【作者】 曲扬；

【导师】 高忠礼；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 脊髓损伤致残率高，目前仍是一治疗难题。近年来，随着对干细胞研究的不断深入，研究者们又看到了脊髓损伤治疗的曙光。虽然神经干细胞治疗脊髓损伤的研究很多，也取得了各种各样的进展，但目前神经干细胞在体内生存率低、分化为神经元较少、迁徙受限仍是难题。姜黄素具有抗炎抗氧化等作用，在神经系统损伤中可发挥神经保护作用，因此本研究联合应用姜黄素和神经干细胞治疗脊髓损伤，以期观察二者是否有协同促进神经功能恢复的作用。研究结果表明：联合姜黄素和神经干细胞在治疗大鼠不同程度的急性脊髓损伤中具有良好的协同作用。与对照组及单纯姜黄素治疗组、单纯神经干细胞治疗组相比较，联合姜黄素与神经干细胞治疗能够显著的提高实验大鼠BBB评分，比目鱼肌重量，能够显著减少损伤脊髓部位空泡形成面积，能够减少ED-1阳性细胞浸润，减少血浆中MDA含量，增加血浆中SOD活性，能够增加脊髓中Brd-u和MAP-2双标阳性细胞的数量，并且双标阳性细胞迁徙距离更远。姜黄素同神经干细胞共培养结果显示，姜黄素能以浓度依赖的方式促进神经干细胞增殖，与对照组相比较有显著差异。姜黄素同脊髓片共培养后，培养液中BDNF在姜黄素加入后12小时显著增加，而将这种滤液加入神经干细胞培养液中后，能够显著的提高神经干细胞分化为神经元的比例，并减少神经元的凋亡。结论：联合姜黄素和神经干细胞治疗不同程度的大鼠脊髓损伤，可以不同程度的促进大鼠神经功能恢复的作用。这种协同作用可能因为姜黄素能够抗炎抗氧化作用，为神经干细胞移植提供良好的脊髓内环境；姜黄素能够促进神经系统BDNF表达，以此促进神经干细胞分化迁徙；姜黄素能够促进神经干细胞增殖；创新点：1从改善脊髓内移植环境出发，联合使用姜黄素和神经干细胞移植治疗脊髓损伤，国内外尚无报道，本研究证实了联合应用的有效性；2从神经干细胞培养方面证实了姜黄素促神经干细胞增殖作用；3脊髓组织片与姜黄素共培养滤过液促进神经干细胞分化，进一步说明姜黄素能通过促进神经系统表达BDNF来发挥作用。更多还原

【Abstract】 Spinal cord injuries lead to serious disability.The treatment always been hotresearch, but the research is slow progress, still, there is no effective therapeuticmethod for spinal cord injury treatment. Spinal cord injury can be divided intoprimary injury and secondary injury, the primary damage is usually caused bymechanical damage such as spinal column fracture or spinal dislocation. The degreeof spinal cord injury from crush injury to complete spinal cord transection. Secondaryinjury refers to the primary level of damage caused by the mechanical damage,including ischemic injury, ischemia-reperfusion injury, lipid peroxidation, edema,energy metabolic disorders, excitatory amino acid accumulation, etc. At later stage ofspinal cord injury, glial scar was formed which influence the recovery of spinal cordfunction seriously.The current treatment of spinal cord injury mainly includes decompressing thespinal cord, alleviating secondary injury, reduce the scar formation, neural stem cellstransplantation and other method. Neural stem cells can differentiate into neurons andglial cells, etc, and to build outstanding contact with the surrounding neurons. Inrecent years, it become a hotspot in research of spinal cord injury. The secondaryinjury made the spinal cord inner environment not conducive to the proliferation anddifferentiation of neural stem cells.The treatment now accepted by most of the doctorsand scholars for the secondary spinal cord damage, is pulsing methylprednisolone.But using large dose of methylprednisolone for a long time can produce manycomplications, such as infection, circulatory failure, gastrointestinal bleeding, femoralhead necrosis.Therefore, it is necessary to find a kind of drugs to reduce the level ofsecondary damage after spinal cord injury, the drugs can generate synergy effect onneural stem cells to promote nerve regeneration. Curcumin is a natural plant extract,the effect of curcumin include anti-inflammatory, antioxidation, antitumor and otherbiological activity, and after long time application almost non-toxic side effects, so inthis study, a combination of curcumin and neural stem cells to treat different degree ofspinal cord injury, to observe the therapeutic effect and to investigate its mechanism.【objective】 To observe the treatment effect that using curcumin combinationwith neural stem cells to treat different level spinal cord injury, and analyze the possible mechanism of the treatment.【methods】 Choosing SD rats as experiment animal of spinal cord injury. UseAllen’s method to made the animal model of different levels spinal cord injury, andaccording to different damage degree, the rats were divided into three group.The rat pregnancy for14days was executed,and fetal rats was take out.Theneural stem cells in hippocampus was cultured. Cell number was counted, andobserve the subculture, The nestin immunohistochemical staining were observed.Pour curcumin into nutrient solution of the culturing neural stem cells, To detectthe curcumin role on the neural stem cells vitality by MTT method, BrdU method wasused to detect the curcumin on proliferation of neural stem cells. The role of curcuminfor formation of neural stem cells spheroids was observedAccording to different degree of spinal cord injury, rats can be divided into A(injury), B (moderate) and C (completely damage) three groups. Each group isdivided into four subgroups, respectively given DMSO, curcumin, neural stem cells,curcumin combined neural stem cells for treatment. Assessed the BBB score weekly.6w after spinal cord injury, soleus weight was weigh out.1w and2w after spinal cordinjury, the content of MDA and the activity of SOD in rat plasma was detected.6wafter spinal cord injury, the spinal cord cavity area in HE staining was calculated. At2weeks end, ED-1immunohistochemical study spinal cord tissue was made to observethe positive cell number.6w after spinal cord injury, Brd-u and MAP-2doubleimmunofluorescence staining was made to observed the migration distance and thenumber of double positive staining cellsTake2weeks of SD rat spinal cord tissue slice culture, to pour curcumin in theculture medium, and Elisa method was used to observe the concentration of BNDF,NGF NT-3at0h,6h,12h and24h after curcumin be poured, the concentrationschanges in was compared with that of control group.Taking curcumin with spinal cord tissue culture filtration liquid, added to themedium of neural stem cells, observe the differentiation of neural stem cells, madecomparative analysis. with FGF-2group and FGF-2combined filtration liquid group【results】This study using the method of Allen successfully produce differentdegree of spinal cord injury animal model. the separation and culture of neural stemcells was succeed, and the nestin immunohistochemical staining was positive incultured cells 48hours after curcumin treated neural stem cells, we found that curcumin in therange50~800ug/ml concentration, can increase the vitality of the neural stem cells,the effect was dose dependent. And curcumin of400ug/ml concentration, the vitalityof neural stem cells is almost equivalent to positive control group, BrdU stainingresults indicate that curcumin with concentration100-400ug/ml can increase BrdUstaining positive cells number in dose dependent manner, and a significant role inpromoting neural stem cells to form cell spheroids.In different degree of spinal cord injury in rats, contrast with control group, onlyinjection of curcumin group,and neural stem cell transplanting group, curcumincombined neural stem cell transplantation can significantly improve the BBB score ofexperimental rats, histopathological examination showed cavity area in spinal cordsignificantly decreased. Around spinal cord injury center, ED-1positive cellssignificantly reduced.In the spinal cord, Brd-u and MAP-2double staining positivecell number was increased significantly and migration distance was farther.Significantly reduce the MDA content in plasma, SOD activity was increasedsignificantly.The Elisa results showed that adding curcumin could enhanced spinal cord tissueexpressing BNDF, The concentration of BDNF reach the peak at12hours, There wassignificant difference compare to control group. And the expression of NGF and NT-3were no significant difference between two groups, and each time point the BNDFexpress no significant difference.The filtrate from curcumin with spinal cord culture can synergy FGF-2, asignificant increase in the MAP-2and DAPI double positive cells, compared withpure filtrate or FGF-2group,there were significant differences between groupsSimply add the filtrate can also stimulate the nerve stem cells differentiate the MAP-2and DAPI double positive cells.【conclusion】1curcumin combination with neural stem cell transplantation,promoted neural functional recovery. in different degree of spinal cord injury2curcumin,in the certain range of concentration can promote neural stem cellproliferation in concentration dependent manner.3curcumin can promote the nervoussystem Express BNDF.4curcumin can promote the differentiation of neural stemcells into neurons by promoting the nervous system express BNDF,.5curcuminreduce inflammatory reaction in the spinal cord, reduce lipid peroxidation, increase the survival rate of neural stem cells and the differentiation to neurons.【Innovative】1From the opinion of improving the environment in the spinalcord transplantation, the combination of curcumin and transplantation of neural stemcells to treat spinal cord injury, have not been reported at home and abroad, this studyconfirmed the validity of the combination application2From the cultivation of neuralstem cells confirmed that curcumin promoting neural stem cell proliferation;3spinalcord tissue slices with curcumin filtration liquid promote neural stem cells, furtherclarification of curcumin can through the expression of BDNF promotes nervoussystem play a role of neuroprotetion.更多还原