氯化镧对肿瘤坏死因子α诱导的核因子κB抑制因子激酶β活化的调控作用  

Regulatory effects of lanthanum chloride on the activation of nuclear factor kappa B inhibitor kinase beta induced by tumor necrosis factor alpha

作　　者：郭菲[1] 何凤[2] 修敏[2] 娄远蕾[1] 谢安[1] 刘芬[1] 李国辉[1] 

机构地区：[1]南昌大学第一附属医院烧伤中心,330006 [2]南昌大学研究生院

出　　处：《中华烧伤杂志》2013年第6期531-536,共6页Chinese Journal of Burns

基　　金：国家自然科学基金（81160193、30960405）;江西省卫生厅课题计划（20121053）

摘　　要：目的分析氯化镧对TNF—α诱导的NF—KB抑制因子（IKB）激酶B（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20ng／mL TNF-α【的无血清RMPI1640培养液恒温培养30min；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、100txmol／L氯化镧的无血清RMPI1640培养液恒温培养4h后，加入含20ng／mLTNF-α的无血清RMPI1640培养液继续培养30min；氯化镧对照组，以含100Ixmol／L氯化镧的无血清RMPI1640培养液恒温培养30min；空白对照组，以无血清RMPI1640培养液恒温培养30rain，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—KB／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．a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—KB／p65蛋白表达以及胞质中IKBtx、IKKD与磷酸化IKKβ、IKKp（p-IKKβ、p-IKKp）蛋白表达；采用NF—KB／p65转录因子试剂盒检测胞核中NF—KB／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—KB／p65表达较强；TNF-α对照组胞核中NF-KB／p65表达较强；氯化镧对照组胞质中NF—KB／p65较空白对照组减弱；3种剂量氯化镧＋TNF一“组间比较，胞核和胞质中的NF—KB／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-“对照组。（2）空白对照组胞核中也有一定量的NF．KB／p65蛋白表达，TNF-α对照组胞核中NF—KB／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．KB／p65蛋白表达随着氯化镧浓度升高逐渐减弱。TNF-α对照组IKBCt表达水平较Objective To investigate the regulatory effects of lanthanum chloride （LaC13 ） on the activation of nuclear factor kappa B inhibitor （IKB） kinase beta （IKK~） induced by tumor necrosis factor alpha （TNF-ct）. Methods （ 1 ） Hela cells were cultured routinely in vitro. One portion of cells were col-lected and divided into TNF-et group （cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-et for 30 min）, low-dose LaC13 ＋ TNF-et group, moderate-dose LaC13 ＋ TNF-et group, high-dose LaCI3 ＋ TNF-ct group, LaC13 group （cultured with serum-free RMPI 1640 medium containing 100 Ixmol/L LaC13 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 LaCI3 ＋ TNF-et group, moderate-dose LaC13 ＋ TNF-et group, high-dose LaC13 ＋ TNF-et group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 p.mol/L LaCI3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-et for 30 rain. There were 3 samples in each group. Cells were collected for detection of intraeellular lo- cation of NF-S：B/p65 protein by immunofluorescence staining. （2） Another portion of cells were collected and divided into TNF-et group, low-dose LaC13 ＋ TNF-et group, moderate-dose LaC13 ＋ TNF-et group, high- dose LaC13 ＋ TNF-et group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-KB/p65 in nuclei, and the protein levels of Is：Bet, phosphorylated Is：Bct （p-Is：Bet） as well as IKKI3 and phosphorylated IKK~ （p-IKK[5） in cytoplasm were determined by Western blotting. The binding activity between NF-S：B/p65 in the nuclear and target gene was determined by NF-S：B/p65 transcription factor kit （ denoted as absorption value）. Data were processed with analysis of vari- ance or LSD- t test. Results （ 1 ） High expression of NF-S：B/p65 was observed in cytoplasm of control group. High e

关 键 词：烧伤 镧 肿瘤坏死因子Α NF-KB NF·KB抑制因子激酶β 

分 类 号：R644[医药卫生—外科学]