中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2010年
5期
455-459
,共5页
黄芬%熊晓昉%游莎%王琼新%曾和松
黃芬%熊曉昉%遊莎%王瓊新%曾和鬆
황분%웅효방%유사%왕경신%증화송
冠状动脉疾病%基质金属蛋白酶2%基质金属蛋白酶9%NF-κB%内脂素
冠狀動脈疾病%基質金屬蛋白酶2%基質金屬蛋白酶9%NF-κB%內脂素
관상동맥질병%기질금속단백매2%기질금속단백매9%NF-κB%내지소
Coronary disease%Matrix metalloproteinase2%Matrix metalloproteinase9%NF-kappa B%Visfatin
目的 探讨内脂素对核因子κB(NF-κB)通路的激活及其在调节人单核细胞基质金属蛋白酶(MMP)-2和MMP-9的表达中的作用,以期进一步了解内脂素与动脉粥样硬化的关系.方法 以健康人外周血单核细胞为研究对象.抽取健康志愿者静脉血,分离、纯化单核细胞后随机分为空白对照组;内脂素组(100、200、400 ng/ml,24 h);Bay11-7082抑制剂组(Bay11-7082 40μmol/ml预孵育30 min,内脂素400 ng/ml刺激24 h).荧光定量PCR检测各组细胞MMP-2和MMP-9 mRNA的表达;Western blot检测MMP-2和MMP-9蛋白的表达;明胶酶谱法检测细胞培养基质中MMP-2和MMP-9的酶活性.内脂素刺激细胞后的核转位分别用Western blot和ELISA进行检测.结果 人外周血单核细胞经不同浓度的内脂素干预培养后24 h单核细胞MMP-2和MMP-9 mRNA及蛋白的表达均明显高于空白对照组(P均<0.05),且呈浓度依赖性.内脂素还上调细胞分泌到细胞培养基中的MMP-2和MMP-9酶活性,内脂素100 ng/ml组MMP-9、MMP-2的酶活性分别为空白对照组的(1.139 ±0.055)和(1.102±0.011)倍(P均<0.05).加入NF-κB抑制剂Bay11-7082后MMP-2、MMP-9的mRNA、蛋白的表达及酶活性均明显低,与内脂素400ng/ml组比较差异均有统计学意义(P均<0.001).内脂素浓度越高,核蛋白NF-κBp65表达越高,内脂素100 ng/ml组相对灰度值为空白对照组的(1.334±0.148)倍(P均<0.05);内脂素浓度越高,NF-κBp65亚单位核内转移越多,内脂素100ng/ml组为0.763±0.056,内脂素400 ng/ml组为1.290±0.065,均大于对照组的0.467±0.046(P均<0.05).结论 内脂素可通过激活NF-κB途径上调人单核细胞中MMP-2和MMP-9表达及活性.这可能是内脂素致动脉粥样硬化及与急性冠状动脉综合征密切相关的机制之一.
目的 探討內脂素對覈因子κB(NF-κB)通路的激活及其在調節人單覈細胞基質金屬蛋白酶(MMP)-2和MMP-9的錶達中的作用,以期進一步瞭解內脂素與動脈粥樣硬化的關繫.方法 以健康人外週血單覈細胞為研究對象.抽取健康誌願者靜脈血,分離、純化單覈細胞後隨機分為空白對照組;內脂素組(100、200、400 ng/ml,24 h);Bay11-7082抑製劑組(Bay11-7082 40μmol/ml預孵育30 min,內脂素400 ng/ml刺激24 h).熒光定量PCR檢測各組細胞MMP-2和MMP-9 mRNA的錶達;Western blot檢測MMP-2和MMP-9蛋白的錶達;明膠酶譜法檢測細胞培養基質中MMP-2和MMP-9的酶活性.內脂素刺激細胞後的覈轉位分彆用Western blot和ELISA進行檢測.結果 人外週血單覈細胞經不同濃度的內脂素榦預培養後24 h單覈細胞MMP-2和MMP-9 mRNA及蛋白的錶達均明顯高于空白對照組(P均<0.05),且呈濃度依賴性.內脂素還上調細胞分泌到細胞培養基中的MMP-2和MMP-9酶活性,內脂素100 ng/ml組MMP-9、MMP-2的酶活性分彆為空白對照組的(1.139 ±0.055)和(1.102±0.011)倍(P均<0.05).加入NF-κB抑製劑Bay11-7082後MMP-2、MMP-9的mRNA、蛋白的錶達及酶活性均明顯低,與內脂素400ng/ml組比較差異均有統計學意義(P均<0.001).內脂素濃度越高,覈蛋白NF-κBp65錶達越高,內脂素100 ng/ml組相對灰度值為空白對照組的(1.334±0.148)倍(P均<0.05);內脂素濃度越高,NF-κBp65亞單位覈內轉移越多,內脂素100ng/ml組為0.763±0.056,內脂素400 ng/ml組為1.290±0.065,均大于對照組的0.467±0.046(P均<0.05).結論 內脂素可通過激活NF-κB途徑上調人單覈細胞中MMP-2和MMP-9錶達及活性.這可能是內脂素緻動脈粥樣硬化及與急性冠狀動脈綜閤徵密切相關的機製之一.
목적 탐토내지소대핵인자κB(NF-κB)통로적격활급기재조절인단핵세포기질금속단백매(MMP)-2화MMP-9적표체중적작용,이기진일보료해내지소여동맥죽양경화적관계.방법 이건강인외주혈단핵세포위연구대상.추취건강지원자정맥혈,분리、순화단핵세포후수궤분위공백대조조;내지소조(100、200、400 ng/ml,24 h);Bay11-7082억제제조(Bay11-7082 40μmol/ml예부육30 min,내지소400 ng/ml자격24 h).형광정량PCR검측각조세포MMP-2화MMP-9 mRNA적표체;Western blot검측MMP-2화MMP-9단백적표체;명효매보법검측세포배양기질중MMP-2화MMP-9적매활성.내지소자격세포후적핵전위분별용Western blot화ELISA진행검측.결과 인외주혈단핵세포경불동농도적내지소간예배양후24 h단핵세포MMP-2화MMP-9 mRNA급단백적표체균명현고우공백대조조(P균<0.05),차정농도의뢰성.내지소환상조세포분비도세포배양기중적MMP-2화MMP-9매활성,내지소100 ng/ml조MMP-9、MMP-2적매활성분별위공백대조조적(1.139 ±0.055)화(1.102±0.011)배(P균<0.05).가입NF-κB억제제Bay11-7082후MMP-2、MMP-9적mRNA、단백적표체급매활성균명현저,여내지소400ng/ml조비교차이균유통계학의의(P균<0.001).내지소농도월고,핵단백NF-κBp65표체월고,내지소100 ng/ml조상대회도치위공백대조조적(1.334±0.148)배(P균<0.05);내지소농도월고,NF-κBp65아단위핵내전이월다,내지소100ng/ml조위0.763±0.056,내지소400 ng/ml조위1.290±0.065,균대우대조조적0.467±0.046(P균<0.05).결론 내지소가통과격활NF-κB도경상조인단핵세포중MMP-2화MMP-9표체급활성.저가능시내지소치동맥죽양경화급여급성관상동맥종합정밀절상관적궤제지일.
Objective To investigate the effects of visfatin on the MMP-2 and MMP-9 expressions in human monocytes and related mechanisms. Methods Human monocytes were isolated from blood, the expressions of MMP-2 and MMP-9 at mRNA and protein levels were detected in visfatin stimulated monocytes (0, 100, 200, 400 ng/ml) in the absence and presence of NF-κB inhibitor specific Bay11-7082 by Realtime PCR or Western blot, the MMP-2 and MMP-9 enzyme activity in the culture media was also detected by Gelatin Zymography. The NF-κB protein level and NF-κBp65 expression in visfatin stimulated cells were measured by Western blot and ELISA, respectively. Results Visftain upregulated MMP-2 and MMP-9 expressions in human monocytes in a dose dependent manner. After treatment with visfatin 400 ng/ml for 24 h, comparing with the free visfatin treatment, the protein expressions of MMP-2 and MMP-9 were up-regulated to 1. 644 ± 0.052 and 3. 578 ± 0. 081 (all P < 0. 001); the enzyme activities of MMP-2 and MMP-9 were enhanced by 1. 661 ± 0. 036 ( P < 0. 001) and 1. 662 ± 0. 100 (P < 0. 001 ). NF-κB was also activated in these cells by visaftin and these effects could be significantly attenuated by Bay 11-7082. Visftain induced a dose-dependent ( 100 - 400 ng) increase of NF-kBp65 nuclear translocation from 0. 763 ±0. 056 to 1. 290 ± 0. 065 at 100 and 400 ng/ml, comparing with free visfatin treatment 0. 467 ± 0.046 (all P<0.05). Bay11-7082 decreased the protein expression of MMP-2 and MMP-9 to 1. 183 ± 0. 030 and 2. 024 ± 0. 056 (all P < 0. 001 comparing with 400 ng/ml visfatin treatment). Conclusion Visfatin enhanced the expression and activity of MMP-2 and MMP-9 in human monocytes via activating NF-κB signaling pathway.
