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目的观察人脐血源基质细胞(hUCBDSCs)经腺病毒介导的缝隙连接蛋白43(Cx43)转染后Cx43基因表达的变化以及转染前后分别对CD4+T分泌IL-4、CD8+T分泌IFN-γ的影响。方法用重组腺病毒Ad-Cx43-GFP转染hUCBDSCs,荧光显微镜报告基因GFP的表达并计算转染效率,RT-PCR法、Western blot法、免疫荧光法检测hUCBDSCs转染前后Cx43基因及蛋白表达情况。将流式细胞仪分选出的CD4+T和CD8+T淋巴细胞分别与未转染以及转染腺病毒载体的hUCBDSCs共培养,共培养72 h后,ELISA双抗体夹心法测上清IL-4,IFN-γ的水平。结果荧光显微镜观察可见hUCBDSCs在转染Ad-Cx43-GFP后24 h即有绿色荧光表达,转染48 h计数绿色荧光细胞的百分率得出转染效率为(87±3.8)%;免疫荧光法、RT-PCR法、Western blot法显示转染Ad-Cx43-GFP后hUCBDSCs Cx43基因及蛋白表达较转染前增强(P<0.05);此外Cx43转染的hUCBDSCs和转染空病毒组比较IL-4分泌增加,IFN-γ分泌降低(P<0.05)。结论 Cx43转染的hUCBDSCs能促进IL-4分泌,抑制IFN-γ分泌,为我们更好地了解其免疫调控作用及为移植物抗宿主病的治疗提供了理论依据。
Objective To observe the changes of Cx43 gene expression in human umbilical cord blood-derived stromal cells (hUCBDSCs) transfected with adenovirus-mediated gap junction protein 43 (Cx43) and the effects of secreting IL-4 and CD8 + T on CD4 + T cells IFN-γ effects. Methods The hUCBDSCs were transfected with the recombinant adenovirus Ad-Cx43-GFP. The expression of GFP was detected by fluorescence microscopy and the transfection efficiency was calculated. The expression of Cx43 gene and protein before and after hUCBDSCs transfection were detected by RT-PCR, Western blot and immunofluorescence . The CD4 + T and CD8 + T lymphocytes sorted by flow cytometry were co-cultured with hUCBDSCs not transfected and transfected with adenovirus vector respectively. After co-cultured for 72 h, the supernatants of IL- 4, the level of IFN-γ. Results Fluorescence microscopy showed that hUCBDSCs expressed green fluorescence at 24 h after transfection with Ad-Cx43-GFP, and the transfection efficiency was (87 ± 3.8)% at 48 h after transfection. The immunofluorescence assay, The expression of Cx43 gene and protein in hUCBDSCs transfected with Ad-Cx43-GFP was higher than that before transfection (P <0.05) by RT-PCR and Western blot. In addition, the expression of Cx43 gene and protein in hUCBDSCs transfected with Cx43 and IL-4 Secretion increased, IFN-γ secretion decreased (P <0.05). Conclusion Cx43-transfected hUCBDSCs can promote the secretion of IL-4 and inhibit the secretion of IFN-γ, which provides a theoretical basis for our better understanding of its immunomodulatory effects and for the treatment of graft-versus-host disease.