中国骨与关节杂志
中國骨與關節雜誌
중국골여관절잡지
Chinese Journal of Bone and Joint
2015年
1期
15-19
,共5页
冀全博%徐小洁%张强%张立%徐亚梦%梁迎春%王涛%李玲%郭靖%叶棋浓%王岩
冀全博%徐小潔%張彊%張立%徐亞夢%樑迎春%王濤%李玲%郭靖%葉棋濃%王巖
기전박%서소길%장강%장립%서아몽%량영춘%왕도%리령%곽정%협기농%왕암
CCAAT增强子结合蛋白α%骨肉瘤%基因,肿瘤抑制%肿瘤细胞,培养的
CCAAT增彊子結閤蛋白α%骨肉瘤%基因,腫瘤抑製%腫瘤細胞,培養的
CCAAT증강자결합단백α%골육류%기인,종류억제%종류세포,배양적
CCAAT-enhancer-binding protein-alpha%Osteosarcoma%Genes,tumor suppressor%Tumor cells,cultured
目的：构建带Myc(pXJ-40)标签的CCAAT增强子结合蛋白α(CCAATenhancerbindingproteinα，C/EBPα)的真核表达载体，并检测其对骨肉瘤细胞生长的影响。方法应用聚合酶链式反应(polymerase chain reaction，PCR )技术从人乳腺文库中扩增出C/EBPα全长编码区基因；将扩增基因克隆到Myc载体中，并将重组质粒转染人胚胎肾293T细胞；Western blotting检测表达情况；另将重组Myc-C/EBPα质粒转染人骨肉瘤细胞系U2OS (实验组)、Myc空载体质粒转染U2OS细胞(对照组)，生长实验检测C/EBPα对肿瘤细胞生长的影响。结果成功构建Myc-C/EBPα真核表达质粒，重组质粒转染人胚胎肾293T细胞后成功表达。生长实验显示Myc-C/EBPα转染的U2OS细胞的生长明显受限，随着培养时间延长，受抑制的程度逐渐增大，培养至第5天，Myc-C/EBPα转染的实验组细胞生长抑制率为58%，且细胞的生长速率OD＝0.495明显低于空白对照组1.179，差异有统计学意义(P＜0.01)。结论 C/EBPα对骨肉瘤细胞的生长具有抑制作用。
目的：構建帶Myc(pXJ-40)標籤的CCAAT增彊子結閤蛋白α(CCAATenhancerbindingproteinα，C/EBPα)的真覈錶達載體，併檢測其對骨肉瘤細胞生長的影響。方法應用聚閤酶鏈式反應(polymerase chain reaction，PCR )技術從人乳腺文庫中擴增齣C/EBPα全長編碼區基因；將擴增基因剋隆到Myc載體中，併將重組質粒轉染人胚胎腎293T細胞；Western blotting檢測錶達情況；另將重組Myc-C/EBPα質粒轉染人骨肉瘤細胞繫U2OS (實驗組)、Myc空載體質粒轉染U2OS細胞(對照組)，生長實驗檢測C/EBPα對腫瘤細胞生長的影響。結果成功構建Myc-C/EBPα真覈錶達質粒，重組質粒轉染人胚胎腎293T細胞後成功錶達。生長實驗顯示Myc-C/EBPα轉染的U2OS細胞的生長明顯受限，隨著培養時間延長，受抑製的程度逐漸增大，培養至第5天，Myc-C/EBPα轉染的實驗組細胞生長抑製率為58%，且細胞的生長速率OD＝0.495明顯低于空白對照組1.179，差異有統計學意義(P＜0.01)。結論 C/EBPα對骨肉瘤細胞的生長具有抑製作用。
목적：구건대Myc(pXJ-40)표첨적CCAAT증강자결합단백α(CCAATenhancerbindingproteinα，C/EBPα)적진핵표체재체，병검측기대골육류세포생장적영향。방법응용취합매련식반응(polymerase chain reaction，PCR )기술종인유선문고중확증출C/EBPα전장편마구기인；장확증기인극륭도Myc재체중，병장중조질립전염인배태신293T세포；Western blotting검측표체정황；령장중조Myc-C/EBPα질립전염인골육류세포계U2OS (실험조)、Myc공재체질립전염U2OS세포(대조조)，생장실험검측C/EBPα대종류세포생장적영향。결과성공구건Myc-C/EBPα진핵표체질립，중조질립전염인배태신293T세포후성공표체。생장실험현시Myc-C/EBPα전염적U2OS세포적생장명현수한，수착배양시간연장，수억제적정도축점증대，배양지제5천，Myc-C/EBPα전염적실험조세포생장억제솔위58%，차세포적생장속솔OD＝0.495명현저우공백대조조1.179，차이유통계학의의(P＜0.01)。결론 C/EBPα대골육류세포적생장구유억제작용。
Objective To construct the eukaryotic expression vector of CCAAT / enhancer binding protein-α ( C/EBPα ) labeled with Myc tag ( pXJ-40 ), and to examine the inhibitory effects of C/EBPα on osteosarcoma cell growth.Methods Human C/EBPα coding gene region was amplified from mammary gland cDNA library by polymerase chain reaction ( PCR ), which was inserted into the pXJ-40-myc vector. Myc-C/EBPα was constructed and transfected into 293T cells. Western blotting analysis was carried out to detect their expressions. In addition, U2OS cells transfected with Myc-C/EBPα were divided into the experimental group, and U2OS cells only transfected with Myc were divided into the control group. The growth of U2OS cells in both groups was identiifed using growth curve experiment.Results The coding region of C/EBPα was successfully ampliifed by PCR and cloned into pXJ-40-myc vector. Myc-C/EBPα was successfully expressed in human 293T cells. Growth curve experiment showed that the growth of U2OS cell lines transfected with Myc-C/EBPα was apparently inhibited. With the extension of the culture time, the inhibitory effects were gradually enhanced. At the 5th day, the inhibition ratio of U2OS cell growth was 58%in the experimental group. The growth rate of U2OS cell lines transfected with Myc-C/EBPα [ optical density ( OD )=0.495 ] was apparently lower than that of the control group ( OD=1.179 ), and the differences between them were statistically signiifcant (P<0.01 ).Conclusions The eukaryotic expression vector Myc-C/EBPα can effectively inhibit osteosarcoma cell growth.
