A potential role for bone morphogenetic protein signalling in glial cell fate determination following adult central nervous system injury in vivo

Bone morphogenetic proteins (BMPs) and their endogenous inhibitors, including noggin, chordin and follistatin, have roles in pattern formation and fate specification of neuronal and glial cells during nervous system development. We have examined their influence on glial reactions in the injured central nervous system (CNS). We show that penetrating injuries to the brain and spinal cord resulted in the upregulation of BMP-2/4, BMP-7, and noggin, with the latter being expressed almost exclusively by reactive astrocytes at the injury site, and we show that astrocytes in vitro produce noggin. As BMPs have been shown to drive cultured NG2-positive oligodendrocyte precursors (OPCs) towards a multipotential phenotype (type II astrocytes), we investigated the effects of inhibiting noggin with a function-blocking antibody (noggin-FbAb). In vitro, BMP-driven conversion of OPCs to type 2 astrocytes was inhibited by noggin, an effect that was reversed by noggin-FbAb. Noggin-FbAb also increased the number of type 2 astrocytes generated from cultured OPCs exposed to an astrocyte feeder layer, consistent with astrocytes producing both BMPs and noggin. In knife cut injuries in vivo, noggin-FbAb treatment resulted in an increase in the number of NG2-positive cells and small GFAP-positive cells in the injury site, and the appearance of glial cells with the morphological and antigenic characteristics of type 2 astrocytes (as generated in vitro), with coexpression of both GFAP and NG2. This potential conversion of inhibitory OPCs to type 2 astrocyte-like cells in vivo suggests that endogenous BMPs, unmasked by noggin antagonism, might be exploited to manipulate cell fate following CNS trauma.     

重组腺病毒介导noggin诱导骨髓问充质干细胞向神经细胞分化

目的 探讨体外分离、培养大鼠间充质干细胞(MSCs)的方法 ,采用重组腺病毒介导的noggin基因体外诱导方法 ,观察MSCs向神经细胞的定向分化效应.方法 原代培养MSCs 24h开始贴壁,呈梭形,集落样生长,纯化后,采用含noggin基因的重组腺病毒(pAdEasy-1-noggin)感染原代培养的MSCs,诱导骨髓MSCs分化为神经细胞.用鉴定神经特异性烯醇化酶(NSE)、神经元核抗原(NeuN)、神经丝蛋白(NF-200)和胶质纤维酸性蛋白(GFAP)的免疫细胞化学方法 对诱导后的细胞进行鉴定.结果 倒置显微镜下观察可见,MSCs经重组腺病毒感染后48h,细胞向神经细胞分化,胞体呈锥形、多角形,由胞体伸出较长的轴突样和树突样突起,且有分支.免疫组织化学鉴定显示,诱导后的细胞能特异性表达NSE、NeuN和NF-200,而GFAP表达阴性.结论 利用密度梯度离心法结合贴壁法可以获得比较纯的MSCs;noggin基因重组腺病毒载体可成功诱导骨髓间充质干细胞向神经元样细胞分化.     

BMP2、Noggin和IGF-1对成骨细胞的调节作用

目的 研究骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)、noggin、胰岛素样生长因子1(insulin like growth factor 1,IGF-1)对成骨细胞增殖的调节作用.方法 将人成骨肉瘤细胞MG63于RPMI 1640培养基中培养,添加不同浓度的BMP2、noggin、IGF-1蛋白进行药物干预,用四甲基偶氮唑盐(methylthiazolyl-tetrazolium,MTT)法研究成骨细胞的增殖率.结果 各药物干预组OD值与对照组相比差异有统计学意义(P＜0.05),对成骨细胞增殖的作用效应呈剂量和时间依赖性,其作用的最佳时间均在72 h.各药物组内之间比较,确定了最佳作用浓度,BMP2为2×10-9 mol/L,对MG63细胞的增殖率达179.59%;noggin为8×10-9 mol/L,对其增殖的抑制率达到15.51%;IGF-1为1×10-8 mol/L,其增殖率达189.80%.结论 BMP2、IGF-1对MG63细胞增殖有促进作用,而noggin则抑制MG63细胞的增殖.