人脐血细胞静脉输注对血管性痴呆大鼠血清神经元特异性烯醇化酶及S-100蛋白的作用

Effects of intravenous administration of human umbilical cord blood cells on neuron specific enolase and S-100 protein in sera of vascular dementia rats

目的:观察静脉输注人脐血细胞对血管性痴呆大鼠的行为学改善以及对血清脑特异性蛋白的影响。方法:实验于2003-03/2005-12在解放军第三军医大学野战外科研究所中心实验室完成。①选择2003-10/2004-12解放军第三军医大学大坪医院妇产科住院的80例健康足月妊娠产妇,非高危妊娠,正常顺产,新生儿四肢活动良好,由产妇及家属填写知情同意书,用于脐血的采集。②选取清洁级11～13个月的老龄SD大鼠90只,随机数字表法分为假手术组、模型对照组、人脐血细胞治疗组,30只/组。③模型对照组、人脐血细胞治疗组大鼠以改良的Pulsinelli’s四血管阻断法建立血管性痴呆动物模型。假手术组处理步骤相同,但不进行椎动脉烧灼和颈总动脉夹闭。人脐血细胞治疗组在造模后3h内通过尾静脉注射给予人脐血细胞,浓度为3×109L-1,注射500μL。模型对照组、假手术组给予等量生理盐水。④各组大鼠分别于造模后2,4,8周采用电脑控制穿梭箱系统对大鼠学习记忆能力进行检测,10只/时相点。用ELISA法测定大鼠血清中神经元特异性烯醇化酶和S-100蛋白含量。结果:实验选取11～13月龄的SD大鼠90只,全部进入结果分析。①造模前后各组大鼠学习记忆能力检测结果:造模前各组大鼠主动回避反应百分率无明显差异(P>0.05)。造模后2,4,8周,模型对照组、人脐血细胞治疗组主动回避反应百分率均明显低于假手术组(P<0.01);但人脐血细胞治疗组高于模型对照组(P<0.01)。②造模后各组大鼠血清神经元特异性烯醇化酶含量检测结果:与模型对照组比较,人脐血细胞治疗组术后2周血清神经元特异性烯醇化酶含量显著降低(P<0.05),术后4,8周明显升高(P<0.05)。③造模后各组大鼠血清S-100蛋白含量检测结果:与模型对照组比较,人脐血细胞治疗组术后各时相点S-100蛋白血清含量均显著降低(P<0.01),与假手术组基本相似(P>0.05)。结论:静脉输注人脐血细胞可明显改善血管性痴呆大鼠的行为学指标,提示人脐血细胞治疗对脑组织细胞具有一定的保护作用。

AIM： To investigate the effect of intravenous administration of human umbilical cord blood cells （HUCBCs） on behavioral improvement and cerebral special protein in serum of vascular dementia rats. METHODS： The experiment was performed at the Central Laboratory, Research Institute of Surgery, Third Military Medical University of Chinese PLA from March 2003 to December 2005. ①A total of 80 healthy pregnancy at term （PAT） puerperants, who hospitalized at the Department of Gynaecology and Obstetrics, Daping Hospital, Third Military Medical University of Chinese PLA from October 2003 to December 2004, were enrolled for cord blood collection. They were not in high-risk pregnancy and had normal spontaneous labor. Neonates had good activity in limbs. The puerperants and their family members all filled in informed consents. ②Totally 90 clean elder SD rats aged 11-13 months were selected and randomly assigned into sham operation group, model control group, HUCBCs treatment group with 30 rats in each group.③Vascular dementia models were established with modified Pulsinelli＇s four-vessel occlusion （4VO） method in the model control group and HUCBCs treatment group, The disposal in the sham operation group was the same to above, but no cautery in vertebral artery and occlusion in common carotid artery. The rats in the HUCBCs treatment group were injected with HUCBCs by vena intravenous injection in 3 hours after establishing models, with the concentration of 3×10^9 L^-1, totally 500 μL. Saline of the same volume was given in the model control group and sham operation group. ④Learning and memory in rats were determined with cybernation suttle box system at weeks 2, 4 and 8 after establishing models in each group with 10 rats in each time phase, respectively. Contents of neuronspecific enolase and S-100 protein in serum were measured with ELISA method. RESULTS： Totally 90 SD rats aged 11-13 months were involved in the result analysis. ①Determination result of learning and memory in rats of each group before and after establishing models： There was no significant difference in active avoidance response （AAR） percentage of rats in each group before establishing models （P 〉 0.05）. AAR percentage in the model control group and HUCBCs treatment group at weeks 2, 4 and 8 after establishing models were significantly lower than those in the sham operation group （P 〈 0.01 ）. But it was higher in the HUCBCs treatment group than that in the model control group （P 〈 0.01 ）. ②Determination result of content of neuronspecific enolase in serum of rats in each group after establishing models： Compared with the model control group, content of neuronspecific enolase in serum decreased obviously at week 2 after operation in the HUCBCs treatment group （P 〈 0.05）, and than increased markedly at weeks 4 and 8 after operation （P 〈 0.05）. ③Determination result of S-100 protein content in serum of rats in each group after establishing models： Compared with the model control group, S-100 protein content in the HUCBCs treatment group at each time phase reduced distinctly （P 〈 0.01）, which was mostly similar with that in the sham operation group （P 〉 0.05 ）. CONCLUSION： Intravenous infusion of HUCBCs can improve significantly the behavioral indexes of vascular dementia rats, which indicates that HUCBCs therapy has certain protection on cerebral histiocytes,