LINGO-1多克隆抗体的制备及其被动免疫治疗大鼠急性脊髓损伤的实验研究
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摘要
研究背景
脊髓损伤(spinal cord injury, SCI)后神经组织自我修复功能极其有限,其最主要的原因是由于崩解的脊髓组织释放出大量的轴突生长抑制因子,形成不利于轴突再生的微环境。目前研究表明,许多轴突生长抑制因子都是通过与神经元细胞膜表面的NgR受体复合体(NgR/p75/LINGO-1及NgR/TAJ(TROY) /LINGO-1)结合后,激活胞内RhoA信号,从而导致生长锥溃变及轴突再生失败。LINGO-1 (LRR and Ig domain-containing, Nogo Receptor-interacting protein, LINGO-1)是构成NgR受体复合体的重要组成部分,同时,其亦存在于少突胶质细胞,在分化、损伤后细胞存活及再髓鞘化等过程中起着负性调节的作用。因此,LINGO-1可作为对抗轴突生长抑制因子抑制作用并促进再生轴突髓鞘化的药物靶点。
一些研究者给予SCI动物主动免疫轴突生长抑制因子产生相应的中和性抗体后,均能达到阻断抑制因子、改善损伤灶局部微环境、促进神经再生和功能恢复的目的。但即使损伤后立即给予主动免疫治疗也至少需要4～6w的时间才能产生保护性抗体。因此,探索早期被动免疫手段促进SCI后功能恢复更适应临床实际需要。
我们的设想是在脊髓损伤后早期,应用LINGO-1特异性抗体阻断损伤处该分子的生物学作用,应当可以削弱下游的轴突抑制信号,阻止损伤局部的神经元凋亡,达到促进轴突再生及神经功能恢复的目的。抗体选择的方面,单克隆抗体有着高度的特异性和优越的质量控制,是抗体治疗的首选,但是其制备周期长、技术要求较高,因此成本相对高昂。而必需明确的首要问题是,阻断靶分子是否能收获有益的治疗效果。因此,基于有效性和经济性的考虑,本研究采用经典的多克隆抗体制备方法获取的足量、高滴度的抗血清作为后续动物实验的干预手段。
本课题通过实验研究初步观察了LINGO-1多克隆抗体对脊髓半横断大鼠模型的早期干预作用,为促进SCI后功能修复提供新的思路。研究内容主要分为以下三个部分：(1) LINGO-1抗血清的制备及鉴定；(2) LINGO-1抗血清对脊髓半横断大鼠模型干预的可行性分析；(3)观察LINGO-1抗血清对脊髓半横断大鼠模型的神经再生及功能恢复的影响。
第一部分：LINGO-1多克隆抗体的制备及鉴定
目的：
表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体。
方法：
1.PCR扩增LINGO-1的76-319位氨基酸编码序列cDNA:根据hLINGO-1aa76-319的cDNA序列设计特异引物,上下游引物的5’端分别引入BamHⅠ、EcoRⅠ酶切位点及保护碱基,以pCMV-SPORT6-LINGO-1质粒为模板,高保真Taq酶作用下PCR扩增hLINGO-1aa76-319 cDNA片段。
2. pET30a(+)-hLINGO-1aa76-319原核表达载体的构建：hLINGO-1aa76-319 cDNA的PCR产物及pET30a(+)载体分别用BamHⅠ、EcoR、双酶切后回收后进行体外连接,转化DH5a感受态细菌,挑取单克隆菌落,摇菌,提取质粒。pET30a(+)-hLINGO-1aa76-319重组质粒经PCR及酶切鉴定后,行核苷酸序列测定和分析。
3.hLINGO-1aa76-319融合蛋白诱导表达条件的优化：将pET30a(+)-hLINGO-1aa76-319重组质粒转化入BL21感受态细菌,挑取菌落PCR阳性的单菌落接种于卡那抗性的LB培养基中,培养至OD为0.6～0.8时分别加入不同终浓度的IPTG诱导,在不同时间点分别收集样本行SDS-PAGE电泳,考马斯亮蓝染色显示蛋白条带,分析确定最佳诱导表达条件。取最高蛋白表达量的菌液适量离心,沉淀重悬于裂菌缓冲液,冰浴超声裂解,分别取上清、沉淀行SDS-PAGE电泳及Western blot检测,以明确目的蛋白在宿主菌中的表达形式并验证融合蛋白是否正确表达。
4.hLINGO-1aa76-319融合蛋白大量表达后的纯化：优化表达条件下,大量培养1000mL菌液,离心后收集菌体沉淀,裂解缓冲液重悬细菌,冰浴后超声破碎,离心获取包涵体,脲溶解后经Ni-NTA螯合树脂层析洗脱回收LINGO-1蛋白。BCA法测定蛋白浓度,SDS-PAGE电泳结果经Bandscan5.0图像分析软件计算蛋白纯度。为了获得纯度更高的蛋白,将全部蛋白初样经SDS-PAGE电泳分离后,切取目的蛋白胶块-80℃冻存。
5.抗hLINGO-1aa76-319多克隆抗体的制备：冻存过夜的蛋白胶块加适量弗氏佐剂研磨。充分乳化后,家兔背部多点皮下注射。每隔7d进行一次加强免疫,第3次免疫注射后的第10d微量取血,4℃冰箱过夜,离心获得血清。以免疫前兔血清作为阴性对照,ELISA测定血清效价。当抗体效价达到预期水平时即可放血收获抗血清,-80℃冰箱保存。
6.抗血清特异性鉴定及效价验证：表达hLINGO-1aa76-319融合蛋白的BL21菌体总蛋白及提取的大鼠脑组织膜蛋白进行SDS-PAGE电泳,制备的LINGO-1多克隆抗体为一抗,Western blot方法验证抗体的特异性。用纯化后hLINGO-1aa76-319融合蛋白包被ELISA反应孔,以免疫前兔血清作为阴性对照,ELISA法检测LINGO-1抗血清的效价。
结果：
1.PCR扩增LINGO-1的76-319位氨基酸编码序列cDNA:PCR产物经琼脂糖凝胶电泳,紫外灯下可见一约732bp条带,与目的片段的大小相符。
2. pET30a(+)-hLINGO-1aa76-319原核表达载体的构建：将hLINGO-1aa76-319PCR扩增产物双酶切后定向插入原核表达质粒pET30a(+)中,提取筛选的重组质粒DNA, PCR反应扩增目的片段,BamHⅠ和EcoRⅠ双酶切鉴定,可得到约5.4kbp及732bp两条带,目的片段大小与预期相符。测序结果显示目标序列与GeneBank(序列号为NM032808)相应序列完全吻合,证实表达载体构建成功,命名为pET30a(+)-hLINGO-1aa76-319。
3.hLINGO-1aa76-319融合蛋白诱导表达条件的优化：重组质粒转化后PCR阳性菌株经IPTG诱导,收集细菌蛋白。SDS-PAGE电泳显示,在37℃、0.4mM IPTG诱导1.5 h的条件下,融合蛋白高表达,经抗His标签的单克隆抗体(monoclonalantibody, mAb)行Western blot鉴定证实该蛋白条带为hLINGO-1aa76-319融合蛋白；菌体沉淀经超声破碎后,上清及沉淀的SDS-PAGE电泳结果进一步证明,该重组蛋白主要以包涵体形式存在。
4.hLINGO-1aa76-319融合蛋白大量表达后的纯化：按步骤三的优化表达条件扩大培养体系,收获包涵体蛋白,纯化后回收重组融合蛋白。BCA法测定蛋白的浓度为4600mg/L,蛋白的纯度为70.8%。
5.抗hLINGO-1aa76-319pAb的制备：免疫前,对动物多次行耳缘静脉滴血法收集162ml血液,获得52ml的对照血清。整个免疫过程中,家兔摄食正常,健康状况良好,无明显不良反应。第3次免疫注射后的10d经耳缘静脉微量取血,ELISA测定血清效价均高于1：312500。4免后,颈动脉采血法收获血液共197ml,制备抗血清61ml,收获的对照血清及抗血清均经过0.22μm滤膜过滤,-80℃冰箱保存。
6. LINGO-1抗血清的效价及特异性鉴定：经ELISA法检测,收获的抗hLINGO-1aa76-319抗血清的效价为1：1562500。表达hLINGO-1aa76-319融合蛋白的BL21菌体总蛋白及提取的大鼠脑组织膜蛋白经Western blot检测,纯化后的抗hLINGO-1aa76-319多克隆抗体为一抗,显色蛋白条带与预期结果吻合,无明显非特异性着色。结果证实所制备的多抗滴度高,特异性良好。
结论：
制备的抗LINGO-1多克隆抗体具备良好的特异性和较高的抗体效价,为进一步研究LINGO-1蛋白的生物学功能奠定了重要的实验基础。
第二部分：LINGO-1多克隆抗体的组织渗透性及其与抗原特异性结合能力分析
目的：
探讨脊髓损伤处局部给予的LINGO-1多克隆抗体的组织渗透性及与抗原的特异性结合能力,为后续的被动免疫治疗研究提供依据。
方法：
将12只成年雌性SD大鼠随机分为正常组、对照组(半横断对照血清组)和实验组(半横断抗血清组)。正常组大鼠不做任何处理,另外两组均行T9脊髓背侧半横断术。半横断术后,使用微量渗透泵分别给予对照血清和LINGO-1抗血清。术后3d和28d取各组T8～T10节段脊髓作冰冻切片,应用间接和直接组织免疫荧光染色法,以两独立样本的t检验进行统计学分析,探讨LINGO-1多抗可否进入脊髓组织并是否与LINGO-1分子(抗原)特异性结合。
结果：
造模大鼠术后清醒时,双侧下肢完全瘫痪,肌张力低,证明模型制作成功。术后3d,正常组兔源性抗体免疫荧光染色阴性,对照组和实验组SCI处可检测出兔源性抗体；术后28d,对照组和实验组SCI处及周围组织仍可以检测到兔源性抗体。
术后3d,对照组切片LINGO-1染色强度明显强于实验组(t=26.491,P=0.000)。多抗预处理后的对照组切片LINGO-1染色强度明显弱于未预处理的对照组切片(t=31.879,P=0.000)。
结论：
成功建立脊髓背侧半横断模型,局部应用抗血清后能有效渗透到损伤的脊髓组织中,并能与脊髓组织中的抗原特异性结合；此外,在脊髓损伤区伤后3-28天的时间窗内均可检测到LINGO-1多克隆抗体,证实该多抗可在损伤局部较长的时间内发挥被动免疫的作用。
第三部分：LINGO-1多克隆抗体对脊髓半横断模型大鼠的被动免疫治疗效果评估
目的：
评价脊髓损伤处局部给予LINGO-1多克隆抗体的治疗效果。
方法：
将49只成年雌性SD大鼠分为正常组(3只)、半横断对照血清组(23只)及半横断抗血清组(23只)。正常组大鼠不做任何处理,另外两组均行T9脊髓半横断术。术后,两组使用微量渗透泵鞘内分别局部给予对照血清和LINGO-1抗血清。术后3d,利用蛋白pull-down技术及Western blot检测方法对各组脊髓损伤处组织行RhoA活性分析,组织免疫荧光双标法检测各组神经元凋亡情况。术后14d,立体定向注射BDA于右侧的感觉运动皮层顺行示踪CST纤维。术后28d,处死动物,收集脊髓损伤处横切和纵切,行BDA免疫组织化学染色。术后1d及每周,对各组动物盲法行BBB行为学评分。活化RhoA蛋白量采用单因素ANOVA检验及LSD多重比较,凋亡神经元计数采用独立样本t检验,最大再生轴突长度采用两独立样本非参数检验,BBB行为学评分采用重复测量ANOVA及独立样本t检验,P≤0.05为差异有统计学意义。
结果：
1.在RhoA活性分析试验中,各组RhoA蛋白总表达量无差异,但活化RhoA蛋白量以对照组最高,实验组次之,正常组最少。各组以RhoA总蛋白为校正标准,获取活化RhoA蛋白光密度指数K为统计量进行统计学分析,经ANOVA分析,结果显示组间比较差异有统计学差异(F=686.711,P=0.000),再经LSD多重比较,结果表明组间两两比较差异均有统计学意义(P=0.000)。对照组的活性RhoA显著高于正常组(P<0.001),实验组的活性RhoA比对照组显著降低(P<0.001),但仍高于正常组(P<0.001)；
2.在凋亡神经元检测中,盲法计数每个样本6张切片的凋亡神经元数目。经两独立样本t检验,结果显示两组间比较差异有统计学意义(t=2.395,P=0.022),证明实验组凋亡细胞数显著少于对照组,与RhoA活性分析试验结果吻合。
3.BDA轴突示踪实验中,对照组和实验组头端距SCI 11～16mm处组织的横切面结果显示,两组CST轴突纤维均特异性着色。而尾端距SCI 11～16mm处组织的横切面结果显示,对照组CST轴突纤维未见着色,实验组中25%可见CST轴突纤维着色(4／16)。各组纵切切片中,对照组标记的CST轴突纤维均在脊髓背侧损伤处上游或者损伤处中止,损伤尾侧未见阳性标记的CST轴突纤维；相反,实验组75%的大鼠损伤尾侧可见阳性标记的CST轴突再生纤维(12／16),再生轴突跨过SCI处最大长度的范围为6.9±1.2mm, Md为7.9mm。
4.脊髓背侧半横断损伤后,大鼠的背侧及背外侧CST轴突被离断,仅脊髓腹侧少量CST轴突纤维残留。术后,各组大鼠双侧下肢完全瘫痪,肌张力低。后肢运动功能采用BBB运动评分量表进行评估,具体统计结果如表3-3所示。脊髓损伤后,对照组大鼠鞘内持续4w给予对照血清后,BBB评分为12.2±1.5,实验组大鼠鞘内持续4w给予对照血清后,BBB评分为14.4±1.4。BBB评分中,11分与13分的差别在于动物的前后肢体间是否存在协调运动。BBB评分大于14分时,动物前后肢体间的协调运动频繁存在,是后肢运动功能恢复良好的一个重要征象。3w时,BBB评分大于14分的实验组动物比率高于对照组(37.5%VS 13.3%),统计学上无显著性差异(P=1.000)；4w时,BBB评分大于14分的实验组动物的比率高于对照组(75.0% VS 20.0%),统计学上有显著性差异(P=0.003)。以上结果表明,LINGO-1抗血清鞘内给药后能促进SCI后运动功能的恢复。
结论：
SCI后鞘内给予LINGO-1多克隆抗血清,可有效的阻断损伤部位轴突再生抑制信号的激活,减少神经元凋亡,显著降低继发性神经细胞损伤,具有保护神经组织,促进轴突再生及运动功能恢复的作用。
Background
The self-repair function of nerve tissue is extremely limited after spinal cord injury, largely due to the micro-environment is not conductive to axon regeneration. It is mainly formed by many axon growth inhibitor released from the collapsing tissue. Recent data indicate that many different axon growth inhibitors could bind the neuronal membrane NgR receptor complex(NgR/p75/LINGO-1 or NgR/TAJ (TROY)/LINGO-1),activate intracellular RhoA signal,and then result in growth cone degeneration and axon regeneration failure.LINGO-1(LRR and Ig domain-containing,Nogo Receptor-interacting protein,LINGO-1) is an important component of the NgR receptor complex,and also present in oligodendrocytes.It functions as a negative regulator of oligodendrocyte differentiation and myelination, neuronal survival and axonal regeneration. The targeted inhibition of LINGO-1 therefore presents a novel therapeutic approach for the treatment of neurological diseases.
Some researchers immunized SCI animals with inhibitor proteins, leading to the production of neutralization antibodys which can block the effect of inhibitors, improve the local micro-environment and promote axonal regeneration and function recovery.But it takes about 6-8 weeks for neutralization antibody production when immunized immediately after injury.It is more adapted to explore means of passive immunization to promote early functional recovery of SCI.
We suppose that the useage of LINGO-1 polyclonal antibody can block the function of LINGO-1,weaken the downstream inhibitory signal,decrease the neuronal apoptosis and promote axonal regeneration and functional recovery. The primary option of antibody therapy is monoclonal antibody for its best specificity and quality control.But It will take much more time and higher ecnomic input for preparation.In addition,the first step for antibody therapy is to find passive immunization of antibody therapy can block the targeted candidate and harvest functional recovery after SCI.For these purposes,we prepared a recombinant human LINGO-1 protein as immunogen and producted a large amount of polyclonal antisera of high titer and good specificity for LINGO-1.
This study planed to explore the intervention of LINGO-1 antisera to spinal cord dorsal hemisection in adult rat,expect to provide new idea for SCI treatment.The reseach is divided into three parts.The first one is the preparation of LINGO-1 polyclonal antisera;the second one is to explore the feasibility of LINGO-1 polyclonal antisera treatment With spinal cord injury model in adult rats;the last is to observe whether the polyclonal antibody can promote the axonal regeneration and function recovery of the animal model.
Part 1 The preparation and identification of LINGO-1 polyclonal antibody
Obejective
To express and purify the LINGO-1 ectodomain fusion protein with poly- histidine tag,then to prepare the rabbit-derived anti-hLINGO-1 polyclonal antisera.
Methods
(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA To design specific primers according to the cDNA sequence hLINGO-1aa76-319,introduce BamH I, EcoR I restriction sites and the protection bases on the 5'end of upstream and downstream primer respectively.Plasmid pCMV-SPORT6-LINGO-1 as a template,to amplificate hLINGO-1 cDNA fragment under high-fidelity Taq enzyme by PCR.
(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-1aa76-319 To treat recycling purified PCR product and vector with BamH I, EcoR I double digestion,recycle and ligate the target gene pieces,then transformate DH5a feel-state bacteria.Pick out monoclonal colonies, shake bacteria, extract plasmid. Identify the recombinant plasmid by PCR, double enzyme digestion and sequencing.Sequences thus obtained were compared to GenBank database using BLAST program,then the validated gene fragments were inserted into pET30a (+) expression vector.
(3) the optimal conditions of hLINGO-1aa76-319 fusion protein induced expression To transform pET30a(+)-hLINGO-1aa76-319 recombinant plasmid into BL21 feel-state bacteria,pick out the positive monoclonal colonies by PCR identification, inoculate in kanamycin-resistant LB medium.Add different final concentration of IPTG for induction,when the OD is between 0.6-0.8.Collect samples at different time-points for SDS-PAGE electrophoresis,then staining by Coomassie brilliant blue for view the protein bands,and determine the optimal induction condition.Centrifuge the bacilli which contain highest amount of protein expression and re-suspended sedimentation by split buffer, get the Supernatant and sedimentation by ice bath ultrasonic lysis, then perform SDS-PAGE and western blotting to verify the expression form of fusion protein.
(4) Purification of fusion protein after substantial expression Cultivate 1000mL bacilli in optimized expression conditions, get sedimentation by Centrifuge the bacilli and re-suspending by split buffer,harvest inclsion bodys by ice bath ultrasonic lysis and centrifugalization.resolve inclsion bodys in urea and recycle the LINGO-1 fusion protein by chelating resin chromatography.The concentration of purified protein was determined by BCA protein assay.The protein pufity was determined by Bandscan 5.0 software.For higher purity protein antigen,all the fusion protein was loaded on the SDS-PAGE electrophoresis.The acrylamide piece with antigen were cut out and preserved at-80℃.
(5) The preparation of hLINGO-1aa76-319 antisera After frozen overnight,glue block grind with Freund's adjuvant adequately.After full emulsification, multi-point injection was done at rabbits back. strengthen the immune response for every 7 days.Harvest sera before immunization for negative control and a small amount of boold 10 days after 3rd immunization. antisera titer was detected by ELISA.When the titer achieved the desired level,collect the blood for antisera preparation and preserve at -80℃.
(6) Identification of antisera specificity and titer detection the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by SDS-PAGE electrophoresis,then the prepared antisera for the first antibody to verify its speciality by western blotting.The ELISA plates were coated overnight at 4℃with 4μg/ml LINGO-1 fusion protein. While sera before immunization for negative control,the antisera titer was detected by ELISA.
Results:
(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA one clear band could be seen around 732bp under UV light after agarose gel electrophoresis,is as the same length as the purpose.
(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-laa76-319 Double-digested hLINGO-laa76-319 PCR products were insert into the prokaryotic expression plasmid pET30a(+) and Extract the screening recombinant plasmid DNA.732bp fragment was got by PCR amplification. 5.4kbp and 732bp fragment were got by BamH I and EcoR I double-digested identification.They were coincident with the expect.The sequencing result was the same as the sequence provided by GeneBank(serial number:NM032808). These results verified that the vector was constructed sucessfully and it was named pET30a(+)-hLINGO-laa76-319.
(3) The optimal conditions of hLINGO-laa76-319 fusion protein induced expression Bacteria BL21 transformed recombinant plasmid,identified by PCR,was inducted by IPTG at different conditions.Collect the total Bacterial protein for SDS-PAGE electrophoresis.The result indicated the fusion protein have highest expression at 37℃,0.4mM IPTG induction for 1.5 h.Then the fusion protein was be confirmed by western blotting with anti-His tag mAb for the first antibody. Bacterial precipitation broken by ultrasonic,the suprenant and sediment were collected for SDS-PAGE electrophoresis.The result confirmed that the fusion protein was in the form of inclusion body.
(4) Purification of fusion protein after substantial expression Expanse culture system according to the optimal condition of step three, harvest inclusion bodys, then recycle the fusion protein after purification.The concentration of protein was 4600 mg/L determining by BCA assay,and the purity was 70.8%.
(5) The preparation of hLINGO-laa76-319 antisera Control sera was harvested before immunization by repeated blood sample method.Rabbits have Normal feeding,be in good health and no significant adverse reactions in whole immune process.serum titers were higher than 1:312500 after 3rd immunization. after fourth immunization,The volumn of blood harvested was 197 ml,and the volumn of prepared antisera was 61 ml.The control sera and antisera were filtered by 0.22 um filter membrane,preserved at-80℃.
(6) Identification of antisera specificity and titer detection The titer of harvested antisera was 1:1562500. the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by western blotting,and the prepared antisera was be the first antibody.The colored protein band was the expected result,these results reprsent that the prepared antisera has high titer and good specificity.
Conclusions
The prepared hLINGO-1aa76-319 antisera has good specificity and high titer,and has laid an important foundation to further study the biological function of LINGO-1 protein.
Part 2 The permeability and specificity of LINGO-1 Polyclonal Antibody in vivo
Objective
To analyzed the permeablilty and specificity to the antigen of LINGO-1 polyclonal antibody which administered locally into the spinal cord injury site of rats.
Methods
Twelve Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group, control group(hemisection+control sera) and experiment group (hemisection +antisera).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively. 3 d and 28 d after hemisection,each T8-T10 segments of each group were harvested for cryosection and immunofluorescence tissue staining,statistical analysis was done by two independent samples t test,to analyzed whether LINGO-1 polyclonal antibody can penetrate into spinal cord tissue and bind with LINGO-1 molecule specifically.
Results
Rat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation.This proved that the model-making was sucessful.Three days after operation,spinal cord injury sites of normal group could not detect rabbit-derived antibody,control group and experiment group could detect penetrating antibody which come from rabbit.Twenty-eight days after operation,the result was the same as before.
Three days after operation,Mean immunofluorescence density of experiment group was significantly lower than control group(P<0.05),and Mean immunofluorescence density of control group,which section pre-treated with LINGO-1 antisera,was also significantly lower than control group which pre-treated with rabbit-derived control antisera(P<0.05).
Conclusions
The spinal cord dorsal hemisection model was successfully set up,which has laid an important foundation to further study the effectiveness of antisera treatment. LINGO-1 polyclonal antisera,administered locally,can detected at the spinal cord injury site in wide time-window and recognized LINGO-1 molecule specifically.It is feasible that the method of passive immuno-therapy for spinal cord injury.
Part 3 Efficacy assessment of LINGO-1 polyclonal antibody on spinal cord hemisection model in rats
Objective
To assess the efficacy of LINGO-1 polyclonal antisera administered locally on spinal cord hemisection model in rats.
Methods
Fourty-nine Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group(n=3), control group(hemisection+control sera, n=23) and experiment group (hemisection +antisera,n=23).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively.
Three days after hemisection,active RhoA of spinal cord tissues were assayed by protein pull-down technology and western blotting, apoptotic neuron death of each group was assessed by Immunofluorescence double-standard method.Fourteen days after hemisection,animals were re-anesthetized and the right sensory-motor cortex exposed via a craniotomy and biotin dextran amine(BDA) in PBS, stereotaxically injected at the motor cotex to label the CST axon.Twenty-eight days after hemisection, animals were killed to collect injury site tissues for cross-section and longitudinal section.BDA Immunohistochemistry staining was taken for tracing CST.Animals were scored using the open field BBB scoring system.Rats were evaluated the day after CST transection(day 2) and weekly thereafter for 4 weeks with observers blinded to the treatment regimen. Quantification of the active RhoA protein was statistically analyzed using the One-Way ANOVA and followed by a post hoc LSD test.Quantification of apoptotic neurons was analyzed by independent-samples t test.The maximum regenerating length was analyzed by two independent sample tests.BBB behavioral scores was analyzed by repeated measures ANOVA followed by independent-samples t test.Differences were considered to be statistically significant when P<0.05.
Results
(1) In active RhoA assay experiment,the RhoA total expression in each group has no difference between each other.But the highest expression of active RhoA is control group,followed by the experimental group,the normal group at least.Acquired the active RhoA densitometry index(K) in each group was for statistics,the total RhoA protein of each group was to be the calibration standards.Accrording to ANOVA analysis,there were significant difference in inter-groups(F=686.711, P=0.000). Then accroding to LSD mutiple-comparisons, there were significant difference between groups(P=0.000).The active RhoA in control group was significantly higher than normal group(P<0.001).The active RhoA in experimental group was significantly lower than normal group(P< 0.001),but still higher than normal group(P<0.001).
(2) In apoptotic neuron detction,six sections in each sample were be taken for counting in blind manner.the result was taken for two independent sample test. there were significant difference between this two groups(t=2.395, P=0.022).This verified that the number of apopototic neuons in experiment group was significantly less than control group and consistent with the result of active RhoA assay.
(3) To evaluate the effect of LINGO-1 antisera on axon regeneration,BDA anterograde tracing was used to label the regenerated CST fibers in both experimental group(n=16) and control (n=15) group. Two weeks after BDA injection, transverse and sagittal sections were collected for BDA staining and the maximum length of labeled fibers, extending caudal to the lesion site, was estimated from serial sections. In transverse sections 11-16 mm rostral to the lesion site, CST fibers were equally labeled in both experimental and control groups.However, in transverse sections 11-16 mm caudal to the lesion site,the labeled fibers could not be detected in control group, while in 25% of experimental group(4 of 16), BDA labeled dorsal axons were still observed in the corresponding area. In longitudinal sections across the lesion site, all of the BDA labeled CST fibers in the control group ceased above or at the lesion sites and the caudal growth of labeled axons was not detected. In contrast, in 75% of experimental group (12of 16), BDA labeled axons sprouted across the lesion area and extended within the caudal part of the spinal cord.The maximum distance of labeled CST axons ranged between 5.7 mm to 8.1 mm distal to the injury(Md=7.9mm).
(4) The dorsal and dorsolateral components of CST were completely interrupted and the ventral portion of the CST left intact.Rat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation. Hindlimb function was quantified using the Basso-Beattie-Bresnahan (BBB) open field scoring method(Fig.3-3). Control animals receiving continuous intrathecal infusion of control sera recovered substantial function over the 4 week duration of the experiment, attaining a mean BBB score of 12.2±1.5. Continuous intrathecal infusion of LINGO-1 antisera for 4 weeks after spinal cord transection resulted in significantly improved BBB scores at 3 and 4 weeks,reaching a mean BBB score of 14.4±1.4.A BBB score of 14 or greater reflects the ability of rats to display consistent coordinated hindlimb-forelimb movement and represents a threshold of recovery that is biologically meaningful.The frequency with which LINGO-1 antisera-treated animals attained a score of 14 or more was greater than for control animals,3 weeks (37.5% VS 13.3%, P=1.000) and 4 weeks (75.0% VS 20.0%, P=0.003) after SCI. Together, these results clearly demonstrate that treatment with LINGO-1 antisera promotes the recovery of function after SCI.
Conlusions
LINGO-1 polyclonal antisera can effectively block the activation of downstream inhibitor signal in spinal cord tissue,reduce neuronal apoptosis, significantly decrease the influence of secondary injury,have the effects of protecting nervous tissue,promoting axonal regeneration and motory function recovery.
脊髓损伤(spinal cord injury, SCI)后神经组织自我修复功能极其有限,其最主要的原因是由于崩解的脊髓组织释放出大量的轴突生长抑制因子,形成不利于轴突再生的微环境。目前研究表明,许多轴突生长抑制因子都是通过与神经元细胞膜表面的NgR受体复合体(NgR/p75/LINGO-1及NgR/TAJ(TROY) /LINGO-1)结合后,激活胞内RhoA信号,从而导致生长锥溃变及轴突再生失败。LINGO-1 (LRR and Ig domain-containing, Nogo Receptor-interacting protein, LINGO-1)是构成NgR受体复合体的重要组成部分,同时,其亦存在于少突胶质细胞,在分化、损伤后细胞存活及再髓鞘化等过程中起着负性调节的作用。因此,LINGO-1可作为对抗轴突生长抑制因子抑制作用并促进再生轴突髓鞘化的药物靶点。
一些研究者给予SCI动物主动免疫轴突生长抑制因子产生相应的中和性抗体后,均能达到阻断抑制因子、改善损伤灶局部微环境、促进神经再生和功能恢复的目的。但即使损伤后立即给予主动免疫治疗也至少需要4～6w的时间才能产生保护性抗体。因此,探索早期被动免疫手段促进SCI后功能恢复更适应临床实际需要。
我们的设想是在脊髓损伤后早期,应用LINGO-1特异性抗体阻断损伤处该分子的生物学作用,应当可以削弱下游的轴突抑制信号,阻止损伤局部的神经元凋亡,达到促进轴突再生及神经功能恢复的目的。抗体选择的方面,单克隆抗体有着高度的特异性和优越的质量控制,是抗体治疗的首选,但是其制备周期长、技术要求较高,因此成本相对高昂。而必需明确的首要问题是,阻断靶分子是否能收获有益的治疗效果。因此,基于有效性和经济性的考虑,本研究采用经典的多克隆抗体制备方法获取的足量、高滴度的抗血清作为后续动物实验的干预手段。
本课题通过实验研究初步观察了LINGO-1多克隆抗体对脊髓半横断大鼠模型的早期干预作用,为促进SCI后功能修复提供新的思路。研究内容主要分为以下三个部分：(1) LINGO-1抗血清的制备及鉴定；(2) LINGO-1抗血清对脊髓半横断大鼠模型干预的可行性分析；(3)观察LINGO-1抗血清对脊髓半横断大鼠模型的神经再生及功能恢复的影响。
第一部分：LINGO-1多克隆抗体的制备及鉴定
目的：
表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体。
方法：
1.PCR扩增LINGO-1的76-319位氨基酸编码序列cDNA:根据hLINGO-1aa76-319的cDNA序列设计特异引物,上下游引物的5’端分别引入BamHⅠ、EcoRⅠ酶切位点及保护碱基,以pCMV-SPORT6-LINGO-1质粒为模板,高保真Taq酶作用下PCR扩增hLINGO-1aa76-319 cDNA片段。
2. pET30a(+)-hLINGO-1aa76-319原核表达载体的构建：hLINGO-1aa76-319 cDNA的PCR产物及pET30a(+)载体分别用BamHⅠ、EcoR、双酶切后回收后进行体外连接,转化DH5a感受态细菌,挑取单克隆菌落,摇菌,提取质粒。pET30a(+)-hLINGO-1aa76-319重组质粒经PCR及酶切鉴定后,行核苷酸序列测定和分析。
3.hLINGO-1aa76-319融合蛋白诱导表达条件的优化：将pET30a(+)-hLINGO-1aa76-319重组质粒转化入BL21感受态细菌,挑取菌落PCR阳性的单菌落接种于卡那抗性的LB培养基中,培养至OD为0.6～0.8时分别加入不同终浓度的IPTG诱导,在不同时间点分别收集样本行SDS-PAGE电泳,考马斯亮蓝染色显示蛋白条带,分析确定最佳诱导表达条件。取最高蛋白表达量的菌液适量离心,沉淀重悬于裂菌缓冲液,冰浴超声裂解,分别取上清、沉淀行SDS-PAGE电泳及Western blot检测,以明确目的蛋白在宿主菌中的表达形式并验证融合蛋白是否正确表达。
4.hLINGO-1aa76-319融合蛋白大量表达后的纯化：优化表达条件下,大量培养1000mL菌液,离心后收集菌体沉淀,裂解缓冲液重悬细菌,冰浴后超声破碎,离心获取包涵体,脲溶解后经Ni-NTA螯合树脂层析洗脱回收LINGO-1蛋白。BCA法测定蛋白浓度,SDS-PAGE电泳结果经Bandscan5.0图像分析软件计算蛋白纯度。为了获得纯度更高的蛋白,将全部蛋白初样经SDS-PAGE电泳分离后,切取目的蛋白胶块-80℃冻存。
5.抗hLINGO-1aa76-319多克隆抗体的制备：冻存过夜的蛋白胶块加适量弗氏佐剂研磨。充分乳化后,家兔背部多点皮下注射。每隔7d进行一次加强免疫,第3次免疫注射后的第10d微量取血,4℃冰箱过夜,离心获得血清。以免疫前兔血清作为阴性对照,ELISA测定血清效价。当抗体效价达到预期水平时即可放血收获抗血清,-80℃冰箱保存。
6.抗血清特异性鉴定及效价验证：表达hLINGO-1aa76-319融合蛋白的BL21菌体总蛋白及提取的大鼠脑组织膜蛋白进行SDS-PAGE电泳,制备的LINGO-1多克隆抗体为一抗,Western blot方法验证抗体的特异性。用纯化后hLINGO-1aa76-319融合蛋白包被ELISA反应孔,以免疫前兔血清作为阴性对照,ELISA法检测LINGO-1抗血清的效价。
结果：
1.PCR扩增LINGO-1的76-319位氨基酸编码序列cDNA:PCR产物经琼脂糖凝胶电泳,紫外灯下可见一约732bp条带,与目的片段的大小相符。
2. pET30a(+)-hLINGO-1aa76-319原核表达载体的构建：将hLINGO-1aa76-319PCR扩增产物双酶切后定向插入原核表达质粒pET30a(+)中,提取筛选的重组质粒DNA, PCR反应扩增目的片段,BamHⅠ和EcoRⅠ双酶切鉴定,可得到约5.4kbp及732bp两条带,目的片段大小与预期相符。测序结果显示目标序列与GeneBank(序列号为NM032808)相应序列完全吻合,证实表达载体构建成功,命名为pET30a(+)-hLINGO-1aa76-319。
3.hLINGO-1aa76-319融合蛋白诱导表达条件的优化：重组质粒转化后PCR阳性菌株经IPTG诱导,收集细菌蛋白。SDS-PAGE电泳显示,在37℃、0.4mM IPTG诱导1.5 h的条件下,融合蛋白高表达,经抗His标签的单克隆抗体(monoclonalantibody, mAb)行Western blot鉴定证实该蛋白条带为hLINGO-1aa76-319融合蛋白；菌体沉淀经超声破碎后,上清及沉淀的SDS-PAGE电泳结果进一步证明,该重组蛋白主要以包涵体形式存在。
4.hLINGO-1aa76-319融合蛋白大量表达后的纯化：按步骤三的优化表达条件扩大培养体系,收获包涵体蛋白,纯化后回收重组融合蛋白。BCA法测定蛋白的浓度为4600mg/L,蛋白的纯度为70.8%。
5.抗hLINGO-1aa76-319pAb的制备：免疫前,对动物多次行耳缘静脉滴血法收集162ml血液,获得52ml的对照血清。整个免疫过程中,家兔摄食正常,健康状况良好,无明显不良反应。第3次免疫注射后的10d经耳缘静脉微量取血,ELISA测定血清效价均高于1：312500。4免后,颈动脉采血法收获血液共197ml,制备抗血清61ml,收获的对照血清及抗血清均经过0.22μm滤膜过滤,-80℃冰箱保存。
6. LINGO-1抗血清的效价及特异性鉴定：经ELISA法检测,收获的抗hLINGO-1aa76-319抗血清的效价为1：1562500。表达hLINGO-1aa76-319融合蛋白的BL21菌体总蛋白及提取的大鼠脑组织膜蛋白经Western blot检测,纯化后的抗hLINGO-1aa76-319多克隆抗体为一抗,显色蛋白条带与预期结果吻合,无明显非特异性着色。结果证实所制备的多抗滴度高,特异性良好。
结论：
制备的抗LINGO-1多克隆抗体具备良好的特异性和较高的抗体效价,为进一步研究LINGO-1蛋白的生物学功能奠定了重要的实验基础。
第二部分：LINGO-1多克隆抗体的组织渗透性及其与抗原特异性结合能力分析
目的：
探讨脊髓损伤处局部给予的LINGO-1多克隆抗体的组织渗透性及与抗原的特异性结合能力,为后续的被动免疫治疗研究提供依据。
方法：
将12只成年雌性SD大鼠随机分为正常组、对照组(半横断对照血清组)和实验组(半横断抗血清组)。正常组大鼠不做任何处理,另外两组均行T9脊髓背侧半横断术。半横断术后,使用微量渗透泵分别给予对照血清和LINGO-1抗血清。术后3d和28d取各组T8～T10节段脊髓作冰冻切片,应用间接和直接组织免疫荧光染色法,以两独立样本的t检验进行统计学分析,探讨LINGO-1多抗可否进入脊髓组织并是否与LINGO-1分子(抗原)特异性结合。
结果：
造模大鼠术后清醒时,双侧下肢完全瘫痪,肌张力低,证明模型制作成功。术后3d,正常组兔源性抗体免疫荧光染色阴性,对照组和实验组SCI处可检测出兔源性抗体；术后28d,对照组和实验组SCI处及周围组织仍可以检测到兔源性抗体。
术后3d,对照组切片LINGO-1染色强度明显强于实验组(t=26.491,P=0.000)。多抗预处理后的对照组切片LINGO-1染色强度明显弱于未预处理的对照组切片(t=31.879,P=0.000)。
结论：
成功建立脊髓背侧半横断模型,局部应用抗血清后能有效渗透到损伤的脊髓组织中,并能与脊髓组织中的抗原特异性结合；此外,在脊髓损伤区伤后3-28天的时间窗内均可检测到LINGO-1多克隆抗体,证实该多抗可在损伤局部较长的时间内发挥被动免疫的作用。
第三部分：LINGO-1多克隆抗体对脊髓半横断模型大鼠的被动免疫治疗效果评估
目的：
评价脊髓损伤处局部给予LINGO-1多克隆抗体的治疗效果。
方法：
将49只成年雌性SD大鼠分为正常组(3只)、半横断对照血清组(23只)及半横断抗血清组(23只)。正常组大鼠不做任何处理,另外两组均行T9脊髓半横断术。术后,两组使用微量渗透泵鞘内分别局部给予对照血清和LINGO-1抗血清。术后3d,利用蛋白pull-down技术及Western blot检测方法对各组脊髓损伤处组织行RhoA活性分析,组织免疫荧光双标法检测各组神经元凋亡情况。术后14d,立体定向注射BDA于右侧的感觉运动皮层顺行示踪CST纤维。术后28d,处死动物,收集脊髓损伤处横切和纵切,行BDA免疫组织化学染色。术后1d及每周,对各组动物盲法行BBB行为学评分。活化RhoA蛋白量采用单因素ANOVA检验及LSD多重比较,凋亡神经元计数采用独立样本t检验,最大再生轴突长度采用两独立样本非参数检验,BBB行为学评分采用重复测量ANOVA及独立样本t检验,P≤0.05为差异有统计学意义。
结果：
1.在RhoA活性分析试验中,各组RhoA蛋白总表达量无差异,但活化RhoA蛋白量以对照组最高,实验组次之,正常组最少。各组以RhoA总蛋白为校正标准,获取活化RhoA蛋白光密度指数K为统计量进行统计学分析,经ANOVA分析,结果显示组间比较差异有统计学差异(F=686.711,P=0.000),再经LSD多重比较,结果表明组间两两比较差异均有统计学意义(P=0.000)。对照组的活性RhoA显著高于正常组(P<0.001),实验组的活性RhoA比对照组显著降低(P<0.001),但仍高于正常组(P<0.001)；
2.在凋亡神经元检测中,盲法计数每个样本6张切片的凋亡神经元数目。经两独立样本t检验,结果显示两组间比较差异有统计学意义(t=2.395,P=0.022),证明实验组凋亡细胞数显著少于对照组,与RhoA活性分析试验结果吻合。
3.BDA轴突示踪实验中,对照组和实验组头端距SCI 11～16mm处组织的横切面结果显示,两组CST轴突纤维均特异性着色。而尾端距SCI 11～16mm处组织的横切面结果显示,对照组CST轴突纤维未见着色,实验组中25%可见CST轴突纤维着色(4／16)。各组纵切切片中,对照组标记的CST轴突纤维均在脊髓背侧损伤处上游或者损伤处中止,损伤尾侧未见阳性标记的CST轴突纤维；相反,实验组75%的大鼠损伤尾侧可见阳性标记的CST轴突再生纤维(12／16),再生轴突跨过SCI处最大长度的范围为6.9±1.2mm, Md为7.9mm。
4.脊髓背侧半横断损伤后,大鼠的背侧及背外侧CST轴突被离断,仅脊髓腹侧少量CST轴突纤维残留。术后,各组大鼠双侧下肢完全瘫痪,肌张力低。后肢运动功能采用BBB运动评分量表进行评估,具体统计结果如表3-3所示。脊髓损伤后,对照组大鼠鞘内持续4w给予对照血清后,BBB评分为12.2±1.5,实验组大鼠鞘内持续4w给予对照血清后,BBB评分为14.4±1.4。BBB评分中,11分与13分的差别在于动物的前后肢体间是否存在协调运动。BBB评分大于14分时,动物前后肢体间的协调运动频繁存在,是后肢运动功能恢复良好的一个重要征象。3w时,BBB评分大于14分的实验组动物比率高于对照组(37.5%VS 13.3%),统计学上无显著性差异(P=1.000)；4w时,BBB评分大于14分的实验组动物的比率高于对照组(75.0% VS 20.0%),统计学上有显著性差异(P=0.003)。以上结果表明,LINGO-1抗血清鞘内给药后能促进SCI后运动功能的恢复。
结论：
SCI后鞘内给予LINGO-1多克隆抗血清,可有效的阻断损伤部位轴突再生抑制信号的激活,减少神经元凋亡,显著降低继发性神经细胞损伤,具有保护神经组织,促进轴突再生及运动功能恢复的作用。
Background
The self-repair function of nerve tissue is extremely limited after spinal cord injury, largely due to the micro-environment is not conductive to axon regeneration. It is mainly formed by many axon growth inhibitor released from the collapsing tissue. Recent data indicate that many different axon growth inhibitors could bind the neuronal membrane NgR receptor complex(NgR/p75/LINGO-1 or NgR/TAJ (TROY)/LINGO-1),activate intracellular RhoA signal,and then result in growth cone degeneration and axon regeneration failure.LINGO-1(LRR and Ig domain-containing,Nogo Receptor-interacting protein,LINGO-1) is an important component of the NgR receptor complex,and also present in oligodendrocytes.It functions as a negative regulator of oligodendrocyte differentiation and myelination, neuronal survival and axonal regeneration. The targeted inhibition of LINGO-1 therefore presents a novel therapeutic approach for the treatment of neurological diseases.
Some researchers immunized SCI animals with inhibitor proteins, leading to the production of neutralization antibodys which can block the effect of inhibitors, improve the local micro-environment and promote axonal regeneration and function recovery.But it takes about 6-8 weeks for neutralization antibody production when immunized immediately after injury.It is more adapted to explore means of passive immunization to promote early functional recovery of SCI.
We suppose that the useage of LINGO-1 polyclonal antibody can block the function of LINGO-1,weaken the downstream inhibitory signal,decrease the neuronal apoptosis and promote axonal regeneration and functional recovery. The primary option of antibody therapy is monoclonal antibody for its best specificity and quality control.But It will take much more time and higher ecnomic input for preparation.In addition,the first step for antibody therapy is to find passive immunization of antibody therapy can block the targeted candidate and harvest functional recovery after SCI.For these purposes,we prepared a recombinant human LINGO-1 protein as immunogen and producted a large amount of polyclonal antisera of high titer and good specificity for LINGO-1.
This study planed to explore the intervention of LINGO-1 antisera to spinal cord dorsal hemisection in adult rat,expect to provide new idea for SCI treatment.The reseach is divided into three parts.The first one is the preparation of LINGO-1 polyclonal antisera;the second one is to explore the feasibility of LINGO-1 polyclonal antisera treatment With spinal cord injury model in adult rats;the last is to observe whether the polyclonal antibody can promote the axonal regeneration and function recovery of the animal model.
Part 1 The preparation and identification of LINGO-1 polyclonal antibody
Obejective
To express and purify the LINGO-1 ectodomain fusion protein with poly- histidine tag,then to prepare the rabbit-derived anti-hLINGO-1 polyclonal antisera.
Methods
(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA To design specific primers according to the cDNA sequence hLINGO-1aa76-319,introduce BamH I, EcoR I restriction sites and the protection bases on the 5'end of upstream and downstream primer respectively.Plasmid pCMV-SPORT6-LINGO-1 as a template,to amplificate hLINGO-1 cDNA fragment under high-fidelity Taq enzyme by PCR.
(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-1aa76-319 To treat recycling purified PCR product and vector with BamH I, EcoR I double digestion,recycle and ligate the target gene pieces,then transformate DH5a feel-state bacteria.Pick out monoclonal colonies, shake bacteria, extract plasmid. Identify the recombinant plasmid by PCR, double enzyme digestion and sequencing.Sequences thus obtained were compared to GenBank database using BLAST program,then the validated gene fragments were inserted into pET30a (+) expression vector.
(3) the optimal conditions of hLINGO-1aa76-319 fusion protein induced expression To transform pET30a(+)-hLINGO-1aa76-319 recombinant plasmid into BL21 feel-state bacteria,pick out the positive monoclonal colonies by PCR identification, inoculate in kanamycin-resistant LB medium.Add different final concentration of IPTG for induction,when the OD is between 0.6-0.8.Collect samples at different time-points for SDS-PAGE electrophoresis,then staining by Coomassie brilliant blue for view the protein bands,and determine the optimal induction condition.Centrifuge the bacilli which contain highest amount of protein expression and re-suspended sedimentation by split buffer, get the Supernatant and sedimentation by ice bath ultrasonic lysis, then perform SDS-PAGE and western blotting to verify the expression form of fusion protein.
(4) Purification of fusion protein after substantial expression Cultivate 1000mL bacilli in optimized expression conditions, get sedimentation by Centrifuge the bacilli and re-suspending by split buffer,harvest inclsion bodys by ice bath ultrasonic lysis and centrifugalization.resolve inclsion bodys in urea and recycle the LINGO-1 fusion protein by chelating resin chromatography.The concentration of purified protein was determined by BCA protein assay.The protein pufity was determined by Bandscan 5.0 software.For higher purity protein antigen,all the fusion protein was loaded on the SDS-PAGE electrophoresis.The acrylamide piece with antigen were cut out and preserved at-80℃.
(5) The preparation of hLINGO-1aa76-319 antisera After frozen overnight,glue block grind with Freund's adjuvant adequately.After full emulsification, multi-point injection was done at rabbits back. strengthen the immune response for every 7 days.Harvest sera before immunization for negative control and a small amount of boold 10 days after 3rd immunization. antisera titer was detected by ELISA.When the titer achieved the desired level,collect the blood for antisera preparation and preserve at -80℃.
(6) Identification of antisera specificity and titer detection the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by SDS-PAGE electrophoresis,then the prepared antisera for the first antibody to verify its speciality by western blotting.The ELISA plates were coated overnight at 4℃with 4μg/ml LINGO-1 fusion protein. While sera before immunization for negative control,the antisera titer was detected by ELISA.
Results:
(1) PCR amplification of LINGO-1 of 76-319 amino acid coding sequence cDNA one clear band could be seen around 732bp under UV light after agarose gel electrophoresis,is as the same length as the purpose.
(2) Construction of Prokaryotic expression vector pET30a(+)-hLINGO-laa76-319 Double-digested hLINGO-laa76-319 PCR products were insert into the prokaryotic expression plasmid pET30a(+) and Extract the screening recombinant plasmid DNA.732bp fragment was got by PCR amplification. 5.4kbp and 732bp fragment were got by BamH I and EcoR I double-digested identification.They were coincident with the expect.The sequencing result was the same as the sequence provided by GeneBank(serial number:NM032808). These results verified that the vector was constructed sucessfully and it was named pET30a(+)-hLINGO-laa76-319.
(3) The optimal conditions of hLINGO-laa76-319 fusion protein induced expression Bacteria BL21 transformed recombinant plasmid,identified by PCR,was inducted by IPTG at different conditions.Collect the total Bacterial protein for SDS-PAGE electrophoresis.The result indicated the fusion protein have highest expression at 37℃,0.4mM IPTG induction for 1.5 h.Then the fusion protein was be confirmed by western blotting with anti-His tag mAb for the first antibody. Bacterial precipitation broken by ultrasonic,the suprenant and sediment were collected for SDS-PAGE electrophoresis.The result confirmed that the fusion protein was in the form of inclusion body.
(4) Purification of fusion protein after substantial expression Expanse culture system according to the optimal condition of step three, harvest inclusion bodys, then recycle the fusion protein after purification.The concentration of protein was 4600 mg/L determining by BCA assay,and the purity was 70.8%.
(5) The preparation of hLINGO-laa76-319 antisera Control sera was harvested before immunization by repeated blood sample method.Rabbits have Normal feeding,be in good health and no significant adverse reactions in whole immune process.serum titers were higher than 1:312500 after 3rd immunization. after fourth immunization,The volumn of blood harvested was 197 ml,and the volumn of prepared antisera was 61 ml.The control sera and antisera were filtered by 0.22 um filter membrane,preserved at-80℃.
(6) Identification of antisera specificity and titer detection The titer of harvested antisera was 1:1562500. the total protein of BL21 expressing the fusion protein and brain membrane protein of rat were perform by western blotting,and the prepared antisera was be the first antibody.The colored protein band was the expected result,these results reprsent that the prepared antisera has high titer and good specificity.
Conclusions
The prepared hLINGO-1aa76-319 antisera has good specificity and high titer,and has laid an important foundation to further study the biological function of LINGO-1 protein.
Part 2 The permeability and specificity of LINGO-1 Polyclonal Antibody in vivo
Objective
To analyzed the permeablilty and specificity to the antigen of LINGO-1 polyclonal antibody which administered locally into the spinal cord injury site of rats.
Methods
Twelve Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group, control group(hemisection+control sera) and experiment group (hemisection +antisera).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively. 3 d and 28 d after hemisection,each T8-T10 segments of each group were harvested for cryosection and immunofluorescence tissue staining,statistical analysis was done by two independent samples t test,to analyzed whether LINGO-1 polyclonal antibody can penetrate into spinal cord tissue and bind with LINGO-1 molecule specifically.
Results
Rat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation.This proved that the model-making was sucessful.Three days after operation,spinal cord injury sites of normal group could not detect rabbit-derived antibody,control group and experiment group could detect penetrating antibody which come from rabbit.Twenty-eight days after operation,the result was the same as before.
Three days after operation,Mean immunofluorescence density of experiment group was significantly lower than control group(P<0.05),and Mean immunofluorescence density of control group,which section pre-treated with LINGO-1 antisera,was also significantly lower than control group which pre-treated with rabbit-derived control antisera(P<0.05).
Conclusions
The spinal cord dorsal hemisection model was successfully set up,which has laid an important foundation to further study the effectiveness of antisera treatment. LINGO-1 polyclonal antisera,administered locally,can detected at the spinal cord injury site in wide time-window and recognized LINGO-1 molecule specifically.It is feasible that the method of passive immuno-therapy for spinal cord injury.
Part 3 Efficacy assessment of LINGO-1 polyclonal antibody on spinal cord hemisection model in rats
Objective
To assess the efficacy of LINGO-1 polyclonal antisera administered locally on spinal cord hemisection model in rats.
Methods
Fourty-nine Sprague-Dawley female rats(200-250g) were randomly divided into three groups:normal group(n=3), control group(hemisection+control sera, n=23) and experiment group (hemisection +antisera,n=23).Normal group was without any treatment,the other two groups were performed T9 dorsal hemisection,completely interrupting the main dorsomedial and dorsolateral corticospinal tract(CST) components.Immediately after CST transection,an intrathecal catheter was inserted into the subarachnoid space at T9 and connected to a primed mini-osmotic pump inserted into the subcutaneous space.Mini-osmotic pumps delivered control sera and LINGO-1 antisera respectively.
Three days after hemisection,active RhoA of spinal cord tissues were assayed by protein pull-down technology and western blotting, apoptotic neuron death of each group was assessed by Immunofluorescence double-standard method.Fourteen days after hemisection,animals were re-anesthetized and the right sensory-motor cortex exposed via a craniotomy and biotin dextran amine(BDA) in PBS, stereotaxically injected at the motor cotex to label the CST axon.Twenty-eight days after hemisection, animals were killed to collect injury site tissues for cross-section and longitudinal section.BDA Immunohistochemistry staining was taken for tracing CST.Animals were scored using the open field BBB scoring system.Rats were evaluated the day after CST transection(day 2) and weekly thereafter for 4 weeks with observers blinded to the treatment regimen. Quantification of the active RhoA protein was statistically analyzed using the One-Way ANOVA and followed by a post hoc LSD test.Quantification of apoptotic neurons was analyzed by independent-samples t test.The maximum regenerating length was analyzed by two independent sample tests.BBB behavioral scores was analyzed by repeated measures ANOVA followed by independent-samples t test.Differences were considered to be statistically significant when P<0.05.
Results
(1) In active RhoA assay experiment,the RhoA total expression in each group has no difference between each other.But the highest expression of active RhoA is control group,followed by the experimental group,the normal group at least.Acquired the active RhoA densitometry index(K) in each group was for statistics,the total RhoA protein of each group was to be the calibration standards.Accrording to ANOVA analysis,there were significant difference in inter-groups(F=686.711, P=0.000). Then accroding to LSD mutiple-comparisons, there were significant difference between groups(P=0.000).The active RhoA in control group was significantly higher than normal group(P<0.001).The active RhoA in experimental group was significantly lower than normal group(P< 0.001),but still higher than normal group(P<0.001).
(2) In apoptotic neuron detction,six sections in each sample were be taken for counting in blind manner.the result was taken for two independent sample test. there were significant difference between this two groups(t=2.395, P=0.022).This verified that the number of apopototic neuons in experiment group was significantly less than control group and consistent with the result of active RhoA assay.
(3) To evaluate the effect of LINGO-1 antisera on axon regeneration,BDA anterograde tracing was used to label the regenerated CST fibers in both experimental group(n=16) and control (n=15) group. Two weeks after BDA injection, transverse and sagittal sections were collected for BDA staining and the maximum length of labeled fibers, extending caudal to the lesion site, was estimated from serial sections. In transverse sections 11-16 mm rostral to the lesion site, CST fibers were equally labeled in both experimental and control groups.However, in transverse sections 11-16 mm caudal to the lesion site,the labeled fibers could not be detected in control group, while in 25% of experimental group(4 of 16), BDA labeled dorsal axons were still observed in the corresponding area. In longitudinal sections across the lesion site, all of the BDA labeled CST fibers in the control group ceased above or at the lesion sites and the caudal growth of labeled axons was not detected. In contrast, in 75% of experimental group (12of 16), BDA labeled axons sprouted across the lesion area and extended within the caudal part of the spinal cord.The maximum distance of labeled CST axons ranged between 5.7 mm to 8.1 mm distal to the injury(Md=7.9mm).
(4) The dorsal and dorsolateral components of CST were completely interrupted and the ventral portion of the CST left intact.Rat bilateral lower limbs were completely paralyzed and low muscle tone when awaked from operation. Hindlimb function was quantified using the Basso-Beattie-Bresnahan (BBB) open field scoring method(Fig.3-3). Control animals receiving continuous intrathecal infusion of control sera recovered substantial function over the 4 week duration of the experiment, attaining a mean BBB score of 12.2±1.5. Continuous intrathecal infusion of LINGO-1 antisera for 4 weeks after spinal cord transection resulted in significantly improved BBB scores at 3 and 4 weeks,reaching a mean BBB score of 14.4±1.4.A BBB score of 14 or greater reflects the ability of rats to display consistent coordinated hindlimb-forelimb movement and represents a threshold of recovery that is biologically meaningful.The frequency with which LINGO-1 antisera-treated animals attained a score of 14 or more was greater than for control animals,3 weeks (37.5% VS 13.3%, P=1.000) and 4 weeks (75.0% VS 20.0%, P=0.003) after SCI. Together, these results clearly demonstrate that treatment with LINGO-1 antisera promotes the recovery of function after SCI.
Conlusions
LINGO-1 polyclonal antisera can effectively block the activation of downstream inhibitor signal in spinal cord tissue,reduce neuronal apoptosis, significantly decrease the influence of secondary injury,have the effects of protecting nervous tissue,promoting axonal regeneration and motory function recovery.
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