天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
8期
778-781
,共4页
朱波%林珊珊%崔春萍%谢明均
硃波%林珊珊%崔春萍%謝明均
주파%림산산%최춘평%사명균
骨肉瘤%细胞系,肿瘤%RNA,小分子干扰%基因沉默%肿瘤移植%小鼠,近交BALB C%低氧诱导因子-2α
骨肉瘤%細胞繫,腫瘤%RNA,小分子榦擾%基因沉默%腫瘤移植%小鼠,近交BALB C%低氧誘導因子-2α
골육류%세포계,종류%RNA,소분자간우%기인침묵%종류이식%소서,근교BALB C%저양유도인자-2α
osteosarcoma%cell line,tumor%RNA,small interfering%gene silencing%neoplasm transplantation%mice,inbred BALB C%hypoxia-inducible factor 2 alpha
目的：探讨低氧诱导因子-2α（HIF-2α）基因沉默对低氧状态下骨肉瘤MG-63细胞的影响。方法Western Blot检测MG-63细胞HIF-2α表达。利用小干扰RNA（siRNA）获得MG-63/siHIF-2α（siHIF-2α）细胞，阴性对照为MG-63/scramble(NC)细胞。Western Blot检测MG-63、NC和siHIF-2α细胞中HIF-2α、血管内皮生长因子（VEGF）、p-Erk/Erk及Mcl-1的表达。低氧下培养NC和siHIF-2α细胞，MTS试剂检测细胞活性，划痕迁移试验检测迁移能力，克隆集落形成试验检测集落形成率，裸鼠皮下移植瘤试验检测体内肿瘤生长情况。结果低氧可诱导MG-63细胞的HIF-2α蛋白表达，并呈时间依赖性(F=2037.412，P＜0.001)。低氧下siHIF-2α细胞的HIF-2α表达低于NC细胞（P＜0.01）。低氧12 h和24 h，siHIF-2α组细胞活性均低于NC组，相对划痕宽度均大于NC组（P＜0.05或P＜0.01）。1000~5000细胞种植数的siHIF-2α组的集落形成率均小于NC组(P＜0.05或P＜0.01)。低氧下siHIF-2α细胞的VEGF、p-Erk/Erk和Mcl-1表达均低于NC细胞（P＜0.01）。裸鼠皮下移植瘤siHIF-2α组肿瘤体积、质量和密度均小于NC组(P＜0.01)。结论阻断HIF-2α信号通路可作为骨肉瘤临床治疗的新策略。
目的：探討低氧誘導因子-2α（HIF-2α）基因沉默對低氧狀態下骨肉瘤MG-63細胞的影響。方法Western Blot檢測MG-63細胞HIF-2α錶達。利用小榦擾RNA（siRNA）穫得MG-63/siHIF-2α（siHIF-2α）細胞，陰性對照為MG-63/scramble(NC)細胞。Western Blot檢測MG-63、NC和siHIF-2α細胞中HIF-2α、血管內皮生長因子（VEGF）、p-Erk/Erk及Mcl-1的錶達。低氧下培養NC和siHIF-2α細胞，MTS試劑檢測細胞活性，劃痕遷移試驗檢測遷移能力，剋隆集落形成試驗檢測集落形成率，裸鼠皮下移植瘤試驗檢測體內腫瘤生長情況。結果低氧可誘導MG-63細胞的HIF-2α蛋白錶達，併呈時間依賴性(F=2037.412，P＜0.001)。低氧下siHIF-2α細胞的HIF-2α錶達低于NC細胞（P＜0.01）。低氧12 h和24 h，siHIF-2α組細胞活性均低于NC組，相對劃痕寬度均大于NC組（P＜0.05或P＜0.01）。1000~5000細胞種植數的siHIF-2α組的集落形成率均小于NC組(P＜0.05或P＜0.01)。低氧下siHIF-2α細胞的VEGF、p-Erk/Erk和Mcl-1錶達均低于NC細胞（P＜0.01）。裸鼠皮下移植瘤siHIF-2α組腫瘤體積、質量和密度均小于NC組(P＜0.01)。結論阻斷HIF-2α信號通路可作為骨肉瘤臨床治療的新策略。
목적：탐토저양유도인자-2α（HIF-2α）기인침묵대저양상태하골육류MG-63세포적영향。방법Western Blot검측MG-63세포HIF-2α표체。이용소간우RNA（siRNA）획득MG-63/siHIF-2α（siHIF-2α）세포，음성대조위MG-63/scramble(NC)세포。Western Blot검측MG-63、NC화siHIF-2α세포중HIF-2α、혈관내피생장인자（VEGF）、p-Erk/Erk급Mcl-1적표체。저양하배양NC화siHIF-2α세포，MTS시제검측세포활성，화흔천이시험검측천이능력，극륭집락형성시험검측집락형성솔，라서피하이식류시험검측체내종류생장정황。결과저양가유도MG-63세포적HIF-2α단백표체，병정시간의뢰성(F=2037.412，P＜0.001)。저양하siHIF-2α세포적HIF-2α표체저우NC세포（P＜0.01）。저양12 h화24 h，siHIF-2α조세포활성균저우NC조，상대화흔관도균대우NC조（P＜0.05혹P＜0.01）。1000~5000세포충식수적siHIF-2α조적집락형성솔균소우NC조(P＜0.05혹P＜0.01)。저양하siHIF-2α세포적VEGF、p-Erk/Erk화Mcl-1표체균저우NC세포（P＜0.01）。라서피하이식류siHIF-2α조종류체적、질량화밀도균소우NC조(P＜0.01)。결론조단HIF-2α신호통로가작위골육류림상치료적신책략。
Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.
