钛颗粒对大鼠成骨细胞影响的mRNA差异显示研究

Abstract

目的观察钛金属颗粒对大鼠成骨细胞基因表达的影响，寻找与假体周围骨溶解有关的新基因。方法在含10%牛血清的DMEM培养基中培养Wistar幼鼠成骨细胞，达80%丰满后加入0.1%钛颗粒,再培养24 h后收获细胞并提取总RNA。用mRNA差异显示技术比较处理组与对照组之间在mRNA表达上的差别。将有差别的表达带回收、扩增、克隆化，用于Reverse Northern杂交、Northern杂交及测序，将测序结果与NCBI数据库比较。结果在处理与对照间找到35条差异表达带，经Reverse Northern杂交得到5个阳性克隆，经测序及NCBI数据库分析，其中被命名为G46T的基因与单核细胞趋化蛋白-3达92%同源，另外4个是新基因。结论钛颗粒可引起鼠成骨细胞基因表达的变化；G46T基因可能与假体周围骨溶解有关。

Objectives To observe the gene expression of rat osteoblasts treated with titanium particles and to identify the novel genes which may be related to the periprosthetic osteolysis. Methods Neonatal rat osteoblasts were grown in DME medium with 10% bovine serum, and 0.1% titanium particles were added to the medium when the cells reached 80% confluency.After 24 hours，the total RNA was isolated.Messenger RNA differential display techniques were used to compare the differences between the treated and the control group. Furthermore，the differentially displayed gene fragments were isolated，reamplified，and cloned for reverse northern hybridization，northern hybridization and sequencing. The sequences were compared with GenBank database using the BLAST network service at the National Center for Biotechnology Information（NCBI）. Results 35 fragments were differentially displayed between tho two groups. Of the 35 fragments that were analyzed by reverse northern hybridization，the 5 positive genes were sequenced and compared with NCBI.One of the five positive genes, named G46T, showed 92% homology with mouse macrophage-derived monocyte chemotactic protein-3. The other 4 positives were unknown genes. Conclusions Titanium particles may alter the gene expression of rat osteoblasts.G46T gene may be related with periprosthetic osteolysis.