Abstract摘 要 创伤性脑损伤（traumatic brain injury，TBI）是指暴力因素作用于头部造成的局部解剖、病理结构的改变，导致患者偏瘫、失语、智力障碍甚至昏迷、死亡，其发病率、致残率较高，危害十分严重。随着我国经济社会的高速发展，交通、建筑、地质勘探、高竞技性体育项目等行业迅速崛起，由生产安全事故及其它意外伤害造成的TBI发病率呈现持续升高的趋势，已达到100/10万，接近发达国家水平。TBI发生后，神经元细胞损伤是导致神经功能障碍的直接原因，由于神经元很难再生与修复，造成TBI缺乏有效的临床治疗手段，且预后较差。 大量的实验研究提示，神经干细胞（neural stem cells，NSC）移植能够改善TBI动物的神经功能恢复，但由于医学伦理学的争议，导致NSC的使用有一定的局限性。基于此，越来越多的学者开始专注于NSC的替代干细胞修复TBI受损神经细胞的研究。已有研究报道，人脱落乳牙牙髓干细胞（stem cells ffrom human exfoliated deciduous teeth，SHED）具有多能干细胞的特性，且能表达神经元细胞表面标志物，并可向神经细胞分化，促进神经细胞的增殖，因此SHED未来有望替代NSC修复TBI受损神经细胞，并在一定程度上恢复神经功能。 目前认为，NSC主要通过在移植区域分化为神经元或旁分泌一些分子来促进TBI的损伤恢复。外泌体是近年来新兴的研究热点，它富含蛋白质、核酸、mRNA、microRNA等生物大分子，在许多生理功能中发挥着重要的作用。有研究提示，NSC部分促神经修复作用可能涉及外泌体，但具体机制尚不完全清楚。 据此，本实验拟选用SD大鼠TBI模型，通过损伤区局部直接注射SHED及SHED外泌体，观察其对损伤神经功能的修复作用，以及能否影响活化的MG，探讨SHED可能发挥的作用，为临床应用SHED治疗TBI提供实验依据。 研究方法： 1. 复苏、培养大鼠神经干细胞（NSC）及永生化人脱落乳牙牙髓干细胞（SHED），使用CM-DiI进行SHED细胞染色。 2. TBI模型的建立及SHED治疗 1）通过“自由落体打击法”构建大鼠TBI模型。 2）于损伤当时，在脑立体定位仪协助下，在损伤灶周围特定标记点给予干预，依据干预手段的不同，动物分为5组（每组6只）：TBI损伤组，TBI+SHED治疗组，TBI+NSC治疗组，TBI+DMEM治疗组，假手术组。 3）治疗效果的检测：用小动物成像系统，追踪SHED在大鼠大脑受损区域内状况；使用BBB评分表进行运动功能评分；苏木精-伊红染色观察脑组织形态；并统计阳性染色细胞数目。 3. SHED外泌体治疗TBI 1）培养SHED细胞，利用外泌体抽提试剂盒收集并使用CM-DiI标记SHED外泌体。 2）给予大鼠TBI模型不同处理，分为3组：TBI损伤组，TBI+SHED治疗组，TBI+SHED外泌体治疗组。 3）治疗效果检测：同前。 研究结果： 1. “自由落体打击法”制备大鼠TBI模型，TBI损伤组大鼠平衡能力及行走功能明显减退，TBI损伤组BBB综合评分显著低于假手术组（P?0.05）；损伤侧大脑皮质可见直径为2 mm损伤灶，脑皮质不连续，提示TBI模型建立成功。 2. 小动物成像结果显示，在48h和2w时，大脑内均有SHED和SHED外泌体存在，荧光强而稳定，但随着时间延长荧光强度略有减弱。 3. BBB评分结果显示，TBI组在48h和2w时BBB评分无明显改变，运动功能障碍无改善；SHED治疗组和SHED外泌体治疗组大鼠运动功能评分在48h与TBI组无明显差异，但在2w时评分显著上调（P?0.05），与NSC治疗组无明显差异。 4. HE染色结果显示，TBI后大鼠右后肢皮层运动功能定位区域脑组织形态有明显损伤和缺损，2w后并无明显恢复；SHED治疗组和SHED外泌体治疗组在2w时损伤区脑皮质有一定程度恢复，连续性变好，与NSC组无统计学差异。 5. IBA-1染色结果显示：TBI组、SHED治疗组、SHED外泌体治疗组的IBA-1+MG数量在48h时无明显差异；但在2w时SHED治疗组、SHED外泌体组较TBI组损伤侧大脑皮质损伤灶IBA-1+MG数量显著降低，与NSC治疗组无明显差异；而TBI组48h和2w时IBA-1+胶质细胞数量之间无明显差异。 结论： 1. TBI大鼠的右侧后肢运动功能减低，损伤部位存在明显MG反应，表现出为MG形态的改变、MG数量上明显增加。 2. 局部注射SHED和SHED外泌体显著抑制损伤灶及周围MG反应性增生，改善大鼠运动神经功能。 3. SHED可能是通过其分泌的外泌体治疗SD大鼠实验性TBI，具有一定的临床应用提示。
ABSTRACT Traumatic brain injury (TBI) refers to the damages to the head caused by violence factors and may results in hemiplegia or aphasia, mental impairment or even coma and death with the high rates of morbidity and disability. With the development of economy, transportation, construction, geological exploration, and high competitive sports industry have been rising rapidly, the incidence of TBI caused by production safety accidents and other injuries showed a rising trend, which has reached 100/100,000, closing to the level of developed countries. After TBI, the direct cause of nerve dysfunction is neuronal cells damage for they are difficult to regenerate and repair, resulting in ineffective clinical treatments of TBI, and poor prognosis. A large number of experimental studies suggest that neural stem cells (NSC) transplantation can improve nerve function in TBI animals. But the use of NSC is limited due to medical ethics controversy. Deciduous teeth dental pulp stem cells (SHED) exfoliated from human shared the characteristics of pluripotent stem cells and have the ability to differentiate into neurons, can express nerve cell surface marker and protect and promote brain cell growth. It may replace neural stem cells in the treatment of TBI. As is knows, NSC carrys out its function through differentiating into neurons or expressing paracrine molecules to promote TBI injury recovery in the transplant area. Exosomes are emerging hot spots in recent years, which is rich in proteins, nucleic acids, such as mRNA and microRNA molecules and has important physiological functions. Studies suggest that exosomes may be involved in repair of NSC, but the exact mechanism is not fully understood. In the present study, we established TBI SD rat mode and observed the repair of damaged nerve function, the activation of MG after direct injection of SHED or SHED exosomes in the damage zone, intended to explore the role of SHED, thus provide experimental evidence for clinical application of SHED for TBI. Method: 1. Recovery and culturing rat neural stem cells and the immortalized human exfoliated deciduous teeth dental pulp stem cells, using the CM-DiI to stain SHED. 2. The establishment of TBI model and SHED treatment 1) TBI rat model was established through "free falling method". 2) Use the brain stereotaxic apparatus to locate the damaged area and give interventions. The groups were TBI group, TBI+SHED group, TBI+NSC group, TBI+DMEM group, and SO group. 3) Therapeutic effect: use small animal imaging system to track SHED in rat brain-damaged region; BBB to assess motor function; HE staining in brain cortex to observe brain recovery; IBA-1 fluorescence staining brain sections, and counting the microglial cells. 3. Treat TBI with SHED exosomes 1) Using exosomes Extraction Kit collects and tags SHED exosomes after culturing SHED cells. 2) TBI rats model were divided into TBI group, TBI+SHED group, TBI+SHED exosomes group. 3) Therapeutic effect: same as before. Results 1) TBI rats model were established by " free falling method ", we observed decreased balance and walking function in injury group, BBB score of injury group significantly decresed compared to the SO Group (P?0.05); diameter of 2 mm damage in the cerebral cortex damage foci is visible, cortex is not continuous, suggesting the establiashment of TBI model is successful; 2) Living imaging of animals in 48 hours and 2 weeks showed SHED and SHED exosomes were visible, fluorescence was strong and stable, but decreased slightly over time; 3) BBB score showed that: no significant change was observed in TBI group after 48h and 2w, implicating no improvement in movement function. In 48h, no significant differences was observed among SHED treatment group, SHED exosomes treatment group and TBI group, but in 2 weeks score obviously up-regulated (P?0.05), and has no significant differences with NSC treatment group; 4) HE results of brain tissue: significant damage and defects were observed in cortex right hind limb movement function regional organizations in rats after TBI and after 2 weeks no obvious recovery was observed; SHED treatment group and SHED exosomes treatment group has certain degree recovery in the cerebral cortex of 2 weeks, continuity gets better, they both have no significant differences with the NSC group; 5) Brain organization slice IBA-1 dyeing results showed: in 48 hours, TBI group, SHED treatment group, and SHED exosomes treatment group of IBA-1+ MG number has unconspicuous difference, but in 2 weeks when compared with TBI group, the IBA-1+ MG number of SHED treatment group and SHED exosomes group have significantly reduced, and they has no obviously differences with NSC treatment group; while the IBA-1+ MG number between TBI group 48 hours and 2 weeks has no obvious difference. Conclusions: 1. In TBI model, motor function of the right hind limb of rat reduced, the damage area of brain tissue has obvious MG reaction, with the significantly upchange of MG number and morphology. 2. Local injection of SHED and SHED exosomes inhibit reactive hyperplasia of MG in damage area and improve the function of motor nerve in rats. 3. The repair function of SHED on TBI may work through exosomes that secreted by SHED. This may has certain clinical implications.