基质金属蛋白酶-9靶向RNA干扰重组载体的构建和效应检测

目的:构建针对人基质金属蛋白酶-9(MMP-9)小干扰RNA(siRNA)表达载体,并观察其对川崎病(KD)血清诱导的血管内皮细胞MMP-9高表达的干扰效果。方法:依据siRNA设计原则设计两对MMP-9基因的特异性干扰序列,体外分别合成两端含BamH Ⅰ和Hind Ⅲ酶切位点的编码短发夹RNA序列的DNA单链,退火后与pSilencer3.1-HI neo线性质粒连接,转化DH5α感受态菌株,筛选阳性克隆,提取质粒进行双酶切及琼脂糖电泳鉴定。阳性重组质粒测序。将构建的两个MMP-9 siRNA质粒以KD血清作为诱导因子,用脂质法转染内皮细胞ECV-304,应用RT-PCR法测定各实验组血管内皮细胞MMP-9mRNA表达、Western blot检测各组MMP-9蛋白表达情况,对所构建的MMP-9 siRNA质粒进行效应检测。结果:DNA测序证实合成的并被克隆入pSilencer3.1-HI neo表达质粒siRNA插入序列与设计完全符合。KD血清组MMP-9蛋白及mRNA表达水平均较正常血清组明显升高(P<0.05);而所构建的两个MMP-9 siRNA及人血丙种球蛋白均能显著抑制KD血清的上述诱导作用(P<0.05)。结论:成功构建的两个MMP-9 siRNA比人血丙种球蛋白更能高效、特异地抑制MMP-9的表达,为进一步研究MMP-9基因对KD冠状动脉病变的影响及其临床应用奠定了基础。

Construction and Identification of Small Interfering RNA Expression Vector Targeting Matrix Metalloproteinase-9

Objective:To construct matrix metalloproteinase-9 (MMP-9) small interfering RNA (siRNA) expression vector and evaluate its expression in vascular endothelial cells(EVC-304) sitmulated by the serum from children with acute phase of Kawasaki disease (KD). Method: Lone DNA with cutting sites of BamHⅠand Hind Ⅲ enzyme was choiced and combined according the sequence of MMP-9. pSilencer3.1-HI neo vector was cut in sites of BamHⅠand Hind Ⅲ enzyme,the fragment of pSilencer3.1-HI neo expressing vector was sub-cloned,and identified by PCR and sequencing.ECV-304 were cultured in six different conditional media:normal serum group,KD serum+ siRNA negative control group,KD serum+MMP-9 siRNA 1 group,KD serum+MMP-9 siRNA 2 group,KD serum+ γ-globulin group,and KD serum group. RT-PCR and Western blot were used to detect MMP-9 mRNA expression and total protein levels of MMP-9 in ECV-304. Results: RT-PCR and sequencing confirmed that it was succeeded in constructing recombinant plasmid pSilencer3.1-HI neo siRNA of MMP-9. The mRNA expression and total protein levels of MMP-9 in ECV-304 cultured with 10% serum from KD patients were all significantly elevated(P<0.05),compared with ECV-304 cultured with 10% serum from normal control children. MMP-9 siRNA and γ-globulin at 100mg/ml could significantly inhibit these elevations (P<0.05).Conclusion: Two recombinant plasmid MMP-9 siRNA of human constructed successfully and lay the foundation for further study RNAi in MMP-9.