内质网滞留型抗明胶酶胞内抗体的基因表达及对肿瘤细胞侵袭和增殖的抑制作用

Gene expression of endoplasmic reticulum-retained anti-gelatinase intrabody and its inhibition of cancer cell invasion and proliferation

目的 :明胶酶 (包括MMP 2和MMP 9)在恶性肿瘤转移扩散中发挥着重要的作用 ,探讨运用胞内抗体技术通过胞内抗体与明胶酶结合 ,阻断明胶酶向细胞表面的分泌 ,对恶性肿瘤细胞侵袭转移抑制的效果。方法 :构建可在真核细胞内质网表达抗明胶酶抗体基因的真核表达载体PcDNA3.1.ER .scFv ,瞬时和稳定基因转染 ,Westernblot检测基因表达 ,免疫沉淀实验检测胞内抗体与靶蛋白结合情况 ,明胶酶谱检测明胶酶的分泌 ,用相关方法观察细胞体外运动、侵袭和增殖。结果 :ER .scFv抗体基因在PG细胞内表达 ,能与靶蛋白MMP 9特异性结合 ,对PG细胞明胶酶分泌具有显著的抑制作用。ER .scFv胞内抗体对PG细胞体外Matrigel侵袭有抑制作用 ,抑制率为 76 .3% (P <0 .0 5 ) ,对PG细胞的体外迁徙没有影响。在体外增殖实验中 ,稳定表达ER .scFv的PG细胞在Matrigel的增殖受到明显抑制。结论 :内质网滞留型胞内抗体技术可以在蛋白加工、分泌这一关键通路中抑制肿瘤细胞明胶酶的活性 ,进而抑制与明胶酶直接相关的侵袭和增殖 。

Objective:This study was designed to block the secretion of gelatinase,and to inhibit tumor invasion and metastasis via intrabody methods.Methods: An expression plasmid PcDNA3.1.ER.scFv coding for an endoplasmic reticulum (ER)-retained, single chain antibody (ER.scFv) against gelatinase was constructed. The intracellular antibody gene was transfected into the human large cell lung cancer PG cells. The expression of PcDNA3.1-ER.scFv was determined by Western blot analysis. Co-immunoprecipitation was used for analyzing the interaction of ER.scFv and target protein in PG cells. Gelatin zymography, cell migration, invasion and proliferation assay were aiso performed.Results:The expression of the intracellular antibody wasdetected. Immunoprecipitation from ER.scFv-transfected cells showed that the single chain antibody possessed the capacity to recognize its cognate target in the context of intracellular expression. By the means of intrabody gene therapy, the function and activity of gelatinase was blocked significantly. The ER.scFv-transfected PG cells were much less invasive than parental or vector control cells, the value of supression is 76.3% (P<0.05), whereas the ER.scFv had no effect in chemotactic motility. In culture on Matrigel the ER.scFv had anti-proliferative effect on PG cell. Conclusion:These findings demonstrate that intracellular antibody technology represents a novel and efficient way to selectively abrogate the activity of gelatinase in the protein trafficking and secretory pathway and effectively inhibits tumor cell invasion and proliferation in vitro. Anti-gelatinase intrabody may be further used in cancer gene therapy.