抗人NRP-1单克隆抗体对结肠癌LOVO细胞株的促凋亡作用

Exacerbated apoptotic effects of anti-human NRP-1 monoclonal antibody in colon cancer cell line LOVOin vitro

目的:研究抗人神经菌毛素1(neuropilin1,NRP-1)单克隆抗体对结肠癌LOVO细胞的促凋亡作用。方法:小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 MAb)并用rProtein A亲和柱纯化,间接ELISA检测抗体的滴度水平。细胞免疫荧光检测NRP-1蛋白在结肠癌细胞株LOVO上的表达,MTT法检测NRP-1 MAb对LOVO的生长抑制作用,DAPI荧光显微镜法观察细胞凋亡形态,ANNEXIN V-FITC/PI流式细胞仪法检测NRP-1 MAb诱导LOVO细胞凋亡,Western blotting检测凋亡相关蛋白caspase-3、cleaved caspase-3蛋白的表达。结果:SDS-PAGE和间接ELISA检测纯化的NRP-1 MAb纯度为95%、效价为2.56×10-5;细胞免疫荧光染色显示NRP-1定位于LOVO细胞膜;MTT法检测和ANNEXIN V-FITC/PI流式细胞仪法检测结果显示,NRP-1 MAb对LOVO细胞有生长抑制作用和促凋亡作用;DAPI染色·荧光显微镜观察发现,NRP-1MAb可以诱导LOVO细胞核浓缩与碎裂;Western blotting检测到在NRP-1 MAb作用下,LOVO细胞裂解液cleaved caspase-3条带信号增强、caspase-3条带信号减弱。结论:纯化的NRP1 MAb能抑制LOVO细胞的生长并诱导其凋亡。

OBJECtIVE To investigate the exacerbated apoptotic effect of anti-human nerve pilifactor 1 （neuropilin-1, NRP- 1） monoelonal antibody （NRP-1 MAb） on colon cancer LOVO cells in vitro. METHODS NRP-1 MAb was prepared from asci- tes and purified by rProtein A affinity column and the titers were measured by indirect ELISA. The NRP-1 expression in colon cancer cell LOVO was detected by immunofluorescence; growth inhibition of LOVO cells treated with different concentration NRP-1 MAb was analyzed by MTT assay; DAPI staining was used to observe the morphology of apoptotic cells with Fluores- cence microscopy; apoptosis rate of LOVO cells was determined by flow cytometry assay with Annexin V-FITC/PI; Western blotting was used to detect Caspase-3 protein expression. RESULTS The results of SDS-PAGE and indirect ELISA showed the purity of NRP-1 MAb was 95% and the titer was 2. 56 × 10-5. Immunofluorescence staining indicated that the NRP-1 was localized on the membrane of LOVO cells. MTT assay and Annexin V-FITC/PI flow cytometry assay indicated that NRP-1 MAb inhibited LOVO cells growth and induced LOVO cells apoptosis. Degradation and fragmentation of nuclear in LOVO cells treated with NRP-1 MAb were observed under a fluorescence microscope by DAPI staining; Western blotting detected that cleaved Caspase-3 band signal enhanced, while Caspase-3 band signal decreased after incubated LOVO cells with the NRP-1 MAb. CONCLUSION NRP-1 MAb can inhibit proliferation and increase apoptosis of the LOVO cells.