骨髓间充质干细胞对缺氧缺血性脑损伤新生大鼠学习记忆功能的重建作用

Effects of bone marrow mesenchymal stem cells on learning and memory functional recovery in neonatal rats with hypoxic-ischemic brain damage

目的研究经不同处理的大鼠骨髓间充质干细胞（rMSCs）对新生鼠缺氧缺血性脑损伤（HIBD）后学习记忆功能重建的作用，探讨rMSCs移植优化方案与MSCs的作用机制。方法全骨髓贴壁筛选法体外扩增rMSCs，Rice法构建新生Wistar大鼠HIBD模型，造模后5d经侧脑室植入不同处理条件的rMSCs（1～2×10^3／只），将HIBD新生鼠分为3组：①PBS对照组，②rMSCs组，③视黄酸（retinoicacid，RA）预诱导组。移植后14、42d取脑组织行免疫组化检测植入的rMSCs的存活、迁移、分化及表达神经细胞标志蛋白的情况，鼠龄7周做穿梭箱实验检测各组模型鼠学习记忆能力。移植后14d，采用神经营养素与受体基因芯片检测各组损伤侧脑组织差异基因表达，用Real—time PCR对差异显著的部分基因进行验证。结果（1）在rMSCs移植后14、42d，RA预诱导组分别检测到少量植入细胞表达神经元中间丝蛋白重链（NF—H）和神经胶质纤维酸性蛋白（GFAP）；（2）穿梭箱实验结果显示rMSCs组（74．60±29．21）次与PBS对照组（94．10±38．18）次比较学习记忆功能有较好恢复，但差异无统计学意义，而RA预诱导组（47．90±21．13）次与PBS对照组和rMSCs组比较学习记忆功能明显恢复（P〈0．05）；（3）基因芯片结果显示，与正常对照组比较，PBS对照组IL-6（高达11．4倍）、肿瘤坏死因子超家族成员之一Fas、脑源性神经营养因子（BDNF）表达明显上调；与PBS对照组比较，rMSCs组三种因子的表达同时降到较低水平，而RA预诱导组的IL-6和Fas下调后水平均高于rMSCs组，BDNF仍保持在PBS对照组水平。Real—time PCR结果显示，IL-6和Fas变化与基因芯片结果基本一致，而RA预诱导组BDNF水平有明显下调，但下调后水平仍略高于rMSCs组。结论在促进HIBD新生鼠学习记忆功能重建中，rMSCs植入有一定作用，RA预诱导的rMSCs植入有明显作用，它们可能通过调节HIBD大鼠脑内的IL-6、Fas和BDNF等因子保持在适宜水平而发挥作用。

Objective Neonatal hypoxic-ischemic brain damage （HIBD）causes acute death and chronic nervous system sequelae in newborn infants and children. Whereas there have been no specific treatment towards it up to now. Studies have shown that bone marrow mesenchymal stem cells （MSCs） have the therapeutic potential in many nervous system diseases and the authors previously found that retinoid acid （RA）, which plays an important role in brain development, could enhance the neural differentiation of rat MSCs （rMSCs） in vitro. This study aimed to examine effects of rMSCs and RA-preinduced rMSC on learning and memory functional recovery after HIBD in neonatal rats in order to explore a new treatment strategy for clinical application, and explore the mechanism of action of rMSCs. Methods Rat MSCs were isolated and purified from the whole bone marrow of juvenile Wistar rats by removing the non-adherent cells in primary and passage cultures. Neonatal hypoxic-ischemic brain damage rat models were built according to the methods described by Rice： the right carotid artery of 7-day-postnatal Wistar rats was ligated under anesthesia, and then the rats were exposed to 8%-9% 02 in a container. At 5 days after hypoxia-ischemia, the HIBD neonatal rats were randomly divided into 3 groups and respectively transplanted with saline, BrdU marked rMSCs （ 1-2 × 10^5 ） or RA-preinduced rMSCs （ 1-2 × 105 ） into their lateral cerebral ventricle. Immunohistochemistry for nestin, neuron-specific enolase （ NSE）, neurofilament protein-heavy chain （ NF- H） and glial fibrillary acidic protein （GFAP） were used to identify cells derived from rMSCs at 14 days and 42 days after transplantation. Shuttle box test was performed to evaluate the condition of learning and memory functional recovery when animals were 7 weeks old. Neurotrophin and receptors cDNA microarray were also employed at 14 days after transplantation to investigate the underlying action mechanisms of rMSCs treatment. Real-time PCR was used to confirm some of the remarkably changed genes. Results （ 1 ） The neonatal rat model of HIBD was successfully established. （2） Immunohistochemistry showed rMSCs-derived cells survived, migrated into the hypoxic-ischemic brain tissue and a few of them expressed protein characteristic of neurons and astrocytes （ NF-H and GFAP） in RA-preinduced group 14 days and 42 days after transplantation, while no positive expression of nestin and NSE were detected. （3） The shuttle box test showed that the average learning times in rats transplanted with saline, rMSC and RA-preinduced rMSCs were（94. 10 ± 38.18 ）, （74. 60 ± 29.21 ） and （47.90 ± 21.13 ）, respectively. The difference between the former two was not significant （ P 〉 0. 05 ） , while the latter one exhibited significant improvement （ P 〈 0. 05）. （4） The cDNA microarray analysis showed that compared with normal control group, IL-6, Fas and BDNF genes of the saline control group significantly up-regulated （ the ratios of the three genes were 11.4, 2.4 and 6. 6 respectively）. Compared with saline group, the three genes in rMSC group were down-regulated （ the ratios were all 0. 1 ）, while the levels of IL-6 and Fas genes （ the ratios were 0. 3 and 0. 4 respectively） in RA-preinduced rMSCs group were higher than rMSCs group after down-regulating, but the level of BDNF remained at the saline group level. Real-time PCR analysis suggested that the results of IL-6 and Fas genes were at equal level with microarray results on the whole, while the level of BDNF gene in RA-preinduced rMSC group was significantly down-regulated （with ratio of 0. 34）, but higher than rMSCs group （the ratio was 0. 25 ） as well. Conclusion Transplantation of rMSC and RA-preinduced rMSCs into lateral cerebral ventricle can improve learning and memory functional recovery after HIBD in neonatal rats, especially RA- preinduced rMSCs. Regulating the levels of IL-6, Fas and BDNF in the brain to maintain at reasonable levels may be the mechanism.