天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (12): 1356-1360.doi: 10.11958/j.issn.0253-9896.2015.12.004

• 细胞与分子生物学 • 上一篇    下一篇

ZHX3 基因沉默对BMSCs 中成骨相关因子表达的影响

张苗苗1，包翠芬2，王艳3，闵鹤鸣4，秦书俭1△   

  1. 1 锦州，辽宁医学院人体解剖与组织胚胎教研室（邮编121000）；2 辽宁省高校分子生物与新药开发重点实验室；3 辽宁医学院附属第一医院神经内科；4 辽宁医学院细胞生物学教研室
  • 收稿日期:2015-08-05 修回日期:2015-09-15 出版日期:2015-12-15 发布日期:2015-12-11
  • 通讯作者: △通讯作者E-mail: mianyizuhua@aliyun.com E-mail:1538702627@qq.com
  • 作者简介:张苗苗（1989），女，硕士在读，主要从事骨组织工程方面研究
  • 基金资助:
    国家自然科学基金资助项目（31170930，81202783）

Effects of ZHX3 gene silence on the expression of osteoblast-related factors in BMSCs

ZHANG Miaomiao1, BAO Cuifen2, WANG Yan3, MIN Heming4 , QIN Shujian1△   

  1. 1 Department of Human Anatomy and Histology and Embryology, 2 Key Lab of Molecular Cell Biology and New Drug Development, 3 Department of Neurology, First Affiliated Hospital of Jinzhou City; 4 Department of Cell Biology, Liaoning Medical University, Jinzhou 121000, China
  • Received:2015-08-05 Revised:2015-09-15 Published:2015-12-15 Online:2015-12-11
  • Contact: △Corresponding Author E-mail：mianyizuhua@aliyun.com E-mail:1538702627@qq.com

PDF (PC)

3927

摘要： 目的探讨锌指蛋白和同源框3（ZHX3）基因沉默对骨髓间充质干细胞（BMSCs）中smad3、smad4、RUNX2 表达的影响。方法构建ZHX3 低表达慢病毒载体并转染大鼠BMSCs（ZHX3 沉默组），同时设空载病毒转染BMSCs （载体对照组）及不做任何处理的BMSCs（空白对照组）。荧光显微镜下测定细胞转染率并采用免疫印迹技术鉴定转染是否成功；采用免疫荧光化学和免疫印迹技术定性定量检测ZHX3 沉默时smad3、smad4、RUNX2 表达情况。结果（1）复苏培养的细胞具有BMSCs 表型。（2）转染后，ZHX3 沉默组和载体对照组均表达绿色荧光，空白对照组不表达荧光，且沉默组ZHX3 基因表达显著低于载体对照组。（3）免疫荧光结果显示，smad3、smad4 的阳性表达位于细胞核和细胞质，RUNX2 的阳性表达主要定位于细胞核。3 组细胞均可见阳性表达细胞，且空白对照组与载体对照组之间荧光强度未见显著差异，但ZHX3 基因沉默组的荧光强度显著低于2 个对照组。（4）免疫印迹检测smad3、 smad4、RUNX2 的条带于空白对照组和载体对照组中无显著差异，但均显著高于ZHX3 沉默组（P＜0.05）。结论 ZHX3 基因沉默后BMSCs 体外成骨能力延迟，可能通过下调smad3、smad4、RUNX2 来发挥作用。　

关键词: DNA 结合蛋白质类, 锌指, 基因, 同源盒, 转录因子, 基因沉默, 间质干细胞, smad3, smad4, RUNX2, 转录抑制因子-锌指蛋白和同源框3

Abstract: Objective To investigate the effects of zinc fingers and homeoboxes 3 (ZHX3) silence on expressions of smad3, smad4 and RUNX2 in bone marrow mesenchymal stem cells (BMSCs). Methods ZHX3 low expression vector (ZHX3 silent group) was constructed and was transfected to rat BMSCs. Empty vector was transfected into BMSCs and was used as vehicle control group, and wild type BMSCs was used as the control group. The cell transfection rate was measured under a fluorescence microscope, and then the successful transfection was identified. The immunocytochemistry and immu⁃ noblotting methods were used to detect the expression levels of smad3, smad4 and RUNX2. Results (1) Cells with BMSCs phenotype can be obtained by recovery culturing. (2) After transfection, the green fluorescent protein was found in ZHX3 si⁃ lence group and vehicle control group. Blank control group showed no significant fluorescence. The expression level of ZHX3 was significantly lower in ZHX3 silence group than that of vehicle control group. (3) Results of immunofluorescence asssay showed that the positive expressions of smad3 and smad4 were located in nucleus and cytoplasm, the positive expression of RUNX2 was mainly located in nucleus. Positive cells were observed in three groups. There was no significant difference in fluorescence intensity between the control group and the vehicle control group, but the fluorescence intensity was significant⁃ ly lower in ZHX3 gene silence group than that of two control groups. (4) There were no significant differences in expressions of smad3, smad4 and RUNX2 betweem control group and the vehicle control group, but they were significantly higher than those of ZHX3 silence group（P < 0.05）. Conclusion ZHX3 gene silence can delay vitro osteogenesis of BMSCs, which may play a role by the down-regulated expression levels of smad3, smad4 and RUNX2.

Key words: DNA-binding proteins, zinc fingers, genes, homeobox, transcription factors, geen silencing, mesenchymal stem cells, smad3, smad4, RUNX2, zinc fingers and homeoboxes 3