人胚胎骨髓间质干细胞移植治疗新生大鼠缺氧缺血性脑病的实验

Transplantation of mesenchymal stem cells in treating newborn rats with hypoxic-ischemic brain encephalopathy

目的:在制作新生大鼠缺氧缺血性脑损伤模型基础上,研究间质干细胞移植治疗缺氧缺血性脑病的应用前景。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。实验中采用L-DMEM分离培养骨髓中间质干细胞。7窝SD1d龄新生大鼠共81只同窝随机分为4组:①正常组,不干预。②模型组:结扎左颈动脉,吸氧3h制成缺氧缺血性模型。③生理盐水组:造模后24h定位注射生理盐水8μL。④间质干细胞组:处理同生理盐水组,注射间质干细胞(1.0～2.0)×106/μL。Morris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间质干细胞在脑内存活和分化情况。结果:①第1周内成活率:第7天,81只实验鼠存活42只,间质干细胞组与正常对照组大鼠存活率分别为75.0%和82.4%,差异不显著;明显高于生理盐水组(35.3%)和模型组(50.0%)(P<0.01)。②水迷宫试验显示间质干细胞组大鼠的空间识别和记忆能力明显优于生理盐水组和模型组(P<0.01);培训后第5天,间质干细胞组识别平台的平均时间为7.5s,与正常组7.0s不存在差异。③病理切片、苏木精-伊红染色和间接免疫实验结果显示:间质干细胞移植组中,进针注射部位存在大量移植细胞,并向周围迁移,皮质层、海马等部位检测到移植细胞;所植入的细胞约6%表达胶质纤维酸性蛋白,约8%表达神经元特异性烯醇化酶。结论:移植人间质干细胞组能有效修复缺氧缺血导致的神经受损,改善其功能,明显提高缺氧缺血性脑损伤大鼠1周内成活率。

AIM： To research the prospect of mesenchymal stem ceils （MSCs） transplantation in treating hypoxic ischemicen encephalopathy （HIE） based on establishing newborn rats into HIE models. METHODS： The experiment was carried out in the research lab of stem ceils of Shenzhen Baoan Blood Center between September 2002 and March 2004. L-DMEM medium was used to separate and culture MSCs. Eightyone newborn SD rats from 7 nests were divided into four groups randomly according to nests： ① normal control group： no intervention. ② model group： The rats were made into HIE models by ligating left carotid artery and 3-hour oxygen intake. ③ saline group： The rats were treated with localized injection of saline （8 μL） at 24 hours after model establishment. ④ MSCs transplantation groups： The rats received the same treatment as those in the saline group, MSCs were injected [（1.0-2.0）×10^6/μL]. The Morris water maze test was performed to evaluate the learning and memory abilities at 42 days after transplantation. The rats were killed under anesthesia at 10 weeks to prepare pathological section, and the survival and differentiation of MSCs in brain were detected with hematoxylin-eosin （HE） staining and indirect immunofluorescence. RESULTS： ① Survival rate in the first week： On the 7^th day, 42 of the 81 rats survived, the survive rates in MSCs group and normal control group had no obvious difference （75.0%, 82.4%）, both were significantly higher than those in the saline group and model group （35.3%, 50.0%）. ② The Morris water maze test showed that the spatial recognition and memory abilities of the rats in the MSCs group were obviously superior to those in the saline group and model group （P 〈 0.01）. On the 5^th day after training, the average time of recognizing platform in the MSCs group （7.5 s） was not different from that in the normal control group （7.0 s）. ③ The results of pathological section, HE staining and indirect immunofluorescence showed that some human MSCs could survive in the HIE rats brain at least 10 weeks. About 6% of them expressed glial fibrillary acidic protein （GFAP）, and 8% of them expressed neuron-specific enolase （NSE）. CONCLUSION： MSCs transplantation can effectively repair hypoxia and ischemia induced neural damage, ameliorate its function, and obviously improve the survival rate within 1 week in HIE rats.