大鼠脊髓损伤后Nogo蛋白受体在浸润巨噬细胞中的表达及作用
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摘要
轴突再生并与靶细胞形成功能性突触结构是脊髓损伤修复的主要目标和关键问题,目前大量促进轴突再生的基础研究显示成体哺乳动物中枢神经系统损伤早期再生失败的主要原因是由于髓鞘抑制物的存在。我们在前期实验中发现,将神经干细胞和(或)嗅鞘细胞移植到受损脊髓局部,可观察到神经干细胞可分化为神经元及其它神经胶质细胞,神经突触形成增加,并观察到脊髓全横断损伤大鼠BBB运动功能评分改善,尽管实验中观察到不少受损神经再生或(和)出芽(待发表资料),但受损局部微环境的改变使轴突再生或出芽的距离极为有限,难以冲破髓鞘抑制因子与胶质瘢痕形成的屏障与远侧断端形成有功能突触。
髓鞘碎片中的某些特异性成分(比如：Nogo66、髓鞘相关糖蛋白MAG、少突胶质细胞髓鞘糖蛋白OMgp等)作用于轴突上的Nogo受体(NgR)及其共受体p75NTR复合物,通过小鸟嘌呤核苷三磷酸酶(small GTPase) Rho途径调节轴突内细胞骨架结构抑制轴突生长,从而使脊髓损伤修复难以获得满意结果。Nogo单克隆抗体IN-1可以拮抗Nogo的抑制作用,但无法与另外两种抑制物MAG和OMgp结合。GrandPre等尝试以竞争性短肽NEPI-40来拮抗NgR的作用,结果显示NEP1-40可对抗Nogo-66诱导的轴突生长抑制,但对除Nogo-66外的其他髓鞘抑制物不敏感,使其在体内应用的效果并不十分理想,表明该短肽并不能封闭NgR所有与抑制分子结合的位点。因此,找到一种有效且靶向性强的髓鞘碎片清理方法成为当务之急。
目前,人们对巨噬细胞在中枢神经系统损伤修复中的作用存在着两种截然相反的观点,一种认为巨噬细胞对轴突生长有抑制作用；而另一种则认为巨噬细胞对轴突的再生起到促进作用。在整个机体组织的修复过程中,炎性反应贯穿始终,而炎性细胞中,巨噬细胞发挥了主要作用,它能清除坏死组织并分泌营养因子,并可在机体组织修复中起着重要作用。Mantovani等认为,巨噬细胞受炎性反应中微环境不同信号影响,可转变为“选择”活化型和“经典”活化型两种。“选择”活化型巨噬细胞由IL-4和糖皮质激素诱导转换而来,可调整免疫炎性反应的过程,清除坏死组织的残片,促进血管再生组织修复和重建；“经典”活化型巨噬细胞可分泌前炎性细胞因子,加重炎症反应,对受损局部细胞造成更进一步的损害。近年来,对于巨噬细胞在脊髓损伤致伤及修复作用报道并不多见。
新近研究表明巨噬细胞在髓鞘碎片吞噬、促进轴突再生及再髓鞘化过程中发挥的作用可能比我们预想的更大。
1998年,Zeev-Brann报道成熟哺乳动物中枢神经系统损伤后的自我修复中出现脊髓衍生炎性细胞,Buss与Schwab等研究发现SCI大鼠髓鞘碎片清理细胞ED-1免疫组织化学染色呈阳性反应,即为小胶质细胞与巨噬细胞。MASAAKI通过鼠脊髓挫伤模型(代表挫伤引起的有髓鞘碎片和髓鞘再生受抑制)与化学性脱髓鞘模型(代表早期髓鞘碎片清除和髓鞘再生的多发性硬化症)的对比,研究了巨噬细胞聚集及其作用,作者使用绿色荧光蛋白转基因小鼠的骨髓细胞分别移植到经放射线照射的脊髓挫伤模型鼠与化学性脱髓鞘模型鼠尾静脉,通过损伤脊髓免疫组化染色研究活化巨噬细胞的浸润和残留髓鞘碎片的不同水平变化,结果显示表达绿色荧光蛋白的活化巨噬细胞构成损伤局部主要细胞群,表明两个模型中的巨噬细胞主要来源于骨髓,很少来源于内在的小胶质细胞。在免疫染色显示在挫伤模型当中,髓鞘碎片长时间持续存在,并且活化巨噬细胞的浸润在挫伤模型中比化学模型中更慢并且数目显著减少,同时伤后2～4天RT-PCR检测趋化因子(MCP-1, GM-CSF)低水平表达,提示活化巨噬细胞浸润的延迟与挫伤性脊髓损伤后髓鞘碎片的持续存在有关,且导致髓鞘再生抑制。
Samuel David等研究了Nogo受体(NgRs)在周围神经损伤中的作用。研究人员损伤鼠坐骨神经,他们发现,一旦巨噬细胞到达受损部位并开始清除工作,它们就表现为表面表达NgR1的活化形式,并进入施旺细胞细胞基底部。当恢复的神经开始产生新的蛋白质髓鞘(表达NgR配体如MAG等),这种受体不但抑制巨噬细胞与髓鞘的结合,还会直接抑制髓鞘的形成,当研究人员再次损伤神经,使髓鞘不能形成时,巨噬细胞继续留在受损神经外层施旺细胞细胞基底部吞噬髓鞘碎片。然而,NgRl基因敲除鼠坐骨神经或使用Y-27632抑制NgR下游Rho相关激酶,可增加巨噬细胞与髓鞘的结合率,证实巨噬细胞表达的NgR参与介导该细胞在周围神经损伤修复中的撤离过程。有关中枢神经系统损伤中巨噬细胞是否表达NgR及二者在中枢神经系统尤其是脊髓损伤修复中的作用有待进一步研究。
脊髓损伤后,活化的巨噬细胞浸润脊髓损伤区,髓鞘碎片的清除延缓导致了轴突延伸的抑制,和由于星形胶质细胞的堆积形成的瘢痕导致了轴突再生的抑制。我们推测,成年哺乳动物脊髓损伤后,活化巨噬细胞迅速清除髓鞘碎片对中枢神经系统的再生与对周围神经系统的再生具有同样重要的意义,但对于巨噬细胞在脊髓损伤中的具体作用仍需深入研究。
研究目的
制备大鼠脊髓损伤模型,分离、培养、鉴定大鼠脊髓损伤局部浸润的巨噬细胞,观察其是否表达NgR；构建大鼠NgR基因慢病毒载体并转染巨噬细胞表达NgR,探讨转染免疫细胞的可行性；比较NgR的表达对巨噬细胞吞噬功能的影响；体外构建神经元损伤模型,将表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养,比较各组巨噬细胞对损伤神经元修复、存活、分化的影响,探讨表达NgRs的巨噬细胞在脊髓损伤修复中的可能作用机制,为进一步动物研究及临床应用提供实验和理论依据。
内容和方法
(一)制备大鼠脊髓损伤模型,第3天及第7天时取材,使用免疫荧光化学染色观察巨噬细胞浸润及NgRs的表达情况。用酶消化法分离,获取损伤脊髓组织局部浸润的巨噬细胞,使用双标免疫荧光化学染色从细胞水平观察巨噬细胞表达NgRs情况；
(二)采用RT-PCR技术获得大鼠NgR基因编码区片段,限制性内切酶酶切和基因重组构建慢病毒载体质粒,在脂质体介导下包装质粒,包膜质粒共转染293T细胞包装生产慢病毒。所获慢病毒感染大鼠巨噬细胞后,PCR检测巨噬细胞中NgR基因的插入和表达。
(三)将髓鞘碱性蛋白(MBP)加入转染NgRs的巨噬细胞、空载组巨噬细胞和未转染NgRs的巨噬细胞培养皿中,Western blot定量检测巨噬细胞吞噬的MBP量,行t检验(Student's t-test),取α=0.05。
(四)制备体外神经元损伤模型,将表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养,各组分别于损伤后30min、1h、6h、12h、24h、48h、72h各时间点提取培养液100 u L,检测LDH含量并采用MTT比色法测量其存活细胞的吸收值。倒置相差显微镜下观察各组不同时间神经元的形态变化,采用重复测量数据方差分析进行统计学分析。
研究结果
(一)在假手术组中,脊髓组织中没有浸润的巨噬细胞；免疫荧光染色显示脊髓损伤3天后浸润的巨噬细胞表面可表达NgR,伤后7天时表达NgR的巨噬细胞的数量明显增多。培养脊髓损伤局部浸润的巨噬细胞,免疫荧光染色显示该细胞NgRs抗原染色阳性；
(二)所获的NgR基因经测序后与Gen Bank报道序列完全一致。重组慢病毒载体质粒经鉴定正确。三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为6.7×107Tu/mL, PCR证实NgR基因插入病毒基因组。感染巨噬细胞后RT-PCR检测试验组NgR-巨噬细胞组更大量表达NgR,与其余2组(Mock-巨噬细胞组、巨噬细胞组)比较差异具有统计学意义；
(三)NgR转染巨噬细胞组的MBP含量比非转染组MBP含量明显增加,差异有显著统计学意义(P<0.05),空载组与非转染组无明显的差异(P>0.05)；
(四)表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养0.5,1,6,12小时后,各组OD值没明显不同。24h后,神经损伤+NgR转染组比其他组有明显的差异(P<0.05),空载组与非转染组没明显的差异(P>0.05),NgR转染组与正常神经组无明显差别(P>0.05)。随时间延长,LDH水平在损伤神经组中下降明显但在NgR转染组下降不明显；培养0.5,1,6,12h后,各组OD值没明显差别；24h后,神经损伤+NgR转染组与其他组比较有明显的差异(P>0.05),空载组与非转染组没明显的差异(P>0.05),NgR转染组与正常神经组无明显差别(P>0.05)。
实验结论
成功制备脊髓损伤模型,从组织学和细胞水平证实损伤脊髓组织局部浸润的巨噬细胞表面有NgRs的表达；成功构建大鼠NgR基因慢病毒载体并转染巨噬细胞表达NgRs,为今后移植基因修饰的巨噬细胞治疗脊髓损伤奠定了基础；表达NgRs的巨噬细胞体外能促进损伤神经元的修复,其机制可能是巨噬细胞通过表达的NgRs介导胞内信号途径调节巨噬细胞的吞噬功能,从而为神经再生创造条件。这为脊髓损伤的治疗研究提供了一个新的解决途径,也为进一步动物研究和临床应用奠定了可靠的实验基础和理论依据。
Back ground
Ax on regeneration and formation of functional synapses with target cells are the main objectives and key issues in repair of spinal cord injury. Large numbers of basic studies on promotion of axon regeneration have shown that failure of early regeneration of the central nervous system in adult mammalians is due to the presence of myelin inhibitors.
We have found in previous experiments that neural stem cells(or) olfactory ensheathing cells could divide into neurons and other glial cells after transplanted into the damaged spinal cord. The nerve synapse formation increased and BBB motor function score improved in all rats with spinal cord injury. Although a lot of damage nerve regeneration and (or) budding (to be published data) was observed in experiments, the distance axonal regeneration or sprouting is extremely limited as the damage to local micro-environment changed.It is difficult to break through the barrier between the myelin-inhibitory factor and glial scar to format function synapses with the distal stump.
Some specific components of the myelin debris (eg:Nogo66, myelin-associated glycoprotein MAG, myelin oligodendrocyte glycoprotein OMgp, etc.) act on the axon on the Nogo receptor (NgR) and its acceptor-p75NTR complex to regulate cytoskeletal structure of axons within the axon growth inhibition through the small guanosine triphosphatase (small GTPase) Rho way.Thus,it is difficult to obtain satisfactory results of spinal cord injury repair. Nogo monoclonal antibody IN-1 can antagonize with Nogo protein, but not with the other two inhibitors MAG and Omgp. Grand Pre used the competing peptide NEPI-40 to antagonize the role of NgR and showed that NEP1-40 may be against the Nogo-66-induced axon growth inhibition, but not sensitive to other myelin inhibitors. This peptide can not be closed all inhibitory molecule binding site of NgR and the effects in vivo is not very satisfactory. Therefore, most urgent task is to find an effective and highly targeted method to clear myelin debris.
At present, there are two opponent views on the role of macrophages in the central nervous system injury. One is that macrophages can inhibit the growth of axons; other is that of macrophages play a catalytic role to the axon regeneration. The inflammatory response appeared through repair process of the tissue. Macrophages play a major role and can remove necrotic tissue and secrete neurotrophic factor, in all the body plays an important role in tissue repair. Mantovani believed that the macrophagecan can chang to "select" activation-type and the "classic" activation of two kinds impacting by the different signals in micro-environment of the inflammatory response. The "select"-activated macrophages are conversed by IL-4 and glucocorticoid-induced and adjust the immune inflammatory reaction process, the debris removal of necrotic tissue, promoting angiogenesis, tissue repair and reconstruction. The "classic"-activated macrophages may secrete pro-inflammatory cytokines and increase the inflammatory reaction, causing further damage to local cell. In recent years, there are rare reports of macrophages in the injured spinal cord injury.
Recent studies have shown that the role of macrophage in phagocytosis of myelin debris and axonal regeneration may be greater than we anticipated.
In 1998, Zeev-Brann reported that there were spinal cord-derived inflammatory cells appearing in mature mammalian central nervous system after injury. Buss and Schwab found that the ED-1 immunohistochemistry staining positive cells were microglia cells and macrophages which cleaned up myelin debris in SCI rats. MASAAKI researched macrophages and its role through the comparement of the rat spinal cord contusion model (on behalf of contusion caused by a myelin debris and myelin regeneration was inhibited) and the chemical demyelination models (on behalf of the early removal of myelin debris and myelin regeneration in multiple sclerosis).
The arthors transplanted green fluorescent protein transgenic mouse bone marrow cells into rat spinal cord contusion model irradiated by the radiation and chemical demyelination models.They studied the infiltration of activated macrophages and myelin debris residual changes in the different levels by immunohistochemical staining of spinal cord. The result showed activated macrophages expressing green fluorescent protein constituted the major cell groups in the damage location, indicating macrophages in the two models primary derived from bone marrow and rarely from intrinsic microglia. The immunohistochemical staining showed myelin debris existed long-time in the contusion model and the activated macrophage infiltration in the contusion model more slowly than that in the chemical model, the number reduction significantly. RT-PCR detection of trend chemokine (MCP-1, GM-CSF) expressed a low level 2 to 4 days after injury, suggesting that the delay infiltration of activation of macrophages post-traumatic spinal cord related to the persistence of myelin debris and lead to remyelination suppression.
Samuel et al [5] studied Nogo receptor (NgRs) in peripheral nerve injury. They induced damage in the sciatic nerve in the thigh of rats and found that macrophages, once arriving and starting to remove myelin debris, they performed for expression of NgR1 activated forms and went into the base of Schwann cells. When the restoration of the nerve start to produce new proteins of myelin (such as NgR ligands), this receptor not only inhibits macrophages with a combination of myelin, but also directly inhibits the formation of myelin. When the researchers damaged the nerve again to inhibit the formation myelin, the macrophages remained in the outer layer of Schwann cells and swallowed myelin debris. However, NgR1 knockout mice or use of Y-27632 to inhibit NgR downstream Rho-associated kinase could increase the combination rate of macrophages and myelin debris, indicating that NgR expression in macrophages participated in the withdrawal process at the end of repair in peripheral nerve injury. But whether macrophages express NgR after CNS injury and what role NgR expression plays in CNS repair remain unclear.
After spinal cord injury, activated macrophages infiltrated to injury areas and the delay of myelin debris clearance inhibit of axon extension. The formation of scar due to the accumulation of astrocytes led to inhibition of axonal regeneration. We speculate that the adult mammalian spinal cord injury, the rapid removal of myelin debris by activated macrophages on the central nervous system regeneration has the same importance as that in the peripheral nervous system, but the specific role of macrophage cells in spinal cord injury still need study.
Objective
To construct the spinal cord injury model and to investigate the isolation, culture methods and evaluation of macrophages expressed with NgRs in vitro in preliminary and reveal the biological characteristics of the cells. To construct the lentiviral vector of rat NgR gene and to transfect macrophages expressing NgR, to explore the feasibility of immune cells transfecting. To compare the effect of NgR expression on macrophage phagocytosis; To construct neuronal injury model in vitro and culture it with NgR-macrophages group, the mock group and the normal macrophage group together, to compare the effect of each group on the repairment, survival, differentiation of the injuried neuron. To explore the possible mechanisms of NgRs expression in macrophages on the spinal cord injury repairment and to give the experimental and theoretical basis for further animal studies and clinical applications.
Methods
1. To construct the spinal cord injury model and draw the materials at the day 3 and 7. To observe the infiltration of macrophages and expression of NgRs by immunofluorescence staining. Primary macrophages were harvested from injured spinal cord and digested with trypsin, and the morphological, biological characteristics and the NgRs antigen positive cells with methods of immunochemistry staining were observed.
2. RT-PCR technique was used to obtain gene-coding region of rat NgR fragments, digestion using restriction enzyme. Construction of gene recombination lentiviral vector plasmid and packaging plasmid mediated by the liposome. The envelope plasmid co-transfected 293T cells and the lentivirus producted. Infection the rat macrophages using the lentivirus and the insertion and expression of NgR gene were detected by PCR.
3. Myelin basic protein (MBP) was added into macrophages (NgR-macrophages group, the mock group and the normal macrophages group) and cultured, and the phagocytic capacity was measured by Western blot. Student's t-test was used for statistical analysis.
4. The neuronal injury model was constructed. Macrophages of different groups were co-cultured with the injured nerve plates. 100μL culture medium in each group was extracted respectively at different time points (30min, 1h,6h,12h,24h,48h,72h after injury), testing LDH content and absorption values of the viable cells using MTT colorimetric. morphological changes of neurons in each group was observed under inverted phase contrast microscope at different times and repeated measures analysis of variance was used for statistical analysis.
Results
1. No macrophage infiltration was seen in the spinal cord of the sham-operated group. Double immunofluorescence labeling of the injured spine in the SCI group showed that NgR positive cells were also CD68 positive macrophages. In addition, immunofluorescence of the tissue sections showed that NgR was on surfaces of these cells, indicating that NgR was expressed in cell surface. To confirm that macrophages expressed NgR, the injured spinal cord at day 7 was isolated and labeled NgR antibody. The results showed that NgR was expressed on cell surfaces in a spotty manner.
2. The result of sequencing showed that the sequence of the cloned NgR gene was consistent with that reported in the Gen Bank. The plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultercentrifugation, and the titer of the replication-defective LV vector particles measured was found to be 6.7×107 TU/mL. After the constructed LV vectors infected the macrophages, the results obtained using RT-PCR showed that the expression of NgR in the NgR-macrophages group(experimental group)was significantly higher than that in the mock group and the macrophages group(control group)at both mRNA and protein level. LV vectors carrying the NgR gene were constructed successfully. The NgR gene-modified macrophages could express NgR to a higher degree.
3. The MBP content in NgR-macrophages group was significantly higher than that in the mock group and the normal macrophages group(P<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05).
4. There was no significant change in optical density (OD) value at 0.5,1,6 and 12 h after culture in each group, and at 24 h a significant difference of decrease was observed in OD value between the injured nerve+NgR-macrophages group and the other groups(p<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05). There was also no significant difference between the NgR-macrophages group and the normal neuron group (P>0.05). With the lapse of time, LDH level decreased markedly in the injured nerve groups, while it did not decrease significantly in the NgR-macrophages group. There was no significant difference of LDH contents decrease in each group after co-culture for 0.5,1,6 and 12h. But 24 h later, LDH contents decreased markedly in the injured nerve+NgR-macrophages group as compared with that of the other groups (p<0.05). There was nos significant difference between the mock group and the normal macrophages group (P>0.05). There was no significant difference between the NgR-macrophages group and the normal nerve group.
Conclusion
The spinal cord injury model was constructed successful. The expression of NgRs on macrophages infiltrating in the injury spinal cord was confirmed From the histological and cellular level.The lentiviral vector of rat NgR gene was successfully constructed and transfected macrophages expression NgRs. This lays the foundation for treatment of spinal cord injury by transplantation of genetically modified macrophages.Macrophages expression NgRs in vitro can promote repairment of the injured neurons. The mechanism may be that macrophages regulate phagocytosis by intracellular signaling pathways through expression NgRs, thus creating conditions for nerve regeneration. Signal transduction pathway mediated by by Nogo receptors might be the key, which could conduct the process of phagocytosis in myelin pieces elimination and be beneficial for neural regeneration.
髓鞘碎片中的某些特异性成分(比如：Nogo66、髓鞘相关糖蛋白MAG、少突胶质细胞髓鞘糖蛋白OMgp等)作用于轴突上的Nogo受体(NgR)及其共受体p75NTR复合物,通过小鸟嘌呤核苷三磷酸酶(small GTPase) Rho途径调节轴突内细胞骨架结构抑制轴突生长,从而使脊髓损伤修复难以获得满意结果。Nogo单克隆抗体IN-1可以拮抗Nogo的抑制作用,但无法与另外两种抑制物MAG和OMgp结合。GrandPre等尝试以竞争性短肽NEPI-40来拮抗NgR的作用,结果显示NEP1-40可对抗Nogo-66诱导的轴突生长抑制,但对除Nogo-66外的其他髓鞘抑制物不敏感,使其在体内应用的效果并不十分理想,表明该短肽并不能封闭NgR所有与抑制分子结合的位点。因此,找到一种有效且靶向性强的髓鞘碎片清理方法成为当务之急。
目前,人们对巨噬细胞在中枢神经系统损伤修复中的作用存在着两种截然相反的观点,一种认为巨噬细胞对轴突生长有抑制作用；而另一种则认为巨噬细胞对轴突的再生起到促进作用。在整个机体组织的修复过程中,炎性反应贯穿始终,而炎性细胞中,巨噬细胞发挥了主要作用,它能清除坏死组织并分泌营养因子,并可在机体组织修复中起着重要作用。Mantovani等认为,巨噬细胞受炎性反应中微环境不同信号影响,可转变为“选择”活化型和“经典”活化型两种。“选择”活化型巨噬细胞由IL-4和糖皮质激素诱导转换而来,可调整免疫炎性反应的过程,清除坏死组织的残片,促进血管再生组织修复和重建；“经典”活化型巨噬细胞可分泌前炎性细胞因子,加重炎症反应,对受损局部细胞造成更进一步的损害。近年来,对于巨噬细胞在脊髓损伤致伤及修复作用报道并不多见。
新近研究表明巨噬细胞在髓鞘碎片吞噬、促进轴突再生及再髓鞘化过程中发挥的作用可能比我们预想的更大。
1998年,Zeev-Brann报道成熟哺乳动物中枢神经系统损伤后的自我修复中出现脊髓衍生炎性细胞,Buss与Schwab等研究发现SCI大鼠髓鞘碎片清理细胞ED-1免疫组织化学染色呈阳性反应,即为小胶质细胞与巨噬细胞。MASAAKI通过鼠脊髓挫伤模型(代表挫伤引起的有髓鞘碎片和髓鞘再生受抑制)与化学性脱髓鞘模型(代表早期髓鞘碎片清除和髓鞘再生的多发性硬化症)的对比,研究了巨噬细胞聚集及其作用,作者使用绿色荧光蛋白转基因小鼠的骨髓细胞分别移植到经放射线照射的脊髓挫伤模型鼠与化学性脱髓鞘模型鼠尾静脉,通过损伤脊髓免疫组化染色研究活化巨噬细胞的浸润和残留髓鞘碎片的不同水平变化,结果显示表达绿色荧光蛋白的活化巨噬细胞构成损伤局部主要细胞群,表明两个模型中的巨噬细胞主要来源于骨髓,很少来源于内在的小胶质细胞。在免疫染色显示在挫伤模型当中,髓鞘碎片长时间持续存在,并且活化巨噬细胞的浸润在挫伤模型中比化学模型中更慢并且数目显著减少,同时伤后2～4天RT-PCR检测趋化因子(MCP-1, GM-CSF)低水平表达,提示活化巨噬细胞浸润的延迟与挫伤性脊髓损伤后髓鞘碎片的持续存在有关,且导致髓鞘再生抑制。
Samuel David等研究了Nogo受体(NgRs)在周围神经损伤中的作用。研究人员损伤鼠坐骨神经,他们发现,一旦巨噬细胞到达受损部位并开始清除工作,它们就表现为表面表达NgR1的活化形式,并进入施旺细胞细胞基底部。当恢复的神经开始产生新的蛋白质髓鞘(表达NgR配体如MAG等),这种受体不但抑制巨噬细胞与髓鞘的结合,还会直接抑制髓鞘的形成,当研究人员再次损伤神经,使髓鞘不能形成时,巨噬细胞继续留在受损神经外层施旺细胞细胞基底部吞噬髓鞘碎片。然而,NgRl基因敲除鼠坐骨神经或使用Y-27632抑制NgR下游Rho相关激酶,可增加巨噬细胞与髓鞘的结合率,证实巨噬细胞表达的NgR参与介导该细胞在周围神经损伤修复中的撤离过程。有关中枢神经系统损伤中巨噬细胞是否表达NgR及二者在中枢神经系统尤其是脊髓损伤修复中的作用有待进一步研究。
脊髓损伤后,活化的巨噬细胞浸润脊髓损伤区,髓鞘碎片的清除延缓导致了轴突延伸的抑制,和由于星形胶质细胞的堆积形成的瘢痕导致了轴突再生的抑制。我们推测,成年哺乳动物脊髓损伤后,活化巨噬细胞迅速清除髓鞘碎片对中枢神经系统的再生与对周围神经系统的再生具有同样重要的意义,但对于巨噬细胞在脊髓损伤中的具体作用仍需深入研究。
研究目的
制备大鼠脊髓损伤模型,分离、培养、鉴定大鼠脊髓损伤局部浸润的巨噬细胞,观察其是否表达NgR；构建大鼠NgR基因慢病毒载体并转染巨噬细胞表达NgR,探讨转染免疫细胞的可行性；比较NgR的表达对巨噬细胞吞噬功能的影响；体外构建神经元损伤模型,将表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养,比较各组巨噬细胞对损伤神经元修复、存活、分化的影响,探讨表达NgRs的巨噬细胞在脊髓损伤修复中的可能作用机制,为进一步动物研究及临床应用提供实验和理论依据。
内容和方法
(一)制备大鼠脊髓损伤模型,第3天及第7天时取材,使用免疫荧光化学染色观察巨噬细胞浸润及NgRs的表达情况。用酶消化法分离,获取损伤脊髓组织局部浸润的巨噬细胞,使用双标免疫荧光化学染色从细胞水平观察巨噬细胞表达NgRs情况；
(二)采用RT-PCR技术获得大鼠NgR基因编码区片段,限制性内切酶酶切和基因重组构建慢病毒载体质粒,在脂质体介导下包装质粒,包膜质粒共转染293T细胞包装生产慢病毒。所获慢病毒感染大鼠巨噬细胞后,PCR检测巨噬细胞中NgR基因的插入和表达。
(三)将髓鞘碱性蛋白(MBP)加入转染NgRs的巨噬细胞、空载组巨噬细胞和未转染NgRs的巨噬细胞培养皿中,Western blot定量检测巨噬细胞吞噬的MBP量,行t检验(Student's t-test),取α=0.05。
(四)制备体外神经元损伤模型,将表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养,各组分别于损伤后30min、1h、6h、12h、24h、48h、72h各时间点提取培养液100 u L,检测LDH含量并采用MTT比色法测量其存活细胞的吸收值。倒置相差显微镜下观察各组不同时间神经元的形态变化,采用重复测量数据方差分析进行统计学分析。
研究结果
(一)在假手术组中,脊髓组织中没有浸润的巨噬细胞；免疫荧光染色显示脊髓损伤3天后浸润的巨噬细胞表面可表达NgR,伤后7天时表达NgR的巨噬细胞的数量明显增多。培养脊髓损伤局部浸润的巨噬细胞,免疫荧光染色显示该细胞NgRs抗原染色阳性；
(二)所获的NgR基因经测序后与Gen Bank报道序列完全一致。重组慢病毒载体质粒经鉴定正确。三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为6.7×107Tu/mL, PCR证实NgR基因插入病毒基因组。感染巨噬细胞后RT-PCR检测试验组NgR-巨噬细胞组更大量表达NgR,与其余2组(Mock-巨噬细胞组、巨噬细胞组)比较差异具有统计学意义；
(三)NgR转染巨噬细胞组的MBP含量比非转染组MBP含量明显增加,差异有显著统计学意义(P<0.05),空载组与非转染组无明显的差异(P>0.05)；
(四)表达NgR的巨噬细胞、空载组巨噬细胞及不表达NgR的普通巨噬细胞与损伤的神经元细胞共同培养0.5,1,6,12小时后,各组OD值没明显不同。24h后,神经损伤+NgR转染组比其他组有明显的差异(P<0.05),空载组与非转染组没明显的差异(P>0.05),NgR转染组与正常神经组无明显差别(P>0.05)。随时间延长,LDH水平在损伤神经组中下降明显但在NgR转染组下降不明显；培养0.5,1,6,12h后,各组OD值没明显差别；24h后,神经损伤+NgR转染组与其他组比较有明显的差异(P>0.05),空载组与非转染组没明显的差异(P>0.05),NgR转染组与正常神经组无明显差别(P>0.05)。
实验结论
成功制备脊髓损伤模型,从组织学和细胞水平证实损伤脊髓组织局部浸润的巨噬细胞表面有NgRs的表达；成功构建大鼠NgR基因慢病毒载体并转染巨噬细胞表达NgRs,为今后移植基因修饰的巨噬细胞治疗脊髓损伤奠定了基础；表达NgRs的巨噬细胞体外能促进损伤神经元的修复,其机制可能是巨噬细胞通过表达的NgRs介导胞内信号途径调节巨噬细胞的吞噬功能,从而为神经再生创造条件。这为脊髓损伤的治疗研究提供了一个新的解决途径,也为进一步动物研究和临床应用奠定了可靠的实验基础和理论依据。
Back ground
Ax on regeneration and formation of functional synapses with target cells are the main objectives and key issues in repair of spinal cord injury. Large numbers of basic studies on promotion of axon regeneration have shown that failure of early regeneration of the central nervous system in adult mammalians is due to the presence of myelin inhibitors.
We have found in previous experiments that neural stem cells(or) olfactory ensheathing cells could divide into neurons and other glial cells after transplanted into the damaged spinal cord. The nerve synapse formation increased and BBB motor function score improved in all rats with spinal cord injury. Although a lot of damage nerve regeneration and (or) budding (to be published data) was observed in experiments, the distance axonal regeneration or sprouting is extremely limited as the damage to local micro-environment changed.It is difficult to break through the barrier between the myelin-inhibitory factor and glial scar to format function synapses with the distal stump.
Some specific components of the myelin debris (eg:Nogo66, myelin-associated glycoprotein MAG, myelin oligodendrocyte glycoprotein OMgp, etc.) act on the axon on the Nogo receptor (NgR) and its acceptor-p75NTR complex to regulate cytoskeletal structure of axons within the axon growth inhibition through the small guanosine triphosphatase (small GTPase) Rho way.Thus,it is difficult to obtain satisfactory results of spinal cord injury repair. Nogo monoclonal antibody IN-1 can antagonize with Nogo protein, but not with the other two inhibitors MAG and Omgp. Grand Pre used the competing peptide NEPI-40 to antagonize the role of NgR and showed that NEP1-40 may be against the Nogo-66-induced axon growth inhibition, but not sensitive to other myelin inhibitors. This peptide can not be closed all inhibitory molecule binding site of NgR and the effects in vivo is not very satisfactory. Therefore, most urgent task is to find an effective and highly targeted method to clear myelin debris.
At present, there are two opponent views on the role of macrophages in the central nervous system injury. One is that macrophages can inhibit the growth of axons; other is that of macrophages play a catalytic role to the axon regeneration. The inflammatory response appeared through repair process of the tissue. Macrophages play a major role and can remove necrotic tissue and secrete neurotrophic factor, in all the body plays an important role in tissue repair. Mantovani believed that the macrophagecan can chang to "select" activation-type and the "classic" activation of two kinds impacting by the different signals in micro-environment of the inflammatory response. The "select"-activated macrophages are conversed by IL-4 and glucocorticoid-induced and adjust the immune inflammatory reaction process, the debris removal of necrotic tissue, promoting angiogenesis, tissue repair and reconstruction. The "classic"-activated macrophages may secrete pro-inflammatory cytokines and increase the inflammatory reaction, causing further damage to local cell. In recent years, there are rare reports of macrophages in the injured spinal cord injury.
Recent studies have shown that the role of macrophage in phagocytosis of myelin debris and axonal regeneration may be greater than we anticipated.
In 1998, Zeev-Brann reported that there were spinal cord-derived inflammatory cells appearing in mature mammalian central nervous system after injury. Buss and Schwab found that the ED-1 immunohistochemistry staining positive cells were microglia cells and macrophages which cleaned up myelin debris in SCI rats. MASAAKI researched macrophages and its role through the comparement of the rat spinal cord contusion model (on behalf of contusion caused by a myelin debris and myelin regeneration was inhibited) and the chemical demyelination models (on behalf of the early removal of myelin debris and myelin regeneration in multiple sclerosis).
The arthors transplanted green fluorescent protein transgenic mouse bone marrow cells into rat spinal cord contusion model irradiated by the radiation and chemical demyelination models.They studied the infiltration of activated macrophages and myelin debris residual changes in the different levels by immunohistochemical staining of spinal cord. The result showed activated macrophages expressing green fluorescent protein constituted the major cell groups in the damage location, indicating macrophages in the two models primary derived from bone marrow and rarely from intrinsic microglia. The immunohistochemical staining showed myelin debris existed long-time in the contusion model and the activated macrophage infiltration in the contusion model more slowly than that in the chemical model, the number reduction significantly. RT-PCR detection of trend chemokine (MCP-1, GM-CSF) expressed a low level 2 to 4 days after injury, suggesting that the delay infiltration of activation of macrophages post-traumatic spinal cord related to the persistence of myelin debris and lead to remyelination suppression.
Samuel et al [5] studied Nogo receptor (NgRs) in peripheral nerve injury. They induced damage in the sciatic nerve in the thigh of rats and found that macrophages, once arriving and starting to remove myelin debris, they performed for expression of NgR1 activated forms and went into the base of Schwann cells. When the restoration of the nerve start to produce new proteins of myelin (such as NgR ligands), this receptor not only inhibits macrophages with a combination of myelin, but also directly inhibits the formation of myelin. When the researchers damaged the nerve again to inhibit the formation myelin, the macrophages remained in the outer layer of Schwann cells and swallowed myelin debris. However, NgR1 knockout mice or use of Y-27632 to inhibit NgR downstream Rho-associated kinase could increase the combination rate of macrophages and myelin debris, indicating that NgR expression in macrophages participated in the withdrawal process at the end of repair in peripheral nerve injury. But whether macrophages express NgR after CNS injury and what role NgR expression plays in CNS repair remain unclear.
After spinal cord injury, activated macrophages infiltrated to injury areas and the delay of myelin debris clearance inhibit of axon extension. The formation of scar due to the accumulation of astrocytes led to inhibition of axonal regeneration. We speculate that the adult mammalian spinal cord injury, the rapid removal of myelin debris by activated macrophages on the central nervous system regeneration has the same importance as that in the peripheral nervous system, but the specific role of macrophage cells in spinal cord injury still need study.
Objective
To construct the spinal cord injury model and to investigate the isolation, culture methods and evaluation of macrophages expressed with NgRs in vitro in preliminary and reveal the biological characteristics of the cells. To construct the lentiviral vector of rat NgR gene and to transfect macrophages expressing NgR, to explore the feasibility of immune cells transfecting. To compare the effect of NgR expression on macrophage phagocytosis; To construct neuronal injury model in vitro and culture it with NgR-macrophages group, the mock group and the normal macrophage group together, to compare the effect of each group on the repairment, survival, differentiation of the injuried neuron. To explore the possible mechanisms of NgRs expression in macrophages on the spinal cord injury repairment and to give the experimental and theoretical basis for further animal studies and clinical applications.
Methods
1. To construct the spinal cord injury model and draw the materials at the day 3 and 7. To observe the infiltration of macrophages and expression of NgRs by immunofluorescence staining. Primary macrophages were harvested from injured spinal cord and digested with trypsin, and the morphological, biological characteristics and the NgRs antigen positive cells with methods of immunochemistry staining were observed.
2. RT-PCR technique was used to obtain gene-coding region of rat NgR fragments, digestion using restriction enzyme. Construction of gene recombination lentiviral vector plasmid and packaging plasmid mediated by the liposome. The envelope plasmid co-transfected 293T cells and the lentivirus producted. Infection the rat macrophages using the lentivirus and the insertion and expression of NgR gene were detected by PCR.
3. Myelin basic protein (MBP) was added into macrophages (NgR-macrophages group, the mock group and the normal macrophages group) and cultured, and the phagocytic capacity was measured by Western blot. Student's t-test was used for statistical analysis.
4. The neuronal injury model was constructed. Macrophages of different groups were co-cultured with the injured nerve plates. 100μL culture medium in each group was extracted respectively at different time points (30min, 1h,6h,12h,24h,48h,72h after injury), testing LDH content and absorption values of the viable cells using MTT colorimetric. morphological changes of neurons in each group was observed under inverted phase contrast microscope at different times and repeated measures analysis of variance was used for statistical analysis.
Results
1. No macrophage infiltration was seen in the spinal cord of the sham-operated group. Double immunofluorescence labeling of the injured spine in the SCI group showed that NgR positive cells were also CD68 positive macrophages. In addition, immunofluorescence of the tissue sections showed that NgR was on surfaces of these cells, indicating that NgR was expressed in cell surface. To confirm that macrophages expressed NgR, the injured spinal cord at day 7 was isolated and labeled NgR antibody. The results showed that NgR was expressed on cell surfaces in a spotty manner.
2. The result of sequencing showed that the sequence of the cloned NgR gene was consistent with that reported in the Gen Bank. The plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultercentrifugation, and the titer of the replication-defective LV vector particles measured was found to be 6.7×107 TU/mL. After the constructed LV vectors infected the macrophages, the results obtained using RT-PCR showed that the expression of NgR in the NgR-macrophages group(experimental group)was significantly higher than that in the mock group and the macrophages group(control group)at both mRNA and protein level. LV vectors carrying the NgR gene were constructed successfully. The NgR gene-modified macrophages could express NgR to a higher degree.
3. The MBP content in NgR-macrophages group was significantly higher than that in the mock group and the normal macrophages group(P<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05).
4. There was no significant change in optical density (OD) value at 0.5,1,6 and 12 h after culture in each group, and at 24 h a significant difference of decrease was observed in OD value between the injured nerve+NgR-macrophages group and the other groups(p<0.05). There was no significant difference between the mock group and the normal macrophages group(P>0.05). There was also no significant difference between the NgR-macrophages group and the normal neuron group (P>0.05). With the lapse of time, LDH level decreased markedly in the injured nerve groups, while it did not decrease significantly in the NgR-macrophages group. There was no significant difference of LDH contents decrease in each group after co-culture for 0.5,1,6 and 12h. But 24 h later, LDH contents decreased markedly in the injured nerve+NgR-macrophages group as compared with that of the other groups (p<0.05). There was nos significant difference between the mock group and the normal macrophages group (P>0.05). There was no significant difference between the NgR-macrophages group and the normal nerve group.
Conclusion
The spinal cord injury model was constructed successful. The expression of NgRs on macrophages infiltrating in the injury spinal cord was confirmed From the histological and cellular level.The lentiviral vector of rat NgR gene was successfully constructed and transfected macrophages expression NgRs. This lays the foundation for treatment of spinal cord injury by transplantation of genetically modified macrophages.Macrophages expression NgRs in vitro can promote repairment of the injured neurons. The mechanism may be that macrophages regulate phagocytosis by intracellular signaling pathways through expression NgRs, thus creating conditions for nerve regeneration. Signal transduction pathway mediated by by Nogo receptors might be the key, which could conduct the process of phagocytosis in myelin pieces elimination and be beneficial for neural regeneration.
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6. Elizabeth J. Fry, Carole Ho, and Samuel David. A Role for Nogo Receptor in Macrophage Clearance from Injured Peripheral Nerve[J]. Neuron,2007,53,649-662.
7 Samuel David, Elizabeth J.Fry, et al. Novel roles for Nogo receptor in inflammation and disease. Trends in Neurosciences 2008; 5(31); 221-226.
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42. Su, Z. et al. Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration. J. Cell Sci.2007,120,1877-1887
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44. Lu, P. et al. Olfactory ensheathing cells do not exhibit unique migratory or axonal growth-promoting properties after spinal cord injury. J. Neurosci.2006,26,11120-11130
2. Elizabeth J. Fry, Carole Ho, and Samuel David. A Role for Nogo Receptor in Macrophage Clearance
from Injured Peripheral Nerve[J]. Neuron,2007,53,649-662.
3. Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66inhibitionof axonal regeneration[J]. Nature,2001,409(6818):341-346.
4. Venkatesh, K. et al. The Nogo-66 receptor homolog NgR2 is a sialic acid-dependent receptor selective for myelin-associated glycoprotein. [J]. Neurosci.2005,25,808-822.
5. Shao Z, Browning JL, Lee X, et al. TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration[J]. Neuron,2005,45(3):353-359.
6.杨明,唐洪涛,鞠学红.中枢神经系统中Nogo与Nogo-R的研究进展[J]。医学综述,2008,30(1)：65-67.
7.李莹,张薇薇.中枢神经系统轴突再生抑制蛋白及其信号转导通路[J]。神经损伤与功能重建,2008,3(4)：281-284.
8. Yamagishi S, Fujitani M, Hata K, et al. Wallerian degeneration involves Rho/Rho-kinase signaling[J]. J Biol Chem,2005,280(21):20384-20388.
9. Madura T, Yamashita T, Kuho T, et al. Activation of Rho in the injured axons following spinal cord injury[J]. EMBO Rep,2004,5(4):412-417.
10. Aepfelbacher, M. et al. Rho is a negative regulator of human monocyte spreading[J]. Immunol. 1996,157,5070-5075.
11. MASAAKI IMAI,1 MASAHIKO WATANABE, KAORI SUYAMA, et al. Delayed accumulation of activated macrophages and inhibition of remyelination after spinal cord injury in an adult rodent model. J Neurosurg Spine 2008,8(1):58-66
12. Samuel David, Elizabeth J. Fry.et al. Novel roles for Nogo receptor in inflammation and disease[J].Trends in Neurosci.2008,31(5):221-226.
13. Stoll, G. et al. Degeneration and regeneration of the peripheral nervous system:from Augustus Waller's observations to neuroinflammation. J. Peripher. Nerv. Syst.2002,7,13-27
14. Perrin, F.E. et al. Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1a and interleukin-1b in Wallerian degeneration. Brain,2005,128,854-866
15. Buss, A. et al. Sequential loss of myelin proteins during Wallerian degeneration in the human spinal cord. Brain,2005,128,356-364
16. Sroga, J.M. et al. Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. J. Comp. Neurol.2003,462,223-240
17. Fleming, J.C. et al. The cellular inflammatory response in human spinal cords after injury. Brain,2006,129,3249-3269
18. Jones, T.B. et al. Inflammatory-mediated injury and repair in the traumatically injured spinal cord. Curr. Pharm. Des.2005,11,1223-1236
19. Dimou, L. et al. Nogo-A-deficient mice reveal strain-dependent differences in axonal regeneration. J. Neurosci.2006,26,5591-5603
20. Schwab, M.E. Nogo and axon regeneration. Curr. Opin. Neurobiol.2004,14,118-124
21. Ji, B. et al. Effect of combined treatment with methylprednisolone and soluble Nogo-66 receptor after rat spinal cord injury. Eur. J. Neurosci.2005,22,587-594
22. Lopez-Vales, R. et al. FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat. J. Neurosci. Res.2005,81,827-836
23. Su, Z. et al. Nogo enhances the adhesion of olfactory ensheathing cells and inhibits their migration. J. Cell Sci.2007,120,1877-1887
24. Raisman, G. and Li, Y. Repair of neural pathways by olfactory ensheathing cells. Nat. Rev. Neurosci. 2007,8,312-319
25. Lu, P. et al. Olfactory ensheathing cells do not exhibit unique migratory or axonal growth-promoting properties after spinal cord injury. J. Neurosci.2006,26,11120-11130
1 Samuel David, Elizabeth J.Fry, et al. Novel roles for Nogo receptor in inflammation and disease. Trends in Neurosciences 2008; 5(31); 221-226.
2 Wong ST, Henley JR, Kanning KC, et al. A p75(NTR)and Nogo receptor complex med iates repulsive signaling by myelin-associated glycoprotein[J]. Nat Neurosci 2002,5(12):1302-1308.
3. Madura T, Yamashita T, Kubo T, et al. Activation of Rho in the injured axons following spinal cord injury[J]. EMBO Rep,2004,5(4):412-417.
4. Satoh J, Tabunoki H, Yarnamura T, et al. TROY and LINGO-1 expression in astrocytes and macropheges /microglia in multiple sclerosis lesions[J]. Neuropathol Appl Neurobiol,2007,33(1):99-107.
5. Park JB, Yiu G, Kaneko S, et al. A TNF receptor family member, TROY, is fl coreceptor with Nogo receptor in med iating the inhibitory activity of myelin inhibitors[J]. Neuron,2005,45(3):345-351.
6. Elizabeth J. Fry, Carole Ho, and Samuel David. A Role for Nogo Receptor in Macrophage Clearance from Injured Peripheral Nerve[J]. Neuron,2007,53,649-662.
7 Samuel David, Elizabeth J.Fry, et al. Novel roles for Nogo receptor in inflammation and disease. Trends in Neurosciences 2008; 5(31); 221-226.
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