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目的将大鼠骨髓间充质干细胞(MSC),经重组腺病毒载体Ad-IL-12转染,构建Ad-IL-12-MSC。方法 Percoll法分离与培养大鼠MSC,流式细胞仪测定细胞表面标志CD29,CD44,CD34。以重组腺病毒载体Ad-IL-12转染MSC,RT-PCR、Western Blott检测转染后IL-12基因mRNA和蛋白的表达。结果分离所得的大鼠MSCs细胞组成比较均一。流式细胞仪检测3种MSCs的表面抗原标志,CD29和CD44,阳性率分别达97.1%和98.2%,而CD34的阳性率仅为4.4%。转染后,Ad-IL-12-MSC细胞IL-12基因在mRNA水平和蛋白质水均有表达。结论 Percoll法分离与培养MSC较为可靠且简单,构建的Ad-IL-12-MSC能成功表达IL-12。
OBJECTIVE: To construct Ad-IL-12-MSCs by transfecting rat bone marrow mesenchymal stem cells (MSCs) with recombinant adenovirus vector Ad-IL-12. Methods Percoll method was used to separate and culture rat MSC. Cell surface markers CD29, CD44 and CD34 were determined by flow cytometry. The recombinant adenovirus Ad-IL-12 was transfected into MSC, and the expression of IL-12 mRNA and protein was detected by RT-PCR and Western Blott. Results The isolated rat MSCs were relatively homogeneous. The surface antigen markers of three kinds of MSCs were detected by flow cytometry. The positive rates of CD29 and CD44 were 97.1% and 98.2% respectively, while the positive rate of CD34 was only 4.4%. After transfection, IL-12 gene in Ad-IL-12-MSC cells was expressed both in mRNA and protein water. Conclusion Percoll method is more reliable and simple to isolate and culture MSCs. The constructed Ad-IL-12-MSC can express IL-12 successfully.