 05或P ＜0.01).烧伤对照组细胞各时相点存活率与正常对照组比较均明显降低(t值为-4.126～-2 363,P值均小于0 05).ITF+烧伤血清组、黏蛋白+烧伤血清组细胞存活率在部分时相点明显高于烧伤对照组(t值为2.120～3.423,P＜0.05或P＜0.01).ITF+黏蛋白+烧伤血清组细胞存活率加菌后15 min为(96.7±2 4)％,明显高于ITF+烧伤血清组[(94 5±3 1)％,t=2.507,P＜0.05];在3h时为(84.0±6 7)％,明显高于黏蛋白+烧伤血清组[(77 1±8 2)％,t=2.934,P＜0.01].(2)ITF+黏蛋白+烧伤血清组培养6、12、24、48 h,TNF-α含量均低于其余3组(t值为-6.914 ～ -2.889,P＜0.05或P＜0.0i).ITF+黏蛋白+烧伤血清组IL-6含量在部分时相点明显低于其余3组(t值为-7.657～-2.580,P＜0.05或P＜0.01).ITF+黏蛋白+烧伤血清组在培养6、12、24、48 h IL-8含量明显低于烧伤对照组和黏蛋白+烧伤血清组(t值为-8.802 ～ -3.640,P值均小于0.01)；在培养12、24 h明显低于ITF+烧伤血清组(t值分别为-2.786、-2.740,P值均小于0.05).结论 ITF能维护肠上皮细胞功能,抵御细菌黏附,降低细胞死亡率,同时能维护细胞免疫稳态,减少炎症介质释放；ITF与黏蛋白联用效果更明显.

abstractsObjective To observe the effect of intestinal trefoil factor (ITF) combined with mucin on immune function of intestinal epithelial cells (IEC) after being treated with burn rat serum.Methods The rat IEC-6 cell lines were divided into control group (C,cultured in DEME medium containing 10％ calf serum),burn control group (BC,cultured in DEME medium containing 10％ burn rat serum),burn serum + ITF group ( B + I,cultured in DEME medium containing 10％ burn rat serum and 25 μg/mL ITF),burn serum + mucin group ( B + M,cultured in DEME medium containing 10％ burn rat serum and 250 μg/mL mucin),and burn serum + ITF + mucin group ( B + I + M,cultured in DEME medium containing 10％ burn rat serum,25 μg/mL ITF,and 250 μg/mL mucin) according to the random number table.Meanwhile,200 μL suspension of E.coli with density of 1 × 108 CFU/mL was added to each culture.At post culture minute (PCM) 15,30 and post culture hour (PCH) 1,2,3,the number of bacteria adherent to IEC-6 was counted after Wright-Giemsa staining,and cell survival rate was calculated after trypan blue staining,with 20 samples in each group at each time point.(2) Other samples of IEC-6 cells without addition of E.coli were divided into BC,B + I,B + M,and B + I + M groups with the same treatment as above.The supernatant contents of IL-6,IL-8,and TNF-α were determined by radioimmunoassay at PCH 3,6,12,24,48,with 6 samples in each group at each time point.Data were processed with t test.Results ( 1 ) Compared with that in C group,count of adherent bacteria to IEC-6 in BC group at each time point was significantly increased (with t values from 2.947 to 8.149,P values all below 0.01 ).Compared with those in BC group,the counts in B + I,B + M,B + I + M groups at the major time points were significantly decreased ( with t values from - 4.733 to - 2.180,P ＜ 0.05 or P ＜ 0.0l ).(2) Compared with that in C group,cell survival rate in BC group at each time point was obviously lowered ( with t values from -4.126 to - 2.363,P values all below 0.05 ).Cell survival rates in B + I and B + M groups at some time points were significantly elevated as compared with those in BC group ( with t values from 2.120 to 3.423,P ＜ 0.05 or P ＜0.01 ).Cell survival rate in B + I + M group at PCM 15 and PCH 3 was respectively (96.7 ± 2.4) ％ and ( 84.0 ± 6.7 ) ％,which was respectively higher than that in B + I and B + M groups [ (94.5 ± 3.1 ) ％,t =2.507,P ＜0.05; (77.1 ±8.2)％,t =2.934,P ＜0.01].(3) The contents of TNF-α in supernatant of B + I + M group at PCH 6,12,24,48 were significantly lower than those in the other 3 groups ( with t values from - 6.914 to - 2.889,P ＜ 0.05 or P ＜ 0.0l ).The contents of IL-6 in supernatant of B + I + M group at some time points were significantly lower than those in the other 3 groups ( with t values from - 7.657 to -2.580,P ＜0.05 orP ＜0.01).The contents of IL-8 in supernatant of B+I+M group at PCH6,12,24,48 were significantly lower than those in BC and B + M groups ( with t values from - 8.802 to - 3.640,P values all below 0.01 ),and those in B + I + M group at PCH 12,24 were lower than those in B + I group (with t value respectively - 2.786,- 2.740,P value all below 0.05 ).Conclusions ITF can maintain immune function and homeostasis of IEC,prevent bacterial adherence,decrease cell death rate,and reduce release of inflammatory mediators.The effect can be strengthened with addition of mucin.