【作者】 徐亮；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 骨科学， 2009， 博士

【摘要】 第一部分肿瘤抑制基因RECK在骨肉瘤组织中表达的初步研究[目的]通过检测RECK基因在人骨肉瘤组织中的表达，探讨RECK基因在骨肉瘤发生及转移过程中的作用。[方法]采用免疫组化学方法检测38例骨肉瘤组织标本及38例正常骨组织中RECK蛋白的表达水平。[结果]38例骨肉瘤组织中有26例RECK蛋白呈弱表达或未见表达，而在所38例正常的骨组织标本中RECK均有表达且呈强表达，骨肉瘤组织、正常骨组织中RECK的吸光度值分别为（0．31±0．07）和（0．58±0．08），骨肉瘤组织RECK的表达明显低于正常骨组织（P＜0．01），差异具有统计学意义；RECK蛋白表达与患者年龄、性别、肿瘤大小、病理分级等均无密切关系，而RECK的低表达或不表达与肺转移有关（P＜0．05，P=0．019）。生存资料分析显示，RECK蛋白高表达患者的平均生存时间为31个月，低表达患者的平均生存时间为15个月，经Log-rank检验，RECK高表达患者与低表达患者的累计生存率差异有统计学意义（P＜0．05，P=0．032）。[结论] RECK基因异常低表达可能参与骨肉瘤侵袭、转移机制，RECK基因可能成为判断骨肉瘤患者预后新的分子标志物及肿瘤治疗的新靶点。第二部分转染RECK基因对人骨肉瘤细胞MMP-2活化及侵袭能力的影响研究[目的]观察转染RECK基因对人骨肉瘤胞系MG-63的MMP-2活化及细胞侵袭力的影响。[方法]以脂质体Lipofectamine^TM 2000介导的方法将含RECK全长基因的真核重组表达质粒pcDNA3-RECK转染入MG-63细胞。RT-PCR、Western blot、流式细胞术检测目的基因的表达；明胶酶谱法、Matrigel侵袭实验分别检测MG-63细胞上清的MMP-2活化比例及细胞侵袭力变化；四甲基偶氮唑蓝（MTT）比色法和细胞生长曲线法观察质粒DNA转染对细胞的毒性情况。[结果]转染后RECK基因在MG-63细胞mRNA和蛋白水平分别有稳定独立表达；明胶酶谱结果显示，正常对照组与空载质粒转染组细胞的MMP-2活化比例分别为（0．72±0．11），（0．80±0．14），二组间差异无显著性意义；重组质粒转染组MMP-2活化比例为（0．23±0．03），与正常对照组、空载质粒转染组比较明显降低，差异有显著性意义（均P＜0．01）。Matrigel侵袭实验结果显示：正常对照组与空载质粒转染组的穿至微孔膜下表面的细胞数分别为（28．18±2．15）和（30．25±2．23），两组间差异无显著性意义（P＞0．05）。重组质粒转染组穿透Matrigel至微孔膜下表面的细胞数为（14．20±1．26），明显低于正常对照组、空载质粒转染组，差异有显著性意义（均P＜0．05）。而MTT法测重组质粒转染组细胞生长曲线与空白对照组、空载质粒转染组无明显差异（均P＞0．05）。[结论] RECK基因过表达可显著减少骨肉瘤细胞MG-63的MMP-2活化及其侵袭能力，RECK基因可能成为肿瘤治疗的新的靶点。更多还原

【Abstract】 Part oneThe preliminary study on the expression of the tumorinhibitory gene RECK in the osteosarcoma tissues[Objective] To detect the the expression of RECK gene in osteosarcoma tissuesexplore its possible roles on the occurrence and metastasis of osteosarcoma.[Methods] The expression level of RECK protein in 38 osteosarcoma tissues and 38normal bone tissues were detected by immunohistochemistry.[Results] Of the 38 osteosarcomas examined,26 were stained weak or negative forRECK,whereas the strong RECK staining was observed in all 38 normal bone tissues.Theabsorbent optical density value of RECK protein in osteosarcoma tissues and normalbone tissuses were （0.31±0.07） and （0.58±0.08）,respectively.The expression of RECK inosteosarcoma tissues was significantly lower than that in normal bone tissuse（P＜0.01）.RECK protein expression was significant correlated with the pulmonary metastasis（P＜0.05,P=0.019）,but had no correlation with age,sex,tumor size and pathologic grades.Ina comparison between the patients with RECK-positive tumors and patients withRECK-negative tumors,the survival time of the RECK-positive group was found to be 31months,whereas that of the RECK-negative group was 15 months,indicating theRECK-positive group had significantly better survival rate than the RECK-negative group（P＜0.05,P=0.032 by Log-rank test）.[Conclusion] The abnoamal low expression of RECK may participate in osteosarcomainvasion and metastasis,and may be a new prognostic molecular marker for osteosarcoma and a new therapeutic target for tumor.Part twoThe study of transfecting RECK gene on MMP-2 activationand invasional ability of human osteosarcoma cell[Objective] To investigate effect of transfecting RECK gene on MMP-2 activation andinvasional ability of human osteosarcoma cell line MG-63.[Methods] The recombinant eukaryotic expression vector pcDNA3-RECK inserted bythe full length cDNA encoding human RECK gene was stably transfected into MG-63 byLipofectamine^TM 2000.RT-PCR,westem blot and flow cytometric analysis were used toassay RECK gene expression.MMP-2 activation ratio of the cell supematant and cellinvasional ability of MG-63 were analyzed by gelatinase zymography and matrigel invasionassay,respectively.The cytotoxicity of plasmid DNA transfection on MG-63 cells weredetermined by MTT assay and cell growth curve method.[Results] The stable and higher expression of RECK in mRNA and protein level weredetected in MG-63,respectively.Gelatinase zymography showed MMP-2 activation ratio intransfecting blank plasmid group and normal control group were （0.72±0.11）,（0.80±0.14）.Nosignificant difference was found between them.However,the MMP-2 activation ratio intransfecting recombinant plasmid group was （0.23±0.03）,which was obviously less thanthose in transfecting blank plasmid group and normal control group（both P value＜0.01）.Mrtrigel invasion assay showed cell number invading through Matrigel in transfecting blankplasmid group and normal control group were （28.18±2.15）and（30.25±2.23）,respectively.Nosignificant difference was found between them.The cell number invading through Matrige intransfecting recombinant plasmid group was （14.20±1.26）,which was dramatically decreasedcompared with those in transfecting blank plasmid group and normal control group（both Pvalue＜0.05）.MTT showed no significant difference was found in transfecting recombinant plasmid group compared with transfecting blank plasmid group and normal control group incomparing cell growth curve （both P value＞0.05）.[Conclusion] Over expression of RECK gene significantly inhibited MMP-2 activationand cell invasional ability of osteosarcoma cell MG-63,suggesting RECK may be a newtarget for osteosarcoma treatment.更多还原