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背景:SOX-9在软骨组织的发生和发育中具有重要作用,可促进间充质细胞凝集,具有转录激活结构域,能直接调控转录。目的:选用SOX-9基因构建pDC316-SOX-9质粒转染兔骨髓基质细胞,观察SOX-9基因对骨髓基质细胞生长特性及产物表达的影响。设计、时间及地点:细胞基因工程体外实验,于2006-06/2007-01在中国科学院北京基因组研究所完成。材料:SPF级6周龄新西兰大白兔8只,用于分离培养骨髓基质细胞。方法:采用脂质体介导法将构建的pDC316-SOX-9质粒转染兔骨髓基质细胞,细胞转染分为SOX-9质粒转染组、空质粒组、只加入等量脂质体的空白对照组。主要观察指标:通过细胞形态、细胞生长活性、免疫组织化学及HE染色、RT-PCR法、ELISA法检测SOX-9基因转染骨髓基质细胞是否成功。结果:pDC316-SOX-9质粒转染4d出现小片的细胞集落,挑取克隆扩增培养后,细胞呈梭形纤维状。随转染时间的延长,空白对照组细胞逐渐死亡,SOX-9质粒转染组、空质粒组的细胞活性均显著延长,在转染2周达高峰,之后逐渐降低。转染第6天,SOX-9质粒转染组骨髓基质细胞免疫组织化学染色呈棕黄色,稳定表达SOX-9蛋白,HE染色可见多个双核细胞,细胞增殖旺盛,与成软骨细胞类似;空质粒组无SOX-9蛋白表达,组胞增殖分裂不旺盛。SOX-9质粒转染组表达SOX-9 mRNA,空质粒组及空白对照组均无SOX-9 mRNA表达。转染后24,48,72h及1,2周,SOX-9质粒转染组细胞上清液中SOX-9含量均明显高于空质粒组、空白对照组(P<0.01)。结论:利用pDC316-SOX-9质粒成功转染兔骨髓基质细胞,增强细胞生长活性,并能持续稳定的分泌SOX-9蛋白,可诱导骨髓基质细胞向软骨方向分化。
BACKGROUND: SOX-9 plays an important role in the development and development of cartilage tissue. It promotes the agglutination of mesenchymal cells and has a transcriptional activation domain that directly regulates transcription. OBJECTIVE: To select the SOX-9 gene to construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells and observe the effect of SOX-9 gene on the growth characteristics and product expression of bone marrow stromal cells. DESIGN, TIME AND SETTING: In vitro cell engineering experiments were performed at the Beijing Genomics Institute, Chinese Academy of Sciences from June 2006 to January 2007. MATERIALS: Eight New Zealand white rabbits, 6 weeks old, were used to isolate and culture bone marrow stromal cells. METHODS: The pDC316-SOX-9 plasmid was transfected into rabbit bone marrow stromal cells by liposome-mediated method. The cells were transfected into SOX-9 plasmid transfected group and empty plasmid group, and only the same amount of liposome Blank control group. MAIN OUTCOME MEASURES: The success of SOX-9 gene transfected bone marrow stromal cells was determined by cell morphology, cell growth activity, immunohistochemistry and HE staining, RT-PCR and ELISA. Results: Small pieces of colonies appeared after 4 days of transfection with pDC316-SOX-9 plasmid. Fibrous cells were spindle-shaped after picked clone culture. With the extension of the transfection time, the cells in the blank control group died gradually. The cell viability of the SOX-9 plasmid transfected group and the empty plasmid group were significantly prolonged, reached its peak at 2 weeks after transfection, and then gradually decreased. On the 6th day after transfection, SOX-9 plasmid transfected bone marrow stromal cells were stained brownish yellow, stably expressing SOX-9 protein. HE staining showed that a large number of binucleated cells were proliferated, similar to chondroblasts; empty No expression of SOX-9 protein in the plasmid group, cell proliferation and division is not strong. SOX-9 mRNA was expressed in SOX-9 plasmid transfected group, and SOX-9 mRNA was not expressed in empty plasmid group and blank control group. The levels of SOX-9 in supernatant of SOX-9 transfection group were significantly higher than that of empty plasmid group and blank control group at 24, 48, 72h and 1, 2 weeks after transfection (P <0.01). Conclusion: The pDC316-SOX-9 plasmid was successfully transfected into rabbit bone marrow stromal cells to enhance the cell growth activity, and the sustained and stable secretion of SOX-9 protein can induce bone marrow stromal cells to differentiate into cartilage.