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Experimental Study Of Combining Curcumin And Neural Stem Cells Therapy For Spinal Cord Injury

Posted on:2015-01-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y QuFull Text:PDF
GTID:1264330428484049Subject:Surgery
Abstract/Summary:PDF Full Text Request
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.
Keywords/Search Tags:Curcumin, neuarl stem cell, BNDF, spinal cord injury, animal experiment
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