自噬基因Beclin1与骨肉瘤发生和发展的相关性研究  

Related research of the role of Autophagy gene Beclin 1 in osteosarcoma development

作　　者：杨诚[1] 贾连顺[1] 刘铁龙[1] 肖建如[1] 

机构地区：[1]上海长征医院骨肿瘤科,200003

出　　处：《中国综合临床》2013年第5期529-532,共4页Clinical Medicine of China

摘　　要：目的探讨自噬基因Beclin1在骨肉瘤发生、发展中的作用，并分析其过表达对人骨肉瘤细胞株MG63体外生长的影响。方法采用逆转录-聚合酶链反应（RT—PCR）及免疫印迹法对人成骨肉瘤细胞MG63及成骨细胞hFOBl-19中Beclin1mRNA及蛋白表达水平进行检测；构建表达融合蛋白的真核表达载体pEGFP／Beclin1，并用脂质体转染人骨肉瘤细胞MG63。用MTT法分析外源性Beclin1过表达对MG63增殖的影响，并用流式细胞仪检测凋亡情况。结果人成骨肉瘤细胞MG63中Beclin1mRNA及蛋白表达水平显著低于人成骨细胞hFOBl．19（0．17±0．06与0．43±0．11，t=29．493，P〈0．01；0．13±0．05与0．25±0．08，t=6．325，P〈0．01）。pEGFP／Beclinl转染人成骨肉瘤细胞MG63后使其mRNA水平表达升高（5．34±0．50）倍，转染成功。正常培养的MG63细胞及空质粒转染的MG63细胞内，凋亡细胞很少，均为（0．10±0．05）％；而转染Beclin1编码序列后凋亡细胞的比例明显升高，为（4．3±0．8）％，差异有统计学意义（t=5．752，P〈0．05）。结论Beclinl在骨肉瘤细胞中表达下降，可能与骨肉瘤的发生、发展密切相关，其过表达可抑制人成骨肉瘤细胞MG63的增殖并诱导其凋亡。Objective To investigate the role of Beclin 1 in the genesis and development of osteosarcoma and the effect of Beclin 1 overexpression on the growth of the in vitro osteosarcoma cell line MG63. Methods Real time-polymerase chain reaction （RT-PCR） and Western blot were used to detect the expressions of Beclin 1 in MG63 and hFOB1.19 at mRNA and protein levels; A eukaryotic clone of plasmid pEGFP/Beclinl fusion with protein EGFP/Beclin 1 was constructed and was transfected into human osteosarcoma cell line MG63 by using lipofectamine 2000. The effect of Beclinl overexpressions on the proliferation of MG63 cells was evaluated by MTF assay. Cell apoptosis was measured by flow cytomerty（FCM）.Results The mRNA and protein expression of Beclinl in human osteosarcoma cell line MG63 was significantly lower than that in the human osteoblast cell line hFOB 1.19 （0. 17 ± 0. 06 vs 0. 43 ± 0. 11, t = 29. 493, P 〈 0.01 ; 0. 13 ± 0. 05 vs. 0. 25 ± 0. 08, t = 6. 325, P 〈 0. 01 ）. The transfection of pEGFP/Beclinl increased the mRNA levels of human osteosarcoma（5.34 ± 0. 50） times in transfected tumor cells MG63. The rate of cell apoptosis was low in control or transfected with lipofectamine 2000 only cells at an average of（0. 10 ± 0. 05） %. The apoptosis rate was significantly higher in pEGFP/Beclinl transfected cells than control cells （ （4. 3 ± 0. 8 ） %, t =5. 752,P 〈 0. 05 ）. Conclusion Compared with control cells, Beclinl is down-regulated in the human osteosarcoma cell line MG63, which indicate the role of Beclin 1 in regulating the malignant behaviors of osteosarcoma. Beclinl overexpressions inhibits cell proliferation and induces apoptosis in MG63 cells.

关 键 词：自噬 骨肉瘤 BECLIN 1 

分 类 号：R738.1[医药卫生—肿瘤]