CD147单抗介导的基因治疗纳米颗粒的肺癌细胞靶向性研究

发布时间：2018-09-06 16:48

【摘要】：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(P0.05),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(P0.05)。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(P0.05)。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。
[Abstract]:Objective: to study the small interfering RNA gene therapy of protein kinase C 蔚 (PKC 蔚) targeting lung cancer cells by targeting the nanoparticles with monoclonal antibody targeting CD147. To observe its inhibitory effect on proliferation and migration of lung cancer cells. Methods: magnetic nanogene vector targeting CD147 protein was prepared. The expression of CD147 in lung cancer cells was observed by laser scanning confocal microscopy. The complexes of CP group CN and LP group were set up, and the cells were transfected according to the total amount of plasmid per 6 hole plate hole of 250 ng. CD147 targeted vector control CA group and untransfected cell control (control) group were used. The endocytosis of nano-contrast agent was observed by laser scanning confocal microscope. The mRNA expression of PKC 蔚 was detected by real-time fluorescence quantitative PCR. Western blot was used to detect the protein expression of PKC 蔚 Ki67 MMP3Cnt1 and GAPDH. The ability of cell proliferation was detected by plate clone formation assay. Transwell method was used to detect cell migration ability. Results: the results of immunofluorescence staining showed that siRNA was highly expressed in A549 cells with high expression of CD147 protein. SiRNA was highly expressed in A549 cells. The relative expression of PKC 蔚 in A549 cells in CN group and LP group. CP group was higher than that in control group (9.76 卤0.18), (98.51 卤0.32), (99.17 卤0.16)% and (99.68 卤0.11), respectively. There was significant difference between CP group and control group (P0.05). The expression of PKC 蔚 Ki-67MMP3 and Wnt1 protein in CP group was significantly lower than that in control group. There was no significant difference in protein expression between CN group and LP group. There was no significant difference between CP group and control group. The number of clone formation in CP group was significantly lower than that in control group. There was no significant difference in the number of effective clones between LP group and CA group compared with control group (P0.05). The number of overmembrane cells in CP group was significantly lower than that in control group. The difference was statistically significant (P0.05). There was no significant difference in the number of LP group and CA group between CN group and control group. Conclusion: CD147 modified nano-gene vector can effectively inhibit the proliferation and migration of lung cancer cells by PKC 蔚 -siRNA gene therapy.
【作者单位】：中山大学附属第三医院急诊科;中山大学附属第三医院心胸外科;
【基金】：广东省科技计划(No.2014A020212533)
【分类号】：R734.2

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