目的 探讨Hedgehog(Hh)信号通路在缺氧肠上皮屏障功能调控的作用.方法 大鼠小肠上皮细胞系IEC-6细胞分为3组:常氧组(21％氧浓度)、缺氧组(2％氧浓度)、缺氧+环巴胺组(用5 mmol/L的环巴胺预处理30 min后,再给予2％氧浓度进行缺氧处理).RT-PCR检测Hh信号通路IHH、PTCH、GLI-1 mRNA的表达变化,电阻测定仪检测跨上皮电阻(TER),Western blot检测IHH、紧密连接蛋白经典表达分子胞质附着蛋白(ZO-1)、咬合蛋白(Occludin)、闭合蛋白(Claudin-1)的表达情况.组间比较采取单因素方差分析,两两比较采用LSD-t检验.结果 RT-PCR检测结果表明:常氧组Hh信号通路IHH、PTCH、GLI-1 mRNA的相对表达量分别为0.056±0.009、0.459±0.087、0.142±0.023；缺氧组分别为0.303±0.052、0.678±0.073、0.483±0.061,两组比较,差异有统计学意义(t=-14.05,-11.85,-6.52,P＜0.05).Western blot检测结果显示:常氧组和缺氧组的IHH相对蛋白表达量分别为0.39±0.06和0.91±0.15,两组比较,差异有统计学意义(t=-8.08,P＜0.05).常氧组、缺氧组和缺氧+环巴胺组的TER分别为(134±5) Ohm/cm2、(100±6)Ohm/cm2、(118±5)Ohm/cm2,3组比较,差异有统计学意义(F=1.04,P＜0.05).与常氧组比较,缺氧组下降约27.7％(t=7.84,P＜0.05)；与缺氧组比较,缺氧+环巴胺组回升约16.4％,但仍低于常氧组(t=4.23,P＜0.05).常氧组胞质附着蛋白-1、咬合蛋白、闭合蛋白-1的表达分别为1.18±0.24、0.80 ±0.13、0.90±0.09,缺氧组分别为0.58±0.08、0.32±0.05、0.50±0.09,缺氧+环巴胺组分别为0.92 ±0.21、0.43 ±0.10、0.82±0.11,3组比较,差异有统计学意义(F=4.95,2.88,10.09,P＜0.05).缺氧组较常氧组分别降低48.7％、40.0％、55.6％(t=12.86,9.35,18.90,P＜0.05)；缺氧+环巴胺组较缺氧组分别提高59.9％、35.2％、65.1％(=5.63,2.92,6.66,P＜0.05).结论 缺氧条件可刺激激活Hh信号通路,从而引起紧密连接蛋白表达降低,导致肠上皮屏障功能损害.
目的 探討Hedgehog(Hh)信號通路在缺氧腸上皮屏障功能調控的作用.方法 大鼠小腸上皮細胞繫IEC-6細胞分為3組:常氧組(21％氧濃度)、缺氧組(2％氧濃度)、缺氧+環巴胺組(用5 mmol/L的環巴胺預處理30 min後,再給予2％氧濃度進行缺氧處理).RT-PCR檢測Hh信號通路IHH、PTCH、GLI-1 mRNA的錶達變化,電阻測定儀檢測跨上皮電阻(TER),Western blot檢測IHH、緊密連接蛋白經典錶達分子胞質附著蛋白(ZO-1)、咬閤蛋白(Occludin)、閉閤蛋白(Claudin-1)的錶達情況.組間比較採取單因素方差分析,兩兩比較採用LSD-t檢驗.結果 RT-PCR檢測結果錶明:常氧組Hh信號通路IHH、PTCH、GLI-1 mRNA的相對錶達量分彆為0.056±0.009、0.459±0.087、0.142±0.023；缺氧組分彆為0.303±0.052、0.678±0.073、0.483±0.061,兩組比較,差異有統計學意義(t=-14.05,-11.85,-6.52,P＜0.05).Western blot檢測結果顯示:常氧組和缺氧組的IHH相對蛋白錶達量分彆為0.39±0.06和0.91±0.15,兩組比較,差異有統計學意義(t=-8.08,P＜0.05).常氧組、缺氧組和缺氧+環巴胺組的TER分彆為(134±5) Ohm/cm2、(100±6)Ohm/cm2、(118±5)Ohm/cm2,3組比較,差異有統計學意義(F=1.04,P＜0.05).與常氧組比較,缺氧組下降約27.7％(t=7.84,P＜0.05)；與缺氧組比較,缺氧+環巴胺組迴升約16.4％,但仍低于常氧組(t=4.23,P＜0.05).常氧組胞質附著蛋白-1、咬閤蛋白、閉閤蛋白-1的錶達分彆為1.18±0.24、0.80 ±0.13、0.90±0.09,缺氧組分彆為0.58±0.08、0.32±0.05、0.50±0.09,缺氧+環巴胺組分彆為0.92 ±0.21、0.43 ±0.10、0.82±0.11,3組比較,差異有統計學意義(F=4.95,2.88,10.09,P＜0.05).缺氧組較常氧組分彆降低48.7％、40.0％、55.6％(t=12.86,9.35,18.90,P＜0.05)；缺氧+環巴胺組較缺氧組分彆提高59.9％、35.2％、65.1％(=5.63,2.92,6.66,P＜0.05).結論 缺氧條件可刺激激活Hh信號通路,從而引起緊密連接蛋白錶達降低,導緻腸上皮屏障功能損害.
목적 탐토Hedgehog(Hh)신호통로재결양장상피병장공능조공적작용.방법 대서소장상피세포계IEC-6세포분위3조:상양조(21％양농도)、결양조(2％양농도)、결양+배파알조(용5 mmol/L적배파알예처리30 min후,재급여2％양농도진행결양처리).RT-PCR검측Hh신호통로IHH、PTCH、GLI-1 mRNA적표체변화,전조측정의검측과상피전조(TER),Western blot검측IHH、긴밀련접단백경전표체분자포질부착단백(ZO-1)、교합단백(Occludin)、폐합단백(Claudin-1)적표체정황.조간비교채취단인소방차분석,량량비교채용LSD-t검험.결과 RT-PCR검측결과표명:상양조Hh신호통로IHH、PTCH、GLI-1 mRNA적상대표체량분별위0.056±0.009、0.459±0.087、0.142±0.023；결양조분별위0.303±0.052、0.678±0.073、0.483±0.061,량조비교,차이유통계학의의(t=-14.05,-11.85,-6.52,P＜0.05).Western blot검측결과현시:상양조화결양조적IHH상대단백표체량분별위0.39±0.06화0.91±0.15,량조비교,차이유통계학의의(t=-8.08,P＜0.05).상양조、결양조화결양+배파알조적TER분별위(134±5) Ohm/cm2、(100±6)Ohm/cm2、(118±5)Ohm/cm2,3조비교,차이유통계학의의(F=1.04,P＜0.05).여상양조비교,결양조하강약27.7％(t=7.84,P＜0.05)；여결양조비교,결양+배파알조회승약16.4％,단잉저우상양조(t=4.23,P＜0.05).상양조포질부착단백-1、교합단백、폐합단백-1적표체분별위1.18±0.24、0.80 ±0.13、0.90±0.09,결양조분별위0.58±0.08、0.32±0.05、0.50±0.09,결양+배파알조분별위0.92 ±0.21、0.43 ±0.10、0.82±0.11,3조비교,차이유통계학의의(F=4.95,2.88,10.09,P＜0.05).결양조교상양조분별강저48.7％、40.0％、55.6％(t=12.86,9.35,18.90,P＜0.05)；결양+배파알조교결양조분별제고59.9％、35.2％、65.1％(=5.63,2.92,6.66,P＜0.05).결론 결양조건가자격격활Hh신호통로,종이인기긴밀련접단백표체강저,도치장상피병장공능손해.
Objective To investigate the regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia.Methods The IEC-6 cells of rats were divided into 3 groups:the normoxia group (21％ oxygen concentration),the hypoxia group (2％ oxygen concentration) and the hypoxia + cyclopamine group (cells pretreated by 5 mmol/L of cyclopamine,and then exposed in an atmosphere with 2％ oxygen concentration).The mRNA expressions of IHH,PTCH and GLI-1 were detected,and the transepithelial electrical resistance (TER) was determined.The protein expressions of tight junction proteins (ZO-1,Occludin,Claudin-1) and IHH were assayed by using the Western blot.All data were analyzed using the one-way analysis of variance or LSD-t test.Results The relative mRNA expressions of IHH,PTCH and GLI-1 were 0.056 ± 0.009,0.459 ± 0.087,0.142 ± 0.023 in the normoxia group,and 0.303 ± 0.052,0.678 ± 0.073,0.483 ± 0.061 in the hypoxia group,with significant difference between the 2 groups (t =-14.05,-11.85,-6.52,P ＜ 0.05).The relative protein expressions of IHH in the normoxia group and the hypoxia group were 0.39 ±0.06 and 0.91 ±0.15,with a significant difference between the 2 groups (t =-8.08,P ＜ 0.05).The TERs of the normoxia group,the hypoxia group and the hypoxia + cyclopamine group were (134 ± 5) Ohm/cm3,(100 ± 6) Ohm/cm3 and (118 ± 5) Ohm/cm3,with significant difference between the 3 groups (F =1.04,P ＜ 0.05).Compared with the normoxia group,the TER of the hypoxia group was decreased by 27.7％ (t =7.84,P ＜ 0.05) ; compared with the hypoxia group,the TER of the hypoxia + cyclopamine group were increased by 16.4％,but it was still significantly lower than the normoxia group (t =4.23,P ＜ 0.05).The expressions of ZO-1,Occludin and Claudin-1 were 1.18 ± 0.24,0.80 ±0.13 and 0.90 ±0.09 in the normoxia group,and 0.58 ±0.08,0.32 ±0.05 and 0.50 ±0.09 in the hypoxia group,and 0.92 ± 0.21,0.43 ± 0.10 and 0.82 ± 0.11 in the hypoxia + cyclopamine group,with significant difference between the 3 groups (F =4.95,2.88,10.09,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in hypoxia group were decreased by 48.7％,40.0％ and 55.6％ when compared with the normoxia group (t =12.86,9.35,18.90,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in the hypoxia + cyclopamine group were increased by 59.9％,35.2％ and 65.1％ when compared with the hypoxia group (t =5.63,2.92,6.66,P ＜ 0.05).Conclusion Hedgehog signal pathway could be activated under hypoxia,and then the expressions of tight junction proteins are decreased,which finally induces the injury of intestinal epithelial barrier function.
