【作者】 黄涛；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 目的构建正、反义人骨唾液酸蛋白基因真核表达载体，为进一步研究人BSP的生物学作用奠定实验基础；检测人骨唾液酸蛋白正、反义真核表达载体在骨肉瘤细胞MG-63中的表达；初步探讨人骨唾液酸蛋白正、反义真核表达载体在骨肉瘤粘附侵袭行为中的作用。方法以RT-PCR方法从人骨膜的总RNA中扩增出hBSP的开放阅读框序列，与克隆载体pUCm-T进行连接，构成克隆质粒。利用引物两端所设计的不同酶切位点将正反义目的片段定向亚克隆到真核表达载体pIRES2-EGFP，将重组后的质粒进行细菌转化，质粒扩增、纯化后，酶切、测序证实其中有目的片段完整插入。分别将正义、反义和空质粒以脂质体介导转染入MG-63细胞，通过RT-PCR检测各组细胞中hBSP mRNA表达，通过Western blot检测各组细胞中hBSP蛋白表达。MTT法测定转染细胞在细胞外基质上的粘附情况，重组基底膜侵袭实验测定BSP对骨肉瘤细胞侵袭力的影响，细胞划痕实验测定转染BSP质粒对瘤细胞迁移情况的影响。结果从人骨膜细胞中成功扩增出hBSP的开放阅读框序列，与pUCm-T连接构建成功BSP克隆质粒，通过亚克隆过程，与pIRES2-EGFP真核表达载体连接，成功构建了人BSP正反、义核酸真核表达载体，转染骨肉瘤细胞后能够有效表达hBSP，与对照组相比，正义转染组高表达hBSP，瘤细胞粘附率提高(P＜0.05)，侵袭力增强(P＜0.05)，迁移速度增快(P＜0.05)。反义转染组低表达hBSP，瘤细胞粘附率降低(P＜0.05)，侵袭力受到抑制(P＜0.01)，迁移速度减慢(P＜0.05)。而空质粒转染组细胞和未处理瘤细胞组细胞在BSP表达上无明显区别，在细胞外基质的粘附能力，侵袭能力，和迁移能力上无显著性差异。结论骨肉瘤细胞中有BSP的表达；过表达BSP能促进骨肉瘤细胞的粘附，侵袭和转移；反义转染人BSP能明显抑制骨肉瘤侵袭转移相关的多个环节；BSP表达的改变可能在骨肉瘤的粘附、迁移过程中起重要作用。更多还原

【Abstract】 Objective To construct sense and antisense human bone sialoprotein(BSP) eukaryotic expression vector for further studying the effective of hBSP , The sense and antisense hBSP eukaryotic expression vector was transfected into MG-63 cells and its expression detected in human osteosarcoma cell line MG-63 cells.To preliminarily study its biological function in adhesion and invasion of osteosarcoma. Methods The open reading frame of Human BSP was amplified by RT-PCR method from human periosteum and inserted into pUCm-T vector to form cloning plasmid.Then the ragment of target was subcloned into eukaryotic expression vector pIRES2-EGFP in orientation thought utilized the difference restriction enzyme cut site of both end of the primers.The recombinant plasmid was identified by restriction enzyme digestion analysis and sequencing analysis after bacterial transformation, amplification and purification. The sense and antisense hBSP eukaryotic expression vector was transfected into MG-63 cells with lipofectamine. The expression of BSP mRNA on the cells was measured by RT-PCR, The expression of BSP on the cells was measured by Western blotting. MTT assay was used to test transfected cells adhesion on extracellular matrix protein. Transwell-chamber method was used to evaluate influence of BSP expression on osteosarcoma cell invasion. Monolayer wound healing assay was used to measure osteosarcoma cell migrationResults The open reading frame of Human BSP was successfully amplified from human periosteum The sense and antisense hBSP eukaryotic vestor was successfully constructed and transfected.Compared with control cells,expression of BSP increased in the cells by sense hBSP eukaryotic vector transfected,adherence ratio of these cells is raise (P<0.05),invasive power of these cells is enhance(P<0.05) and migration rate of these cells is increase(P<0.05). Expression of BSP decreased in the cells by antisense hBSP eukaryotic vector transfected, adherence ratio of these cells is degrade (P<0.05) , the ability of invasive of these cells is been inhabited(P<0.05) and migration rate of these cells is decrease(P<0.05).. Conclusion Osteosarcoma cells can express BSP. Up-expression of BSP can enhance adherenc,invasive and migration of osteosarcoma cell, antisense hBSP eukaryotic vector transfected have inhibitory effects on many aspects or steps associated with metastasis of osteosarcoma, The change of BSP expression may play an important role in osteosarcoma adhesion and migration.更多还原