CD147单抗介导的基因治疗纳米颗粒的肺癌细胞靶向性研究

CD147 monoclonal antibody-mediated nanoparticles for gene therapy to target lung cancer cells

目的:本研究采用靶向CD147的单克隆抗体对纳米基因载体颗粒进行靶向修饰后,进行针对肺癌细胞的蛋白激酶Cε(protein kinase Cε,PKCε)小干扰RNA基因治疗,观察其对肺癌细胞增殖和迁移能力的抑制效果。方法:制作可靶向CD147蛋白的磁性纳米基因载体。激光扫描共聚焦显微镜观察肺癌细胞CD147表达量。分别设立CP组、CN组和LP组复合物,按每6孔板孔质粒总量250 ng进行细胞转染。另设CD147靶向载体对照CA组和未转染细胞的对照(control)组。激光扫描共聚焦显微镜观察纳米造影剂的细胞内吞效果。实时荧光定量PCR检测PKCε的mRNA表达。Western blot法检测PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白表达。平板克隆形成实验检测细胞的增殖能力。Transwell法检测细胞的迁移能力。结果:免疫荧光法染色观察证实,人肺癌A549细胞的胞膜高表达CD147蛋白。CP组细胞中siRNA高效进入A549细胞,质粒内吞效率大于CN组和LP组。CP组、CN组、LP组和CA组的A549细胞中PKCε的mRNA相对表达量分别为control组的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%,CP组与control组间的差异有统计学显著性(P<0.05),CN组、LP组与control组间的差异无统计学显著性。CP组PKCε、Ki-67、MMP3及Wnt1蛋白的表达量明显降低,CN组和LP组与对照组之间的蛋白表达量的差异无统计学显著性。CP组的克隆形成数量明显少于control组,差异具有统计学显著性(P<0.05)。CN组、LP组和CA组的有效克隆数量与control组相比差异没有统计学显著性。CP组的过膜细胞数量明显少于control组,差异具有统计学显著性(P<0.05)。CN组、LP组和CA组的数量与control组相比差异没有统计学显著性。结论:靶向CD147修饰的纳米基因载体,可以对肺癌细胞进行高效的PKCε-siRNA基因治疗,实现对肺癌细胞增殖和迁移能力的高效抑制。

AIM： In this study,CD147 antibody was used to carry out targeted modification of nanoparticles for protein kinase Cε（ PKCε）-siRNA gene therapy to target lung cancer cells. The inhibitory effects of the nanoparticles on the proliferation and invasion of the lung cancer cells were observed. METHODS： The magnetic nanoparticles targeting CD147 protein were assembled as gene vector. The expression of CD147 in the lung cancer cells was observed under laser scanning confocal microscope. The cells were divided into CP group,CN group and LP group as the experimental groups.Targeted nanoparticles were used as CA group. Non-transfected cells were used as control group. The cell transfection was carried out with 250 ng plasmids / well in 6-well plate. The effect of nanocontrast agent on the cell endocytosis was observed under laser scanning confocal microscope. The mRNA expression of PKCε was detected by RT-q PCR. The protein expression of Ki67,MMP3,PKCε,Wnt1 and GAPDH was determined by Western blot. The cell proliferation ability was detected with colony formation assay. The cell invasion ability was detected by Transwell method. RESULTS： The expression ofCD147 protein in the human lung cancer A549 cells was confirmed by immunofluorescence staining. The endocytosis of siRNA into the A549 cells in CP group was observed with the highest efficiency as compared with CN group and LP group.The relative mRNA expression of PKCε in the A549 cells of CP group,CN group,LP group and CA group were（ 9. 76 ±0. 18） %,（ 98. 51 ± 0. 32） %,（ 99. 17 ± 0. 16） % and（ 99. 68 ± 0. 11） %,respectively. The difference between CP group and control group was statistically significant（ P〈0. 05）. No significant difference among CN group,LP group and control group was observed. The protein expression of PKCε,Ki-67,MMP3 and Wnt1 in CP group was significantly reduced,and the protein expression levels among CN group,LP group and control group had no significant difference. The colony number in CP group was significantly smaller than that in control group（ P〈0. 05）. The effective colony numbers in CN group,LP group and CA group had no significant difference as compared with control group. The number of the invading cells in CP group was significantly less than that in control group（ P〈0. 05）. The numbers of the invading cells in CN group,LP group and CA group had no significant difference as compared with control group. CONCLUSION： Nanogene vector targeting CD147 can carry PKCε-siRNA to conduct gene therapy efficiently on the lung cancer cells to achieve effective inhibitory effects on the proliferation and invasion of the lung cancer cells.