论文部分内容阅读
目的探讨内质网滞留型胞内抗体对Ⅳ型胶原酶分泌及其对人巨细胞肺癌PG细胞侵袭的抑制作用。方法构建了编码胞浆和内质网滞留型抗Ⅳ型胶原酶抗体的表达载体pcDNA3.1-CP.scFv和pcDNA3.1-ER.scFv。对人巨细胞肺癌PG细胞系进行基因转染。以Western blot检测pcDNA3.1-CP.scFv和pcDNA3.1-ER.scFv的表达,明胶酶谱检测PG细胞Ⅳ型胶原酶分泌情况,Matrigel实验检测细胞侵袭。结果CP.scFv和ER.scFv胞内抗体在PG细胞内表达,ER.scFv对Ⅳ型胶原酶分泌具有显著的抑制作用,对基质金属蛋白酶-9和基质金属蛋白酶-2的抑制率分别为85.7%和51.2%;而CP.scFv对Ⅳ型胶原酶分泌无抑制作用。ER.scFv编码基因转染的PG细胞与野生型和空白质粒组比较,对体外侵袭有明显的抑制,抑制率为76.3%;而CP.scFv编码基因转染的PG细胞未显示出有抑制作用。结论内质网滞留型胞内抗体技术可以在蛋白加工、分泌通路中抑制Ⅳ型胶原酶的活性,进而抑制肿瘤侵袭,可能在肿瘤基因治疗中具有重要的应用前景。
Objective To investigate the effect of endoplasmic reticulum immobilized intracellular antibodies on the secretion of type Ⅳ collagenase and its effect on the invasion of PG cells in human giant cell lung cancer. Methods The expression vectors pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasmic and endoplasmic reticulum anti-type Ⅳ collagenase antibody were constructed. Transfection of human giant cell lung cancer PG cell lines. Western blot was used to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was used to detect the type Ⅳ collagenase secretion in PG cells. Matrigel assay was used to detect cell invasion. Results The intracellular antibodies of CP.scFv and ER.scFv were expressed in PG cells. ER.scFv had a significant inhibitory effect on the secretion of type Ⅳ collagenase and the inhibitory rates of MMP-9 and MMP-2 were 85.7 % And 51.2% respectively; while CP.scFv had no inhibitory effect on the secretion of type Ⅳ collagenase. PG cells transfected with ER.scFv gene significantly inhibited the invasion in vitro compared with the wild-type and blank plasmid groups, and the inhibition rate was 76.3%. However, the PG cells transfected with the CP.scFv gene showed no inhibitory effect . Conclusion Endoplasmic reticulum retention intracellular antibody technology can inhibit the activity of type Ⅳ collagenase in protein processing and secretion pathway, and then inhibit tumor invasion, which may have important application prospect in gene therapy of cancer.