新生大鼠缺氧缺血性脑损伤后骨髓基质细胞移植时间的选择

目的：众多研究已证实骨髓基质细胞移植治疗缺氧缺血性脑损伤是有效的，但移植时间点的选择尚无定论。在新生大鼠缺氧缺血性脑损伤后不同时间点进行骨髓基质细胞移植，通过对其远期行为学及组织学检测，探讨最佳的移植时间窗。方法：实验于2005—06／2006—06在中南大学湘雅医院中心实验室完成。①动物：选取清洁级7d龄SD大鼠50只，随机数字表法分为5组：正常对照组、模型对照组、24h，72h、7d细胞移植组，10只/组。另取4周龄SD大鼠10只用于骨髓基质细胞的培养。实验过程中对动物的处置符合动物伦理学标准。②实验方法：除正常对照组外，其余组大鼠均建立缺氧缺血性脑损伤模型。在脑立体定位仪下，24h，72h，7d细胞移植组分别于造模后对应时间点，将体外培养3-5代且经Hochest33324标记24h的鼠骨髓基质细胞2uL脑内移植于左侧海马，约10^5个细胞。③实验评估：各组大鼠40d日龄时进行迷宫觅水测试，记录觅水时间、错误次数、重复次数。行为学测试后取脑组织片行尼氏染色，计数左侧海马CA1区神经元数。在荧光显微镜下观察骨髓基质细胞的存活、增殖，用免疫组化法检测骨髓基质细胞神经元特异性烯醇化酶的阳性率。结果：50只大鼠均进入结果分析。①放射臂迷宫实验检测：模型对照组觅水时间、错误次数、重复次数均高于正常对照组和各细胞移植组（P〈0．01），24h细胞移植组上述3项指标均低于72h，7d细胞移植组（P〈0．05）。②左侧海马CA1区神经元尼氏染色结果：模型对照组神经元数较正常对照组明显减少（P〈0．01），且排列紊乱，丢失明显；24h，72h，7d细胞移植组神经元数均较模型对照组显著增加（P〈0．01），排列整齐；24h细胞移植组神经元数较72h，7d细胞移植组明显增多（P〈0．05）。③骨髓基质细胞体内增殖和神经

Time window choice for bone marrow stromal cells transplantation in the treatment of hypoxic-ischemic brain damage in neonatal rats

AIM： Many researches have shown that transplantation of bone marrow stromal cells （BMSCs） can reduce hypoxic-ischemic brain damage （HIBD）, but the therapeutic window is unknown. This study is designed to conduct BMSCs transplantation on neonate rats following HIBD at different time points, and investigate the optimal therapeutic time window by assessment of long-lasting ethology and histology.
METHODS： This study was completed in the Central Laboratory of Xiangya Hospital of Central South University from June 2005 to June 2006.①Animals： Fifty 7-day-old SD rats of cleaning grade were divided into 5 groups at random： normal control group, model control group, 24-hour, 72-hour, 7-day transplantation groups （n=10）. Another 10 4-week-old SD rats were prepared for culture of BMSCs. The rat treatments followed the animal ethics standard.②Experimental methods： Except the normal control group, the rats of other four groups were use to establish HIBD model. By means of stereotaxis instrument, 1 × 10^5 BMSCs （2 μL） of passage 3 stained with Hochest33324 for 24 hours were transplanted into the left hippocampus at 24 hours, 72 hours and 7 day after induction of HIBD in the corresponding groups.③Experimental assessment： Radial arm maze test was performed at 40 days, and the time taken to visit the 3 baited arms, number of error and number of repetition were recorded. After ethology test, Nissl staining of brain sections was used to count the neurons of CAI area in the left hippocampus. The survival and proliferation of BMSCs were observed under fluorescence microscope, and imrnunofluorescence was used to identify the expressions of neuron specific encolase （NSE） of the transplanted BMSCs.
RESULTS： All of 50 rats were involved in the result analysis.①Radial arm maze test： The time taken to visit the 3 baited arms, number of error and number of repetition in the model control group were higher than that of other groups （P 〈 0.01）, and the rats of 24-hour transplantation gro