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目的 通过腺病毒介导CTLA4Ig在骨髓基质细胞 (BMSCs)中的表达 ,探讨CTLA4Ig基因修饰的BMSCs支持CD34+ 细胞扩增的功能变化 ,为该基因修饰的BMSCs联合造血干细胞移植 (HSCT) ,达到预防移植物抗宿主病 (GVHD )和纠正预处理损伤的造血微环境 (HIM)积累实验依据。方法 以CTLA4Ig 重组腺病毒按感染复数 (Multiplicityofinfection ,MOI) 5 0转染BMSCs ,以RT PCR检测目的基因转录 ;免疫磁珠 (MACS)阳性选择分选骨髓CD34+ 细胞 ,流式细胞仪检测其纯度 ;通过骨髓基质细胞支持CD34+ 扩增的细胞总数及集落形成细胞数 (CFC)的变化 ,比较转染和未转染BMSCs在支持CD34+ 细胞扩增方面的差异。结果 RT PCR在转染组及转染后传两代BMSCs中检测到CTLA4Ig基因的转录 ;通过观察扩增后细胞总数及CFC数的变化 ,发现CTLA4Ig基因转染组与未转染组BMSCs支持CD34+ 细胞扩增的能力也无显著差异 (P >0 .0 5 )。结论 CTLA4Ig 重组腺病毒有效介导目的基因对BMSCs的转染 ,在MOI为 5 0时对其支持造血的功能无显著影响
OBJECTIVE: To investigate the function of CTLA4Ig in bone marrow stromal cells (BMSCs) mediated by adenovirus and to explore the functional changes of CTLA4Ig gene-modified BMSCs in supporting the expansion of CD34 + cells. The aim of this study was to evaluate the effect of CTLA4Ig on bone marrow stromal cells (HSCT) Anti-host disease (GVHD) and hematopoietic microenvironment (HIM) accumulation to correct preconditioning damage are based on experimental evidence. METHODS: BMSCs were transfected with CTLA4Ig recombinant adenovirus at multiplicity of infection (MOI) of 50 and RT-PCR was used to detect the transcription of the target gene. The positive cells were sorted by immunomagnetic beads (MACS), and the purity of CD34 + cells was detected by flow cytometry. Differences in the proliferation of CD34 + cells between transfected and non-transfected BMSCs were compared by counting the number of CD34 + cells and the number of colony forming cells (CFCs) in bone marrow stromal cells. Results RT-PCR showed that the CTLA4Ig gene was detected in the transfected and post-transfection BMSCs. By observing the total number of cells and the number of CFCs after transfection, we found that CTLA4Ig transfection group and non-transfected BMSCs support CD34 + There was no significant difference in cell proliferation (P> 0.05). Conclusions CTLA4Ig recombinant adenovirus can effectively mediate the transfection of target gene into BMSCs, and has no significant effect on the function of supporting hematopoiesis when the MOI is 50