混合初级皮层文化从产后第一天准备Sprague-Dawley老鼠的神经突结果有无hMNP-conditioned媒体(CM)或MN分化媒体(没有空调)如前所述罗西et al。
20.]。短暂,皮质机械分离,使胰蛋白酶化,旋转在1500 g 5分钟。颗粒在Neurobasal resuspended媒体(表达载体,卡尔斯巴德,CA)。40000个细胞被镀到poly-L-lysine涂布室幻灯片神经突产物(
23]。播种后,室幻灯片是美联储在未来2天Neurobasal媒体补充10 ng / mL FGF直到神经元附件。
2.6。在体外生长因子分泌试验
确定hMNP-conditioned媒体曾对神经突神经营养作用产物,MN分化媒体条件48小时的hMNPs表示皮质神经元和删除。初级皮层神经元喂养与MN分化媒体或hMNP-CM稀释1:1 MN分化媒体(
n
=
4/组)。MN分化培养基(没有空调)包括渗透性改编DMEM: F12混合物(260 mOsm)补充Glutamax, B27补充(Gibco-Invitrogen,卡尔斯巴德,CA)、胰岛素10毫克/毫升,亚硒酸钠1 ng / mL,转铁蛋白10毫克/毫升(西格玛奥德里奇,圣路易斯,密苏里州),MgSO40.5毫米和bFGF 5 ng / ml(微孔、Billerica MA) (
20.]。
老鼠被分为两组治疗(与hMNPs移植;
n
=
25)和车辆控制(vehicle-injected;
n
=
25)组织进行评估,最后阶段的蛋白表达和组织学评价供者细胞表型。此外,1组的动物(由15老鼠hMNP治疗和车辆控制SOD1 G93A老鼠)分析了组织学量化和1组动物的分子分析进行了分析(由10老鼠hMNP治疗和车辆控制SOD1 G93A老鼠)。
SMA模型。在产后第一天,Smn−−/;SMNΔ7+ / +;SMN2+ / +(以下称为Δ7SMN)幼崽cryoanesthetized,执行和椎板切除术在木材上地区。glass-pulled提示附加到汉密尔顿注射器是获得立体定向仪,20000 hMNPs(10000细胞/ uL)或体积相当于汽车将双边移植到脊髓(
24]。老鼠分为治疗(与hMNPs移植;
n
=
11)和车辆控制(vehicle-injected;
n
=
4)组织进行评估的结束阶段进行组织学分析。动物幸存移植后14天。
老鼠被分为两组治疗(与hMNPs移植;
n
=
30.)和车辆控制(vehicle-injected;
n
=
30.)组织进行评估,最后阶段的蛋白表达和组织学评价供者细胞表型。此外,动物被分为2组;第一组是老鼠(由20 hMNP-treated和车辆控制SCI老鼠)组织学量化分析和分子分析第二组动物进行了分析(组成的
n
=
10老鼠hMNP-treated和车辆控制SCI老鼠)。动物被移植后存活3个月。
2.8。免疫组织化学分析hMNPs
动物通过transcardiac牺牲和4%多聚甲醛灌注。组织低温贮藏在27%蔗糖,声带被分为10月1毫米块和嵌入化合物。组织是cryosectioned 20
μm和安装到gelatin-coated幻灯片。组织部分被封锁有10%是因为在PBS和permeabilized Triton x - 100年的0.1%。确定移植hMNPs的命运,部分处理了多个标记。主要抗体Ku80一夜之间被添加在4°C (1: 300;Abcam), Islet-1 (1: 200;Abcam),生物素化的NeuN (1: 200;微孔),我(1:200年,圣克鲁斯生物技术、圣克鲁斯,CA),和聊天(1:100;微孔)。驴antirabbit共轭与生物素(1:250;英杰公司)、链霉亲和素594 (1:250; Invitrogen), Streptavidin 488 (1 : 250; Invitrogen), or goat antirabbit Alexa Fluor-488/-594 (1 : 250; Invitrogen) was used for 2 hours at room temperature as secondary antibodies.
2.9。神经元的分析
的脊髓神经保留的所有三个动物模型进行了分析。每十张幻灯片,间隔200
μ分开,是为了处理可视化脊髓的长度(
20.]。包含IHC染色进行组织幻灯片使用鼠标anti-NeuN (1: 200;微孔)主要抗体识别神经元和兔子anti-Ku80 (1: 300;Abcam)主要抗体识别人类细胞核。串行部分是与人类的核抗原标记,确保没有包含在分析人类细胞。运动神经元的分析、鼠标anti-ChAT (1: 100;微孔)主要抗体和兔anti-Ku80 (1: 300;Abcam)主要抗体。抗体在PBS稀释,孵化一夜之间在4°C。向量ABC生物素装备和Vectastain民建联基质包被用于二次检测。 Hoechst was used as a nuclear counterstain. Following IHC staining, the tissue was imaged using an Olympus AX-80 microscope. Images were taken of transverse sections of the ventral horns using MicroFire software. NeuN-positive neurons were quantified up to 2 mm cranial and 2 mm caudal to the injury epicenter or transplant site. The number of neurons was quantified using ImageJ software. Each image was opened individually and changed to an 8-bit image. Each image was then inverted, then the brightness and contrast was adjusted to the Auto setting, which made the gray areas darker and neurons were more easily visible. Then, threshold was adjusted from Default to MaxEntropy setting. Finally, the Nucleus Counter plug-in was used to detect pixels, the smallest particle size set to 400 and largest particle size set to 2000. Background subtraction and watershed filter were used in the plug-in. The plug-in then showed the black and white image of the neurons with a number indicating that it was counted and a summary table of the total counts. If a cell double stained positive for both NeuN and Ku80, then that cell was not used in quantification. Statistical analysis was performed using a Student’s
t以及(2跟踪分布和两个样本方差相等)。
hMNP内生神经元移植备件。(一)比较NeuN阳性细胞在颅(
n
=
13hMNP;
n
=
15车辆)和尾(
n
=
14hMNP;
n
=
15车辆)横向部分在SCI的动物。(b)聊天比较阳性细胞在颅(
n
=
10hMNP;
n
=
12车辆)和尾(
n
=
9hMNP;
n
=
12车)的横向部分腹侧角在SCI的动物。(c)的比较NeuN阳性细胞在颅横向部分SOD1 G93A动物(
n
=
5hMNP;
n
=
5车辆)。(d)的比较NeuN阳性细胞在颅(
n
=
3hMNP;
n
=
3车辆)和尾(
n
=
3hMNP;
n
=
2车)的横向部分Δ7SMN动物。NeuN染色组织学部分腰椎脊髓hMNP (e)和车辆(f)注入SOD1 G93A老鼠清楚显示不同的神经元数量。数据表示为平均值±标准错误。
*
P
<
。
05年。酒吧= 50
μm。