中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
4期
835-837
,共3页
熊健%陈廖斌%鲁厚庚%佘远举%许永涛
熊健%陳廖斌%魯厚庚%佘遠舉%許永濤
웅건%진료빈%로후경%사원거%허영도
干扰素应答因子1%骨肉瘤细胞%p53%凋亡
榦擾素應答因子1%骨肉瘤細胞%p53%凋亡
간우소응답인자1%골육류세포%p53%조망
Interferon regulatory factor-1%Osteosarcoma cells%p53%Apoptosis
目的 观察干扰素应答因子1(IRF-1)基因对不同p53状态骨肉瘤细胞凋亡的调节作用,并探讨其机制.方法 利用U2OS(p53+/+)和Saos2(p53-/-)骨肉瘤细胞作为研究模型,用不同剂量阿霉素处理后,Western blot检测IRF-1表达的变化.进一步通过过表达IRF-1基因或敲除IRF-1基因,检测其对骨肉瘤细胞凋亡的影响.并通过实时定量逆转录聚合酶链反应(RT-qPCR)法检测IRF-1对p53下游靶基因B细胞淋巴瘤/白血病-2相关X蛋白(bax)和p21的调节作用.结果 不同剂量阿霉素处理U2OS和Saos2后,Western blot结果显示IRF-1的表达明显增高.进一步过表达IRF-1后,乳酸脱氢酶(LDH)法检测细胞死亡率明显增高(P＜0.05);干涉IRF-1基因后,全量LDH检测细胞死亡率则明显降低(P＜0.05).RT-qPCR检测显示,IRF-1对p53下游靶基因bax和p21在p53-/-SaOS-2细胞中具有明显的促进作用(P＜0.05),而对于p53+/+U2OS中的作用并不明显(P＞0.05).结论 IRF-1可通过调节p53下游靶基因bax和p21的表达,从而显著促进阿霉素诱导的骨肉瘤细胞凋亡.
目的 觀察榦擾素應答因子1(IRF-1)基因對不同p53狀態骨肉瘤細胞凋亡的調節作用,併探討其機製.方法 利用U2OS(p53+/+)和Saos2(p53-/-)骨肉瘤細胞作為研究模型,用不同劑量阿黴素處理後,Western blot檢測IRF-1錶達的變化.進一步通過過錶達IRF-1基因或敲除IRF-1基因,檢測其對骨肉瘤細胞凋亡的影響.併通過實時定量逆轉錄聚閤酶鏈反應(RT-qPCR)法檢測IRF-1對p53下遊靶基因B細胞淋巴瘤/白血病-2相關X蛋白(bax)和p21的調節作用.結果 不同劑量阿黴素處理U2OS和Saos2後,Western blot結果顯示IRF-1的錶達明顯增高.進一步過錶達IRF-1後,乳痠脫氫酶(LDH)法檢測細胞死亡率明顯增高(P＜0.05);榦涉IRF-1基因後,全量LDH檢測細胞死亡率則明顯降低(P＜0.05).RT-qPCR檢測顯示,IRF-1對p53下遊靶基因bax和p21在p53-/-SaOS-2細胞中具有明顯的促進作用(P＜0.05),而對于p53+/+U2OS中的作用併不明顯(P＞0.05).結論 IRF-1可通過調節p53下遊靶基因bax和p21的錶達,從而顯著促進阿黴素誘導的骨肉瘤細胞凋亡.
목적 관찰간우소응답인자1(IRF-1)기인대불동p53상태골육류세포조망적조절작용,병탐토기궤제.방법 이용U2OS(p53+/+)화Saos2(p53-/-)골육류세포작위연구모형,용불동제량아매소처리후,Western blot검측IRF-1표체적변화.진일보통과과표체IRF-1기인혹고제IRF-1기인,검측기대골육류세포조망적영향.병통과실시정량역전록취합매련반응(RT-qPCR)법검측IRF-1대p53하유파기인B세포림파류/백혈병-2상관X단백(bax)화p21적조절작용.결과 불동제량아매소처리U2OS화Saos2후,Western blot결과현시IRF-1적표체명현증고.진일보과표체IRF-1후,유산탈경매(LDH)법검측세포사망솔명현증고(P＜0.05);간섭IRF-1기인후,전량LDH검측세포사망솔칙명현강저(P＜0.05).RT-qPCR검측현시,IRF-1대p53하유파기인bax화p21재p53-/-SaOS-2세포중구유명현적촉진작용(P＜0.05),이대우p53+/+U2OS중적작용병불명현(P＞0.05).결론 IRF-1가통과조절p53하유파기인bax화p21적표체,종이현저촉진아매소유도적골육류세포조망.
Objective Detect the effects of interferon regulatory factor-1 (IRF-1) gene on the apoptosisregulation in osteosarcoma with different p53 status and disclose the underlying mechnisms preliminarily.Methods To observe the effects of IRF-1 gene on the apoptosis effects induced by chemotherapeutic agent adriamycin in steosarcoma with different p53 status,p53 +/+ U2OS and p53-/-SaOS-2 were used.Western blotting analysis was firstly used to detect the expression of IRF-1 protein in adriamycin treated p53 +/+ U2OS and p53-/-SaOS-2 cells.And then IRF-1 gene was further overexpressed or knocked out for further detection the apoptosis effect induced by adriamycin respectively.Meanwhile,the target genes of p53,B cell lymphoma/leukemia-2 associated X protein (bax) and p21,were detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR).Results Western blotting showed that the expression of IRF-1 has been improved significantly when p53 +/+ U2OS and p53-/-SaOS-2 were treated with adriamycin for 24 h.The significant increase in the death rate of cells has been detected by the release of lactate dehydrogenase (LDH) Activity (P ＜ 0.05).When IRF-1 gene were overexpressed with transient transfection,adriamycin-induced apoptosis effect were increased significantly (P ＜ 0.05),while the knockdown of IRF-1 gene by small interfering RNA (siRNA) transfection were decreased significantly (P ＜ 0.05).RT-qPCR were used to further detected the expression of downstream target genes bax and p21,which do great favor to p53-dependent apoptosis.The results showed that bax and p21 were both upregulated significantly in p53-/-osteosarcoma cells (P ＜ 0.05),but not observed in p53 +/+ osteosarcoma cells (P ＞ 0.05).Conclusion IRF-1 plays a crucial role in adrymycin induced apoptosis by modulating p53 target genes,especially to the cancer cells with p53 gene deficient.
