CD147慢病毒载体对兔外周血单核巨噬细胞的沉默作用

目的 设计合成干扰细胞外基质金属蛋白酶诱导因子(CD147)基因表达并筛选出能高效抑制兔外周血单核/巨噬细胞中CD147表达的shRNA慢病毒表达载体.方法 根据兔源CD147mRNA的序列构建慢病毒表达载体,实验分为6组:空白对照组,阴性对照组(NC),构建4对干扰CD147基因表达的shRNA慢病毒组(A、B、C、D),并转染兔外周血单核/巨噬细胞,72h后通过荧光显微镜观察转染效果,采用实时荧光定量PCR(RT-PCR)和Elisa,观察CD147mRNA和蛋白表达情况.结果 转染后72h收集细胞,空白对照组CD147mRNA相对表达量为(0.98±0.09),CD147蛋白表达(119.69±16.40)pg/ml,NC对照组(1.00±0.05)pg/ml及(115.66±13.88)pg/ml,4对shRNA慢病毒载体组分别为A组(0.42±0.03)pg/ml及(58.74±5.11)pg/ml、B组(0.56±0.04)pg/ml及(70.99±7.42)pg/ml、C组(0.63±0.08)pg/ml及(82.79±7.71)pg/ml、D组(0.53±0.04)pg/ml及(69.71±5.84)pg/ml.设计合成的4对shRNA慢病毒载体中A组可以特异性抑制巨噬细胞中CD147mRNA和蛋白表达,分别减少57.72%和50.92%(P<0.05).结论 成功构建了针对CD147基因的shRNA慢病毒载体,并从中筛选出能特异且高效阻断CD147表达的shRNA.

Silencing effect of CD147 lentiviral vector on monocytes and macrophagocytes in peripheral blood of rabbits

Objective To study the expression of matrix metalloproteinase inducer(CD147) and screen the lentivirus vectors that highly suppress the CD147-expressed shRNA in monocytes and macrophagocytes in peripheral blood of rabbits.Methods Lentivirus vectors were constructed according to the sequence of CD147 mRNA from rabbits.The animals were divided into blank control group,negative control(NC) group,and 4 CD147 shRNA lentivirus groups(groups A-D).Seventy-two hours after monocytes and macrophagocytes were transfected into shRNA,the transfection results were observed by fluorescence microscopy.The expression of CD147 mRNA and protein was assayed by fluorescence quantitative RT-PCR and ELISA,respectively.Results RT-PCR showed that the expression level of CD147 mRNA and protein was(0.98±0.09)pg/ml and(119.69±16.40)pg/ml,(1.00±0.05)pg/ml and(115.66±13.88)pg/ml,(0.42±0.03)pg/ml and(58.74±5.11)pg/ml,(0.56±0.04)pg/ml and(70.99±7.42)pg/ml,(0.63±0.08)pg/ml and(82.79±7.71)pg/ml,(0.53±0.04)pg/ml and(69.71±5.84)pg/ml,respectively,in control group,NC group,and 4 shRNA lentivirus vector groups,72h after transfection.The CD147 shRNA lentivirus vectors inhibited the expression of CD147 mRNA and protein in macrophagocytes of about 57.72% and 50.92%(P<0.05).Conclusion The successfully designed and synthesized CD147 shRNA lentivirus vectors can specifically and effectively block the CD147-expressed shRNA.