干细胞贴膜治疗脊髓损伤

Stem cell membrane transplantation for treatment of spinal cord injury

背景:目前人们较多采用的成体干细胞移植,移植方式多形成再次损伤,且体内神经元分化的效率不高。目的:在以往分步诱导分化高效获得神经元前体细胞的基础上,制成干细胞贴膜,观察其贴敷移植治疗脊髓损伤功能恢复的效果。设计、时间及地点:随机对照动物实验,于2008-05/10在郑州大学医学院实验中心完成。材料:酶、化学法制作膜样基质;分离培养、扩增羊膜间充质干细胞,BrdU标记,调整部分细胞浓度为1×1011L-1成细胞悬液备用。余分步诱导并制成干细胞贴膜。方法:健康成年雄性SD大鼠88只,体质量220～250g,以自制打击器制作急性脊髓损伤模型。模型成功后6h,抽签法随机分为4组,每组22只。干细胞贴膜组:在T11进行椎板切除术,打开硬膜充分显露损伤脊髓,止血,将干细胞贴膜贴敷在损伤脊髓表面,手术显微镜下固定于硬膜,分层缝合肌肉皮肤;细胞移植组:损伤脊髓头尾端移植1×1011L-1细胞悬液共5μL,余同上;基质组:贴敷膜样基质在损伤脊髓表面,余同上;对照组:仅行椎板切除术,并打开硬膜。主要观察指标:术后第1天及其后1周1次共10周,进行BBB评分和斜板实验等运动功能检测;术后2,4,6周观察移植细胞存活及形态学变化,免疫组织化学SP法检测胶质纤维酸性蛋白、突触素及神经元特异性烯醇化酶的表达。结果:①运动功能检测:各组BBB积分在4周及以后达到稳定,以10周时BBB评分12及以上为重要的恢复指标,则干细胞贴膜组为100%,细胞移植组为33%,基质组和对照组仅为17%。斜板实验的顺位实验,干细胞贴膜组第10周时的角度与第4周相比有显著改善(P=0.027),而同组同时间点的BBB分值间差异无显著性意义(P=0.286)。②干细胞贴膜组和细胞移植组远端损伤边缘BrdU阳性细胞计数差异无显著性意义(P=0.089)。干细胞贴膜组BrdU阳性细胞多呈神经元形态,细胞移植组BrdU阳性细胞多呈小胶质细胞形态。③各组突触素、胶质纤维酸性蛋白表达均逐渐增强,神经元特异性烯醇化酶表达则呈减少趋势。干细胞贴膜组各时间点的突触素、神经元特异性烯醇化酶表达高于其他各组(P<0.05);胶质纤维酸性蛋白表达则低于其他各组且保持稳定(P<0.05)。细胞移植组、基质组和对照组不同时间点的胶质纤维酸性蛋白表达差异有显著性意义且逐渐增高(P<0.05)。结论:干细胞贴膜上的神经元前体细胞可以在早期整合到损伤脊髓中,长期存活,并且具有高效的神经元分化特性;干细胞贴膜脊髓表面贴敷移植有助于脊髓损伤区突触素的表达、神经网络重建和神经功能恢复;干细胞贴膜移植较单纯干细胞移植更能有效促进神经功能恢复。

BACKGROUND： Adult stem cell （ASC） transplantation is commonly used presently. However, the present transplanted method could result in injury again, and efficiency of neuron differentiation in vivo was low. OBJECTIVE： Based on the previous study of neuron precursor cell collection by step-by-step induction and differentiation, to design stem cell membrane, and to observe functional recovery after attachment transplantation for treating spinal cord injury. DESIGN, TIME AND SETTING： The randomized, controlled, animal study was performed at the Experimental Center, Medical College, Zhengzhou University from May te October 2008. MATERIALS： Membrane-like matdx was established by enzyme and chemical method. Amnion-derived mesenchymal stem cells （ADMSCs） were isolated, cultured and amplified, and then labeled by BrdU. Cells at 1×10^11/L were used for further use. The remaining cells were induced and prepared into stem cell membrane. METHODS： Totally 88 healthy adult male Sprague Dawley rats, weighing 220 250 g, were used to create acute spinal cord injury models by self-designed shape-suitable impinge. 6 hours after modeling, rats was equally and randomly divided into 4 groups. Rats in the stem cell membrane group received laminectomy at T11. The dura mater was opened to fully expose damaged spinal cord and to stop bleeding. The stem cell membrane was covered on the damaged spinal cord, which was then fixed on the dura mater under a microscope. Muscle and skin were sutured. In the stem cell transplantation group, 5 μL cell suspension （1 ×10^11/L） was infused into the damaged spinal cord. The following procedures were the same as above mentioned. In the matrix group, membrane-like matrix was coated on the damaged spinal cord. The following procedures were the same as above mentioned. Rats in the control group only underwent laminectomy, and the dura mater was opened. MAIN OUTCOME MEASURES： One day postinjury and weekly thereafter for 10 weeks, behavioral analysis was performed by Basse-Beattie-Bresnahan （BBB） scale, the ability to maintain body position on an inclined plane. 2, 4, 6 week postinjury, cell survival and morphology were observed. Immunocytochemistry was performed employing the following antibodies： glial fibrillary acidic protein （GFAP）, synaptophysin and neurone specific enolase （NSE）, RESULTS： BBB gradually stabilized from 4 weeks after injury in each group. BBB scoring over 12 points was important recovery index at 10 weeks, and 100% in the stem cell membrane group, 33% in the stem cell transplantation group, 17% in the matrix group, and 17% in the control group. During inclined plane test, when facing upwards, the angle in the stem cell membrane group was significantly improved at 10 weeks compared with that at 4 weeks （P = 0.027）, and no significant difference in BBB score was detected at the same time point in the same group （P = 0.286）. BrdU-positive cells did not show statistically significant different between the stem cell membrane and stem cell transplantation groups （P=0.089）. The cells was neuron-like in the stem cell membrane group and microglia-like in the stem cell transplantation group. Synaptophysin, GFAP expression enhanced and NSE expression decreased gradually in each group. Synaptophysin and NSE expression was higher, but GFAP expression was lower in the stem cell membrane group than other groups at each interval （P 〈 0.05）. GFAP expression was gradually increased in the stem cell membrane, matrix and control groups at various time points （P 〈 0.05）. CONCLUSION： Neuronic precursor cells on stem cell membrane could integrate into injured cord and survive for a long time, and have a property to differentiate into neuron high-efficiency in vivo. Stem cell membrane transplantation on the injured cord contributes to synaptophysin expression, neural network reconstruction and neural functional recovery in the damaged cord. Stem cell membrane transplantation can show better functional recovery compared with stem cell transplantation alone.