慢病毒介导骨形态发生蛋白7基因转染诱导大鼠骨髓间充质干细胞向神经元样细胞的分化

文题释义：骨形态发生蛋白7：骨形态发生蛋白不仅决定着胚胎早期神经与非神经的命运，还与Wnt、Shh等其他信号通路协同作用，在神经干细胞的增殖、分化以及神经系统各亚型细胞的形成中发挥着重要作用。骨形态发生蛋白7是骨形态发生蛋白家族的重要成员之一，通过静脉或脑室注射骨形态发生蛋白7蛋白，可改善脑缺血大鼠的神经功能，而且骨形态发生蛋白7能够促进神经树突生长，说明骨形态发生蛋白7可能在神经系统发生与修复中具有重要的调节作用，起到神经保护作用。骨髓间充质干细胞：其具有来源广泛、取材容易、免疫排斥反应弱、避免伦理争议等优点，应用于治疗脊髓损伤具有重要的临床意义，为一种理想的组织工程干细胞。但利用骨髓间充质干细胞治疗脊髓损伤存在诸多问题，例如向神经细胞分化效率低、移植进入体内后分化形成的神经细胞能否与内源性神经细胞形成突触联系等。背景：骨髓间充质干细胞具备向神经元样细胞分化的潜能，已经被列为治疗脊髓损伤的首选干细胞，但其分化效率低，寻找一种具有高效诱导能力的因子尤为重要。基于文献查阅及课题组研究基础，推测骨形态发生蛋白7(bone morphogenetic protein 7，BMP-7)基因可能在促进骨髓间充质干细胞向神经元样细胞分化上发挥着重要作用。目的：探讨骨形态发生蛋白7慢病毒载体转染诱导大鼠骨髓间充质干细胞向神经元样细胞分化的作用。方法：采用全骨髓贴壁法培养SD大鼠骨髓间充质干细胞，以感染复数为50，25，10，1进行LV-GFP转染，转染后3 d在荧光倒置显微镜下观察各组GFP表达情况，确定最佳感染复数。取第3代骨髓间充质干细胞，分为空白对照组(常规培养)、LV-GFP组、LV-BMP-7-GFP组，以最佳感染复数转染24，48，72，96，120 h后，采用MTT法检测细胞存活率；转染3 d后，采用免疫细胞化学实验检测神经细胞标记物神经丝蛋白200、突触素1表达。结果与结论：①LV-GFP转染后3 d，感染复数为1组未见GFP阳性细胞，感染复数为10，25，50组可见GFP阳性细胞，其中感染复数为10组平均荧光强度高于其余组(P < 0.05)，为最适感染复数；②空白对照组、LV-GFP组神经丝蛋白200、突触素1表达呈阴性；LV-BMP-7-GFP组神经丝蛋白200、突触素1表达呈阳性，胞体和轴突被染成亮棕黄色；③结果表明，LV-BMP-7转染可促进骨髓间充质干细胞向神经元样细胞分化。ORCID: 0000-0001-5909-5524(张文)中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Neuronal differentiation of rat bone marrow mesenchymal stem cells via lentivirus-mediated bone morphogenetic protein 7 transfection

BACKGROUND:Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells,which have been listed as the preferred stem cells for the treatment of spinal cord injury.However,due to their low differentiation efficiency,it is particularly important to find a factor with high induction ability.Based on literature review and our previous studies,it is speculated that bone morphogenetic protein 7(BMP-7)gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells.OBJECTIVE:To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection.METHODS:Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method,and then were transfected with LV-GFP when multiplicities of infection were 50,25,10,and 1.Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection,to confirm the best multiplicity of infection.Passage 3 bone marrow mesenchymal stem cells were divided into blank control group(routine culture),LV-GFP group,and LV-BMP-7-GFP group,followed by transfection at the best multiplicity of infection.After 24,48,72,96,and 120 hours of transfection,MTT assay was used to detect cell survival rate in each group.Immunocytochemical assay was used to detect the expression of nerve cell markers(neurofilament protein 200,synaptophysin-1)after 3 days of transfection.RESULTS AND CONCLUSION:(1)After 3 days of LV-GFP transfection,GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10,25,and 50,whereas no GFP-positive cells were found when the multiplicity of infection was 1.The average fluorescence intensity was the highest when the multiplicity of infection was 10(P<0.05),indicating that multiplicity of infection=10 had the best infection effect.(2)Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group,but positive in the LV-BMP-7-GFP group.The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown.In summary,lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.