【作者】 饶以华；

【导师】 方加胜；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 脊髓损伤(spinal cord injury, SCI)常导致脊髓损伤平面以下肢体不可逆的神经功能丧失,给家庭和社会带来沉重负担。目前统计资料显示脊髓损伤发病率约为80/100万,并以每年7/1万的速率在增加。脊髓损伤康复一直是临床治疗难点。破解脊髓损伤奥秘及其寻找有效治疗途径是医学工作者的神圣使命。脊髓损伤导致损伤病变部位神经元及神经传导束路的损害。神经传导束功能障碍,引起损伤平面以下的所有功能失去大脑的支配,导致严重的临床症状,表现为截瘫伴随着生活自理能力的丧失。研究者通过移植胚胎中枢神经组织、雪旺氏细胞、嗅鞘细胞等替代缺失的神经细胞,改善局部微环境来促进神经功能的恢复。由于存在移植物的来源限制、组织移植物引起的免疫排斥反应等问题,限制了其临床应用。随着神经生物学和干细胞技术的发展,通过干细胞移植以增加神经细胞数量、减少胶质瘢痕和空洞形成,为治疗中枢神经系统损伤带来了新的曙光。已有研究报道胚胎干细胞、神经干细胞、骨髓基质干细胞等移植有助于神经功能恢复,然而,伦理道德及取材等多方面的问题使干细胞移植治疗中枢神经系统损伤进展缓慢。脂肪基质干细胞(adipose derived stem cells, ADSCs)由于其完全不存在上述限制,使其获得了广泛的应用前景。由于脂肪干细胞可以在体外长期培养、大量扩增产生充足的细胞来源,具有多向分化潜能,移植后通过迁移修复损伤神经组织,并能适应局部的微环境而分化为合适的细胞类型,也可长期低温冻存而保持其生物学特性不变,因此脂肪干细胞是组织工程理想的种子细胞。目前国内外对脂肪干细胞治疗脊髓横断损伤模型尚未见报道。干细胞移植治疗脊髓损伤有着巨大的潜力,但是真正应用临床面临诸如问题,如移植干细胞替代受损脊髓细胞的机制,如何创造适合神经修复的微环境,如何促进和诱导干细胞定向分化。同时干细胞移植的安全性仍有争议,如移植的细胞在体内分化方向如及是否分化形成肿瘤。因此脂肪干细胞治疗脊髓横断损伤的作用及相应修复机制还有待深入研究。研究包括以下三个部分。第一章大鼠脑匀浆上清对脂肪干细胞诱导分化成神经细胞的研究目的：分离脂肪干细胞,体外传代培养、鉴定及扩增；应用大鼠脑匀浆上清诱导其分化,为实验研究奠定基础。方法：从SD大鼠腹股沟脂肪组织分离得到脂肪干细胞,用含血清培养基对其培养、扩增、传代并纯化。分别用β-巯基乙醇(β-mercaptoethanol, BME)和大鼠脑匀浆上清诱导脂肪干细胞分化,观察细胞变化,并利用免疫荧光及免疫组化技术对分化后细胞鉴定,同时对ADSCs进行冻存复苏等实验。结果：从大鼠腹股沟脂肪组织获取大量ADSCs,电镜下细胞呈低分化状态,体外诱导后细胞可表达神经细胞标记物(GFAP,NSE),冻存复苏后ADSCs保持干细胞特性。结论：脂肪干细胞具有自我更新及自我分化潜能；脑匀浆上清可诱导其向神经细胞分化；体外可以长期培养,冻存复苏后仍保持干细胞特性。第二章脊髓横断损伤后脊髓内源性神经干细胞的实验研究目的：建立大鼠脊髓横断伤模型,观察大鼠脊髓损伤后脊髓内源性神经干细胞的激活情况,检测其特异性标志物Nestin mRNA的表达,寻找干细胞移植治疗脊髓损伤的合适时间窗。方法：制作大鼠脊髓横断伤模型；术前和术后腹腔注射5-溴脱氧尿核苷(Bromodeoxyuridine, BrdU)标记脊髓内源性神经干细胞；取假手术对照组及脊髓横断伤组大鼠脊髓组织冰冻切片,应用免疫荧光标记技术观察大鼠脊髓组织中脊髓内源性神经干细胞的激活后分布情况；取假手术对照组及大鼠脊髓横断伤组术后不同时间点的大鼠脊髓组织标本,检测Nestin mRNA在脊髓组织中的表达。结果：大鼠脊髓损伤后,损伤部位脊髓组织中证实BrdU阳性细胞存在,免疫荧光双标显示,BrdU阳性细胞为神经元和星形胶质细胞,而假手术对照组相同部位BrdU阳性细胞为阴性。RT—PCR结果显示,脊髓损伤后1d，1w,2w,3w,4w Nestin mRNA的表达水平呈上升趋势,4w后表达水平下降,至6w Nestin mRNA几乎无表达。结论：大鼠脊髓横断伤模型可靠并且重复方便；通过脊髓横断伤大鼠模型能够检测到脊髓内源性神经干细胞的激活后分布、增殖、迁移及分化情况,提示脊髓损伤后脊髓组织中的微环境能够激活神经干细胞,并存在适合干细胞移植的时间窗。第三章脂肪干细胞移植治疗脊髓横断损伤的研究目的：研究ADSCs移植对大鼠脊髓横断伤模型运动功能的影响及其可能机制。方法：成年S-D大鼠,随机分为假手术对照组(n=10)、脊髓(胸10)横断损伤对照组(n=20)和脂肪干细胞移植组(n=30)。移植后1d、1w、2w、3w、4w,每组各取3只动物(正常对照组共3只)横断损伤处脊髓组织,用RT-PCI法检测GAP-43 mRNA及BDNF mNRA表达。术后10周,取3只脂肪干细胞移植组大鼠脊髓组织进行免疫荧光染色观察ADSCs移植后的存活、迁移和分化情况。三组实验动物术后均采用BBB评分法定期评估运动功能。所得数据进行统计学处理。结果：ADSCs移植后能在宿主体内存活、迁移并分化为GFAP和CAPase阳性神经细胞。ADSCs移植治疗组脊髓组织内GAP-43mRNA和BDNF mRNA的表达上调,较脊髓横断伤组明显增高(P<0.05)且持续时间延长(达3周)。ADSCs移植治疗组大鼠后肢的自主运动功能BBB评分明显好于脊髓横断伤组,但两组动物评分均明显低于假手术对照组(P<0.01)。结论：ADSCs移植到损伤脊髓后,能在宿主体内存活、迁移并分化为星形胶质细胞和少突胶质细胞。促进GAP-43 mRNA及BDNF mNRA高表达的和持续时间的延长,从而促进神经轴突的修复,改善运动功能。更多还原

【Abstract】 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更多还原