金属硫蛋白1H在儿童和青少年骨肉瘤血清中的表达及其对细胞增殖的影响  

Expression of metallothionein 1H in children and adolescents osteosarcoma and effect on cell proliferation

作　　者：侯新芳[1] 李帅[1] 吴晨[1] 徐淑宁[1] 李克[1] 王居峰[1] 

机构地区：[1]郑州大学附属肿瘤医院内科,河南省肿瘤医院内科,450008

出　　处：《中华实用儿科临床杂志》2017年第3期208-211,共4页Chinese Journal of Applied Clinical Pediatrics

摘　　要：目的检测金属硫蛋白1H（MT1H）在儿童和青少年骨肉瘤血清中的表达，分析MT1H与临床病理特征的关系，并探讨MT1H对人骨肉瘤细胞U2OS增殖能力的影响及其机制。 方法采用酶联免疫吸附试验（ELISA）检测MT1H在儿童和青少年骨肉瘤和骨良性疾病中表达情况，分析MT1H表达和骨肉瘤临床病理特征之间的关系。在体外将MT1H基因转染人骨肉瘤细胞U2OS，采用反转录聚合酶链反应（RT－PCR）和Wes－tern blot方法检测MT1H mRNA和蛋白的表达情况，采用四甲基偶氮唑蓝（MTT）法检测细胞增殖，采用Western blot方法检测核因子（NF）－κB、抑制性κB（IκB）－α蛋白表达情况。 结果MT1H在骨良性疾病和骨肉瘤血清中的表达水平分别为（0.51±0.52） μg/L和（2.17±0.78） μg/L，其在骨肉瘤血清中的表达水平明显升高（t＝－8.966，P〈0.05）。MT1H在Ⅰ～ⅡA期、ⅡB～Ⅲ期骨肉瘤血清中的表达水平分别为（1.98±0.69） μg/L、（2.45±0.82） μg/L，其表达水平呈逐渐升高趋势，差异有统计学意义（t＝－2.343，P〈0.05）。转染MT1H基因后，骨肉瘤细胞MT1H mRNA和蛋白表达均升高（均P〈0.05）。MTT结果显示，与空白对照组、空质粒转染组相比，MT1H真核质粒转染骨肉瘤细胞系U2OS 72 h后，细胞增殖加快。空白对照组、空质粒转染组、MT1H转染组A值分别为0.38±0.03、0.36±0.03、0.42±0.03；与空白对照组、空质粒转染组相比，MT1H转染组A值升高（F＝4.213，P〈0.05）。Western blot检测显示，空白对照组、空质粒转染组、MT1H转染组细胞中NF－κB的相对表达量分为0.56±0.05、0.53±0.05、0.92±0.07，IκB－α蛋白的相对表达量分别为0.64±0.06、0.62±0.09、0.34±0.08，与空白对照组、空质粒转染组相比，MT1H转染组NF－κB蛋白表达上调、IκB－α蛋白表达下调（F＝44.581、14.927，均P〈0.05）。 结论MT1H在儿童和青少年骨肉瘤血清中表�Objective To detect the expression levels of metallothioneinl H （MT1 H） in children and adolescents osteosarcoma serums, and to analyze its relationship with clinicopathological features, and to explore the effect of MT1H on cell proliferation of osteosarcoma cells and its mechanism. Methods Enzyme - linked immuno sorbent assay （ELISA） was performed to detect the expression of MT1H in children and adolescents osteosarcoma serums and non- neoplastic disease serums. MT1H vector was transfected into the osteosarcoma U2OS cells. Reverse transcription -poly- merase chain reaction（ RT -PCR） and Western blot were used to detect the expression of the mRNA and protein of MT1 H, respectively. Methylthiazolyldiphenyl- tetrazolium bromide（MTT） was used to detect the cell growth. Western blot was performed to detect the expression of nuclear factor （ NF ） -κB, and inhibitor of KB （ IKB ） -α protein. Results The expressions of MT1H in osteosarcoma serums and nonneoplastic disease serums was （0. 51±0.52） μg/L and （2.17 ± 0.78） μg/L, respectively,with a significant difference between the 2 groups （t = -8. 966, P〈0. 05）.The expression of MT1H in stage Ⅰ-ⅡA and 11B - HI was （1.98 ±0.69）μg./L and （2.45 ±0.82） μg/L,respectively, showing a gradual increase depending on clinical staging（ t = -2. 343 ,P 〈 0.05 ）. The expressions of MT1H mRNA and protein were elevated in osteosarcoma U2OS cells after MT1H vector transfection （ all P 〈 0.05 ）. MTF assay showed that, the A value in blank control group, blank vector group, MT1H vector group were 0.38± 0.03, 0.36 ±0.03,0.42± 0.03, respectively, the cell proliferation in the MT1H vector group was significantly promoted when compared with these in the blank vector group and blank control group（ F = 4.213 ,P 〈 0.05 ）from the third day. West- eru blot showed that, the relative expression of NF -κB in blank control group, blank vector group, MT1H vector group were 0.56±0.05,0.53 ±0.05,0.92 ±0.07, respectively, t

关 键 词：骨肉瘤 金属硫蛋白1H 酶联免疫吸附试验 增殖 核因子-κB通路 

分 类 号：R738.1[医药卫生—肿瘤]