摘要
目的:观察局灶性脑缺血大鼠移植永生化神经前体细胞在脑内的存活情况,为进一步研究细胞移植及转基因治疗脑缺血提供有意义的数据及图像资料。方法:实验于2005-10/2006-03在华中科技大学同济医学院附属同济医院麻醉学实验室进行。①选取健康雄性SD大鼠18只,随机数字表法分为假手术组、脑缺血对照组、细胞移植组,6只/组。②脑缺血对照组、细胞移植组采用线栓法建立大鼠右侧大脑中动脉缺血模型。假手术组仅分离血管,不进行脑缺血处理。③应用无血清Neurobasal培养基进行INPC培养,移植前加入BrdU,使终浓度为10μmol/L,标记3d,然后取出细胞爬片,无水甲醇固定10min,SP法检测BrdU标记细胞阳性率。④将BrdU标记3d的永生化神经前体细胞用胰蛋白酶消化,离心重悬后使细胞密度达到2×1010L-1,细胞移植组于大鼠脑缺血后3d取5μL永生化神经前体细胞移植到右侧纹状体缺血半暗带区,以前囟为零点,即A:-0.5mm,R:2.5mm,V:-4.5mm。脑缺血对照组仅注射等量磷酸盐缓冲液。⑤各组分别于缺血后6h,1d,3d及细胞移植后1周对大鼠进行Longa神经功能评价(0～4分,分数越高表示神经功能损伤越严重),缺血后6h神经功能评分≥1分视为模型成功。⑥细胞移植后1周处死各组大鼠,取脑组织制作冰冻切片,通过免疫组织化学SP法检测BrdU的表达,观察移植的永生化神经前体细胞在脑内缺血半暗带区的存活情况。结果:18只大鼠均进入结果分析。①体外检测移植细胞BrdU标记阳性率:永生化神经前体细胞加入BrdU标记3d后,免疫组化检测阳性细胞胞核呈棕黄色,细胞阳性率达95%以上。②造模后不同时间点各组大鼠神经功能评价:缺血后6h,脑缺血对照组与细胞移植组每只大鼠神经功能Longa评分均≥1分,表明造模成功,且Longa评分均明显高于假手术组[(1.70±0.48)分,(1.60±0.52)分,0分,P<0.05]。缺血后6h,1d,3d及细胞移植后1周脑缺血对照组与细胞移植组Longa评分均基本相似(P>0.05)。③移植细胞的存活情况:细胞移植后1周,细胞移植组在永生化神经前体细胞移植侧针道附近可检测到BrdU免疫染色阳性细胞,而假手术组、脑缺血对照组、细胞移植组的移植对侧BrdU免疫染色均呈阴性。结论:永生化神经前体细胞植入局灶性脑缺血大鼠缺血半暗带区后,主要存活于移植侧针道附近。
AIM： To investigate the survivorship of immortalized neural progenitor ceils （INPC） transplanted into the brain of rats with focal cerebral ischemia, so as to provide significant data and image data for further investigation on cell transplantation and transgene treatment of cerebral ischemia. METtlODS： The experiment was conducted from October 2005 to March 2006 in the Laboratory of Anesthesiology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. ① Eighteen male SD rats were randomly divided into： the sham-operation group, cerebral ischemia control group and cell transplantation group with 6 rats in each group.② Rats in the cerebral ischemia control group and cell transplantation group were established into models by middle cerebral artery occlusion （MCAO） in the right side, while rats in the sham-operation group were only separated the vessels without treatment of cerebral ischemia.③ INPC was cultured in neurobasal without serum, and BrdU was input before transplantation at the concentration of 10 μmol/L. It was labeled for 3 days, and then the cell-covered plate was fixed with absolute methanol for 10 minutes, and BrdU labeled positive rate was detected with SP method. ④ BrdU labeled INPCs of 3 days were digested with trypsinization, the density of which was 2×10^10 L^-1 after centrifugalization. 5 μL of INPCs were obtained at 3 days before cerebral ischemia to be transplanted into the ischemia in penumbra zone of right striate body by taking the bregma as the zero, i.e. A： -0.5 mm,R：2.5 mm,V：-4.5 mm. Rats in the cerebral ischemic control group were only injected with phosphate buffer （PB） solution at the same dose. ⑤ Longa neurofuntional evaluation was conducted in rats of each group respectively at 6 hours, 1 day and 3 days after ischemia and one week after transplantation （0-4 points, the higher the score was, the severer the neurofucntional injury was）. The neurofunctional score equal or greater than 1 point at 6 hours after ischemia was considered as successful model. ⑥ Rats were executed at one week after transplantation to obtain the brain tissues for frozen sections, and the expression of BrdU was detected by immunohistochemical SP method. The survivorship of INPCs in semi-dark band of cerebral ischemia was studied. RESULTS： A total of 18 rats were involved in the analysis of results. ①BrdU labeled positive rate of transplanted cells detected in vitro： Three days after BrdU labeled INPC, immunohistochemical detection showed that the nucleus of positive cell was in brown, and the positive rate was more than 95%. ② Neurofuctional evaluation on rats of each group at different time points after the modeling： At 6 hours after ischemia, the Longa scores of rats in the cerebral ischemic control group and cell transplantation group were greater than one point, which indicated successful modeling, moreover, the Longa scores were significantly higher than that of the sham-operation group [（1.70±0.48） points, （1.60±0.52） points, zero,P 〈 0.05]. At 6 hours, 1 day and 3 days after ischemia and one week after cell transplantation, the Longa score in the cerebral ischemic control group was similar to that in the cell transplantation group （P 〉 0.05）. ③ The survivorship of transplanted ceils： At one week after transplantation, BrdU immune positive ceils could be detected in the periphery of pinholes of INPC transplanted side in the cell transplantation group, whereas they were negative in the sham-operation group, cerebral ischemic control group and cell transplantation group. CONCLUSION： INPC can survive in penumbra zone surrounding the injection site in rats with focal cerebral ischemia.
出处
《中国临床康复》
CSCD
北大核心
2006年第45期36-38,I0004,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金(30300328)~~
