CAJ | 학술논문

目的探讨结核分枝杆菌感染对人星形胶质细胞基质金属蛋白酶(MMP)-9表达的影响及其机制.方法分别用结核分枝杆菌(MTB)直接感染人星型胶质细胞系U251细胞,MTB感染外周血单个核细胞(PBMC)后的条件培养基(CoMTB)、含RPMI-1640的MTB条件培养基(CoTB)及未感染MTB的PBMC条件培养基(CoMCON)刺激U251细胞,ELISA检测细胞上清液中MMP-9含量,明胶酶谱法检测MMP-9活性,Western印迹法检测MMP-9、核因子(NF)-кB及核因子-кB抑制蛋白(IкB)α和IкBβ的表达.组间及配对数据比较采用t检验,多组间比较先行方差齐性检验,方差齐采用方差分析,不齐采用秩和检验.结果 MTB直接感染U251细胞后MMP-9几乎检测不到,CoMTB刺激U251细胞后,MMP-9含量从12 h的(400.1±12.6) pg/mL逐步升至72 h的(2900.3±108.4) pg/mL,呈时间依赖性；而CoMCON和CoTB组刺激72 h后MMP-9含量分别为(300.8±40.4) pg/mL和(400.2±42.8) pg/mL,差异有统计学意义(t=9.514,P=0.000;t=9.476,P=0.000).明胶酶谱法和Western印迹法检测CoMTB组上清液中MMP-9的表达,随刺激时间延长,其活性和表达量逐渐增强,与ELISA法一致.CoMTB刺激U251细胞后NF-кB P65表达增加,1h时是CoMCON组的2倍(t=6.78,P=0.041),2h时是CoMCON组的5倍(t=9.23,P=0.008),CoMTB组IкBα和IкBβ蛋白0.5h开始降解并持续至6h,而CoMCON组未见降解.结论 PBMC-神经胶质细胞网络在结核感染的基质降解中起重要作用,CoMTB可能通过激活NF-кB信号通路上调MMP-9的表达.
목적 탐토결핵분지간균감염대인성형효질세포기질금속단백매(MMP)-9표체적영향급기궤제.방법 분별용결핵분지간균(MTB)직접감염인성형효질세포계U251세포,MTB감염외주혈단개핵세포(PBMC)후적조건배양기(CoMTB)、함RPMI-1640적MTB조건배양기(CoTB)급미감염MTB적PBMC조건배양기(CoMCON)자격U251세포,ELISA검측세포상청액중MMP-9함량,명효매보법검측MMP-9활성,Western인적법검측MMP-9、핵인자(NF)-кB급핵인자-кB억제단백(IкB)α화IкBβ적표체.조간급배대수거비교채용t검험,다조간비교선행방차제성검험,방차제채용방차분석,불제채용질화검험.결과 MTB직접감염U251세포후MMP-9궤호검측불도,CoMTB자격U251세포후,MMP-9함량종12 h적(400.1±12.6) pg/mL축보승지72 h적(2900.3±108.4) pg/mL,정시간의뢰성；이CoMCON화CoTB조자격72 h후MMP-9함량분별위(300.8±40.4) pg/mL화(400.2±42.8) pg/mL,차이유통계학의의(t=9.514,P=0.000;t=9.476,P=0.000).명효매보법화Western인적법검측CoMTB조상청액중MMP-9적표체,수자격시간연장,기활성화표체량축점증강,여ELISA법일치.CoMTB자격U251세포후NF-кB P65표체증가,1h시시CoMCON조적2배(t=6.78,P=0.041),2h시시CoMCON조적5배(t=9.23,P=0.008),CoMTB조IкBα화IкBβ단백0.5h개시강해병지속지6h,이CoMCON조미견강해.결론 PBMC-신경효질세포망락재결핵감염적기질강해중기중요작용,CoMTB가능통과격활NF-кB신호통로상조MMP-9적표체.
Objective To explore the impact of Mycobacterium tuberculosis (M.tb) infection on the expression of matrix metalloproteinase (MMP)-9 in human astrocyte cell line and its mechanism.Methods Human astrocyte cell line U251 cells were stimulated by direct M.tb infection,conditioned mediums of M.tb-infected peripheral blood mononuclear cells (PBMC,CoMTB),incubation M.tb monocyte free medium (CoTB) and PBMC not infected with M.tb (CoMCON),respectively.Enzymelinked immunosorbent assay (ELISA) and Gelatin zymography were used to detect cell supernatant content and activity of MMP-9,respectively.Western blot assay was used to detect the protein expressions of MMP-9,nuclear factor (NF)-кB,and inhibitory subunits of NF-кB (IкBα and IкBβ).Comparisons between two groups or paired results were performed by student t-test,and comparison among multiple groups was performed by homogeneity test of variance (analysis of variance rather than rank sum test).Results U251 cells stimulated with direct M.tb infection,expressed undetectable of MMP-9,while after stimulation by CoMTB,MMP-9 content gradually increased from (400.1 ±12.6)pg/mL at 12 h to (2900.3±108.4) pg/mL at 72 h time-dependently.MMP-9 contents in CoMCON and CoTB groups at 72 h were (300.8±40.4) pg/mL and (400.2±42.8) pg/mL,respectively,with statistical significance (t=9.514,P =0.000; t =9.476,P=0.000).Activity and expression of MMP-9 increased over time in supernatant of CoMTB group measured by Gelatin zymography and Western blot assay,which were consistent with the results of ELISA.NF-кB P65 began to increase after 30 min of CoMTB,compared with CoMCON.NF-кB P65 expression inducing a 2-fold increase after 1 h and 5-fold incoase after 2 h,and continued to increase until 6 h.Degradation of IкBα and IкBβ in CoMTB group started from 0.5 h,and persisted until 6 h,which was not observed in CoMCON group.Conclusions The results suggest that PBMC-dependent cytokine network plays an important role in the development of a matrix-degrading environment.CoMTB may increase the expression and secretion of MMP-9 through activation of NF-кB signaling pathway.