慢病毒介导沉默P75神经营养因子受体联合神经生长因子过表达促进大鼠骨髓间充质干细胞的增殖

文题释义：P75神经营养因子受体(P75 Neurotrophin receptor，P75^NTR)：属于一种Ⅰ型糖蛋白，在非神经系统组织及多种肿瘤细胞中表达。p75^NTR与Trk受体通过不同的信号传递对细胞增殖与凋亡发挥着双重的生物效应。神经生长因子：在神经生长因子信号通路中，p75^NTR可以与神经生长因子相结合，引起神经酰胺的释放而激活JNK通路来调节细胞的增殖和凋亡。  摘要背景：P75神经营养因子受体(P75 Neurotrophin receptor，P75^NTR)是神经生长因子(nerve growth factor，NGF)的受体之一。在各种细胞组织中发挥着促进增殖或凋亡的双重作用，且P75^NTR在骨折不愈合处存在高表达，而过量的NGF可关闭P75^NTR受体，从而挽救受损的细胞。因此研究沉默P75^NTR联合NGF过表达共转染对骨髓间充质干细胞增殖的影响，可为临床治疗骨折不愈合提供新思路。目的：观察慢病毒介导沉默P75^NTR联合NGF过表达共转染对SD大鼠骨髓间充质干细胞增殖的影响。方法：体外培养至第3代SD大鼠骨髓间充质干细胞分为空白对照组、阴性对照组、沉默p75^NTR组、NGF过表达组、沉默p75^NTR联合NGF过表达组。将慢病毒介导沉默P75^NTR、过表达NGF转染至大鼠骨髓间充质干细胞，倒置荧光学显微镜观察慢病毒转染第3天细胞形态变化，流式细胞仪检测转染效率及Western blot方法检测P75^NTR和NGF的表达，最后用MTT法和CCK-8法检测各组细胞增殖活性。结果与结论：①共转染慢病毒后的细胞生长、分布良好；双基因慢病毒载体的转染效率已超过70%；②与空白对照组和阴性对照组比较，沉默P75^NTR联合NGF过表达组P75^NTR蛋白表达明显下调，NGF蛋白表达明显增加；③与空白对照组与阴性对照组相比，沉默P75^NTR联合NGF过表达组、沉默P75^NTR组、NGF过表达组细胞增殖明显加快，差异有显著性意义(P < 0.05)，其中沉默P75^NTR联合NGF过表达组增殖最快；④结果显示：沉默P75^NTR联合NGF过表达共转染可促进SD大鼠骨髓间充质干细胞的增殖。ORCID: 0000-0003-3826-7213(王宁) 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

Lentivirus-mediated P75 neurotrophin receptor silencing combined with nerve growth factor overexpression promotes proliferation of rat bone marrow mesenchymal stem cells

BACKGROUND:P75 Neurotrophin receptor(P75NTR)is one of the receptors for nerve growth factor(NGF).P75NTR plays a dual role in promoting proliferation or apoptosis in various cell tissues,and is highly expressed at fracture nonunion sites.However,excessive NGF can shut down P75^NTR receptor,thereby saving damaged cells.Therefore,the study regarding co-transfection of silenced P75NTR and NGF overexpression is of great significance for the proliferation of bone marrow mesenchymal stem cells and provides new ideas for clinical treatment of fracture nonunion.OBJECTIVE:To observe the effect of lentivirus-mediated silencing of P75^NTR combined with NGF overexpression on the proliferation of bone marrow mesenchymal stem cells in Sprague-Dawely rats.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured to the third generation in vitro and were divided into blank control group,negative control group,silent P75^NTR group,NGF overexpression group,and silent p75NTR combined with NGF overexpression group.Lentivirus-mediated silencing of P75^NTR and overexpression of NGF were transfected into rat bone marrow mesenchymal stem cells to induce P75^NTR silencing and NGF overexpression.Inverted fluorescence microscopy was used to observe changes in cell morphology on day 3 after transfection.Flow cytometry was used to detect transfection efficiency and western blot method was applied to detect the expression of P75^NTR and NGF.Finally,the cell proliferation activity was detected by MTT method and cell counting kit-8 method.RESULTS AND CONCLUSION:Cell growth and distribution were good after co-transfection of lentivirus.The transfection efficiency of the double-gene lentiviral vector exceeded 70%.Compared with the blank control and negative control groups,the expression of P75^NTR protein was significantly down-regulated,and the expression of NGF was profoundly up-regulated in the silent P75^NTR combined with NGF overexpression group.Compared with the blank control and negative control groups,cell proliferation was significantly increased in the other three groups(P<0.05),and the fastest proliferation was observed in the silent P75^NTR combined with NGF overexpression group.To conclude,silencing P75^NTR combined with NGF overexpression co-transfection can promote the proliferation of bone marrow mesenchymal stem cells from Sprague-Dawley rats.