The Experimental Research On Transplantation Of Adipose Derived Stem Cells For Rat Spinal Cord Complete Transection Models

Spinal cord injury (SCI) usually leads to the loss of unreversible neurologic deficit under the damaged level, and seriously bring burdens to the society and families. Statistical datas show SCI incidence is approximately 80/1,000,000 and increases by a 7/10,000 ratio per year presently. The restoration of SCI still is a huge task for the human being. Trying to make certain the mechanism of SCI and find an ideal method is the common destination for the human being. SCI not only causes the loss of local nerve cells,but also breaks off descending and ascending tracts. The discontinue of descending and ascending tract gives rise to dysfunction in the lower damaged-level regions and then presents the severe symptoms such as paraplegia without self-care ability because of the loss of dominating from the brain. SCI can't significantly regenerate under no condition of suitable micro-environment. The researchers already have been done lots of experiments and they applied embryonic stem cells, neural stem cells, bone marrow stem cells, etc. to improve the outcome of neurological function. Their results were excited, but such stem cells grafting encounters lots of puzzles including the limited implants sources, ethnic and immune elimination due to implants. Because possessing these natural advantages of abundant resource, avoiding of ethnic,etc., ADSCs has been presenting exciting prospect in curing SCI. Based on produce enough cells sources, variety of differentiation abilities, recovering injuried neural tissues through migration after transplantation and being able to adapt to the local micro-environment and differentiate into the suitable cell types meanwhile maintaining the biological characteristics of stem cell after cryopreservation undergoing the long time freezing storage, ADSCs is a kind of ideal seed cell for histological engineering to treat SCI. Presently, there still hasn't the report of application of ADSCs grafting to cure SCI. Certainly there are many problems which refer to the effects and mechanism of ADSCs grafted to treat SCI, how to create suitalbe micro-environment for neurological function recovery, regulating and controlling of differentiation of ADSCs, and the safety of ADSCs grafting.So we have a long way to achieve the destination of application of ADSCs grafting for patients. The research included following three parts.PARTⅠthe study of adiposed stem cells differentiate into neurocytes induced by supernate of rat brainObjective:To establish isolation, cultivation and identification system of ADSCs in-vitro and apply the supernate of rat brain to induce ADSCs. Methods:The ADSCs of rat were isolated, and cultured,expanded, purified, and passed generation in vitro by added serum culturing,and the proliferating ability of ADSCs was detected with BrdU. Then differentiation was induced byβ-mercaptoethanol and the supernate of rat brain respectively and the differentiated neurocytes were identified by immunofluorescence technology. Meanwhile the research on long-term cultivation, passage, cryopreservation and revitalization had been undergoing. Results:Lots of ADSCs, gained from the inguen region, could self-proliferate and combine with BrdU, and then differentiate byβ-mercaptoethanol and rat brain supernate successfully. The differentiated cells present some specific antigens, such as GFAP and CNPase of neurocytes. The ADSCs can be perserved for a long time under low temperature.The survival ratio attain 80-95% after cryopreservation and revitalization.Conclusion:ADSCs have the ability of self-renewal capacity and the multi-differentiation potential, and can differentiate into neurocytes induced by supernate of rat brain. ADSCs can be perserved for a long time under low temperature, and after cryopreservation ADSCs still keep stem cells characteristics. PARTⅡThe study on the endogenous neural stem cells in spinal cord of rats with experimental spinal cord complete transection injuryObjective:To build the rat model of spinal cord complete transection injury, observe the activation of endogenous neural stem cells(NSCs) in SCI, detect the expression of Nestin mRNA of spinal cord tissue, and find a suitable time window for stem cells grafting after spianl cord injury. Methods:Adult Sprague-Dawley rats were divided into sham operation control group and SCI group at random. And then manufacturing the rat model of spinal cord complete transection injury, Evaluation of SCI rats was performed before and after the operation. The Bromodeoxyuridine (BrdU) were intraperitoneal injected before and after the operation at certain time to mark the endogenous NSCs. The rat spinal cord tissue were extracted at a certain time point after SCI respectively, and underwent frozen section, and then dyed by double-marked immunofluorescence staining method to detect the distribution, migration and differentiation of endogenous NSCs. RT-PCR method was applied to detect the expression change of the Nestin mRNA. Result:The BrdU-positive cells in the rat spinal cord tissue increased after SCI and the results of double-marked immunofluorescence staining for BrdU and neuronal specific nuclear antibody(glial fibrillary acidic protein(GFAP) showed the celltypes of BrdU positive expression were neuroctyes. However the BrdU positive staining cell in the same regions of normal spinal cord tissue was rarely observed.The results of RT-PCR showed that the expression of Nestin mRNA gradually up-regulated in the rat spinal cord tissue 24 hours after SCI, decreased 4 weeks after SCI and hardly be detected 6 weeks after SCI. Conclusion:The rat model of spinal cord complete transection injury is reliable and repeatible conveniencely. We successfully observed the activation of endogenous NSCs and it's distribution, migration and differentiation in the injuried spinal cord tissue on the rat SCI model.The experimental results suggested that there lies a appropriate local micro-environment to activate the endogenous NSCs after SCI, that there lies a suitable time window for stem cells transplantation to cure SCI.PARTⅢThe detect on adiposed stem cells transplanting for rat spinal cord complete transection injury modelsObjective:To explore the impact of ADSCs grafting on movement function improvement in rat spinal cord complete transection models and it's possible mechanism. Methods:60 adult Sprague-Dawley rats were divided into sham operation normal control group (G1, n=10), spinal cord transection injury group (G2, n=20) and adiposed stem cells grafting after spinal cord transection injury group (G3, n=30) at random. Spinal cord tissue; gained from 3 rats respectively each group(G1=3 together) from SCI area 1 day and 1,2,3,4 weeks after the surgery,was detected GAP-43 mRNA and BDNF mRNA expression by RT-PCR method. Spinal cord tissue of 3 rats in ADSCs grafting group was used to observe survival, migration and differentiation of grafted ADSCs by immunofluorescence 10 weeks after surgery. The BBB scores were applied to assess rats' behaviors at the regular intervals after the surgery and then the results were analyzed statistically. Results:Grafted ADSCs could survive, migrate in the injuried area and differentiate into GFAP and CAPase positive neuroctyes. GAP-43 mRNA and BDNF mRNA expression in spinal cord tissue of ADSCs grafting group was sharply higher and sustained longer(maximum 3 weeks) than the spinal cord transection injury groups. BBB scores of animals of the ADSCs grafting group were higher than that of the spinal cord transection injury group(P<0.05), however both BBB scores were obviously lower compared to sham...