【摘要】 目的:脐血有核细胞中富含多系前体细胞,具备改善受损神经系统功能的潜能。为此建立阻塞性脑卒中动物模型,探讨脐血有核细胞移植对其治疗的可行性。方法:实验于2004-09/2005-05在深圳市宝安区人民医院动物实验室完成。①实验材料:脐带血取自足月新生儿,由深圳宝安血站研究室提供,产妇及其家属均签署知情同意书。SD清洁级成年大鼠80只,随机取20只作为正常对照组,另60只以电凝法建立阻塞性脑卒中模型。剔除运动功能障碍不典型鼠后,随机取5只作为模型观察,余鼠按1∶1随机分为细胞移植组和模型对照组,当出现单只为尾数时,将其分配在细胞移植组。②实验方法:无菌抽取脐血20mL,加入乙二胺四乙酸抗凝,Ficoll法分离脐血有核细胞。造模后第10天,细胞移植组大鼠将头部固定,按前囟尾侧3.0mm,中线旁1.5mm,深度1.2mm注入1011L-1脐血有核细胞悬液5μL,1μL/min。模型对照组注射等量无细胞脐血清,正常对照组不给予任何干预。③实验评估:造模后4周,5只模型观察大鼠制作脑切片,行苏木精-伊红染色镜检。各组分别于细胞移植前、细胞移植后2,6周进行横木行走实验和触觉刺激试验,检测其运动和触觉功能的恢复。处死各组大鼠制作病理切片,免疫荧光检测脐血有核细胞在脑内生存和分化情况。结果:正常对照组、细胞移植组、模型对照组各20只、24只、24只进入结果分析。①阻塞性脑卒中模型大鼠病理特征:造模大鼠出现行为障碍,缺血部位出现中风囊,病理切片可见缺血坏死区等阻塞性脑卒中所致脑组织病理损伤。②运动和触觉功能测试:细胞移植前,细胞移植组与模型对照组大鼠的横木行走能力、撕胶纸能力均基本相似(P=0.05),且明显低于正常对照组(P<0.01)。移植后第2周,细胞移植组大鼠两项能力均明显强于模型对照组(P<0.05),但仍低于正常对照组(P<0.01)。移植后第6周,细胞移植组大鼠的横木行走能力明显改善,与正常对照组基本相似(P>0.05);撕胶纸能力仍低于正常对照组(P<0.01)。③阳性细胞免疫荧光检测:细胞移植组进针注射部位存在大量抗人RNP/CD45双阳性完整细胞,并向大脑中线、皮层梗死部位迁移。所植入的细胞约2.95%表达胶质纤维酸性蛋白,3.41%表达神经特异性烯醇化酶。结论:移植人脐血有核细胞能有效改善阻塞性脑卒中大鼠的运动和触觉功能缺陷。更多还原

【Abstract】 AIM:Cord blood karyocytes are richly composed of various precursor cells and can ameliorate injured nervous system.This article is designed to establish middle cerebral artery occlusion(MCAO)rats and explore the feasibility of cord blood karyocyte transplantation.METHODS:The experiment was carried out at Animal Laboratory,Shenzhen Baoan People’s Hospital from September 2004 to May 2005.①Cord blood was collected from full-term newborns of Researching Laboratory of Shenzhen Baoan Blood Station.Puerperants and their family members signed the informed consent.A total of 80 SD clean-grade adult rats were randomly divided into normal control group(n =20)and model group(n =60).The rats in the model group were established obstructive stroke models by electrocoagulation.After removing atypical motor dysfunction rats,5 rats were randomly selected as observational models.Other rats were randomly assigned into cell transplantation group and model control group according to the ratio of 1:1.If the rat number was odd,the left rat should be divided into cell transplantation group.②20 mL cord blood was collected sterilely,and then anticoagulated with ethylene diamine tetraacetic acid.Cord blood karyocytes were isolated by Ficoll method.At day 10 after establishing models,heads of rats in the cell transplantation group were fixed.1011 L-1 cord blood karyocyte suspension 5 μL was injected at 3.0 mm anterior fontanel tail side,1.5 mm from median line and 1.2 mm depth,1 μL/min.Cord blood serum without cells of the same volume was injected into rats of model control group,whereas rats in the normal control group did not receive any intervention.③Four weeks after establishing models,brain section was made by 5 observational model rats,and then received haematoxylin-eosin(HE)staining.Rats in each group received beam walking test(BWT)and tactile stimulation test before transplantation and 2 and 6 weeks after transplantation to measure the motor and tactile functions.Rats of each group were killed to make pathological sections,and then survival and differentiation of cord blood karyocytes were determined by immunofluorescence method.RESULTS:Totally 20,24 and 24 rats in the normal control group,cell transplantation group and model control group respectively were involved in the result analysis.①Pathological feature of obstructive stroke rats:Behavioral disturbance and stroke bags appeared in rats of the cell transplantation group.Pathological section showed pathological injury of brain tissue induced by cell necrosis.②Motor and tactile functions:Before transplantation,beam walking and adhesive paper tear capabilities of rats in the cell transplantation group and model control group were similar(P =0.05),and were obviously lower than the normal control group(P < 0.01).Two weeks after transplantation,these two capabilities in the rats of cell transplantation group were markedly better than the rats of model control group(P < 0.05),but still lower than the normal control group(P < 0.01).Six weeks after transplantation,beam walking capability was remarkably improved in rats of the cell transplantation group,and similar to that in rats of the normal control group(P > 0.05).Adhesive paper tear capability was still lower than that in rats of normal control group(P < 0.01).③Immunofluorescence detection in positive cells:A mass of anti-human RNP/CD45 double positive cells appeared in cell transplantation group,and immigrated toward brain middle and cortex infarcted regions.The expressive rate of glial fibriliary acidic protein(GFAP)in transplanted cells was 2.95%,and the positive expressive rate of neuron-specific enolase(NSE)was 3.41%.CONCLUSION:Human cord blood karyocytes possess the potential to improve the motor and tactile functional defect of MCAO rats.更多还原