人脐带间充质干细胞对新生大鼠缺氧缺血性脑损伤的神经修复作用

目的探讨人脐带间充质干细胞（hUCMSCs）对新生大鼠缺氧缺血性脑损伤（HIBD）的神经修复作用，为hUCMSCs治疗新生儿HIBD寻找实验依据。方法将7日龄SD大鼠制成HIBD动物模型，分为HIBD后24h、72h2个时段干预组，每个时段选用3种不同的干预方法，分别为：静脉注入干细胞、腹腔注入干细胞、静脉注入干细胞＋腹腔注入神经节苷脂，同时设立HIBD模型鉴定组和HIBD后24h生理盐水对照组。将hUCMSCs进行Dil标记，分别通过颈静脉或腹腔将标记的hUCMSCs注入模型鼠体内。选用Morris水迷宫测试方法，了解干细胞干预后实验大鼠学习记忆力改善情况。采用免疫组化方法，了解干细胞在大鼠脑组织内的定值、分化。结果HIBD后24h、72h静脉注入hUCMSCs组大鼠水迷宫测试逃避潜伏时间分别为（56．0±6．9）s、（76．4±6．7）s，较生理盐水组（98．8±9．0）明显缩短（P〈0．05），且24h组较72h组逃避潜伏时间明显缩短（P〈0．05）；HIBD后24h、72h静脉注入hUCMSCs＋腹腔注入神经节苷脂组大鼠逃避潜伏时间分别为（38．3±7．5）s、（50．0±7．2）s，较24h、72h单纯静脉注入hUCMSCs大鼠逃避潜伏时间（56．0±6．9）s和（76．4±6．7）s明显缩短（P〈0．05）。进入大鼠体内的hUCMSCs能进入脑内并显示神经元前体细胞特性；hUCMSCs静脉移植组缺血区胶质纤维酸性蛋白阳性细胞（1276．7±551．8）个，明显低于对照组阳性细胞（2789．0±940．6）个（P〈0．01）。结论静脉移植的hUCMSCs可以向脑内迁移并分化为神经元前体细胞，抑制神经胶质细胞增生，明显改善HIBD大鼠行为。大鼠HIBD制模后24h输注hUCMSCs较72h输注脑功能改善情况更好，移植hUCMSCs联合神经节苷脂对神经修复有促进作用。

Human umbilical cord mesenchymal stem cells improve neural behavior of newborn rats with hypoxic-iscbemic brain damage

Objective To investigate the rule of human umbilical cord mesenchymal stem cells （hUCMSCs） in neural recovery of neonatal rats with hypoxic-ischemic brain damage （HIBD） of brain. Methods Hypoxic-ischemic brain injuries were induced in 7 day old Sprague-Dawley （SD） rats through right carotid artery ligation followed by exposure to hypoxic environment. Rats were separated into 24 hr and 72 hr post HIBD interventional groups. Dil labeled HUCMSCs were introduced to each group of rats through intravenous （i. v. ） injection, intra-abdominal injection or i.v. HUCMSCs ＋ intra-abdominal ganglioside injection routes. Two control groups were established： HIBD rats without treatment and with normal saline injections. Learning and memory abilities of rats were evaluated by Morris Water Maze Test. Stem cells distribution and differentiation in brain tissue were determined by immunohistochemistry. Results （1） Morris Water Maze Test showed that animals implanted with the hUCMSCs had a significantly shorter mean latency to escape time than those in the saline control group （ P 〈 0. 05 ） ; Comparing the latency time between different transplantation time points indicated that animals receiving cell grafts at 24 hour post HIBD showed a more significant improvement than those implanted at 72 hour （56.0±6.9 s vs. 76.4 ±6.7 s, P 〈0.05; P 〈0.05）. hUCMSC transplantation combined with ganglioside （ GM1 ） treatment greatly improved the animal behavior by further decrease the latency time in both 24 hour post HIBD （38.3 ± 7.5 s with GM1 vs. 56.0 ± 6.9 s with hUCMSC alone, P 〈 0.05）, and 72 hour post HIBD groups （50. 0 ±7.2 s with GM1 vs. 76. 4 ±6.7 s with hUCMSC alone （P 〈0. 05）. （2） Under laser confocal microscope, some of the Dil positive cells were also found immune-reactive to Hoechst （stains nuclei of neural cells） and DCX stains （stains neuron precursor cells）, which revealed that hUCMSCs grafts have been migrated to brain and differentiated into neuronal precursors. （3） Furthermore, hUCMSCs grafts through jugular vein injection was found a more efficient route when compared to grafts through abdominal injection, because lower expression level of GFAP, a pathological marker with increasing activity in HIBD, was observed in rat brains with hUCMSC injection through jugular vein （ P 〈 0. 01 ）. Conclusions Early transplantation of hUCMSCs via jugular vein is a promising way to improve the behavior of hypoxic-ischemic rats and decrease the gliosis. The transplanted cells could differentiate into neurons. Ganglioside treatment further enhances the recovery of neurological functions on the base of hUCMSCs transplantation.