骨骼肌细胞Syncytin-1过表达对脊髓前角运动神经元-骨骼肌细胞-施万细胞共培养模型的影响

Effect of Syncytin-1 overexpression in skeletal muscle cells on co-culture model of spinal cord anterior horn motor neurons, Schwann cells, and skeletal muscle cells

目的探讨骨骼肌细胞Syncytin-1过表达对脊髓前角运动神经元-骨骼肌细胞-施万细胞共培养模型炎性因子、钠离子依赖性中性氨基酸转运体1(ASCT1)和神经保护因子的影响。方法(1)体外原代培养脊髓前角运动神经元、骨骼肌细胞、施万细胞,免疫荧光染色分别检测细胞胆碱乙酰转移酶(ChAT)、α-平滑肌肌动蛋白(α-SMA)、钙结合蛋白B(S100B)的表达进行鉴定。(2)将Syncytin-1重组质粒及空载质粒分别转染骨骼肌细胞后24 h,与另外2种细胞混合,分别建立3种细胞的共培养模型(Syncytin-1重组质粒转染组:Syncytin-1重组质粒转染的骨骼肌细胞、脊髓前角运动神经元、施万细胞混合;空载质粒转染组:空载质粒转染的骨骼肌细胞、脊髓前角运动神经元、施万细胞混合)。于倒置显微镜下观察共培养细胞形态及连接的变化。共培养48 h后应用酶联免疫吸附实验(ELISA)检测2组共培养细胞上清液中肿瘤坏死因子-α(TNF-α)、诱导型一氧化氮合酶(iNOS)、血管内皮生长因子(VEGF)的浓度,采用实时荧光定量PCR(qRT-PCR)和Western blotting分别检测2组共培养细胞Syncytin-1、ASCT1、TNF-α、iNOS、VEGF mRNA和蛋白的表达。结果(1)免疫荧光染色检测显示:脊髓前角运动神经元中ChAT阳性表达细胞、骨骼肌细胞中α-SMA阳性表达细胞、施万细胞中S100B阳性表达细胞均占95%以上,且定位在细胞质内。(2)Syncytin-1重组质粒转染组及空载质粒转染组共培养细胞的数量及形态未见明显差异。与空载质粒转染组比较,Syncytin-1重组质粒转染组共培养细胞上清液TNF-α、iNOS及VEGF的浓度均增高,细胞TNF-α、iNOS、Syncytin-1、VEGF mRNA和蛋白的表达均增高,ASCT1 mRNA和蛋白的表达均降低,差异均有统计学意义(P<0.05)。结论骨骼肌细胞Syncytin-1过表达可以引起共培养细胞炎性因子释放、ASCT1表达降低、神经保护因子VEGF表达增高。

Objective To explore the effect of Syncytin-1 overexpression in the skeletal muscle cells on changes of sodium-dependent neutral amino acid transporter 1(ASCT1),inflammatory factors and neuroprotective factors in co-culture model of spinal cord anterior horn motor neurons,Schwann cells,and skeletal muscle cells.Methods(1)Spinal cord anterior horn motor neurons,skeletal muscle cells,and Schwann cells were primarily cultured in vitro;the expressions of choline acetyltransferase(ChAT),α-smooth muscle actin(α-SMA)and calcium-binding protein B(S100B)in the neurons,muscle cells,and Schwann cells were detected by immunofluorescence staining,respectively.(2)Plasmids containing Syncytin-1 or control plasmids were transfected into the skeletal muscle cells,respectively;24 h after that,these transfected skeletal muscle cells were co-cultured with spinal cord anterior horn motor neurons and Schwann cells,respectively(Syncytin-1 recombinant plasmid transfection group or control plasmid transfection group);changes of morphology and junction of co-culture cells were observed under inverted microscope.Forty-eighty h after co-culture,enzyme-linked immunosorbent assay(ELISA)was used to detect the tumor necrosis factorα(TNF-α),inducible nitric oxide synthase(iNOS)and vascular endothelial growth factor(VEGF)concentrations in the supernatant of co-culture cells;real-time quantitative(qRT)-PCR and Western blotting were used to detect the Syncytin-1,ASCT1,TNF-α,iNOS,and VEGF mRNA and protein expressions in the co-culture cells Results(1)Immunofluorescent staining showed that more than 95%spinal cord anterior horn motor neurons were with positive CHAT expression,more than 95%skeletal muscle cells were with positiveα-SMA expression,and more than 95%Schwann cells were with positive S100B expression;all of which were localized in the cytoplasm.(2)There were no obvious differences in number or morphology of co-culture cells between the Syncytin-1 recombinant plasmid transfection group and control plasmid transfection group.As compared with the control plasmid transfection group,Syncytin-1 recombinant plasmid transfection group had significantly increased concentrations of TNF-α,iNOS and VEGF in the supernatant of co-cultured cells,and statistically increased mRNA and protein expressions of TNF-α,iNOS,syncytin-1 and VEGF,and significantly decreased ASCT1 mRNA and protein expressions(P<0.05).Conclusion Syncytin-1 overexpression in the skeletal muscle cells may decrease the ASCT1 expression,induce the inflammatory factor release,and increase the neuroprotective factor VEGF expression.