中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2011年
3期
195-198
,共4页
杨春涛%凌宏忠%曾凡钦%张辉%杨战利%傅璐%叶锋%莫利球%韩艳芳%冯鉴强
楊春濤%凌宏忠%曾凡欽%張輝%楊戰利%傅璐%葉鋒%莫利毬%韓豔芳%馮鑒彊
양춘도%릉굉충%증범흠%장휘%양전리%부로%협봉%막리구%한염방%풍감강
细胞低氧%角蛋白细胞%炎症%NF-κB
細胞低氧%角蛋白細胞%炎癥%NF-κB
세포저양%각단백세포%염증%NF-κB
Cell hypoxia%Keratinocytes%Inflammation%NF-kappa B
目的 探讨核因子-κB(NF-κB)亚基P65磷酸化是否参与化学性缺氧模拟剂氯化钴(CoCl2)诱导的永生化人皮肤角质形成细胞(HaCaT)毒性作用及炎症反应.方法 用2 mmol/L CoCl2处理HaCaT细胞,建立化学性缺氧诱导HaCaT细胞损伤的体外模型.RNA干扰法下调NF-κB亚基P65的表达.细胞计数试剂盒-8比色法检测细胞存活率;ELISA法检测培养基中IL-6和IL-8的含量;Western印迹法检测总量P65及磷酸化P65的蛋白表达.结果 CoCl2处理HaCaT细胞0～4 h,可促进NF-κB亚基P65的磷酸化,在0.5 h时P65亚基开始磷酸化,1.5 h时P65亚基的磷酸化水平达到高峰,约为对照组的6.6倍,而4 h基本恢复到正常水平.CoCl2处理HaCaT细胞0～6 h,可时间依赖性地降低细胞活力,2、4和6 h的细胞存活率与对照组比较,P值分别＜0.05、＜0.01及＜0.01.CoCl2处理6 h,还引起IL-6和IL-8的释放显著增加.RNA干扰法下调P65的表达后,CoCl2处理引起的HaCaT细胞毒性作用被明显减弱,即使细胞存活率升高了11%左右,下调P65表达还明显抑制了CoCl2处理引起的IL-6和Il-8释放增多.结论 磷酸化NF-κB亚基P65介导CoCl2诱导的HaCaT细胞毒性及炎症反应.
目的 探討覈因子-κB(NF-κB)亞基P65燐痠化是否參與化學性缺氧模擬劑氯化鈷(CoCl2)誘導的永生化人皮膚角質形成細胞(HaCaT)毒性作用及炎癥反應.方法 用2 mmol/L CoCl2處理HaCaT細胞,建立化學性缺氧誘導HaCaT細胞損傷的體外模型.RNA榦擾法下調NF-κB亞基P65的錶達.細胞計數試劑盒-8比色法檢測細胞存活率;ELISA法檢測培養基中IL-6和IL-8的含量;Western印跡法檢測總量P65及燐痠化P65的蛋白錶達.結果 CoCl2處理HaCaT細胞0～4 h,可促進NF-κB亞基P65的燐痠化,在0.5 h時P65亞基開始燐痠化,1.5 h時P65亞基的燐痠化水平達到高峰,約為對照組的6.6倍,而4 h基本恢複到正常水平.CoCl2處理HaCaT細胞0～6 h,可時間依賴性地降低細胞活力,2、4和6 h的細胞存活率與對照組比較,P值分彆＜0.05、＜0.01及＜0.01.CoCl2處理6 h,還引起IL-6和IL-8的釋放顯著增加.RNA榦擾法下調P65的錶達後,CoCl2處理引起的HaCaT細胞毒性作用被明顯減弱,即使細胞存活率升高瞭11%左右,下調P65錶達還明顯抑製瞭CoCl2處理引起的IL-6和Il-8釋放增多.結論 燐痠化NF-κB亞基P65介導CoCl2誘導的HaCaT細胞毒性及炎癥反應.
목적 탐토핵인자-κB(NF-κB)아기P65린산화시부삼여화학성결양모의제록화고(CoCl2)유도적영생화인피부각질형성세포(HaCaT)독성작용급염증반응.방법 용2 mmol/L CoCl2처리HaCaT세포,건립화학성결양유도HaCaT세포손상적체외모형.RNA간우법하조NF-κB아기P65적표체.세포계수시제합-8비색법검측세포존활솔;ELISA법검측배양기중IL-6화IL-8적함량;Western인적법검측총량P65급린산화P65적단백표체.결과 CoCl2처리HaCaT세포0～4 h,가촉진NF-κB아기P65적린산화,재0.5 h시P65아기개시린산화,1.5 h시P65아기적린산화수평체도고봉,약위대조조적6.6배,이4 h기본회복도정상수평.CoCl2처리HaCaT세포0～6 h,가시간의뢰성지강저세포활력,2、4화6 h적세포존활솔여대조조비교,P치분별＜0.05、＜0.01급＜0.01.CoCl2처리6 h,환인기IL-6화IL-8적석방현저증가.RNA간우법하조P65적표체후,CoCl2처리인기적HaCaT세포독성작용피명현감약,즉사세포존활솔승고료11%좌우,하조P65표체환명현억제료CoCl2처리인기적IL-6화Il-8석방증다.결론 린산화NF-κB아기P65개도CoCl2유도적HaCaT세포독성급염증반응.
Objective To explore whether the phosphorylation of NF-κB P65 subunit is involved in the cytotoxicity to and inflammation in an immortal human keratinocyte cell line HaCaT during cobalt chloride (CoCl2-induced chemical hypoxia. Methods HaCaT cells were treated with CoCl2 of 2 mmol/L to set up a chemical hypoxia-induced cell model of injury. Then, RNA interference was used to down-regulate the expression of P65 in CoCl2-induced HaCaT cells. After additional culture, cell viability was tested by cell counting kit8 (CCK-8), the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) were detected by ELISA kits, phosphorylated and total P65 protein was measured by Western blot. Results The exposure of HaCaT cells to 2 mmol/L CoCl2 for 0 to 4 hours enhanced the phosphorylation of P65, which began at 0.5 hour, peaked at 1.5 hours, and restored to the normal level at 4 hours, and the level of P65 phosphorylation was about 6.6 times that in the untreated control group. The CoCl2 of 2 mmol/L decreased the cell viability of HaCaT cells in a time dependent manner, and a significant difference was observed in the viability of HaCaT cells between CoCl2-treated and untreated HaCaT cells at 2, 4, and 6 hours (P ＜ 0.05, 0.01, 0.01 ). The release of IL-6 and IL-8 from HaCaT cells was also promoted by CoCl2 treatment. The knockdown of P65 expression with siRNA markedly suppressed the CoCl2-induced cytotoxicity to and increase in the release of IL-6 and IL-8 from HaCaT cells,despite of an increment in cell viability by about 11%. Conclusion The phosphorylated P65 subunit mediates CoCl2-induced cytotoxicity and inflammatory injury to HaCaT cells.
