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目的:观察人脐血有核细胞在缺氧缺血性脑损伤新生大鼠脑内生存和抗原表达。方法:实验于2002-09/2004-03在深圳宝安血站干细胞研究实验室内完成。①实验中采用羟乙基淀粉沉淀法分离脐血有个核细胞。②5窝F344新生1d龄大鼠共64只,同窝随机分为3组:正常组:不干预。模型组:结扎左颈动脉,吸入体积分数为0.08的低氧3h制成缺氧缺血性脑病模型。人脐血有核细胞组:造模后24h定位注射人脐血有核细胞(1.0～2.0)×106。③Morris水迷宫试验评估移植后第42天大鼠学习和记忆能力;第10周麻醉状态下处死实验鼠,制作病理切片,苏木精-伊红染色和间接免疫荧光检测人脐血有核细胞在脑内存活和分化情况。结果:64只大鼠,实验过程中死亡20只,正常组、模型组、脐血有核细胞组分别存活17,11,16只,进入结果分析。①缺氧缺血性脑病模型制作结果:模型组出现行为障碍,病理切片可见细胞变性坏死、胶质细胞吞噬、血管套、胶质细胞结节形成等典型缺氧缺血所致脑组织病理损伤。②第42天水迷宫试验显示:人脐血有核细胞组大鼠的空间识别和记忆能力明显尤于模型组(P<0.01),与正常对照组差异无显著性。③病理切片、苏木精-伊红染色和间接免疫实验结果显示:人脐血细胞组,进针部位存在大量移植细胞,成团或散在分布,呈方向性向周围迁移;左侧大脑血管壁,可见大量细胞迁移环绕;所植入的细胞中胶质纤维酸性蛋白表达率约为4.59%,神经元特异烯醇化酶阳性表达率约为2.68%。结论:人脐血有核细胞在缺氧缺血脑损伤新生大鼠脑内可以部分存活,表达胶质纤维酸性蛋白、神经元特异烯醇化酶抗原,并有效改善缺氧缺血性新生大鼠的功能缺陷。
Objective: To observe the survival and antigen expression of human umbilical cord blood cells in neonatal rats with hypoxic-ischemic brain damage. Methods: The experiment was completed in Shenzhen Baoan Blood Station Stem Cell Research Laboratory from September 2002 to March 2004. ① experiments using hydroxyethyl starch precipitation cord blood cells have a nuclear cell. ② 5 F344 neonate 1d old rats a total of 64, littermates were randomly divided into 3 groups: normal group: no intervention. Model group: Ligation of left carotid artery, inhalation volume fraction of 0.08 hypoxia 3h hypoxic-ischemic encephalopathy model. Human umbilical cord blood cells group: Human umbilical cord blood cells (1.0 ~ 2.0) × 106 were injected into the umbilical cord blood after 24h of modeling. Morris water maze test was used to assess the learning and memory abilities of rats on the 42nd day after transplantation. At week 10, the rats were sacrificed and the pathological sections were made. The hematinocytes were detected by hematoxylin-eosin staining and indirect immunofluorescence Brain survival and differentiation. Results: Sixty-four rats died of 20 rats during the experiment. The normal group, the model group and the umbilical cord blood nucleated cell group survived 17, 11 and 16, respectively, and entered into the result analysis. ① Hypoxic-ischemic encephalopathy model made results: behavioral disorders in the model group, the pathological changes can be seen cell degeneration and necrosis, glial cell phagocytosis, vascular sets, glial cell nodules and other typical hypoxic-ischemic brain tissue caused by pathological damage . ② On the 42nd day, the water maze test showed that the spatial identification and memory ability of the human umbilical cord blood mononuclear cells group was significantly more than that of the model group (P <0.01), but no significant difference with the normal control group. ③ Pathological sections, hematoxylin-eosin staining and indirect immunization results showed that there were a large number of transplanted cells in the human umbilical cord blood cells group and the needle insertion site, which were distributed in groups or scattered, and migrated to the periphery in the directional direction. The left cerebral vascular wall was visible Surrounded by a large number of cells, the expression of glial fibrillary acidic protein in the implanted cells was about 4.59% and the positive rate of neuron specific enolase was about 2.68%. CONCLUSION: Human umbilical cord blood cells can partially survive in the brain of neonatal rats with hypoxic-ischemic brain damage, express glial fibrillary acidic protein and neuron-specific enolase antigen, and effectively improve hypoxic-ischemic neonatal rats The functional deficiencies.