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目的构建针对细胞外基质金属蛋白酶诱导因子(CD147)的慢病毒干扰载体,探讨干扰CD147表达后对白血病细胞系SHI-1细胞体外增殖、侵袭、转移能力的影响。方法设计针对CD147的干扰序列片段,构建于慢病毒载体pGCSIL-GFP上,重组慢病毒载体与包装载体共转染293T细胞,收集病毒上清,测定病毒滴度。病毒上清感染SHI-1细胞,RT-PCR法检测CD147、MMP-2及MMP-9 mRNA表达,Western blotting法检测CD147蛋白水平;MTT法检测细胞体外增殖能力,体外SHI-1细胞与骨髓基质细胞共培养跨Matrigel基质胶检测SHI-1细胞的体外侵袭能力。结果成功构建慢病毒干扰载体,获得重组慢病毒,滴度达1×109TU/mL。病毒感染SHI-1细胞后,SHI-1/CD147i细胞的CD147 mRNA较感染阴性对照病毒的SHI-1/NC细胞下调86.7%,CD147蛋白表达下降91%,SHI-1/CD147i细胞MMP-2及MMP-9 mRNA表达较SHI-1/NC细胞分别下降78.3%和70.6%。SHI-1/CD147i细胞体外增殖能力较SHI-1/NC及SHI-1细胞明显下降;与骨髓基质细胞共培养24 h后,SHI-1、SHI-1/NC、SHI-1/CD147i细胞移行至Transwell下层的细胞占接种细胞数比例分别为(18.2±2.5)%、(16.5±2.7)%、(4.5±1.2)%,SHI-1/CD147细胞移行能力显著低于SHI-1和SHI-1/NC细胞。结论干扰SHI-1细胞的CD147表达后,通过下调MMPs表达抑制SHI-1细胞体内外增殖及移行能力,CD147可能成为治疗急性白血病的靶点。
Objective To construct a lentivirus vector targeting extracellular matrix metalloproteinase inducer (CD147) and investigate the effect of interfering CD147 expression on proliferation, invasion and metastasis of leukemia cell line SHI-1 in vitro. Methods The interfering sequence fragment targeting CD147 was designed and constructed on the lentiviral vector pGCSIL-GFP. The recombinant lentiviral vector and the packaging vector were co-transfected into 293T cells, and the virus supernatant was collected to determine the virus titer. The expression of CD147, MMP-2 and MMP-9 mRNA were detected by RT-PCR and CD147 protein levels by Western blotting. The proliferative ability of SHI-1 cells and bone marrow stromal cells Cell co-cultures were performed to detect the in vitro invasiveness of SHI-1 cells across Matrigel matrigel. Results The lentiviral vector was successfully constructed and the recombinant lentivirus was obtained with a titer of 1 × 109TU / mL. The CD147 mRNA of SHI-1 / CD147i cells was down-regulated by 86.7%, the expression of CD147 protein was down 91%, the expression of MMP-2 in SHI-1 / CD147i cells was significantly lower than that in SHI-1 / NC cells infected with negative control virus Compared with SHI-1 / NC cells, MMP-9 mRNA expression decreased by 78.3% and 70.6% respectively. The proliferation of SHI-1 / CD147i cells in vitro was significantly lower than that in SHI-1 / NC and SHI-1 cells. SHI-1, SHI-1 / NC and SHI-1 / CD147i cells were transplanted with bone marrow stromal cells The number of cells inoculated into the lower layer of Transwell was (18.2 ± 2.5)%, (16.5 ± 2.7)% and (4.5 ± 1.2)% respectively. The migration ability of SHI-1 / CD147 cells was significantly lower than that of SHI- 1 / NC cells. Conclusions After interfering with the expression of CD147 in SHI-1 cells, CD147 may be the target of treating acute leukemia by down-regulating the expression of MMPs and inhibiting the proliferation and migration of SHI-1 cells in vitro and in vivo.