CN115478053A - 神经干细胞源“溶瘤”外泌体及其用途 - Google Patents
神经干细胞源“溶瘤”外泌体及其用途 Download PDFInfo
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Abstract
本发明涉及生物医学领域，涉及一种神经干细胞源“溶瘤”外泌体及其用途。具体涉及到一种以溶瘤腺病毒感染神经干细胞后分离获得的包含病毒组分且具有溶瘤病毒功能的外泌体的设计、分离及其应用。
Description
技术领域
本发明涉及生物医药领域,特别涉及一种用于肿瘤治疗的神经干细胞源“溶瘤”外泌体及其用途。
背景技术
溶瘤病毒疗法是通过选择性地在肿瘤细胞中复制，破坏并溶解细胞，同时表达治疗基因来杀死肿瘤细胞，并伴随肿瘤裂解释放细胞因子和肿瘤抗原进一步调节肿瘤免疫微环境的治疗方法。与其他肿瘤疗法相比，溶瘤病毒(Oncolytic Viruses)具有杀伤效率高、靶向精确、副作用和耐药少、成本低等优点，这些都使得溶瘤病毒疗法与手术治疗、放疗、化疗和靶向治疗相比，成为一种更有前景的癌症治疗方法[1]。目前，有多种溶瘤病毒被用于肿瘤的免疫治疗临床试验，与其他溶瘤病毒相比，溶瘤腺病毒具有高度的遗传稳定性和低致病性，它们相对来说更容易稳定地维持在高滴度和高纯度，这使得它们特别适合于应用到疾病治疗中，无论是基因和癌症治疗还是疫苗的开发，因此溶瘤腺病毒成为其中使用最多的病毒[2]。
结合基因治疗和溶瘤病毒治疗的双重优势，以溶瘤腺病毒为载体介导的基因治疗被广泛应用到临床实验当中，例如DNX-2401(Delta-24-RGD)被用来进行恶性胶质瘤的治疗[3,4],VCN-01被用来进行胰腺癌的治疗[5]，Ad4-H5-Vtn作为流感疫苗也进入到临床研究中[6]。这些临床研究都展示出溶瘤腺病毒作为一种基因治疗的载体在未来多种疾病治疗中的潜力。然而溶瘤腺病毒也依然存在一些限制，阻碍其进一步的应用和发展。一方面是给药方式限制，病毒无法通过静脉注射给药，因为静脉注射后腺病毒无法专一靶向病灶，这会导致能够到达肿瘤部位并行使功能的病毒数量骤减[7]；另外病毒无法穿透生理屏障如血脑屏障等，对于恶性胶质瘤等疾病的治疗效果大大受限，出于这些限制，目前包括溶瘤腺病毒在内的绝大多数溶瘤病毒的给药方式是瘤内注射，但是这种给药方式也存在很多问题导致效果受限，病毒在瘤内的滞留性差，可能会在周围正常组织中非特异性脱落，这会导致治疗效果差，脱落的病毒还会被机体的免疫系统识别并清除，甚至引发严重的炎症反应[8,9]；另一方面是免疫调节功能的限制，单纯的病毒治疗所能够引起的免疫调节能力有限，难以引发高效的抗肿瘤免疫，另外免疫抑制肿瘤微环境的存在也进一步限制了能够抗肿瘤的T细胞反应，针对这个限制，目前常用的解决方式是改造病毒的基因组，使其表达具有免疫调节能力的小分子，或者与抗体联合使用，从而提升抗肿瘤免疫的水平[10,11]。
尽管溶瘤腺病毒构建策略日趋成熟，然而对于病毒骨架的改造仍然存在很多问题，病毒的基因组能够插入的片段大小有限，且这种改造方式很可能会影响病毒的包装，另外，传统的病毒改造方式很难同时改善两种使用限制，而如何同时提升病毒的体内靶向性以及免疫调节能力，进一步优化病毒的使用效果至关重要。本发明旨在提供一种新的模型，在保留病毒原有使用优势的基础上，同时提升体内靶向性及免疫调节能力，进一步提升病毒载体的应用前景。
外泌体是通过核内体凹陷形成多泡体(MVBs)，并且通过多泡体和脂膜融合释放到细胞外基质的一种囊泡[12,13]，参与了各种生物过程，包括病毒感染、免疫反应、哺乳动物的发育和繁殖[14]，是细胞间实现信息交流的一种手段。近年来外泌体也因为其能够携载多种化疗药物或治疗基因，易修饰，且具有特定的细胞向性，能够穿透生理屏障，免疫原性低而安全性高，血液稳定性好等优点，成为了一种备受关注的药物载体[15-17]。本发明的目的就是利用外泌体这一理想的载体，构建出具有腺病毒组分和功能的外泌体，呈递一种新的“病毒”模式，在实施“溶瘤”功能的同时，利用外泌体的天然靶向性和免疫调节能力，改善溶瘤腺病毒给药靶向性不足和免疫调节能力不足的问题，提升溶瘤腺病毒的使用效果。研究报道病毒在感染过程中，会将遗传物质和蛋白导入到外泌体中，随着外泌体被周围的细胞吸收，将病毒的组分导入到受体细胞中，进而促进病毒的感染[18]。由病毒基因组组成的外泌体可以通过进入对病毒敏感的细胞而增强病毒传播，同时避开免疫识别[19]。
综合这些研究，我们的设计就是利用病毒感染细胞的生理过程，用病毒感染的方法进行病毒基因和蛋白的传递，筛选出具有“溶瘤”功能的外泌体，这种设计避免了外泌体内容物的外流，保留了外泌体本身的生理功能。我们通过溶瘤腺病毒感染神经干细胞并分离含有病毒组分，具有溶瘤病毒功能的外泌体，称为“溶瘤”外泌体。以神经干细胞作为模式细胞系，使这种“溶瘤”外泌体具有了神经干细胞系类似的功能，与溶瘤腺病毒相比，其能够突破血脑屏障并具有更好的胶质瘤靶向性，同时也提升了在肿瘤微环境中的免疫调节能力，发挥出更好的抗肿瘤效果。
发明内容
本发明提供了一种新型“溶瘤病毒”模式：“溶瘤”外泌体(Adexo)。这种策略利用了病毒的感染过程，筛选出具有“溶瘤”功能的外泌体，该策略摒弃原有的病毒结构，是以包含病毒组分的外泌体作为初始感染单位，在被感染的细胞内再生成具有生物活性的子代病毒颗粒。
本发明提供的“溶瘤”外泌体除了具有溶瘤腺病毒的溶瘤效果以及治疗基因的治疗效果外，还通过静脉给药在小鼠的胶质瘤原位模型中展示出了更好的靶向性及治疗效果。
本发明提供的“溶瘤”外泌体还具有亲本细胞的免疫调节能力，在肿瘤微环境中通过T细胞和B细胞的激活展示出更好的抗肿瘤免疫调节功能，进一步提升了治疗效果。
本发明提供的“溶瘤”外泌体既具备作为抗肿瘤药物的功能，也展示出作为疫苗的应用潜力。
本发明提供的新型修饰模式，可以根据不同的疾病，选择不同类型的亲本细胞为之服务，同时所使用的病毒也可进行不同修饰，根据使用目的表达不同的治疗基因，是一种有广泛应用潜力的新型外泌体模型。
附图说明
图1.“溶瘤”外泌体分离原理示意图。
图2.“溶瘤”外泌体的电镜表征。
图3.“溶瘤”外泌体的溶瘤功能验证。
图4.“溶瘤”外泌体静脉给药后的胶质瘤靶向效果。
图5.“溶瘤”外泌体的胶质瘤治疗流程及治疗效果,(A)种瘤流程及给药策略；(B)治疗后的裸鼠生存期和(C)脑组织的H&E染色结果和肿瘤相对大小量化。
图6.“溶瘤”外泌体在免疫全能小鼠肿瘤模型(B16)中的抑制抑瘤效果。
图7.“溶瘤”外泌体在肿瘤微环境中的免疫调节活性检测。
具体实施方式
实施例1“溶瘤”外泌体的分离及筛选
1、外泌体的分离
用含有10％FBS的DMEM培养基(Gibco)培养C17.2细胞，待细胞生长至70-80％时，换用无外泌体的10％FBS培养基继续培养72h，收集细胞上清，进行外泌体纯化，具体的纯化步骤如下：先300×g，4℃离心10min取上清，将上清继续用2000×g，4℃离心40min，将离心后的上清10,000×g，4℃离心1h后取上清，最后100,000×g，4℃离心2h后得到外泌体，沉淀用无菌的PBS重悬，BCA试剂盒(Takara)定量后储存于-80℃条件下备用。
2、溶瘤外泌体的筛选
对于病毒感染之后收取的外泌体，沉淀用少量无菌PBS重悬后，要通过碘克沙醇(Thermo Fisher)密度梯度离心进行后续的筛选：
(1)病毒感染细胞72h后，收取上清，进行外泌体分离。4℃条件下，300×g，离心5min，然后2,000×g,离心40min，10000×g，离心1h，取上清；
(2)保留的上清最后于4℃，100,000×g，离心2h，用PBS重悬沉淀，得到外泌体；
(3)稀释60％OptiPrepTM密度梯度储备液，稀释成不连续的6个密度梯度浓度。然后按照浓度从高到低的浓度顺序加入13.5mL的一次性SW40的超离管(Thermo Fisher)中，形成不连续的密度梯度层，在最上层加入用PBS重悬好的外泌体。35,000×g，4℃离心3h；
(4)3h离心结束后，分离出目的外泌体。
实施例2“溶瘤”外泌体的电镜表征
(1)取10μL样品滴至铜网(中兴百瑞)，静止5min；
(2)沿铜网用滤纸吸收多余的样品，随后再滴加2％磷钨酸(阿拉丁)溶液负染5min，结束后再用滤纸吸附多余的负染液；
(3)PBS滴加洗三次，每次5min；
(4)在透射电镜中用80KV的电压观察样品形态。
实施例3“溶瘤”外泌体的溶瘤功能验证
将293细胞(ATCC)铺到6孔板(NEST)中，待细胞生长至90％的密度，把10％血清浓度的DMEM培养基(Gibco)替换为2％血清浓度，将等量的Ad和Adexo加至细胞培养上清中，称为感染，24h后拍摄荧光图像，待细胞全部病变至漂起后，收取细胞，用200μL空培养基重悬，冻融裂解三次，释放细胞内子代病毒，12000rpm离心10min，用上清再次感染293细胞，24h后再次拍摄荧光图像，视为二次感染。
实施例4“溶瘤”外泌体的体内靶向效果评价
选取4-6周裸鼠，在颅内注射5×105个U87(ATCC)细胞，10天后，在第11天和13天给药，共分为四组，分别是PBS，Ad，Adexo以及exo，每只鼠静脉注射病毒，剂量为1×1010vp，Adexo和Ad通过qRT PCR(Takara)对病毒进行统一定量，exo组的加入量需要先将exo和Adexo通过BCA定量，根据加入的Adexo的量进行换算，加入等质量的exo。第二次给药后第二天，处死小鼠，取脑组织进行荧光成像。
实施例5“溶瘤”外泌体的胶质瘤治疗效果评价
选取4-6周裸鼠，与颅内注射5×105个U87细胞，10天后，开始进行治疗，共分为四组，每组6只鼠，分别在第11天，第13天，第15天，于静脉注射1×1010vp病毒，后跟踪记录生存期并观察小鼠状态，直至达到判定标准后，处死小鼠，取小鼠脑部组织进行组织切片及H&E染色，并对肿瘤面积大小进行统计并分析显著性差异。
H&E染色：
(1)从小鼠体内分离组织，放入预冷的4％多聚甲醛(阿拉丁)中固定，每24h更换一次多聚甲醛，共固定3天；
(2)将固定好的组织放入20％蔗糖(辽宁泉瑞试剂有限公司)中，4℃放置，将沉底后换新的20％蔗糖溶液，再次沉底后换成30％蔗糖溶液；
(3)将脱水后的组织用OCT(日本樱花)包埋，-80℃过夜；
(4)使用切片机进行切片，切片后用载玻片(Genview)贴取切下来的组织；
(5)载玻片置于60℃，烘片1h；
(6)取出后用组化笔(迈新试剂)圈出组织位置；
(7)置于PBS中复水，每次5min，进行3次；
(8)苏木素(北京鼎国昌盛生物技术有限公司)：5min；
(9)分化液(碧云天)：30s；
(10)返蓝液(北京索莱宝科技有限公司)：2min；
(11)伊红(北京鼎国昌盛生物技术有限公司)：2-5min(至细胞质颜色清楚)；
(12)中性树脂(爱必信)封片并用显微镜拍摄。
实施例6“溶瘤”外泌体在免疫全能小鼠体内肿瘤抑制实验
选取4-6周Balb/c小鼠，皮下荷瘤B16(1×106)(ATCC)，12只鼠一组，平均瘤体积达到100mm3时，在第0，3，6天瘤内(1×109vp)注射PBS，Ad，Adexo或exo，持续监测瘤体积，在第13天进行免疫细胞检测。
实施例7“溶瘤”外泌体瘤内免疫细胞的调节分析
1.肿瘤细胞分离：
(1)取肿瘤至于预先置于24孔板(NEST)中；
(2)剪刀剪碎后，加入胶原酶(罗氏)至终浓度0.04mg/mL(0.5mg/mL储液加入80mL至1mL中)，加入DnaseI(Thermo Fisher)至10U/mL(2,000U/mL,1:200使用,即1mL加入5μL),37℃消化1h吹悬后，取上清400×g，5min离心,吸取并留存上清，沉淀用含10％BSA的1640培养基(Gibco)重悬。上清用于剩余组织残渣的消化，继续消化1h后筛网轻轻研磨，离心取沉淀；
(3)获得的细胞悬液过筛网至新的离心桶(NEST)中，350×g离心5min；
(4)2mL含10％BSA的1640培养基重悬后进行细胞计数，标记所需管数后，剩余细胞置于冰上暂存。
2.封闭Fc受体
每管加入100μL block-Fc(Biolegend)，冰上放置5-10min。
3.细胞表面抗原染色
(1)将制备获得的脾细胞及肿瘤相关细胞用PBS+10％FBS洗一遍(500μL PBS重悬，350×g离心5min)，吸弃上清；
(2)分别加入对应编号的染色体系100μL/管,空白对照各加入100μL stainingbuffer(Biolegend)；
(3)避光孵育20min；
(4)PBS洗2遍；
(5)挑出细胞内染色进行后续步骤，其余各管均用100μL staining buffer重悬后，避光放置。
4.细胞内染色
(1)将细胞内染色各管细胞，每管加入400μL||fixation buffer(ebioscience)混匀，室温避光30min；
(2)350×g离心5min，弃掉上清；
(3)用400μL 1×Staining Perm Wash Buffer(ebioscience)重悬细胞，避光放置8min，350×g离心8min，重复两次；
(4)加入细胞内染色抗体(Biolegend)，室温避光30min；
(5)1mL 1×Intracellular Staining Perm Wash Buffer(ebioscience)洗2次，200μL staining buffer重悬，流式上机检测。
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Claims (6)
1.一种神经干细胞源“溶瘤”外泌体，其特征在于：以溶瘤病毒感染神经干细胞后分离得到的含有病毒组分的外泌体具有优良的抗肿瘤作用。
2.如权利要求1所述的神经干细胞源“溶瘤”外泌体，其特征在于：其中所述的外泌体衍生自溶瘤腺病毒感染的神经干细胞。
3.如权利要求2所述的神经干细胞源“溶瘤”外泌体，其特征在于：其中所述的溶瘤病毒为衣壳修饰的溶瘤腺病毒。
4.如权利要求1所述的神经干细胞源“溶瘤”外泌体，其特征在于：其可用于治疗的肿瘤类型包括但不限于脑胶质瘤、黑色素瘤、结直肠癌等。
5.如权利要求1所述的神经干细胞源“溶瘤”外泌体，其特征在于：其给药方式包括瘤内注射、静脉注射、颅内注射等途径。
6.权利要求2-5任一项所述的神经干细胞源“溶瘤”外泌体用于制备肿瘤治疗药物和疫苗的用途。
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