【作者】 徐盛明；

【导师】 袁文； 陈德玉； 叶晓健； 何成；

【作者基本信息】 第二军医大学 ， 外科学， 2007， 博士

【摘要】 背景:脊髓损伤(spinal cord injury ,SCI)后轴突再生差,是中枢神经系统(central nervous system ,CNS)损伤后神经功能难以恢复的重要原因。目前大多数学者认为,中枢神经系统轴突内在特性并不是不能再生的,外部微环境中的抑制因子可能具有更为重要的作用。研究表明,脊髓损伤后髓磷脂相关抑制物作用是抑制中枢神经轴突再生的关键因素之一。在促进中枢神经损伤轴突再生的研究中,发现三种中枢神经髓鞘来源的主要髓磷脂相关轴突生长抑制物Nogo-A、髓磷脂相关糖蛋白(myelin-associated glycoprotein ,MAG)、少突胶质细胞糖蛋白(oligodendrocyte myelin glycoprotein ,OMgp),在脊髓损伤后对轴突生长起抑制作用。进一步研究证明,Nogo-A、MAG、Omgp通过位于神经元表面的共同高亲合受体Nogo受体(Nogo receptor,NgR)发挥作用,三种髓磷脂相关轴突生长抑制物及其NgR在神经组织及损伤模型中高表达,能够抑制轴突再生、促进生长锥溃变,影响脊髓损伤的修复。NgR具有高亲和性、高效性(同时与3个抑制蛋白结合)以及神经特异性,阻断NgR的功能可以同时减轻Nogo-A,MAG,Omgp三者对轴突生长的抑制作用。在针对脊髓损伤髓磷脂相关抑制物的研究中,目前主要通过细胞外应用Nogo-A抗体IN-1、髓磷脂或cDNA疫苗、抗NgR竞争性肽段等。虽然不同程度上对抗了髓磷脂相关抑制因子的作用,促进轴索再生。但IN-1只能阻断Nogo的轴突生长抑制作用,无法同时阻断MAG与Omgp的作用;髓磷脂或cDNA疫苗难以通过血脑屏障到达损伤脊髓的局部。尽管分子途径的NgR抗体或抗NgR竞争性肽段封闭Nogo蛋白与NgR的结合,可能同时减轻这三个髓磷脂相关抑制因子的作用。但因其易受环境因素的影响,且肽段到达损伤部位的效率不高、作用时间短暂而限制了其在SCI中的实际应用。如果在基因水平下调NgR的表达,既可直接减轻髓磷脂相关抑制物的抑制作用,又避免分子途径应用的不足。近两年来发展迅速的RNA干扰技术,提供了这种可行性。RNA干扰(RNAi)是由双链RNA介导的、在转录后mRNA水平关闭相应序列基因表达的过程。在此过程中,基因可以进行正常转录,但不能正常积累mRNA,从而使基因在转录后mRNA水平失活。RNA干扰技术是近年兴起的一项新技术,其具有特异性强,效率高,对正常基因序列影响小等特点,是基因治疗上的一大突破。为此,发明此项技术的两位美国科学家斯坦福大学的Andrew Fire和麻萨诸塞州医学院的Craig Mello获得了2006年的诺贝尔医学奖。通过RNA干扰技术直接沉默相关基因的表达来克服神经再生抑制因子的作用无疑是一种合理的选择。应用基因治疗的RNA干扰技术下调脊髓损伤中NgR的表达,既可同时减轻脊髓损伤后三种髓磷脂相关抑制物的作用,又可以克服分子途径存在作用时间短、特异性差、易受干扰等缺点。2002年Krichevshy和Kosik将RNA干扰成功地应用到大鼠脑皮质和海马神经元,有效的抑制了神经元内源基因的表达,同时转染外源基因也得到表达;2003年,Hommel等首次利用针对酪氨酸羟化酶(tyrosine hydroxylase,TH)mRNA特异序列合成的shRNA,构建由U6启动子驱动的载体,并将之注射至中脑黑质区,结果显示12d后载体介导的RNAi能有效地减少中脑多巴胺能神经元THmRNA的表达。这些结果提示,RNA干扰在原代神经元是可以被激活的,而且还可以用于神经元的基因功能和基因治疗的研究。我们在前期实验中成功地应用化学合成NgR特异性小干扰RNA(siRNA)下调原代皮层神经元NgR的表达水平,并筛选出基因沉默效率较高的小干扰RNA序列siNgR199。在接下来的实验中将NgR特异性小干扰RNA序列设计成小发卡式RNA结构,再将其克隆入质粒载体,在体外、体内研究中均证实转染后能够直接在神经元内长期、稳定表达小干扰RNA。但质粒载体转染时需与转染试剂配比使用,影响体内应用时的转染效率。而慢病毒载体能将外源基因整合入非分裂细胞的基因组内,神经元细胞转染率明显高于质粒载体及腺相关病毒载体和逆转录病毒载体,RNAi效应持续时间长,体内应用可长期表达而无明显免疫反应,目前尚无慢病毒载体本身导致的副反应的报道。因此,本研究拟构建NgR特异性小干扰RNA慢病毒重组体,优化NgR特异性小干扰RNA的合成、递呈,通过病毒转染技术体外进行大鼠原代皮层神经元细胞RNA干扰,验证其有效性并确定最佳感染复数(MOI);最后在大鼠脊髓横断损伤模型证实慢病毒介导的NgR特异性siRNA能够成功长期下调NgR蛋白表达,一定程度上促进损伤大鼠神经功能恢复和神经纤维再生。目的:1.在前期实验的基础上,设计、构建介导NgR特异性小干扰RNA序列的慢病毒重组体。2.验证于大鼠原代皮层神经元细胞培养中慢病毒重组体可以成功转染神经元细胞,引发内源性NgR基因沉默。并确定体内最佳转染浓度。3.观察大鼠脊髓横断损伤模型应用慢病毒重组体沉默NgR基因后对神经轴突再生和神经功能恢复的影响,为进一步应用临床试验提供理论依据。方法:1.按照E1bashir等设计原则和siRNA表达载体的要求,设计构建NgR特异siRNA199慢病毒重组体,并进行酶切鉴定及基因测序;通过转染293T细胞确定最佳转染浓度和感染复数(MOI值)。2.体外培养大鼠原代皮层神经元细胞,培养后第7天,进行不同滴度的重组慢病毒转染,荧光显微镜下观察慢病毒转染情况,确定最佳MOI值及活体应用病毒量,应用实时荧光定量PCR检测内源性NgR基因沉默效果;3.建立大鼠脊髓横断损伤模型;51只成年雌性S-D大鼠随机分为假手术对照组(6只)、生理盐水组(15只)、空慢病毒组(15只)、慢病毒重组体组(15只),脊髓横断损伤后7天皮层体感运动区分点微量注射给药。损伤后8周(处死前1周)皮层体感运动区分点微量注射BDA示踪剂顺行示踪。4.伤后每周对大鼠的后肢运动功能进行神经功能行为学评分(BBB评分);采用双盲、双人独立观察记录法。给药10天后荧光显微镜下观察报告基因GFP表达情况;实时荧光定量PCR(RT-PCR)检测注射区皮层神经元细胞NgR mRNA表达情况;免疫组化观察NgR蛋白表达状况,及脊髓损伤神经修复情况。9周后观察脊髓损伤区神经修复、再生情况。结果:1.构建的NgR特异性siRNA199慢病毒重组体经酶切鉴定,产物琼脂糖凝胶电泳DNA片段约6.4Kbp,经基因测序仪测序,结果与设计序列完全相符,目的基因序列准确无误,慢病毒重组体构建成功。2.慢病毒重组体实现体外大鼠原代皮层神经元转染,96小时后神经细胞开始表达绿色荧光,146小时表达绿色荧光最强;适合高效感染的MOI值为3;转染192h后收集细胞进行实时荧光定量PCR检测,结果显示目的基因抑制效率为61%,取得较满意的效果。3.损伤后1周在大鼠脊髓横断损伤模型中行皮层体感运动区分点微量注射给药,给药10d后取注射区脑组织行RT-PCR检测NgR mRNA表达水平,结果显示重组慢病毒注射组NgR mRNA的表达水平明显低于生理盐水注射组与空慢病毒注射组,差异有统计学意义(P<0.05);注射区脑组织冰冻切片贴片行免疫组化检测NgR蛋白表达情况,生理盐水组及空慢病毒组可见清晰阳性细胞,重组慢病毒组NgR染色阳性颗粒明显减少,从而说明在活体内大脑皮层微量注射慢病毒重组体转染皮层神经元细胞后成功实现了NgR基因的沉默。4. BBB评分结果:手术大鼠在损伤后最初都表现为丧失所有后肢运动功能,BBB评分为0分。损伤后4周内无明显改变,第5周起,各组动物均出现不同程度的后肢运动,BBB评分最高为3分。随后逐渐上升,至第8周时最高达到8分;各实验组大鼠的神经功能均有一定程度的恢复,但各组间BBB评分提高程度无统计学显著差异(P>0.05)。5.组织学和神经纤维染色检查结果:重组慢病毒组脊髓横断损伤部位有更多髓鞘组织形成及胶质细胞增生;BDA顺行神经示踪观察见重组慢病毒组有少量的轴突生长通过损伤区域,要明显好于生理盐水组及空慢病毒组。结论:我们设计、构建的NgR特异性siRNA199慢病毒重组体,能够在大鼠体外原代皮层神经元转染和体内皮层运动区注射后稳定地实现RNA干扰,较长时间下调神经元的内源性NgR mRNA表达,并在一定程度上促进脊髓神经轴突再生和大鼠后肢运动功能的恢复。本研究证明,慢病毒载体介导的RNA干扰可能成为脊髓损伤治疗的有效手段之一,虽然短期在功能恢复上未发现明显优势,但在组织学上已有所体现,远期治疗效果较乐观。其可能的机制是:慢病毒重组体能稳定地在皮层神经元中进行RNA干扰,长期下调NgR mRNA表达,阻断NgR与配体(Nogo-A、MAG、Omgp)的结合,从而对抗三种髓磷脂相关抑制物的轴突再生抑制作用。更多还原

【Abstract】 BackgroundThe axon is hard to regenerate after spinal cord injury. and it’s the most important reason that the functional recovery is very limited. Now,most researchers belive that it is mainly for the inhibitors in the microenvironment that play an important role inhibiting the axonal regeneration ability.Recent studies shows that the myelin-associated inhibitors is one of the crucial factor to inhibit axonal regeneration of CNS(central nervous system).It was found through research that the inhibitors of growth present in CNS myelin sheath, these myelin inhibitors include NogoA (also known as reticulon 4), MAG (myelin-associated glycoprotein) and OMgp (oligodendrocyte myelin glycoprotein) inhibit neurite outgrowth. Further studies indicated that these myelin inhibitors bind a common receptor, the Nogo receptor (NgR). NgR is a neuronal specific receptor with high effect and affinity by binding Nogo-A, MAG and Omgp,mediating the inhibition of axonal regrowth and induction of growth cone collapse. Being the convergence of the three myelin inhibitors, suppressing Nogo receptor protein can decrease Nogo-A, MAG and Omgp’s inhibition at the same time. In the research of the myelin-associated inhibitors in CNS,anti-NogoA antibody IN-1 , myelin or associated cDNA vaccine and a NogoA-derived peptide, NEP1-40, have been delivered to rats with spinal cord injury.Functional recovery and axonal regeneration were improved after treated with them. But there are some deficiencies in these studies, IN-1 only inhibits Nogo-A, myelin or associated cDNA vaccine can not pass the blood brain barrier efficiently. Meanwhile,other myelin inhibitors’function can not be decreased in this method. Although NEP1-40 can decrease Nogo-A, MAG and Omgp’s inhibition at the same time, it is not stable enough in the environment,and effect as a transient way.If Nogo receptor gene expression can be suppressed, we can find a attractive strategy to avoid deficiencies in IN-1, vaccine and NEP1-40 treatment .A gene therapy ,RNA interference, known as the small RNA induced gene silencing is a possible method. RNA interference (RNAi) is a post transcriptional gene silencing process targeting homologous mRNA for degradation,and induced by Double-stranded RNA (dsRNA). In this process, targeted gene can be transcripted as normal,but mRNA can not be accumulated for it’s degradation.It is reasonable to suppressed axonal regeneration inhibitors’gene or silencing NgR gene to suppress myelin inhibitors’function,especially the latter is a good choice. Recent studies shows that RNAi is effective at suppressing specific gene expression in a number of primary cells including neurons. In 2000, Krichevsky and Kosik found that the rat hippocampus and forebrain can be effectively suppressing endogenous and heterologous genes induced by small RNA interfering. In 2003,Hommel and Sears knocked down localized gene, encoding the dopamine synthesis enzyme tyrosine hydroxylase in the brain, by using viral vector-mediated RNA interfering.These experiments indicated that neurons can get gene silence by RNA interfering.The condition of axonal regeneration after SCI is that the neuron can express growth gene and be supplied with neurotrophy and axon growth substantia basilaris, and inhibiting factor of axon growth must be prevented. Therapeutic regimens about SCI might be foetus neural graft, peripheral nerve tissue or cell graft, and gene therapy. Foetus neural graft is restricted by poor source and ethics problem though foetus neural graft has a good therapeutic effect to SCI for low rejection and strong fissionability. Peripheral nerve tissue has a poor prostecdtive efficacy to SCI for high rejection. Gene therapy has a lower rejection than peripheral nerve tissue and can act a long-term effectiveness after single therapy. For this reason, the most of scholar consider that gene therapy is one of the best Therapeutic regimens about SCI .RNA interference (RNAi) is a post transcriptional gene silencing process targeting homologous mRNA induced by Double-stranded RNA (dsRNA), and it can specially silence internal source or extrinsic source target gene. Nowadays, RNA interference has been applied with many kinds of creature including plants, eumycete, virus, mammalia. Applying vitro synthesis siRNA is the latest development of RNAi and establish a foundation for RNA drug treatment. In mammalian cell, vitro synthesis siRNA can specially degradatiohn homologous mRNA and silence target protein expressing, and can prevent from non-specificity degradation and cell death.In our prophase study, we had successfully inhibited the expression of endogenous NgR genes effectively in cultured rat neuron by applying chemical synthetic siRNA, and sieved out a high-performance RNA interference chi sequence siNgR199. Then we designed siNgR199 to little hare cadette frame and cloned into plasmid or viral vector, they might long-term interfere with target gene in neuron after infection. The plasmid has low transducing rate. Lentiviral vector is more suitable to infect unseparated nerve cell than adeno-associated viral vector and other viral vectors because its high tropism to stem cell and other cells in vitro culture. Lentiviral vector has powerful tropism to neuron but no counter transport ability. As a vector, Lentiviral vector is perfect to mediate RNAi in CNS.In this study, we first constructed lentivirus siNgR recombinant to optmize the process of synthetizing and transmitting siNgR. We selected siNgR199 as homologous NgR RNA succession, because it had the greater specific effect on NgR gene silence. Then, we applied recombinant to infect rat cortical neuron in vitro for checking the effectivity of recombinant silencing target gene. At last, we verified the capability of recombinant about inducing nerve fiber regeneration and promoting functional recovery in rat SCI model.Objective:1.To construct a recombinant of RNA interference with lentivirus vector and siNgR199;2.To check the effectivity of the recombinant silencing NgR gene in the rat cortical Cells and optimize the dose and time dependent in vitro ;3.To investigate the effect of the recombinant in inducing nerve axon regeneration and promoting functional recovery of nerve in rat SCI model.Methods:First, we selected siNgR199 which was designed into a short hairpin RNA construction as homologous NgR RNA succession, and cloned it into lentivirus vector using the E1bashir’s criteria and the RNAi vector rule; Then, we evaluated the recombinant by enzyme cutting and gene sequencing test.Second, we introducted recombinant into the cultured nerve cell from the rat cerebral cortex with different concentrations, studying the transfection of recombinant by observing the marker cGFP gene expressing, evaluating the effect of suppressing NgR gene with real-time PCR. Third, we created a transection model of SCI in rat, and saline, lentivirus vector and recombinant was injected into hindlimb motor area of cerebral cortex after 7 days, 8 weeks after administration, nerve tracer agent was also injected into hindlimb motor area of cerebral cortex .Fourth, every week postinjury we assessing the hindlimb motor functional recovery.9 weeks after administration, the rats were killed for histologic studies after the last assess of the functional recovery.Results:1. We had successfully constructed a recombinant targeting NgR-specific siNgR199 with Lentiviral vector. The recombinant molecular weight and gene sequence is correct by checking with enzyme cutting and gene sequencing test.2. we introducted the recombinant into the neuron cultures, finding that the marker cGFP gene began to express 96h after transfection, and expressed powerfully 146h after transfection. The best MOI value for effective transfection is 3. NgR mRNA had been suppressed 61% at 192h after transfection by checking with real-time PCR test.3. we finded that NgR mRNA expressed more little in rats injected with recombinant than in rats injected with saline or lentivirus vector 8d after administration, and NgR masc-grana was also more little in rats injected with recombinant than other groups. There is statistical difference between them(P<0.05).These results demonstrated that NgR protein was suppressed effectively by injecting recombinant in vivo.4. We assessed the functional recovery using BBB score, all rats was 0 in the first day postinjury, until 5 weeks after injury,it became 3. The functional recovery improved slowly in the last 2 weeks finally up to 8. All rats were observed to have the hindlimb functional recovery, and the BBB scores were no statistical difference between each group(P>0.05). Moreover, we finded that some nerve fiber passed the injured region and more medullary sheath and glial cell were present in the spinal cord injured region in these rats injected with recombinant.Conclusions:We had successfully constructed a recombinant targeting NgR-specific siNgR199 with Lentivirs vector. The recombinant can effectively suppress NgR mRNA expression in rats’cortical nerver cells in vitro or vivo, and can promote neurological functional recovery and axon regeneration in rat SCI model.which indicates that lentivirus mediating RNAi may be a potential therapeutic method for SCI. The possible mechanism is that the recombinant stably express siNgR199 in cortical nerver cells, and long-term induce silencing NgR gene, decreasing the combination of NgR with Nogo,MAG and OMgp, which result in promoting the regeneration of axon at the lesion site after SCI.更多还原