中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
5期
961-963
,共3页
陈辉星%陈燕凌%洪海杰%唐南洪%王百林
陳輝星%陳燕凌%洪海傑%唐南洪%王百林
진휘성%진연릉%홍해걸%당남홍%왕백림
肠上皮细胞%防御素%免疫抑制剂%核因子-κB
腸上皮細胞%防禦素%免疫抑製劑%覈因子-κB
장상피세포%방어소%면역억제제%핵인자-κB
Intestinal epithelial cells%Defensin%Immunosuppressant%Nuclear factor-κB
目的 探讨4种不同免疫抑制剂(他克莫司、环孢素A、雷帕霉素、雷公藤多甙)对Caco-2和HT-29细胞人β防御索1和2(hRD-1/2)的表达影响及机制.方法 通过实时定量聚合酶链反应(Real-time PCR)和Western blot检测终质量浓度为10 μmol/L的4种不同免疫抑制剂对白细胞介素-1β(IL-1β,20μg/L)诱导的人肠上皮细胞hBD-1/2表达的影响,应用凝胶电泳迁移率实验法(EMSA)和酶联免疫吸附试验(ELISA)检测Caco-2细胞核子-κB(NF-κB)的表达.结果 肠上皮细胞组成性表达hBD-1；他克莫司、环孢素A和雷帕霉素组在Caco-2细胞中hBD-2 mRNA促进率分别为(20.58±1.02)％、(139.49±6.97)％和(425.97 ±21.29)％,雷公藤多甙组hBD-2 mRNA抑制率为(48.59±2.42)％ (P＜0.05).HT-29细胞中上述指标分别为(62.52±3.25)％、(20.56±1.10)％、(46.79±2.28)％和(82.73±7.88)％(p＜0,05).Caco-2细胞中hBD-2蛋白的表达与mRNA的表达趋势一致.EMSA和ELISA检测活化的NF-κB显示他克莫司、环孢素A和雷帕霉素组促进NF-κB的活化,雷公滕多甙组抑制NF-κB的活化.并与Ser276磷酸化有关.结论 他克莫司、环孢素A和雷帕霉素促进人肠系细胞在IL-1β3诱导下表达hBD-2,雷公藤多甙抑制hBD-2的表达.其机制与NF-κB信号途径有关.
目的 探討4種不同免疫抑製劑(他剋莫司、環孢素A、雷帕黴素、雷公籐多甙)對Caco-2和HT-29細胞人β防禦索1和2(hRD-1/2)的錶達影響及機製.方法 通過實時定量聚閤酶鏈反應(Real-time PCR)和Western blot檢測終質量濃度為10 μmol/L的4種不同免疫抑製劑對白細胞介素-1β(IL-1β,20μg/L)誘導的人腸上皮細胞hBD-1/2錶達的影響,應用凝膠電泳遷移率實驗法(EMSA)和酶聯免疫吸附試驗(ELISA)檢測Caco-2細胞覈子-κB(NF-κB)的錶達.結果 腸上皮細胞組成性錶達hBD-1；他剋莫司、環孢素A和雷帕黴素組在Caco-2細胞中hBD-2 mRNA促進率分彆為(20.58±1.02)％、(139.49±6.97)％和(425.97 ±21.29)％,雷公籐多甙組hBD-2 mRNA抑製率為(48.59±2.42)％ (P＜0.05).HT-29細胞中上述指標分彆為(62.52±3.25)％、(20.56±1.10)％、(46.79±2.28)％和(82.73±7.88)％(p＜0,05).Caco-2細胞中hBD-2蛋白的錶達與mRNA的錶達趨勢一緻.EMSA和ELISA檢測活化的NF-κB顯示他剋莫司、環孢素A和雷帕黴素組促進NF-κB的活化,雷公滕多甙組抑製NF-κB的活化.併與Ser276燐痠化有關.結論 他剋莫司、環孢素A和雷帕黴素促進人腸繫細胞在IL-1β3誘導下錶達hBD-2,雷公籐多甙抑製hBD-2的錶達.其機製與NF-κB信號途徑有關.
목적 탐토4충불동면역억제제(타극막사、배포소A、뢰파매소、뢰공등다대)대Caco-2화HT-29세포인β방어색1화2(hRD-1/2)적표체영향급궤제.방법 통과실시정량취합매련반응(Real-time PCR)화Western blot검측종질량농도위10 μmol/L적4충불동면역억제제대백세포개소-1β(IL-1β,20μg/L)유도적인장상피세포hBD-1/2표체적영향,응용응효전영천이솔실험법(EMSA)화매련면역흡부시험(ELISA)검측Caco-2세포핵자-κB(NF-κB)적표체.결과 장상피세포조성성표체hBD-1；타극막사、배포소A화뢰파매소조재Caco-2세포중hBD-2 mRNA촉진솔분별위(20.58±1.02)％、(139.49±6.97)％화(425.97 ±21.29)％,뢰공등다대조hBD-2 mRNA억제솔위(48.59±2.42)％ (P＜0.05).HT-29세포중상술지표분별위(62.52±3.25)％、(20.56±1.10)％、(46.79±2.28)％화(82.73±7.88)％(p＜0,05).Caco-2세포중hBD-2단백적표체여mRNA적표체추세일치.EMSA화ELISA검측활화적NF-κB현시타극막사、배포소A화뢰파매소조촉진NF-κB적활화,뢰공등다대조억제NF-κB적활화.병여Ser276린산화유관.결론 타극막사、배포소A화뢰파매소촉진인장계세포재IL-1β3유도하표체hBD-2,뢰공등다대억제hBD-2적표체.기궤제여NF-κB신호도경유관.
Objective To investigate the effect of tacrolimus (FK506),cyclosporin A (CsA),rapamycin (Rapa) and Glucosida Tripterygii TOTA (GTT) on human β-defensin (hBD-1 and hBD-2) expression in intestinal epithelial cells (Caco-2 and HT-29 cells).Methods Quantitative real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the effects of the final concentration of 10 μmol/L of FK506,CsA,Rapa and GTT on hBD-1 and hBD-2 expression in human intestinal epithelial cells stimulated by interleukin (IL)-1 β (20 μg/L).Nuclear factor-κB (NF-κB)binding activity in the Caco-2 cells was examined by using electrophoretic mobility shift assay (EMSA) and an enzyme linked immunosorbent assay (ELISA)-based assay with immobilized oligonucleotide.Results As compared with the IL-1β alone,the addition of FK506,CsA or Rapa in the culture medium in Caco-2 cells significantly increased the mRNA level of hBD-2 by (20.58 ± 1.02)％,(139.49 ± 6.97)％ and (425.97 ± 21.29)％ respectively (P ＜0.05).Meanwhile,the mRNA level of hBD-2 was decreased by (48.59 ± 2.42)％ in Caco-2 cells cultured with GTT (P＜ 0.05).In HT-29 cells,the value was (62.52±3.25)％,(20.56±1.10)％,(46.79 ±2.28)％ and (82.73 ±7.88)％ respectively (P＜0.05).Western blotting analysis revealed that the same results.However,GTT attenuated the expression of hBD-2.As compared with the control group,either FK506,CsA and rapamycin promnoted the activation of NF-κB,but GTr decreased the activation of NF-κB (P ＜ 0.05),which was related to the phosphorylation of Ser276.Conclusion The results presented in this paper show that FK506,CsA and Rapa activate NF-κB in intestinal epithelial cells,resulting in efficient induction of hBD-2 production in vitro.
