重组慢病毒介导的OPCML基因的体外转导及其对卵巢上皮性癌细胞的抑制作用

目的探讨重组慢病毒介导的体外转导OPCML基因的可行性及其对卵巢上皮性癌(卵巢癌)细胞的抑制作用。方法采用PCR技术,用基因特异性引物将OPCML基因的cDNA从CD1小鼠脑组织中扩增,并将OPCML基因克隆到慢病毒载体表达质粒pWPI GFP上,构建慢病毒载体表达质粒pWPI OPCML;将pWPI OPCML或pWPI GFP(空载体对照)质粒和包装质粒pCMVdR8.74、pMDG共同转染人胚胎肾上皮细胞系293T细胞,获得携带OPCML和绿色荧光蛋白(GFP)基因的重组慢病毒WPI OPCML或仅携带GFP基因的重组慢病毒WPI GFP;用重组慢病毒WPI OPCML和WPI GFP分别转导靶细胞,即人卵巢癌细胞系A2780、OCC1细胞和小鼠正常卵巢上皮细胞系CD1细胞,以未转染任何基因者为空白对照。通过细胞增殖实验、细胞聚合力实验、细胞周期分析和体内成瘤实验观察OPCML基因对卵巢癌细胞的抑制作用。结果(1)OPCML基因能被重组慢病毒高效地转导入靶细胞,转导效率几乎达100%,并稳定表达;蛋白印迹法检测显示,靶细胞中OPCML和GFP蛋白均有表达。(2)细胞增殖实验显示,A2780细胞中,转导OPCML基因72h后的A2780/OPCML细胞为(7.6±1.0)×105个,明显少于未转导基因的A2780细胞或仅转导空载体的A2780/pWPI细胞[分别为(20.0±2.6)×105和(18.1±1.7)×105个;P<0.01];但OCC1和CD1细胞中,OPCML基因对细胞的增殖无明显的影响(P>0.05)。(3)细胞周期分析显示,OPCML基因对A2780细胞的细胞周期有明显的阻滞作用(转导前、后G0～G1期细胞分别为67%、75%;P<0.05),但对OCC1、CD1细胞则无明显阻滞作用(P>0.05)。(4)细胞聚合力实验显示,与空载体对照相比,OPCML基因能明显增强细胞的黏附力(P<0.05)。(5)移植瘤实验显示,将A2780/OPCML细胞注射到裸鼠皮下,4只裸鼠中仅1只裸鼠有肿瘤生长,其肿瘤重量为10mg,显著低于注射A2780、A2780/pWPI细胞的裸鼠[各4只裸鼠均有肿瘤生长,其肿瘤重量分别为(280±53)及(677±323)mg;P<0.01]。免疫组化法检测显示,肿瘤组织中OPCML蛋白有表达。结论用重组的慢病毒载体作为转基因的工具,具有高效性,同时能使目的基因达到持续稳定的表达。OPCML基因能够增加A2780细胞的黏附能力,抑制A2780细胞的增殖和体内成瘤,提示OPCML基因可能是一个新的抑癌基因。

*OPCML gene transferred by recombinant lentiviruses in vitro and its inhibition to ovarian cancer cells

OBJECTIVE: To study the inhibition of OPCML on ovarian cancer cell lines using a lentiviral vector system for efficient gene transduction. METHODS: The murine OPCML cDNA was amplified by PCR from CD1 murine brain cDNA using gene specific primers, and subcloned into the lentiviral vector, pWPI-GFP, to generate the lentiviral expression vector, pWPI-OPCML. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pWPI-OPCML, with the packaging plasmids pCMV-dR8.74 and pMDG. The resulting recombinant lentiviruses which carried OPCML or control viruses (only carrying GFP), were then used to infect the human ovarian cancer cell lines A2780 and OCC1 in addition to normal CD1 mouse ovarian surface epithelial cells. The infected cells were then characterized by cell proliferation assays, cell aggregation assays, cell cycle analysis by flow cytometry and tumorigenicity assays following injection into nude mice. RESULTS: (1) The efficiency of infection of the cell lines using the lentiviral vectors was almost 100% allowing the stable expression of OPCML in nearly all cells. Stable expression of OPCML (60 000) and GFP (27 000) proteins was confirmed by western blot analysis. (2) A2780 cells expressing OPCML [(7.6 +/- 1.0) x 10(5)] grew slowly compared to A2780 parental [(20.0 +/- 2.6) x 10(5)] or control virus infected cells [(18.1 +/- 1.7) x 10(5), P < 0.01], but the expression of OPCML had no effect on the proliferation rates of OCC1 and the normal CD1 cells when compared to their respective parental or controls (P > 0.05). (3) Flow cytometry based cell cycle assays showed that the expression of OPCML could arrest A2780 cells (G(0) approximately G(1) 67% vs 75%, P < 0.05); but not OCC1, CD1 cells. (4) The rate of aggregation of single cell suspensions was measured and found to be increased in all cell lines expressing OPCML indicating the increased cell surface adhesion mediated by OPCML. (5) A2780 cells expressing OPCML only formed a single tumor in 1/4 mice (10 mg) which was significantly smaller than controls [4/4; A2780 (280 +/- 53) mg and A2780/pWPI (677 +/- 323) mg; P < 0.01]. Expression of OPCML in tumor was confirmed by immunohistochemistry. CONCLUSIONS: The use of lentiviral vectors allowed the efficient expression of OPCML in nearly 100% of target cells. Expression of the OPCML cDNA resulted in an increase of cell adhesion in all cell lines tested, and decreased the proliferation and tumorigenicity of the A2780 ovarian cancer cell line. This indicates that the OPCML may be a new tumor suppressor gene.