中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2009年
9期
837-840
,共4页
RNA%小分子干扰%黏膜皮肤淋巴结综合征%内皮细胞%基质金属蛋白酶9
RNA%小分子榦擾%黏膜皮膚淋巴結綜閤徵%內皮細胞%基質金屬蛋白酶9
RNA%소분자간우%점막피부림파결종합정%내피세포%기질금속단백매9
RNA%small interfering%Mucocutaneous lymph node syndrome%Endothelial cells%Matrix metalloproteinase 9
目的 观察川崎病急性期血清对血管内皮细胞摹质金属蛋白酶-9(MMP-9)表达的影响以及MMP-9小干扰RNA(siRNA)对上述过程的调节作用.方法 构建MMP-9 siRNA质粒,脂质法转染血管内皮细胞(ECV-304).ECV-304细胞培养分为川崎病血清+siRNA阴性对照组、正常血清组、川崎病血清+MMP-9 siRNA1组、川崎病血清+MMP-9 siRNA2组、川崎病血清+人血丙种球蛋白组、川崎病血清组.应用逆转录聚合酶链式反应检测MMP-9 mRNA的表达,免疫印迹法检测MMP-9蛋白的表达.结果 川崎病血清组和川崎病血清+siRNA阴性对照组的MMP-9 mRNA、MMP-9蛋白表达分别是2.49±0.03、1.20±0.04和2.45±0.03、1.15±0.03,均较正常血清组(1.21±0.03、0.52±0.03)明显增高(均P<0.01).川崎病血清+MMP-9 siRNA1组(0.78±0.03、0.31±0.02)、川崎病血清+MMP-9 siRNA2组(0.74±0.04、0.28±0.04)及川崎病血清+人血丙种球蛋白组(1.58±0.03、0.88±0.04)的MMP-9 mRNA、MMP-9蛋白表达与川崎病血清组及川崎病血清+siRNA阴性对照组比较均明显减少(均P<0.01).结论 川崎病急性期血清能诱导血管内皮细胞的MMP-9表达增高,可能参与川崎病冠状动脉损伤的发生.构建的MMP-9 siRNA能显著抑制MMP-9 mRNA和蛋白的表达.
目的 觀察川崎病急性期血清對血管內皮細胞摹質金屬蛋白酶-9(MMP-9)錶達的影響以及MMP-9小榦擾RNA(siRNA)對上述過程的調節作用.方法 構建MMP-9 siRNA質粒,脂質法轉染血管內皮細胞(ECV-304).ECV-304細胞培養分為川崎病血清+siRNA陰性對照組、正常血清組、川崎病血清+MMP-9 siRNA1組、川崎病血清+MMP-9 siRNA2組、川崎病血清+人血丙種毬蛋白組、川崎病血清組.應用逆轉錄聚閤酶鏈式反應檢測MMP-9 mRNA的錶達,免疫印跡法檢測MMP-9蛋白的錶達.結果 川崎病血清組和川崎病血清+siRNA陰性對照組的MMP-9 mRNA、MMP-9蛋白錶達分彆是2.49±0.03、1.20±0.04和2.45±0.03、1.15±0.03,均較正常血清組(1.21±0.03、0.52±0.03)明顯增高(均P<0.01).川崎病血清+MMP-9 siRNA1組(0.78±0.03、0.31±0.02)、川崎病血清+MMP-9 siRNA2組(0.74±0.04、0.28±0.04)及川崎病血清+人血丙種毬蛋白組(1.58±0.03、0.88±0.04)的MMP-9 mRNA、MMP-9蛋白錶達與川崎病血清組及川崎病血清+siRNA陰性對照組比較均明顯減少(均P<0.01).結論 川崎病急性期血清能誘導血管內皮細胞的MMP-9錶達增高,可能參與川崎病冠狀動脈損傷的髮生.構建的MMP-9 siRNA能顯著抑製MMP-9 mRNA和蛋白的錶達.
목적 관찰천기병급성기혈청대혈관내피세포모질금속단백매-9(MMP-9)표체적영향이급MMP-9소간우RNA(siRNA)대상술과정적조절작용.방법 구건MMP-9 siRNA질립,지질법전염혈관내피세포(ECV-304).ECV-304세포배양분위천기병혈청+siRNA음성대조조、정상혈청조、천기병혈청+MMP-9 siRNA1조、천기병혈청+MMP-9 siRNA2조、천기병혈청+인혈병충구단백조、천기병혈청조.응용역전록취합매련식반응검측MMP-9 mRNA적표체,면역인적법검측MMP-9단백적표체.결과 천기병혈청조화천기병혈청+siRNA음성대조조적MMP-9 mRNA、MMP-9단백표체분별시2.49±0.03、1.20±0.04화2.45±0.03、1.15±0.03,균교정상혈청조(1.21±0.03、0.52±0.03)명현증고(균P<0.01).천기병혈청+MMP-9 siRNA1조(0.78±0.03、0.31±0.02)、천기병혈청+MMP-9 siRNA2조(0.74±0.04、0.28±0.04)급천기병혈청+인혈병충구단백조(1.58±0.03、0.88±0.04)적MMP-9 mRNA、MMP-9단백표체여천기병혈청조급천기병혈청+siRNA음성대조조비교균명현감소(균P<0.01).결론 천기병급성기혈청능유도혈관내피세포적MMP-9표체증고,가능삼여천기병관상동맥손상적발생.구건적MMP-9 siRNA능현저억제MMP-9 mRNA화단백적표체.
Objective To investigate the matrix metalloproteinase-9 (MMP-9) expression in vascular endothelial cells stimulated by the serum obtained from children with Kawasaki disease (KD) during the acute phase in the absence and presence of MMP-9 small interfering RNA (siRNA). Methods MMP-9 siRNA plasmids were constructed and transduced into vascular endothelial cells (ECV-304) by liposomal transfection. ECV-304 were cultured in 6 different conditional media: KD serum + siRNA negative control, normal serum, KD serum + MMP-9 siRNA1 (pSilencer3.1-MMP1), KD serum + MMP-9 siRNA2 (pSilencer3.1-MMP2), KD serum + γ-globulin, and KD serum. RT-PCR and Western blot were used to detect MMP-9 expression at mRNA and protein levels in ECV-304. Results The mRNA and protein expression of MMP-9 in ECV-304 cultured with 10% serum from KD patients (2.49±0.03, 1.20±0.04) and KD serum + siRNA negative control plasmid (2.45±0.03, 1.15±0.03)were significantly higher than those cultured with 10% serum from normal control children (1.21±0.03, 0.52±0.03, respectively; all P<0.01) and the increased MMP-9 expression could be significantly inhibited by MMP-9 siRNA1, MMP-9 siRNA2 and γ-globulin (100 mg/ml, all P < 0.01) . Conclusions The increase of MMP-9 expression in vascular endothelial cells induced by the serum from KD patients might take part in the formation of coronary artery lesions. Two customized MMP-9 siRNA plasmids (pSilencer3.1-MMP1 and pSilencer3.1-MMP2) can significantly inhibit both MMP-9 mRNA and protein expression.
