中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2013年
6期
531-536
,共6页
烧伤%镧%肿瘤坏死因子α%NF-κB%NF-κB抑制因子激酶β
燒傷%鑭%腫瘤壞死因子α%NF-κB%NF-κB抑製因子激酶β
소상%란%종류배사인자α%NF-κB%NF-κB억제인자격매β
Burns%Lanthanum%Tumor necrosis factor-alpha%NF-kappa B%NF-kappa B inhibitor kinase beta
目的 分析氯化镧对TNF-α诱导的NF-κB抑制因子(IκB)激酶β(IKKβ)活化的调控作用. 方法 (1)体外培养Hela细胞,按照随机数字表法分为TNF-α对照组,以含20 ng/mLTNF-α的无血清RMPI 1640培养液恒温培养30 min;小剂量氯化镧+TNF-α组、中等剂量氯化镧+TNF-α组、大剂量氯化镧+TNF-α组,3组细胞分别以含5、25、100 μmol/L氯化镧的无血清RMPI1640培养液恒温培养4h后,加入含20 ng/mL TNF-α的无血清RMPI 1640培养液继续培养30 min;氯化镧对照组,以含100 μmol/L氯化镧的无血清RMPI 1640培养液恒温培养30 min;空白对照组,以无血清RMPI 1640培养液恒温培养30 min,各组样本数均为3.收集各组细胞,免疫细胞荧光染色观察NF-κB/p65蛋白转位情况.(2)另取Hela细胞,同上分为5组,即TNF-α对照组、小剂量氯化镧+TNF-α组、中等剂量氯化镧+TNF-α组、大剂量氯化镧+TNF-α组、空白对照组并行相同处理,各组样本数均为3,采用蛋白质印迹法检测胞核中NF-κB/p65蛋白表达以及胞质中IκBα、IKKβ与磷酸化IκBα、IKKβ(p-IκBα、p-IKKβ)蛋白表达;采用NF-κB/p65转录因子试剂盒检测胞核中NF-κB/p65蛋白与靶基因结合的活性,数据以吸光度值表示.对数据进行方差分析、LSD-t检验. 结果 (1)空白对照组胞质中NF-κB/p65表达较强;TNF-α对照组胞核中NF-κB/p65表达较强;氯化镧对照组胞质中NF-κB/p65较空白对照组减弱;3种剂量氯化镧+TNF-α组间比较,胞核和胞质中的NF-κB/p65表达量随着氯化镧浓度的升高而减弱,总体表达明显弱于TNF-α对照组.(2)空白对照组胞核中也有一定量的NF-κB/p65蛋白表达,TNF-α对照组胞核中NF-κB/p65蛋白表达强于空白对照组;3种剂量氯化镧+ TNF-α组间比较,胞核中NF-κB/p65蛋白表达随着氯化镧浓度升高逐渐减弱.TNF-α对照组IκBα表达水平较空白对照组明显下降,而p-IκBα明显上升;3种剂量氯化镧+TN F-α组间比较,随着氯化镧浓度的升高,IκBα表达水平逐步升高,而p-IκBα水平逐渐降低.IKKβ的表达在各组无明显差异.空白对照组p-IKKβ几乎不表达,TNF-α对照组p-IKKβ水平明显升高;3种剂量氯化镧+TNF-α组间比较,随着氯化镧浓度的升高,p-IKKβ水平逐渐下降.TNF-α对照组NF-κB/p65蛋白与靶基因结合的活性为0.39 ±0.03,明显高于空白对照组(0,t=-7.23,P＜0.01);小剂量氯化镧+TNF-α组、中等剂量氯化镧+TNF-α组、大剂量氯化镧+TNF-α组NF-κB/p65蛋白与靶基因结合的活性分别为0.17 ±0.03、0.15±0.03和0,均显著低于TNF-α对照组(t值分别为-6.54、-5.92、-7.23,P值均小于0.01). 结论 氯化镧能通过阻断Hela细胞中IKKβ磷酸化,进而阻断NF-κB信号通路的活化.
目的 分析氯化鑭對TNF-α誘導的NF-κB抑製因子(IκB)激酶β(IKKβ)活化的調控作用. 方法 (1)體外培養Hela細胞,按照隨機數字錶法分為TNF-α對照組,以含20 ng/mLTNF-α的無血清RMPI 1640培養液恆溫培養30 min;小劑量氯化鑭+TNF-α組、中等劑量氯化鑭+TNF-α組、大劑量氯化鑭+TNF-α組,3組細胞分彆以含5、25、100 μmol/L氯化鑭的無血清RMPI1640培養液恆溫培養4h後,加入含20 ng/mL TNF-α的無血清RMPI 1640培養液繼續培養30 min;氯化鑭對照組,以含100 μmol/L氯化鑭的無血清RMPI 1640培養液恆溫培養30 min;空白對照組,以無血清RMPI 1640培養液恆溫培養30 min,各組樣本數均為3.收集各組細胞,免疫細胞熒光染色觀察NF-κB/p65蛋白轉位情況.(2)另取Hela細胞,同上分為5組,即TNF-α對照組、小劑量氯化鑭+TNF-α組、中等劑量氯化鑭+TNF-α組、大劑量氯化鑭+TNF-α組、空白對照組併行相同處理,各組樣本數均為3,採用蛋白質印跡法檢測胞覈中NF-κB/p65蛋白錶達以及胞質中IκBα、IKKβ與燐痠化IκBα、IKKβ(p-IκBα、p-IKKβ)蛋白錶達;採用NF-κB/p65轉錄因子試劑盒檢測胞覈中NF-κB/p65蛋白與靶基因結閤的活性,數據以吸光度值錶示.對數據進行方差分析、LSD-t檢驗. 結果 (1)空白對照組胞質中NF-κB/p65錶達較彊;TNF-α對照組胞覈中NF-κB/p65錶達較彊;氯化鑭對照組胞質中NF-κB/p65較空白對照組減弱;3種劑量氯化鑭+TNF-α組間比較,胞覈和胞質中的NF-κB/p65錶達量隨著氯化鑭濃度的升高而減弱,總體錶達明顯弱于TNF-α對照組.(2)空白對照組胞覈中也有一定量的NF-κB/p65蛋白錶達,TNF-α對照組胞覈中NF-κB/p65蛋白錶達彊于空白對照組;3種劑量氯化鑭+ TNF-α組間比較,胞覈中NF-κB/p65蛋白錶達隨著氯化鑭濃度升高逐漸減弱.TNF-α對照組IκBα錶達水平較空白對照組明顯下降,而p-IκBα明顯上升;3種劑量氯化鑭+TN F-α組間比較,隨著氯化鑭濃度的升高,IκBα錶達水平逐步升高,而p-IκBα水平逐漸降低.IKKβ的錶達在各組無明顯差異.空白對照組p-IKKβ幾乎不錶達,TNF-α對照組p-IKKβ水平明顯升高;3種劑量氯化鑭+TNF-α組間比較,隨著氯化鑭濃度的升高,p-IKKβ水平逐漸下降.TNF-α對照組NF-κB/p65蛋白與靶基因結閤的活性為0.39 ±0.03,明顯高于空白對照組(0,t=-7.23,P＜0.01);小劑量氯化鑭+TNF-α組、中等劑量氯化鑭+TNF-α組、大劑量氯化鑭+TNF-α組NF-κB/p65蛋白與靶基因結閤的活性分彆為0.17 ±0.03、0.15±0.03和0,均顯著低于TNF-α對照組(t值分彆為-6.54、-5.92、-7.23,P值均小于0.01). 結論 氯化鑭能通過阻斷Hela細胞中IKKβ燐痠化,進而阻斷NF-κB信號通路的活化.
목적 분석록화란대TNF-α유도적NF-κB억제인자(IκB)격매β(IKKβ)활화적조공작용. 방법 (1)체외배양Hela세포,안조수궤수자표법분위TNF-α대조조,이함20 ng/mLTNF-α적무혈청RMPI 1640배양액항온배양30 min;소제량록화란+TNF-α조、중등제량록화란+TNF-α조、대제량록화란+TNF-α조,3조세포분별이함5、25、100 μmol/L록화란적무혈청RMPI1640배양액항온배양4h후,가입함20 ng/mL TNF-α적무혈청RMPI 1640배양액계속배양30 min;록화란대조조,이함100 μmol/L록화란적무혈청RMPI 1640배양액항온배양30 min;공백대조조,이무혈청RMPI 1640배양액항온배양30 min,각조양본수균위3.수집각조세포,면역세포형광염색관찰NF-κB/p65단백전위정황.(2)령취Hela세포,동상분위5조,즉TNF-α대조조、소제량록화란+TNF-α조、중등제량록화란+TNF-α조、대제량록화란+TNF-α조、공백대조조병행상동처리,각조양본수균위3,채용단백질인적법검측포핵중NF-κB/p65단백표체이급포질중IκBα、IKKβ여린산화IκBα、IKKβ(p-IκBα、p-IKKβ)단백표체;채용NF-κB/p65전록인자시제합검측포핵중NF-κB/p65단백여파기인결합적활성,수거이흡광도치표시.대수거진행방차분석、LSD-t검험. 결과 (1)공백대조조포질중NF-κB/p65표체교강;TNF-α대조조포핵중NF-κB/p65표체교강;록화란대조조포질중NF-κB/p65교공백대조조감약;3충제량록화란+TNF-α조간비교,포핵화포질중적NF-κB/p65표체량수착록화란농도적승고이감약,총체표체명현약우TNF-α대조조.(2)공백대조조포핵중야유일정량적NF-κB/p65단백표체,TNF-α대조조포핵중NF-κB/p65단백표체강우공백대조조;3충제량록화란+ TNF-α조간비교,포핵중NF-κB/p65단백표체수착록화란농도승고축점감약.TNF-α대조조IκBα표체수평교공백대조조명현하강,이p-IκBα명현상승;3충제량록화란+TN F-α조간비교,수착록화란농도적승고,IκBα표체수평축보승고,이p-IκBα수평축점강저.IKKβ적표체재각조무명현차이.공백대조조p-IKKβ궤호불표체,TNF-α대조조p-IKKβ수평명현승고;3충제량록화란+TNF-α조간비교,수착록화란농도적승고,p-IKKβ수평축점하강.TNF-α대조조NF-κB/p65단백여파기인결합적활성위0.39 ±0.03,명현고우공백대조조(0,t=-7.23,P＜0.01);소제량록화란+TNF-α조、중등제량록화란+TNF-α조、대제량록화란+TNF-α조NF-κB/p65단백여파기인결합적활성분별위0.17 ±0.03、0.15±0.03화0,균현저저우TNF-α대조조(t치분별위-6.54、-5.92、-7.23,P치균소우0.01). 결론 록화란능통과조단Hela세포중IKKβ린산화,진이조단NF-κB신호통로적활화.
Objective To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).Methods (1) Hela cells were cultured routinely in vitro.One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min),low-dose LaCl3 + TNF-α group,moderate-dose LaCl3 + TNF-α group,high-dose LaCl3 + TNF-α group,LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 μmol/L LaCl3 for 30 min),and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table.Cells in low-dose LaCl3 + TNF-α group,moderate-dose LaCl3 + TNF-α group,high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5,25,100 μmol/L LaC13 for 4 h,and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min.There were 3 samples in each group.Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining.(2) Another portion of cells were collected and divided into TNF-α group,low-dose LaCl3 + TNF-α group,moderate-dose LaCl3 + TNF-α group,highdose LaCl3 + TNF-α group,and control group with the same treatment as above.There were 3 samples in each group.The protein levels of NF-κB/p65 in nuclei,and the protein levels of IκBα,phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting.The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value).Data were processed with analysis of variance or LSD-t test.Results (1) High expression of NF-κB/p65 was observed in cytoplasm of control group.High expression of NF-κB/p65 was observed in nuclei of TNF-α group.The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group.In groups treated with LaCl3 and TNF-α,NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3,which were all lower than those in TNF-α group.(2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group.The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group.In groups treated with LaCl3 and TNF-α,the expressions of NF-κB/p65 protein in nuclei were decreased along with an inerease in the concentration of LaCl3.The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group.Along with the increase in the concentration of LaCl3,the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α.There were no statistical differences in expression levels of IKKβ among the 5 groups.The expression of p-IKKβ could be hardly observed in control group,but it was obviously increased in TNF-α group.The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3.The absorption value in TNF-α group was 0.39 ± 0.03,which was higher than that in control group (0,t =-7.23,P ＜ 0.01).The absorption values in low-dose LaCl3 + TNF-α group,moderate-dose LaCl3 + TNF-α group,and high-dose LaCl3 + TNF-α group were respectively 0.17 ±0.03,0.15 ±0.03,and 0,which were obviously lower than that in TNF-α group (with t values respectively-6.54,-5.92,-7.23,P values all below 0.01).Conclusions LaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.
