CN114807233B - 一种巨噬细胞特异性usp13过表达的重组腺相关病毒及其应用 - Google Patents
一种巨噬细胞特异性usp13过表达的重组腺相关病毒及其应用 Download PDFInfo
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Abstract
本发明涉及一种巨噬细胞特异性USP13过表达的重组腺相关病毒,构建方法:体外克隆USP13基因,将USP13基因和pAAV‑Lyz2载体分别用限制性内切酶BamHI和EcoRI双酶切,然后DNA连接,得到含有USP13的重组穿梭质粒,扩增、提纯后,将重组穿梭质粒和pHelper和pAAV‑RC质粒混合,共转染AAV‑293细胞,培养后收获细胞,反复冻融后过滤,获得病毒液,纯化后即得。该病毒带有巨噬细胞特异性启动子Lyz2和USP13基因表达序列,可在体内巨噬细胞中特异性过表达USP13蛋白并靶向修饰下游IκBα蛋白泛素化水平,抑制下游NF‑κB通路,促进巨噬细胞M2极化,改善脊髓损伤功能。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种巨噬细胞特异性USP13过表达的重组腺相关病毒及其在制备脊髓损伤治疗药物中的应用。
背景技术
脊髓损伤(Spinal Cord Injury,SCI)发病率高、致死致残率极高,常导致损伤节段以下肢体严重感觉及运动功能障碍。随着人口老年化水平迅速上升及交通运输业的飞速发展,SCI发病率逐年递增。日益增长的SCI不仅对患者身心造成严重伤害,同时也是家庭及整个社会的沉重负担。然而,到目前为止,仍没有有效的解决及治疗方法。
血脊髓屏障是由连续排列的脊髓血管内皮细胞通过细胞间紧密连接构成,正常情况下其可作为自然的生理屏障阻止外周的炎症细胞及因子的侵袭。然而,SCI后5分钟之内血脊髓屏障破裂,大量外周来源的巨噬细胞、炎症细胞通过破损的血管内皮细胞间隙进入中枢脊髓损伤中心,导致损伤灶附近严重的炎症级联反应、胶质细胞激活、神经元细胞死亡,最终加剧SCI发展。外周浸润的巨噬细胞及中枢来源的小胶质细胞在SCI后显著激活,根据其表面标志物的不同,可分为促炎M1型以及抑炎M2型。M1型巨噬细胞分泌炎症细胞因子和趋化因子,进而维持和加重炎症反应,增加神经细胞的兴奋性和毒性,导致神经元死亡。SCI约1周后,巨噬细胞开始向M2型极化,M2型巨噬细胞进行组织碎片的吞噬、细胞营养因子的释放、促进组织修复和抑制炎症反应,参与继发性SCI的修复过程。总之,SCI后的微环境变化是一个非常复杂的动态过程,目前已知的是,极化的巨噬细胞在此过程中扮演着非常重要的角色,因此,如何调控巨噬细胞的极化对于SCI的预后极为重要。
泛素化是常见的蛋白质翻译后修饰方式之一,其参与了体内包括胚胎发育、细胞分化与***、转录调控、炎症免疫反应、肿瘤发生发展等诸多生理以及病理过程。而在神经***中,泛素化修饰也参与了神经***发育、干细胞分化、神经轴突再生、胶质细胞激活等过程。泛素化修饰是一个可逆反应,而去泛素化酶(deubiquitinating enzymes,DUBs)主要通过将泛素分子从目标底物靶蛋白上切割下来从而介导这一过程。由于其独特的结构和功能的多样性,近年来,DUBs在诸多疾病模型中的作用不断被发现和报道。然而,其在脊髓损伤中的功能作用鲜有报道。
腺相关病毒(adeno-associated virus,AAV)属于细小病毒科内的依赖细小病毒属,是用于治疗多种人类疾病的基因传递工具。开发理想的AAV衣壳、优化基因组设计和利用革命性生物技术方面的最新进展为临床上基因治疗领域的发展做出了重大贡献。AAV作为理想的治疗载体在基因置换、基因沉默和基因编辑方面广受欢迎,其中两种基于AAV的疗法已在欧洲或美国获得了监管部门的批准。由于有血脊髓屏障的存在,传统药物干预很难抵达中枢神经***发挥相应的作用,而AAV却不受血脊髓屏障的限制,同时具有易获得、高安全性、低免疫原性、强靶向性,能够长期稳定表达外源基因等优点。
因此,通过构建一种重组腺相关病毒在脊髓损伤后靶向调控损伤灶内的巨噬细胞的IκBα蛋白泛素化水平,抑制下游NF-κB信号通路来促进巨噬细胞M2极化以改善脊髓损伤功能预后,具有重要的意义。
发明内容
本发明的目的在于解决现有技术的不足,提供一种巨噬细胞特异性USP13过表达的重组腺相关病毒,其带有巨噬细胞特异性启动子Lyz2和USP13基因表达序列,可在体内巨噬细胞中特异性过表达USP13蛋白并靶向修饰下游IκBα蛋白泛素化水平,抑制下游NF-κB通路,促进巨噬细胞M2极化,改善脊髓损伤功能。
技术方案
一种巨噬细胞特异性USP13过表达的重组腺相关病毒,构建方法包括如下步骤:
(1)体外克隆USP13基因;
(2)将USP13基因和pAAV-Lyz2载体分别用限制性内切酶BamH I和EcoR I进行双酶切,然后进行DNA连接反应,得到含有目的基因USP13的重组穿梭质粒,对其进行扩增、提纯;
(3)将含有目的基因USP13的重组穿梭质粒和含腺相关病毒基因组DNA的pHelper和pAAV-RC质粒按摩尔比1:1:1混合,共转染AAV-293细胞,培养后收获细胞,反复冻融后过滤,获得病毒液,纯化后,得到巨噬细胞特异性USP13过表达的重组腺相关病毒。
步骤(1)中,所述USP13基因的序列如SEQ ID NO:1所示。
SEQ ID NO:1:
atgcagcgccggggcgccctgttcagcgtgccgggcggcggcgggaagatggctgcaggggacctgggcgagctgctggtgcctcatatgcccacgatccgcgtgcccaggtcgggggaccgcgtctacaagaacgagtgcgccttctcctacgactccccgaactctgaaggtgggctctacgtatgcatgaatacctttttggcctttggaagggaacacgtagaaagacactttcgaaaaactggacagagcgtatacatgcacctgaagaggcacatgcgagagaaggtaagaggagcctctggtggagctttacccaaaaggaggaattccaagatatttttagatctagatatggatgacgatttaaatagtgacgattacgaatatgaagacgaagccaaacttgttatattcccagaccactatgaaatagcccttcctaacattgaggagttaccagccctggtaacaattgcttgtgatgcagtgctcagctcaaagtccccttacaggaagcaggatccagacacatgggaaaacgaagtgccagtatcgaagtatgccaacaaccttgtgcaactggacaacggggtcaggattcctcccagtggctggaagtgtgcccgatgtgacctgcgggagaacctctggttgaatctgactgacggctctgttctgtgtgggaagtggttttttgacagctcagggggcaacggccacgcactggagcattacagggacatgggctatcctctggccgtgaagctgggcaccatcacacctgatggggcagatgtttattcttttcaagaagaggggcctgtttcggatcctcatttggccaaacacttagcacattttgggatcgacatgctccacacgcaagggacagagaacggtctccgggacaatgacatcaaaccgagagtcagcgagtgggaagtgatccaggagtcaggaactaagctgaagccgatgtacggcccagggtacacgggcctgaagaacctgggcaacagttgctacctcagttctgtcatgcaggccatcttcagcatcccagagttccagagagcgtatgtaggaaacctcccaaggatatttgactactcaccgttagatccaacgcaggacttcaacacacaaatgactaagttgggacatggcctcctctctggccagtactcgaagcctccagtgaaatctgagctcattgaacaggtgatgaaggaggagcacaagcctcagcagaatgggatctctccacgcatgttcaaggcctttgtcagcaagagccacccggaattctcctccaacagacagcaggatgcccaggagtttttcttgcatttggtcaatctggtagagaggaatcgcattggctcagaaaacccaagtgatgttttccggtttttggtggaggagcgaattcaatgctgtcagaccagaaaggttcgctacacggagagggtggactacctaatgcagttacctgtggccatggaggcagcaaccaacaaagatgagctgatcacctatgaactcatgcggagggaagcagaagccaacagaagacccctacctgagctggtgcgagccaagatcccattcagtgcctgccttcaggcctttgctgaaccagacaatgtggatgatttctggagcagcgctctgcaggccaagtctgcaggggtcaaaacttctcgctttgcctcattccctgaatacttggtagtgcagataaagaagttcacttttggtcttgactgggttcccagaaaatttgatgtttctattgatatgccagacctactagatatcagccatctcagagccaggggcttgcagccaggggaagaggagcttcctgacatcagcccccccatagtcattcctgatgactcaaaagaccgcttgatgaaccagttgatagacccctcagacattgatgagtcttcggtgatgcagctggctgagatgggcttccctttggaagcctgcaggaaggctgtgtacttcacggggaacaccggagctgaggtggccttcaactggattatcgtgcacatggaggagcctgactttgctgaaccactggccatacctgggtatggaggggctggggcctctgtctttggtgctactggattggacaaccaacctcctgaggaaatcgtagctattatcacctcgatgggattccagcgaaatcaggcagtgcaggctctacaagcaacgaatcataacctggaaagagcactggactggatcttcagccaccccgagtttgaagaggacagtgactttgtgatcgagatggagaacaatgcaaatgccaacatcgtgtctgaggccaagccagagggacccagagtgaaggatgggtctggaatgtacgagttgtttgctttcatcagtcacatgggaacatctacaatgagtggccattatgtttgccatatcaagaaagagggacgatgggtgatctacaatgaccacaaagtttgtgcctcagaaaggccccccaaagacctgggctatatgtacttttaccgcaggataccaagctaa
进一步,步骤(1)中,所述体外克隆USP13基因的方法为:提取并体外培养原代巨噬细胞,收集细胞后提取总RNA,逆转录得到cDNA,然后以cDNA为模板,进行PCR扩增反应,得到USP13基因;所述PCR扩增的引物序列为:前引物:5’-CGTCCAGGTCCTGTTCTCC-3’,后引物:5’-TACCCATGTAGTCCTCCGCA-3’。
进一步,步骤(3)中,所述纯化方法为:将病毒液离心后弃去大部分上清,加入核酸酶消化去除残留的质粒DNA,37℃孵育后离心,取上清加入到超滤管中,然后加入碘克沙醇梯度液超速离心,收集病毒层。
上述巨噬细胞特异性USP13过表达的重组腺相关病毒在制备脊髓损伤治疗药物中的应用。
进一步,所述药物还包括药学上可接受的载体。所述药学上可接受的载体包括稀释剂和赋形剂。
本发明的有益效果:
本发明成功构建了一种巨噬细胞特异性USP13过表达的重组腺相关病毒,该病毒带有巨噬细胞特异性启动子Lyz2和USP13基因表达序列,可在体内巨噬细胞中特异性过表达USP13蛋白并靶向修饰下游IκBα蛋白泛素化水平,抑制下游NF-κB通路,促进巨噬细胞M2极化,改善脊髓损伤功能,为脊髓损伤的临床治疗提供了新的策略。
附图说明
图1为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的BMS评分结果;
图2为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的转棒实验测试结果;
图3为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的肌电图及波幅和潜伏期统计图;
图4为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的M1和M2相关基因的qRT-PCR定量检测结果;
图5为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠iNOS和Arg1免疫荧光染色及统计图;
图6为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的NF免疫荧光染色及轴突数量统计图;
图7为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的存活神经元免疫荧光染色及数量统计图;
图8为USP13免疫沉淀复合物中IκBα特异性肽段鉴定图;
图9为USP13和IκBα的免疫共沉淀结果;
图10为敲低USP13后巨噬细胞IκBα、胞质p65和核内p65蛋白表达水平的检测结果及敲低USP13后巨噬细胞p65细胞定位检测结果;
图11为敲低USP13后巨噬细胞IκBα蛋白的泛素化水平检测结果。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案进行详细说明。
实施例1
构建巨噬细胞特异性USP13过表达的重组腺相关病毒,具体步骤如下:
(1)体外克隆USP13基因:
1)提取并体外培养原代巨噬细胞:将4周龄C57BL/6小鼠处死后75%酒精浸泡消毒。取出小鼠,移入超净台,暴露股骨与胫骨,去除股骨、胫骨两端关节面,用冰PBS溶液冲洗骨髓腔,用70μm无菌过滤器过滤后加入5mL红细胞裂解液去除红细胞。细胞悬液1000rpm离心5分钟后,用含20ng/mL巨噬细胞集落刺激因子的完全培养基洗涤两次并重悬,接种于培养皿中,37℃,5%CO2培养箱中培养。
2)收集细胞后用Trizol试剂(日本Takara公司)裂解细胞提取总RNA,采用逆转录试剂盒(日本Takara公司)行逆转录,得到cDNA,逆转录反应程序:37℃15分钟,85℃5秒钟;反应体系:2μL 5×PrimeScript RT Master Mix,500ng RNA,补加DEPC水至10μL。
3)以cDNA为模板,设计引物进行PCR扩增反应,得到USP13基因,USP13基因的核苷酸序列如SEQ ID NO:1所示;
所述PCR扩增的引物序列为:前引物:5’-CGTCCAGGTCCTGTTCTCC-3’,后引物:5’-TACCCATGTAGTCCTCCGCA-3’。
PCR扩增反应体系:1μL上述cDNA模板,5μL 10×Reaction buffer,3μL 25mMMgCl2,3μL 2.5μM dNTP,1μL前引物,1μL后引物,1μL Taq DNA polymerase,35μL dH2O。
PCR扩增反应程序:94℃预变性3min,94℃变性1min,60℃退火45s、72℃延伸45s,重复35个循环,4℃保存。
(2)将USP13基因和pAAV-Lyz2载体(和元生物)分别用限制性内切酶BamH I和EcoRI(和元生物)进行双酶切,然后混合,加入T4连接酶,4℃过夜,得到含有目的基因USP13的重组穿梭质粒;
对重组穿梭质粒进行扩增、提纯,方法为:冰上融化DH5α感受态细胞,加2μL重组穿梭质粒于100μL感受态细胞中,冰上放置30min,随后42℃水浴热激90s,迅速转移至冰上3-5min,加入1mL无抗生素的LB液体培养基(胰蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,用NaOH调pH为7.4),于37℃震荡培养1h使细菌恢复正常生长状态,并表达质粒编码的抗生素抗性基因,菌液摇匀后取100μL均匀涂在含抗生素的LB筛选平板上,正面向上放置半小时,待菌液完全被培养基吸收后,倒置于37℃恒温孵育箱中培养过夜,次日挑选单克隆菌落,接种于LB液体培养基中,37℃摇菌过夜扩增,收集菌液,离心裂解,抽提纯化质粒,双酶切鉴定。
(3)将含有目的基因USP13的重组穿梭质粒和含腺相关病毒基因组DNA的pHelper和pAAV-RC质粒按摩尔比1:1:1混合溶于500μL Opti-MEM培养基(Gibco),轻轻混匀后静置5min,得到质粒稀释液;将Obio转染试剂(和元生物)溶于500μL Opti-MEM培养基,轻轻混匀后静置5min,得到转染试剂稀释液;将上述转染试剂稀释液滴加到质粒稀释液中,静置20min形成稳定的转染复合体,随后转染进AAV-293细胞,6h后换用新鲜的培养基,转染72小时后,细胞刮刀收集细胞和上清液至离心管,取细胞裂解液在液氮浴和37℃水浴反复冻融,过滤后获得病毒液,将病毒液进行纯化,得到巨噬细胞特异性USP13过表达的重组腺相关病毒。
所述纯化方法为:将病毒液离心后弃去大部分上清,加入核酸酶消化去除残留的质粒DNA,37℃孵育后离心,取上清加入到超滤管中,然后加入碘克沙醇梯度液,48000rpm超速离心2.5h,收集病毒层,于-80℃保存。
通过定量PCR法检测基因组中AAV载体的基因组拷贝数来测定AAV的病毒颗粒数:准备样品和标准品,梯度稀释标准品质粒和待测样品至原浓度的10-5,10-6,10-7,10-8,每个梯度做两个副孔,每个反应孔中加入5μL的模板,上机,退火温度设置为60℃,根据CT值计算样品中的AAV拷贝数。
实施例2
小鼠脊髓损伤造模、病毒注射及功能恢复检测:
准备8周龄C57BL/6小鼠(南京市江宁区青龙山动物繁殖场),分对照组AAV-Con及实验组AAV-USP13,每组各12只。造模过程及处理步骤如下所述:C57BL/6小鼠术前6小时禁食水,异氟烷(深圳瑞沃德生命科技有限公司)吸入麻醉后对小鼠背部皮肤进行备皮及碘伏消毒;取背部正中切口,逐层顿性分离皮下组织、筋膜、肌肉及椎旁组织,暴露T8及邻近节段;用弯钳小心摘除T8椎板,暴露脊髓,注意止血;将小鼠固定于脊髓打击器(深圳瑞沃德生命科技有限公司)上,以5g重量打击头从6.5cm高处垂直下落打击脊髓,生理盐水冲洗后可观察到明显的脊髓出血、水肿。脊髓损伤后,立即将实施例1获得的巨噬细胞特异性USP13过表达的重组腺相关病毒以尾静脉方式注射进小鼠体内(5×1011vg;250μL)。对于对照组(AAV-Con组),注射等量无USP13过表达序列的对照病毒(即AVV载体),其余步骤不变。然后逐层缝合切口并消毒,保温至苏醒后放回笼中,术后每天给予人工排尿直至膀胱功能恢复正常,并给与抗生素与止痛药处理。
在损伤后的1、3、7、14、21、28天进行BMS评分,评估小鼠后肢运动功能恢复情况;在损伤后第28天利用转棒实验评估小鼠后肢感觉及后肢平衡恢复情况;肌电图测定运动诱发电位的潜伏期和波幅,评估神经传导功能。进一步将小鼠安乐死后取出脊髓,Trizol法提取总RNA后进行qRT-PCR检测M1和M2相关指标的mRNA相对表达量;同时,将组织于4%多聚甲醛(武汉塞维尔)中固定24h,梯度乙醇脱水,二甲苯透明后进行石蜡包埋并切片通过iNOS或Arg1免疫荧光染色观巨噬细胞M1和M2极化情况;通过NF免疫荧光染色观察轴突再生情况;通过NeuN免疫荧光染色观察损伤灶以远不同距离的存活神经元数量;上述所有行为学检测及病理学实验方法以及测试结果如下。
(1)BMS评分
于造模前及造模后1天、3天、7天、14天、21天、28天对各组小鼠进行BMS评分。评分由两名熟悉评分细则且不知晓分组情况的研究人员分别独立完成,将小鼠置于旷场内观察其自由活动4分钟得出评分。每只小鼠的最终评分取两名评分人员的平均值。评分细则见表1。
表1:BMS评分量表
图1为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的BMS评分结果,*P<0.05,**P<0.01,***P<0.001,从图中可以看出,两组小鼠在脊髓损伤后后肢运动功能立即丧失,但是AAV-USP13组小鼠在损伤后BMS评分均显著高于AAV-Con组小鼠,表明尾静脉注射USP13过表达的重组腺相关病毒能够改善小鼠脊髓损伤后运动功能恢复。
(2)转棒实验
利用转棒疲劳仪(深圳瑞沃德生命科技有限公司)在脊髓损伤后28天检测小鼠平衡和运动协***况。将小鼠放在匀加速(0-40rpm)的转棒疲劳仪上。记录小鼠从棒上掉落时的棒的转速和小鼠持续站在棒上的时间。测试结果见图2。
图2为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的转棒实验测试结果,其中,图2A为小鼠待在棒上的最长时间,图2B为小鼠能够维持在棒上的最大转速,*P<0.05,**P<0.01,***P<0.001。可以看出,与对照组相比,AAV-USP13尾静脉注射显著延长了小鼠待在转棒上的时间,同时提高耐受的转速,表明AAV-USP13尾静脉注射能改善后肢运动功能和躯体平衡能力。
(3)肌电图
于造模后28天进行肌电图检测。麻醉小鼠后,将刺激电极置于暴露脊髓的头端,记录电极***股二头肌屈肌深处1.5mm处。参比电极置于后肢肌腱远端,接地线置于皮下。用0.5mA,0.5ms,1Hz的刺激诱发电位,计算肌电图波幅及潜伏期来评价后肢功能。测试结果见图3。
图3为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的肌电图及波幅和潜伏期统计图,其中,图3A为AAV-Con组和AAV-USP13组小鼠的肌电图波形,图3B和图3C分别为运动诱发电位的波幅和潜伏期统计图,*P<0.05,**P<0.01,***P<0.001。可以看出,与AAC-Con组相比,注射AAV-USP13的小鼠运动诱发电位具有更短的潜伏期和更高的波幅,表明尾静脉注射重组腺相关病毒能够改善脊髓损伤小鼠的后肢神经传导功能。
(4)RNA提取及qRT-PCR
Trizol法提取总RNA后进行qRT-PCR检测M1(iNOS、TNF-α、IL-1β为巨噬细胞M1极化的标志物)和M2(Arg1、CD206、YM1/2是巨噬细胞M2极化的标志物)相关指标的mRNA相对表达量:
将小鼠安乐死后取出脊髓,加入适量Trizol试剂,匀浆机充***解组织,-80℃放置一天,提取总RNA,室温下静置5分钟,使核酸与蛋白复合物完全分离,加入0.2mL氯仿,用手剧烈颠倒震荡混匀,然后室温静置10min,4℃下12000rpm离心10分钟,小心吸取上层水相,转移至新的EP管中,加入等体积的异丙醇,上下颠倒混匀,室温静置10分钟。4℃下12000rpm离心10分钟,弃上清,用-20℃预冷的75%乙醇洗涤RNA沉淀,4℃下12000rpm离心10分钟,弃上清,至少洗涤2次。室温放置干燥RNA,加入20-100μL无RNase水溶解RNA,Nanodrop测定RNA浓度。于37℃15分钟,85℃5秒钟条件下进行逆转录,逆转录反应体系为:2μl5×PrimeScript RT Master Mix,500ng RNA,补加DEPC水至10μl。以GAPDH作为mRNA的内参,每个样本设置3个副孔,使用2-ΔΔCT法计算目的基因相对表达量。PCR反应体系:10μl2×TB Green Premix Ex Taq,0.4μL前后引物,0.4μL 50×ROX Reference Dye,2μl cDNA模板,6.8μl灭菌水。PCR反应程序:95℃预变性30秒钟,95℃变性5秒钟,60℃退火、延伸30秒,循环40次。所用引物如下:
iNOS forward:5’-GCTCGCTTTGCCACGGACGA-3’
iNOS reverse:5’-AAGGCAGCGGGCACATGCAA-3’
TNF-αforward:5’-CCCTCCTGGCCAACGGCATG-3’
TNF-αreverse:5’-TCGGGGCAGCCTTGTCCCTT-3’
IL-1βforward:5’-GCCTCGTGCTGTCGGACCCATAT-3’
IL-1βreverse:5’-TCCTTTGAGGCCCAAGGCCACA-3’
Arg1 forward:5’-CTATGTGTCATTTGGGTGGA-3’
Arg1 reverse:5’-TCTGGGAACTTTCCTTTCAG-3’
CD206 forward:5’-CAAGGAAGGTTGGCATTT-3’
CD206 reverse:5’-CCTTTCAGTCCTTTGCAAGC-3’
YM1/2forward:5’-CAGGGTAATGAGTGGGTTGG-3’
YM1/2reverse:5’-CACGGCACCTCCTAAATTGT-3’。
图4为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的M1和M2相关基因的qRT-PCR定量检测结果。其中,iNOS、TNF-α、IL-1β为巨噬细胞M1极化的标志物,Arg1、CD206、YM1/2是M2极化的标志物,*P<0.05,**P<0.01,***P<0.001,可以看出,与AAV-Con组相比,注射AAV-USP13的小鼠脊髓M1极化相关基因mRNA水平显著下降,而M2相关基因的mRNA相对表达量显著升高,表明尾静脉注射重组腺相关病毒能够促进小鼠脊髓损伤后体内巨噬细胞M2极化。
(5)免疫荧光染色
将组织于4%多聚甲醛(武汉塞维尔)中固定24h,梯度乙醇脱水,二甲苯透明后进行石蜡包埋并切片,石蜡切片经过二甲苯脱蜡,梯度酒***化后,用柠檬酸钠修复液(武汉塞维尔)高温高压修复,然后用5%BSA(美国赛默飞)溶液室温封闭1小时阻断非特异性结合。滴加一抗,湿盒中4℃孵育过夜,然后PBS洗三遍,每遍5分钟,接着室温避光孵育荧光二抗2小时,继续PBS洗三遍,每遍5分钟,复染DAPI并封片后拍照。通过iNOS或Arg1免疫荧光染色观巨噬细胞M1和M2极化情况;通过NF免疫荧光染色观察轴突再生情况;通过NeuN免疫荧光染色观察损伤灶以远不同距离的存活神经元数量。测试结果见图5-7。所用抗体信息见表2。
表2:抗体信息
图5为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠iNOS和Arg1免疫荧光染色及统计图,其中,图5A为iNOS,图5B为Arg1,*P<0.05,**P<0.01,***P<0.001。可以看出,与AAC-Con组相比,注射AAV-USP13的小鼠脊髓M1巨噬细胞显著减少,而M2巨噬细胞数量明显增多。
图6为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的NF免疫荧光染色及轴突数量统计图,其中图6A为AAV-Con组和AAV-USP13组小鼠脊髓石蜡切片NF的免疫荧光染色,图6B为损伤灶以远不同距离的NF阳性的轴突数量统计图,可以看出,与AAV-Con组相比,AAV-USP13组小鼠脊髓NF阳性的轴突数量明显增多,表明原位注射重组腺相关病毒促进小鼠脊髓损伤后轴突再生。
图7为重组腺相关病毒尾静脉注射治疗脊髓损伤小鼠的存活神经元免疫荧光染色及数量统计图,其中图7A为AAV-Con组和AAV-USP13组小鼠脊髓石蜡切片NeuN免疫荧光染色,图7B为损伤灶以远不同距离的存活神经元数量统计图。可以看出,与AAV-Con组相比,AAV-USP13组小鼠脊髓Z1(距损伤灶边缘0-250μm)、Z2(距损伤灶边缘250-500μm)、Z3区域(距损伤灶边缘1000-1250μm)存活神经元的数量明显增多,而Z4(距损伤灶边缘2000-2250μm)区域的神经元数量无明显差别。
实施例3
为了进一步研究USP13促进脊髓损伤小鼠巨噬细胞M2极化的机制,我们随后进行了蛋白免疫共沉淀串联质谱分析了所有可以与USP13结合的蛋白。从中,我们鉴定出了IκBα蛋白的特异性肽段,随后我们分别进行了USP13和IκBα的蛋白免疫共沉淀实验。为了进一步验证USP13可以靶向调控IκBα的泛素化,我们通过shRNA技术敲低巨噬细胞中的USP13水平(shUSP13),随后利用Western Blot实验检测IκBα及其下游p65的蛋白表达量,利用蛋白免疫共沉淀实验检测IκBα的泛素化水平,利用细胞免疫荧光检测巨噬细胞内p65细胞定位。实验方法如下:
(1)蛋白提取及Western blot
巨噬细胞总蛋白提取:弃去巨噬细胞原有DMEM培养基,PBS洗三遍,加入适量裂解液。按照1mL裂解液、10μL磷酸酶抑制剂、10μL PMSF及1μL蛋白酶抑制剂配置蛋白裂解液。将细胞及蛋白裂解液放置于冰上裂解10分钟,用细胞刮刀将裂解物全部刮下后收集于EP管中;4℃,12000rpm,5分钟离心,将上清转入新的EP管中;吸取部分蛋白裂解液行BCA蛋白浓度测定;余下体积按4:1比例加入5×Loading Buffer后于100℃煮沸;待室温冷却后根据实验安排放入-20℃保存或行Western Blot实验。
电泳与转膜:按照实验需求配制不同浓度的分离胶及浓缩胶;加入样品,80V跑浓缩胶;待蛋白样品至分离胶时调节电压至120V;待蛋白至底时停止电泳。裁剪适当大小的PVDF膜,顺序叠放滤纸、凝胶及PVDF膜制备转膜三明治,赶尽其中的气泡;夹紧转膜夹放入转膜槽之中;将转膜槽放入冰盒中,加入预冷的转膜液,300mA恒定电流转膜,转膜时间视情况而定。转膜后取出PVDF膜,放入5%BSA封闭液中封闭2小时。
抗体孵育及检测:封闭结束后将孵育相应的一抗,4℃过夜;次日TBST洗膜三遍,孵育相应二抗,常温2小时;洗膜、配置曝光液,将曝光液涂抹均匀至PVDF膜,放入凝胶成像***,拍照分析。
(2)免疫共沉淀(Co-IP)
巨噬细胞总蛋白提取同上;加入1μg与后续IP同种属IgG和20μl充分重悬的Protein A/G-agarose beads(美国Santa Cruz公司),4℃缓慢摇动1小时,后2500rpm离心5分钟,收集上清用于后续的免疫沉淀。加入5μg用于IP的相应一抗,4℃缓慢摇动过夜。次日加入20μl充分重悬的Protein A/G-agarose beads,4℃缓慢摇动2小时。后2500rpm离心5分钟,小心吸除上清,留下沉淀,裂解液洗涤沉淀5次。完成最后一次洗涤后根据余下体积按比例加入5×Loading Buffer后100℃煮沸;待室温冷却后根据实验安排放入-20℃保存或行Western Blot实验。
(3)细胞免疫荧光
去除细胞培养板中的培养基,PBS洗三遍,4%多聚甲醛固定15分钟,0.3%TritonX-100破膜15分钟,后10%山羊血清封闭1小时;加入相应的适当比例一抗在4℃过夜;第二天彻底洗去未结合的一抗,用相应的适当比例荧光二抗在常温中孵育2小时;后彻底洗去未结合的二抗,染核,荧光显微镜下观察。
(4)免疫沉淀质谱联用(IP/MS)
免疫沉淀步骤同上,于南京医科大学分析测试中心行IP质谱检测并分析。
(5)细胞转染
巨噬细胞生长至合适密度时进行转染,步骤如下:5μL shRNA或质粒(Genebaybiotech公司)加P3000试剂(美国赛默飞公司)溶于250μL Opti-MEM培养基,轻轻混匀后静置5min,然后将lipo3000转染试剂(美国赛默飞公司)溶于250μL Opti-MEM培养基,轻轻混匀后静置5min。将上述两种转染试剂进行混合,静置10min形成稳定的转染复合体,随后转染进细胞,6h后换液,换用新鲜的培养基。48小时后提取蛋白进行后续操作。
结果见图8-11。
图8为USP13免疫沉淀复合物中IκBα特异性肽段鉴定图。通过免疫共沉淀串联质谱,我们鉴定出了IκBα特异性肽段LVDDR,表明USP13可以和IκBα蛋白结合。
图9为USP13和IκBα的免疫共沉淀结果,其中,图9A为利用USP13抗体进行免疫沉淀,IκBα抗体进行免疫印迹检测,图9B为利用IκBα抗体进行免疫沉淀,USP13抗体进行免疫印迹检测。可以看出,USP13抗体可以共沉淀IκBα蛋白,IκBα抗体也可以共沉淀USP13蛋白,说明USP13和IκBα可以相互结合。
图10为敲低USP13后巨噬细胞IκBα、胞质p65和核内p65蛋白表达水平的检测结果及敲低USP13后巨噬细胞p65细胞定位检测结果,其中,图10A为敲低USP13后巨噬细胞IκBα、胞质p65和核内p65蛋白表达水平的检测结果,图10B为敲低USP13后巨噬细胞p65细胞定位图,可以看出,当敲低USP13后,IκBα蛋白水平也相应降低,同时,IκBα调控的经典蛋白p65在胞质内的表达降低,在核内的表达升高,表明p65蛋白从胞质转移到核内,NF-κB通路被激活。
图11为敲低USP13后巨噬细胞IκBα蛋白的泛素化水平检测结果。利用IκBα抗体进行免疫沉淀,Ub抗体进行免疫印迹检测IκBα的泛素化水平。可以看出敲低USP13后,IκBα上结合的泛素分子更多,泛素化水平升高。说明USP13可以调控IκBα蛋白的泛素化水平。
序列表
<110> 江苏省人民医院(南京医科大学第一附属医院)
<120> 一种巨噬细胞特异性USP13过表达的重组腺相关病毒及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2577
<212> DNA
<213> 脊髓损伤(Spinal Cord Injury)
<400> 1
atgcagcgcc ggggcgccct gttcagcgtg ccgggcggcg gcgggaagat ggctgcaggg 60
gacctgggcg agctgctggt gcctcatatg cccacgatcc gcgtgcccag gtcgggggac 120
cgcgtctaca agaacgagtg cgccttctcc tacgactccc cgaactctga aggtgggctc 180
tacgtatgca tgaatacctt tttggccttt ggaagggaac acgtagaaag acactttcga 240
aaaactggac agagcgtata catgcacctg aagaggcaca tgcgagagaa ggtaagagga 300
gcctctggtg gagctttacc caaaaggagg aattccaaga tatttttaga tctagatatg 360
gatgacgatt taaatagtga cgattacgaa tatgaagacg aagccaaact tgttatattc 420
ccagaccact atgaaatagc ccttcctaac attgaggagt taccagccct ggtaacaatt 480
gcttgtgatg cagtgctcag ctcaaagtcc ccttacagga agcaggatcc agacacatgg 540
gaaaacgaag tgccagtatc gaagtatgcc aacaaccttg tgcaactgga caacggggtc 600
aggattcctc ccagtggctg gaagtgtgcc cgatgtgacc tgcgggagaa cctctggttg 660
aatctgactg acggctctgt tctgtgtggg aagtggtttt ttgacagctc agggggcaac 720
ggccacgcac tggagcatta cagggacatg ggctatcctc tggccgtgaa gctgggcacc 780
atcacacctg atggggcaga tgtttattct tttcaagaag aggggcctgt ttcggatcct 840
catttggcca aacacttagc acattttggg atcgacatgc tccacacgca agggacagag 900
aacggtctcc gggacaatga catcaaaccg agagtcagcg agtgggaagt gatccaggag 960
tcaggaacta agctgaagcc gatgtacggc ccagggtaca cgggcctgaa gaacctgggc 1020
aacagttgct acctcagttc tgtcatgcag gccatcttca gcatcccaga gttccagaga 1080
gcgtatgtag gaaacctccc aaggatattt gactactcac cgttagatcc aacgcaggac 1140
ttcaacacac aaatgactaa gttgggacat ggcctcctct ctggccagta ctcgaagcct 1200
ccagtgaaat ctgagctcat tgaacaggtg atgaaggagg agcacaagcc tcagcagaat 1260
gggatctctc cacgcatgtt caaggccttt gtcagcaaga gccacccgga attctcctcc 1320
aacagacagc aggatgccca ggagtttttc ttgcatttgg tcaatctggt agagaggaat 1380
cgcattggct cagaaaaccc aagtgatgtt ttccggtttt tggtggagga gcgaattcaa 1440
tgctgtcaga ccagaaaggt tcgctacacg gagagggtgg actacctaat gcagttacct 1500
gtggccatgg aggcagcaac caacaaagat gagctgatca cctatgaact catgcggagg 1560
gaagcagaag ccaacagaag acccctacct gagctggtgc gagccaagat cccattcagt 1620
gcctgccttc aggcctttgc tgaaccagac aatgtggatg atttctggag cagcgctctg 1680
caggccaagt ctgcaggggt caaaacttct cgctttgcct cattccctga atacttggta 1740
gtgcagataa agaagttcac ttttggtctt gactgggttc ccagaaaatt tgatgtttct 1800
attgatatgc cagacctact agatatcagc catctcagag ccaggggctt gcagccaggg 1860
gaagaggagc ttcctgacat cagccccccc atagtcattc ctgatgactc aaaagaccgc 1920
ttgatgaacc agttgataga cccctcagac attgatgagt cttcggtgat gcagctggct 1980
gagatgggct tccctttgga agcctgcagg aaggctgtgt acttcacggg gaacaccgga 2040
gctgaggtgg ccttcaactg gattatcgtg cacatggagg agcctgactt tgctgaacca 2100
ctggccatac ctgggtatgg aggggctggg gcctctgtct ttggtgctac tggattggac 2160
aaccaacctc ctgaggaaat cgtagctatt atcacctcga tgggattcca gcgaaatcag 2220
gcagtgcagg ctctacaagc aacgaatcat aacctggaaa gagcactgga ctggatcttc 2280
agccaccccg agtttgaaga ggacagtgac tttgtgatcg agatggagaa caatgcaaat 2340
gccaacatcg tgtctgaggc caagccagag ggacccagag tgaaggatgg gtctggaatg 2400
tacgagttgt ttgctttcat cagtcacatg ggaacatcta caatgagtgg ccattatgtt 2460
tgccatatca agaaagaggg acgatgggtg atctacaatg accacaaagt ttgtgcctca 2520
gaaaggcccc ccaaagacct gggctatatg tacttttacc gcaggatacc aagctaa 2577
Claims (4)
1.巨噬细胞特异性USP13过表达的重组腺相关病毒在制备治疗脊髓损伤的药物中的应用,所述巨噬细胞特异性USP13过表达的重组腺相关病毒的构建方法包括如下步骤:
(1)体外克隆USP13基因,所述USP13基因的序列如SEQ ID NO:1所示;
(2)将USP13基因和pAAV-Lyz2载体分别用限制性内切酶BamH I和EcoR I进行双酶切,然后进行DNA连接反应,得到含有目的基因USP13的重组穿梭质粒,对其进行扩增、提纯;
(3)将含有目的基因USP13的重组穿梭质粒和含腺相关病毒基因组DNA的pHelper和pAAV-RC质粒按摩尔比1:1:1混合,共转染AAV-293细胞,培养后收获细胞,反复冻融后过滤,获得病毒液,纯化后,得到巨噬细胞特异性USP13过表达的重组腺相关病毒。
2.如权利要求1所述的应用,其特征在于,步骤(1)中,所述体外克隆USP13基因的方法为:提取并体外培养原代巨噬细胞,收集细胞后提取总RNA,逆转录得到cDNA,然后以cDNA为模板,进行PCR扩增反应,得到USP13基因;所述PCR扩增的引物序列为:前引物:5’-CGTCCAGGTCCTGTTCTCC-3’,后引物:5’-TACCCATGTAGTCCTCCGCA-3’。
3.如权利要求1所述的应用,其特征在于,所述药物还包括药学上可接受的载体。
4.如权利要求3所述的应用,其特征在于,所述药学上可接受的载体包括稀释剂和赋形剂。
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