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目的探讨在脱氧胆酸(deoxycholic acid,DCA)诱导Barrett食管(Barrett’s esophagus,BE)形成中,Krüppel样锌指转录因子4(Krüppel-like factor 4,KLF4)与同源异型框转录因子2(caudalrelated homeodomain transcription factor 2,CDX2)的作用。方法采用免疫组化S-P法检测49例人正常食管组织和22例BE组织中KLF4、CDX2的表达;体外培养Het-1A细胞,依据DCA处理时间分为0 h组(未经DCA处理)、4 h组、8 h组、12 h组并进行DCA处理。分别设置空白对照组、阴性siRNA对照组、阴性siRNA+DCA处理12 h组、KLF4-siRNA干扰组、KLF4-siRNA干扰+DCA处理12 h组,进行相应处理。设置空白对照组、阴性病毒对照组、阴性病毒+DCA处理12 h组、KLF4过表达病毒组,进行相应处理。待上述各实验组处理结束后,运用Real-time PCR、Western blot检测各组KLF4、CDX2、MUC2mRNA及蛋白的表达。结果免疫组化检测发现:在22例BE组织中,KLF4阳性表达15例,阳性率68.18%,阳性染色主要集中在细胞核;CDX2阳性表达16例,阳性率72.73%,阳性染色也主要集中在细胞核。与人正常食管鳞状上皮组织比较,BE组织中KLF4、CDX2表达均增高(χ2=18.642,P<0.05;χ2=23.678,P<0.05)。Real-time PCR及Western blot检测结果显示:随DCA处理时间的增加,Het-1A细胞KLF4、CDX2、MUC2的表达逐渐升高(P<0.05);KLF4-siRNA干扰组及KLF4-siRNA干扰+DCA处理12 h组中,KLF4、CDX2、MUC2表达均下调,且不再随DCA处理的刺激而升高(P<0.05);此外,阴性病毒+DCA处理12 h组及KLF4过表达病毒组中,KLF4、CDX2、MUC2表达均上调,且KLF4过表达病毒组3个基因蛋白表达上调更为显著(P<0.05)。结论 DCA通过KLF4上调CDX2,间接引起BE标志性分子MUC2的表达上调,从而促进正常食管上皮向BE转化。
OBJECTIVE: To investigate the effect of Krüppel-like factor 4 (KLF4) and caudalrelated genes on the formation of Barrett’s esophagus (BE) induced by deoxycholic acid (DCA) homeodomain transcription factor 2, CDX2). Methods Immunohistochemical SP method was used to detect the expression of KLF4 and CDX2 in 49 human normal esophageal tissues and 22 cases of BE tissues. Het-1A cells were cultured in vitro and were divided into 0 h group (without DCA treatment), 4 h group, 8 h group, 12 h group and DCA treatment. The control group, negative siRNA control group, negative siRNA + DCA group for 12 h, KLF4-siRNA interference group, KLF4-siRNA interference + DCA group for 12 h were set up for the corresponding treatment. The control group, negative control group, negative control group + DCA for 12 h and KLF4 overexpression group were set up and treated accordingly. After the above experimental groups were over, the expression of KLF4, CDX2 and MUC2 mRNA and protein in each group were detected by Real-time PCR and Western blot. Results Immunohistochemical results showed that in 22 cases of BE tissue, 15 cases were positive for KLF4, the positive rate was 68.18%. The positive staining was mainly in the nucleus. The positive expression rate of CDX2 in 16 cases was 72.73%. The positive staining was mainly in nucleus . Compared with human normal esophageal squamous epithelium, the expression of KLF4 and CDX2 in BE tissues were increased (χ2 = 18.642, P <0.05; χ2 = 23.678, P <0.05). The results of Real-time PCR and Western blot showed that the expression of KLF4, CDX2 and MUC2 in Het-1A cells gradually increased with the increase of DCA treatment time (P <0.05); the interference of KLF4-siRNA and KLF4-siRNA + DCA The expression of KLF4, CDX2 and MUC2 in the untreated group was down-regulated at 12 h, and no longer increased with the stimulation of DCA treatment (P <0.05). In addition, The expression of KLF4, CDX2 and MUC2 were up-regulated, and the expression of three genes in KLF4 overexpression group was up-regulated more significantly (P <0.05). Conclusion DCA up-regulates CDX2 through KLF4, which indirectly leads to the up-regulation of MUC2, a marker of BE, and promotes the normal esophageal epithelium to transform to BE.