广西医学
廣西醫學
엄서의학
Guangxi Medical Journal
2015年
9期
1222-1225
,共4页
活性氧%转化生长因子 β1%基质金属蛋白酶%血管平滑肌细胞%大鼠
活性氧%轉化生長因子 β1%基質金屬蛋白酶%血管平滑肌細胞%大鼠
활성양%전화생장인자 β1%기질금속단백매%혈관평활기세포%대서
Reactive oxygen species%Transforming growth factor-β1%Matrix metalloproteinase%Vascular smooth muscle cell%Rat
目的：探讨活性氧在转化生长因子-β1（TGF-β1）刺激血管平滑肌细胞（VSMC）产生基质金属蛋白酶9（MMP9）中的作用。方法取一批原代培养大鼠主动脉VSMC分为6组，分别按0、5、0、0．05、0．5、5 mmol／L给予抗氧化剂N-乙酰半胱氨酸（NAC）预干预细胞1 h，再用不同浓度TGF-β1（0、0、10、10、10、10 ng／ml）作用24 h，明胶酶谱法检测培养基上清中MMP的活性，裂解细胞蛋白质印迹检测细胞中MMP9的表达。另取一批VSMC原位装载2′，7′-二氯荧光黄双乙酸盐荧光探针，分为两组，A组予TGF-β1（10 ng／ml）干预，B组予NAC（5 mmol／L）预作用1 h后再予10 ng／ml TGF-β1干预，倒置荧光显微镜观察细胞内荧光强度。最后取一批 VSMC 分为6组：（1）对照组；（2）0．1 mmol／L 过氧化氢（H2O2）组；（3）1 mmol／L H2O2组；（4）5 mmol／LNAC组；（5）5 mmol／L NAC＋1 mmol／L H 2O 2组；（6）5 mmol／L NAC＋1 mmol／L H 2O 2＋10 ng／ml TGF-β1组。分别作用24 h后检测培养基上清中蛋白酶的活性和细胞中MMP9的表达。结果 NAC 显著抑制 VSMC 在 TGF-β1作用下MMP9的表达。 TGF-β1作用30 min及60 min后细胞内荧光强度明显增加；而NAC预作用后荧光强度明显减弱。1 mmol／L H2 O2使VSMC表达MMP9显著增高，NAC显著抑制这种作用。结论活性氧参与TGF-β1刺激VSMC表达的MMP9的过程。
目的：探討活性氧在轉化生長因子-β1（TGF-β1）刺激血管平滑肌細胞（VSMC）產生基質金屬蛋白酶9（MMP9）中的作用。方法取一批原代培養大鼠主動脈VSMC分為6組，分彆按0、5、0、0．05、0．5、5 mmol／L給予抗氧化劑N-乙酰半胱氨痠（NAC）預榦預細胞1 h，再用不同濃度TGF-β1（0、0、10、10、10、10 ng／ml）作用24 h，明膠酶譜法檢測培養基上清中MMP的活性，裂解細胞蛋白質印跡檢測細胞中MMP9的錶達。另取一批VSMC原位裝載2′，7′-二氯熒光黃雙乙痠鹽熒光探針，分為兩組，A組予TGF-β1（10 ng／ml）榦預，B組予NAC（5 mmol／L）預作用1 h後再予10 ng／ml TGF-β1榦預，倒置熒光顯微鏡觀察細胞內熒光彊度。最後取一批 VSMC 分為6組：（1）對照組；（2）0．1 mmol／L 過氧化氫（H2O2）組；（3）1 mmol／L H2O2組；（4）5 mmol／LNAC組；（5）5 mmol／L NAC＋1 mmol／L H 2O 2組；（6）5 mmol／L NAC＋1 mmol／L H 2O 2＋10 ng／ml TGF-β1組。分彆作用24 h後檢測培養基上清中蛋白酶的活性和細胞中MMP9的錶達。結果 NAC 顯著抑製 VSMC 在 TGF-β1作用下MMP9的錶達。 TGF-β1作用30 min及60 min後細胞內熒光彊度明顯增加；而NAC預作用後熒光彊度明顯減弱。1 mmol／L H2 O2使VSMC錶達MMP9顯著增高，NAC顯著抑製這種作用。結論活性氧參與TGF-β1刺激VSMC錶達的MMP9的過程。
목적：탐토활성양재전화생장인자-β1（TGF-β1）자격혈관평활기세포（VSMC）산생기질금속단백매9（MMP9）중적작용。방법취일비원대배양대서주동맥VSMC분위6조，분별안0、5、0、0．05、0．5、5 mmol／L급여항양화제N-을선반광안산（NAC）예간예세포1 h，재용불동농도TGF-β1（0、0、10、10、10、10 ng／ml）작용24 h，명효매보법검측배양기상청중MMP적활성，렬해세포단백질인적검측세포중MMP9적표체。령취일비VSMC원위장재2′，7′-이록형광황쌍을산염형광탐침，분위량조，A조여TGF-β1（10 ng／ml）간예，B조여NAC（5 mmol／L）예작용1 h후재여10 ng／ml TGF-β1간예，도치형광현미경관찰세포내형광강도。최후취일비 VSMC 분위6조：（1）대조조；（2）0．1 mmol／L 과양화경（H2O2）조；（3）1 mmol／L H2O2조；（4）5 mmol／LNAC조；（5）5 mmol／L NAC＋1 mmol／L H 2O 2조；（6）5 mmol／L NAC＋1 mmol／L H 2O 2＋10 ng／ml TGF-β1조。분별작용24 h후검측배양기상청중단백매적활성화세포중MMP9적표체。결과 NAC 현저억제 VSMC 재 TGF-β1작용하MMP9적표체。 TGF-β1작용30 min급60 min후세포내형광강도명현증가；이NAC예작용후형광강도명현감약。1 mmol／L H2 O2사VSMC표체MMP9현저증고，NAC현저억제저충작용。결론활성양삼여TGF-β1자격VSMC표체적MMP9적과정。
Objective To explore the effect of reactive oxygen species ( ROS) on inducing matrix metalloproteinase ( MMP) 9 from vascular smooth muscle cells(VSMC) with stimulation of transforming growth factor-β1(TGF-β1).Methods VMSCs of primary culture from rats were obtained and divided into 6 groups.The 6 groups were pretreated for one hour with various concentrations (0,5,0,0.05,0.5 and 5 mmol/L,respectively) of N-acetylcysteine(NAC),then were treated with various concentrations (0,0,10,10,10 and 10 ng/ml, respectively) of TGF-β1 for 24 hours.Gelatin zymography was used to detect the activity of MMP in the supernatant liquid of culture medium.Western Blot was used to determine the expression of MMP 9 in VMSCs.Additional VSMCs were incubated in situ with probes of 2′,7′-Dichlorofluorescin diacetate ,and were divided into two groups .TGF-β1 ( 10 ng/ml ) intervention was conducted in Group A , while Group B was given pretreatment of NAC (5 mmol/L) for one hour and then was intervened with TGF-β1(10 ng/ml).Inverted fluorescence microscope was used to observe the intracellular fluorescence intensity .Another few VSMCs were divided into 6 groups,including control group,H2 O2 (0.1 mmol/L) group,H2 O2 (1 mmol/L) group,NAC (5 mmol/L) group,NAC(5 mmol/L) +H2 O2 (1 mmol/L) group and NAC(5 mmol/L)+H2 O2 (1 mmol/L) +TGF-β1(10 ng/ml) group.After 24 hours of tretatment ,the activity of MMP in the supernatant liquid of culture medium and MMP 9 expression in VMSCs were analyzed respectively .Results NAC significantly inhibited the expression of MMP9 from VMSC with stimulation of TGF-β1.The intracellular fluorescence intensity increased 30 and 60 minutes after the pretreatment with TGF-β1,but the fluorescence intensity reduced after the pretreatment with NAC.H2 O2 of 1 mmol/L induced VSMC to express more MMP9 significantly ,which was significantly inhibited by NAC .Conclusion ROS participates in the process of TGF-β1′s stimulating the expression of MMP9 from VMSC.
