血管生成素样蛋白4基因对骨肉瘤增殖及迁移作用的影响

The effects of ANGPTIA gene on the proliferation and migration of osteosarcoma

目的应用腺病毒改良载体系统构建的携带ANGPTL4和siANGPTL4基因的重组腺病毒，通过体内外实验研究ANGPTIA基因对骨肉瘤细胞株143B增殖和迁移能力的影响。方法应用腺病毒AdEasy系统，采用同源重组法构建携带ANGPTLA和siANGPTIA基因片段的重组腺病毒Ad-ANGPTIA和Ad-siANGPTL4，有限稀释法检测病毒滴度；重组腺病毒转染143B细胞，RT-PCR检测重组腺病毒体外转染的作用，结晶紫染色法、细胞计数、划痕实验观测ANGPTIA基因对143B细胞体外增殖和迁移能力的影响；采用胫骨肿瘤细胞注射法建立裸鼠143B骨肉瘤动物模型，Xenogen生物发光成像系统动态监测骨肉瘤原位生长情况。结果成功构建携带ANGPTLA和siANGPTL4基因的重组腺病毒Ad．ANGPTLA和Ad-siANGPTL4，病毒滴度达1．2×10^11～2．6×10^11pfu／mt；根据加入的重组腺病毒携带的基因不同将实验细胞分为阴性对照组、RFP组、ANGPTL4组及siANGPTL4组，各组细胞ANGPTL4基因表达的相对强度分别为157．39±9．87、156．90±8．95、185．29±8．24、129．28±7．28，ANGPTIA组和siANGPTIA组中基因相对表达强度显著增加或降低（P〈0．05）。结晶紫染色、细胞计数及划痕实验显示，ANGPTL4基因过度表达可促进143B细胞增殖和迁移；该基因抑制后143B细胞增殖和迁移明显减缓（P〈0．05）。成功建立裸鼠143B骨肉瘤动物模型，成瘤率达100％；Xenogen生物发光成像系统动态监测发现ANGPTL4基因过度表达可促进骨肉瘤的生长；该基因抑制后肿瘤生长明显减缓（P〈0．05）。结论成功构建了Ad-AN—GPTL4和Ad-siANGPTIA重组腺病毒并感染143B细胞使其内源性表达的ANGPTL4基因增加或沉默；该基因的表达增强或沉默能促进或抑制骨肉瘤143B细胞在体内外的增殖和迁移。

Objective To construct a recombinant adenovirus with angiopoietin-like 4 and siangiopoietin-like 4 genes by modified AdEasy system, and investigate the effects of ANGPTL4 gene on the proliferation and migration of osteosarcoma 143B ceils in vitro and in vivo. Methods The recombinant adenovirus with ANGPTL4 and siANGPTIA genes was constructed by modified AdEasy system, and the viral titre was determined by limiting dilution assay. After 143B cells were transfected by recombinant adenovirus, the transfected efficiency of Ad-ANGPTL4 and Ad-siANGPTL4 was identified by RT-PCR method through detecting the relative expression levels of the genes. The effects of ANGPTL4 gene on proliferation and migration of the osteosarcoma cell line 143B were determined by Crystal Violet staining, ceil count and wound healing assay in vitro. Moreover, the transfected 143B cells were injected into the right tibial medullary cavity of the nude mouse to establish osteosarcoma animal model. The effects of ANGPTL4 gene on osteosarcoma growth were monitored by Xenogen bio/umineseence imaging system in vivo. Results The recombinant adenovirus with ANGPTL4 and siANGPTIA genes was constructed successfully and the viral titre was 1.2×10^11～2.6×10^11pfu/mt. After transfected, the relative expression intensities of ANGPTIA gene in 143B cells were 157.39 ± 9.87, 156.90 ± 8.95, 185.29 ± 8.24, and 129.28 ± 7.28 in negative control, RFP, ANGPTL4 and siANGPTIA groups, respectively, and the differences were significant （P（ 0. 05）. The proliferation and migration abilities of 143B cells were improved by over expression of ANGPTL4 gene and reduced by ANGPTIA gene silencing（P〈0. 05）. The nude mice model with osteosarcoma was estabilished successfully. The osteosarcoma with ANGPTL4 over expression grew rapidly, however, the growth of the tumor was inhibited obviously by low expression of ANGPTIA（P 〈0. 05）. Conclusions Recombinant adenovirus carrying ANGPTIA and siANGPTL4 genes could be constructed by AdEasy system with high titre and efficient transfection. It can promote or inhibit the proliferation and migration of 143B cells in vitro and in vivo by up-regulating or silencing ANGPTIA gene expression.