人脐血有核细胞在缺氧缺血新生大鼠脑内的生存及其抗原表达

Survival and antigen expression of human cord blood karyocytes in brain of hypoxic-ischemic newborn rats

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。

AIM： To observe the survival and antigen expression of human cord blood karyocytes in hypoxie ischemicen encephalopathy （HIE） newborn rats. METHODS： The experiment was carried out at Laboratory of Stem Cell Research, Shenzhen Baoan Blood Station from September 2002 to March 2004. ① Hespan sedimentation was used to separate karyocytes from human cord blood. ②A total of 64 F344 newborn rats aged 1 day from 5 nests were divided into three groups randomly according to nests： normal group, no intervention; model group： the hypoxic ischemic models were made by left carotid artery ligation with hypoxia of 0.08 volume fraction for 3 hours; human cord blood karyocytes group： human cord blood karyocytes （1.0-2.0） ×10^6 were stereotaxically injected 24 hours after establishing models. ③On day 42, the Morris water maze test was performed to evaluate the pups＇ spatial learning and memorying abilities. All pups were then killed at week 10 under drugged state. Pathological diagnosis, HE staining and indirect immunofluorescence were used to evaluate the living and differentiation state of human cord blood karyocytes in HIE rats＇ brain. RESULTS： Of the 64 rats, 20 rats died in the experiment, and 17, ll and 16 rats in the normal group, model group and human cord blood karyocytes group, respectively were involved in the result analysis. ①Result of HIE group： The rats in the model group appeared behavioral disturbance. Pathological section appeared cell degeneration, necrosis, phagncytosis of glial cells, vascular cannula, tuberculation of glial cells induced by typical hypoxia and ischemia. ②Morris water maze test showed that at day 42 the spatial recognition and memory ability of the rats in human cord blood karyocytes group was better significantly than that in the model group （P 〈 0.01）, which had insignificant difference with normal control group. ③Pathological diagnosis, HE staining and indirect immunofluorescence showed that a mass of transplanted cells in the entering needle part, distributed in a mass or scatteredly, migrated towards around in the human cord blood monocyte group; Blood wall of left brain showed a plenty of cell migration; Expressive rate of glial fibriliary acidic protein in transplanted cells was 4.59%, and the positive expressive rate of neuron-specific enolase was 2.68%. CONCLUSION： Human cord blood karyocytes can survive partially at the hypoxic-ischemic damaged brain of newborn rats, express glial fibriliary acidic protein and neuron-specific enolase antigen, and effectively improve the functional defect of hypoxic ischemic newborn rats.