CAJ | 학술논문

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ANGPTL4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ANGPTL4和siANGPTL4基因片段的重组腺病毒Ad-ANGPTL4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ANGPTL4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ANGPTL4和siANGPTL4基因的重组腺病毒Ad-ANGPTL4和Ad-siANGPTL4,病毒滴度达1.2×1011～2.6×1011 pfu/ml；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,ANGPTL4组和siANGPTL4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-siANGPTL4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.
목적 응용선병독개량재체계통구건적휴대ANGPTL4화siANGPTL4기인적중조선병독,통과체내외실험연구ANGPTL4기인대골육류세포주143B증식화천이능력적영향.방법 응용선병독AdEasy계통,채용동원중조법구건휴대ANGPTL4화siANGPTL4기인편단적중조선병독Ad-ANGPTL4화Ad-siANGPTL4,유한희석법검측병독적도；중조선병독전염143B세포,RT-PCR검측중조선병독체외전염적작용,결정자염색법、세포계수、화흔실험관측ANGPTL4기인대143B세포체외증식화천이능력적영향；채용경골종류세포주사법건립라서143B골육류동물모형,Xenogen생물발광성상계통동태감측골육류원위생장정황.결과 성공구건휴대ANGPTL4화siANGPTL4기인적중조선병독Ad-ANGPTL4화Ad-siANGPTL4,병독적도체1.2×1011～2.6×1011 pfu/ml；근거가입적중조선병독휴대적기인불동장실험세포분위음성대조조、RFP조、ANGPTL4조급siANGPTL4조,각조세포ANGPTL4기인표체적상대강도분별위157.39±9.87、156.90±8.95、185.29±8.24、129.28±7.28,ANGPTL4조화siANGPTL4조중기인상대표체강도현저증가혹강저(P＜0.05).결정자염색、세포계수급화흔실험현시,ANGPTL4기인과도표체가촉진143B세포증식화천이；해기인억제후143B세포증식화천이명현감완(P＜0.05).성공건립라서143B골육류동물모형,성류솔체100％；Xenogen생물발광성상계통동태감측발현ANGPTL4기인과도표체가촉진골육류적생장；해기인억제후종류생장명현감완(P＜0.05).결론 성공구건료Ad-AN-GPTL4화Ad-siANGPTL4중조선병독병감염143B세포사기내원성표체적ANGPTL4기인증가혹침묵；해기인적표체증강혹침묵능촉진혹억제골육류143B세포재체내외적증식화천이.
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 cells in vitro and in vivo.Methods The recombinant adenovirus with ANGPTL4 and siANGPTL4 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,cell 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 bioluminescence imaging system in vivo.Results The recombinant adenovirus with ANGPTL4 and siANGPTL4 genes was constructed successfully and the viral titre was 1.2 × 1011-2.6 × 1011 pfu/ml.After transfected,the relative expression intensities of ANGPTL4 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 siANGPTL4 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 ANGPTL4 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 ANGPTL4(P ＜0.05).Conclusions Recombinant adenovirus carrying ANGPTL4 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 ANGPTL4 gene expression.