内质网滞留型抗Ⅳ型胶原酶胞内抗体对肿瘤细胞侵袭和增殖的抑制作用

Inhibitory Effect of Endoplasmic Reticulum-Retained Anti-type ⅣCollagenase In trabody on Invasion and Proliferation of Cancer Cell

背景与目的:侵袭转移是恶性肿瘤细胞的重要特性,Ⅳ型胶原酶(包括基质金属蛋白酶2和基质金属蛋白酶9)在恶性肿瘤转移扩散中发挥着极其重要的作用。我们通过胞内抗体技术,阻断Ⅳ型胶原酶的分泌,以期达到抑制恶性肿瘤细胞侵袭转移的效果。方法:构建编码可在内质网滞留的抗Ⅳ型胶原酶单链抗体的表达载体pcDNA3.1-ER.scFv,并转染入人巨细胞肺癌PG细胞系内。Westernblot检测pcDNA3.1-ER.scFv的表达,免疫共沉淀实验分析胞内抗体ER.scFv在PG细胞内与靶蛋白相互作用情况,明胶酶谱检测Ⅳ型胶原酶的分泌,以及体外侵袭和增殖实验。结果:ER.scFv在PG细胞内表达,而且能够识别和结合靶蛋白基质金属蛋白酶9。通过胞内抗体的基因转染,显著抑制了Ⅳ型胶原酶的功能和活性。ER.scFv转染的PG细胞较野生型和空白质粒组,侵袭能力明显降低,抑制率为76.3%(P<0.05),在Matrigel上进行细胞培养,ER.scFv对PG细胞有明显的抗增殖效果。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Ⅳ型胶原酶的活性,进而抑制了肿瘤侵袭和增殖,在肿瘤基因治疗中具有应用前景。

BACKGROUND &OBJECTIVE: Invasion and metastasis are significant ch ar acteristics of cancer cells, Type Ⅳcollagenase (matrix metalloproteinase-2, an d-9) plays an important role in cancer invasion and metastasis. This study was designed to block the secretion of type Ⅳcollagenase via intracellular antibody (intrabody) methods, and inhibit cancer invasion and metastasis. METHODS: We co nstructed expression plasmid pcDNA3.1-ER.scFv coding for an endoplasmic reticul um (ER)-retained, single chain antibody (ER.scFv) against the type Ⅳcollagenas e. The intrabody gene was transfected into human giant cell pulmonary carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-ER.sc Fv. Co-immunoprecipitation was used to analyse the interaction between ER. scFv and target protein in PG cells. The secretion of type Ⅳcollagenase was detecte d by gelatin zymography. Cell behavior was examined by invasion and proliferatio n assay in vitro. RESULTS: ER. scFv expressed in PG cells, and can recognize and combine its cognate target protein matrix metalloproteinase-9. Intrabody gene transfection significantly blocked the function and activity of type Ⅳcollegena se. The ER.scFv-transfected PG cells were less invasive than parental or vector control cells, the suppression rate of ER.scFv-transfected PG cells was 76.3% (P< 0.05). ER. scFv showed anti-proliferative effect on PG cells cultured on Ma trigel. CONCLUSIONS: ER-retained intrabody technology may restrain the activity of type IV collagenase in protein processing and secretory pathway, and inhibit s cancer cell invasion and proliferation. Anti-type Ⅳcollagenase intrabody may be further used in cancer gene therapy.