CN110623981B - NK细胞外泌体hsa-miR-330-5p在抗菌中的应用 - Google Patents
NK细胞外泌体hsa-miR-330-5p在抗菌中的应用 Download PDFInfo
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Abstract
本发明公开了经跨膜IL‑21激活的NK细胞(aNK)分泌的外泌体,及其包含的多种miRNA在抗菌中和抗肿瘤的应用,为治疗细菌感染和抗肿瘤提供了新的思路。此方法:(1)不同于抗生素治疗,外泌体抗菌具有较低的副作用;(2)可通过联合使用增强抗菌治疗效果;(3)外泌体可低温储存,miRNA可以人工合成,成本低,可以实现大规模的生产;(4)外泌体和多种miRNA对多种细菌均有抑制效果,具有宽泛的抗菌谱;(5)miRNA作用于多种肿瘤细胞共有的信号通路,能抑制多种肿瘤,具有宽泛的抗肿瘤应用。
Description
本申请为申请号为2017113093652、申请日为2017年12月11日、发明名称“NK细胞外泌体及相关miRNA在抗菌和抗肿瘤中的应用”的分案申请。
技术领域
本发明涉及通过经跨膜IL-21激活的NK细胞(aNK)分泌的外泌体(exosome),以及相关的miRNA在抗菌中和抗肿瘤的应用。
背景技术
外泌体是一类由细胞分泌的携带胞质组分的纳米级别的膜性小泡,由机体的多种细胞分泌,广泛分布于唾液、血浆、乳汁等体液中。外泌体中含有蛋白质、mRNA和miRNA等多种生物活性物质。外泌体以膜融合的方式将miRNA和蛋白质传递给其他细胞,作为细胞之间相互交流的桥梁。
NK细胞是固有免疫系统中主要的效应细胞,已应用于肿瘤的治疗,其通过释放穿孔素和颗粒酶杀伤被感染或发生癌变的细胞。NK细胞分泌的外泌体含有部分NK细胞的活性物质,也具有抗肿瘤的能力。
NK细胞外泌体应用于临床治疗最大的瓶颈在于因为NK细胞在体外无法很好的扩增,从而无法获取足够数量的外泌体。为了解决这一问题,我们采用人工抗原呈递细胞(genetically-engineered artificial antigen-presenting cells,aAPCs)表达的跨膜IL-21来刺激NK细胞的增殖,aNK细胞的扩增数量足以满足临床治疗的需求。经过14-18天的激活和扩增,大量的外泌体由aNK细胞释放进入培养液中,这部分aNK细胞外泌体拥有典型的NK细胞特征蛋白,并对多种肿瘤细胞均具有杀伤能力。
在过去数十年中,抗生素的广泛使用使得微生物耐药频率和其与相关的严重感染性疾病的以令人担忧的速度增加。在革兰氏阳性菌中,最重要的耐药性病原体是耐甲氧西林的金黄色葡萄球菌、耐β-内酰胺的肺炎球菌和多药耐药性肺炎球菌,以及耐万古霉素的肠球菌。肺炎克雷伯氏菌、大肠杆菌和奇异变形杆菌等革兰氏阴性菌中的β-内酰胺酶耐药性是造成革兰氏阴性菌耐药性的重要原因。因此临床治疗迫切需要新颖的抗菌剂,尤其具有新颖作用机制的抗菌剂。
本发明发现激活后的aNK细胞,其外泌体不仅富含NK细胞的胞浆成分,还富含大量的miRNA。这些miRNA在实验中可以显著的抑制细菌的生长,具有出色的抗菌功能,同时发现这些miRNA在实验中可以抑制肿瘤细胞的生长,具有抗肿瘤的功能。
发明内容
本发明的目的在于提供aNK细胞分泌的外泌体,及其包含的miRNA在抗菌和抗肿瘤中的应用。
本发明的目的是通过以下技术方案实现的:一种经跨膜IL-21激活的NK细胞分泌的外泌体在抗菌中的应用。
进一步地,所述外泌体包含一条或一条以上选自SEQ ID NO.1~SEQ ID NO.2144所示序列的miRNA。
进一步地,将所述外泌体单独用于抗菌,或将所述外泌体与aNK细胞共同用于抗菌。
SEQ ID NO.1~SEQ ID NO.1411所示的任一条miRNA。
SEQ ID NO.1~SEQ ID NO.2144所示的任一条miRNA在抗菌中的应用。
进一步地,用于制备抗菌制剂。
进一步地,针对的细菌包括但不限于:大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、肺炎克雷伯菌(Klebsiella pneumoniae)、绿脓杆菌(Pseudomonas aeruginosa)、屎肠球菌(Enterococcus faecium)、表皮葡萄球菌(Staphylococcus epidermidis)、粪肠球菌(Enterococcus faecalis)、B族链球菌(Streptococcus Gr.B)、鲍曼不动杆菌(Acinetobacter baumannii)、奇异变形杆菌(Proteus mirabilis)、溶血性葡萄球菌(Staphylococcus haemolyticus)、人葡萄球菌(Staphylococcus hominis)、阴沟肠杆菌(Enterobacter cloacae)、结核分枝杆菌(Mycobacterium tuberculosis)、白色念珠菌(Candida albicans)、嗜麦芽窄食单胞菌(Stenotrophomonasmaltophilia)、头葡萄球菌(Staphylococcus capitis)、脆弱拟杆菌(Bacteroidesfragilis)、粘质沙雷菌(Serratiamarcescens)、摩氏摩根氏菌(Morganellamorganii)、流感嗜血杆菌(Haemophilusinfluenzae)、医院不动杆菌(Acinetobacter nosocomialis)、克氏柠檬酸杆菌(Citrobacter koseri)、咽峡炎链球菌(Streptococcus anginosus)、痤疮丙酸杆菌(Propionibacterium acnes)、大芬戈尔德菌(Finegoldia magna)、里昂葡萄球菌(Staphylococcus lugdunensis)、产气肠杆菌(Enterobacter aerogenes)、星状链球菌(Streptococcus constellatus)、草绿色链球菌(Streptococcus viridans)中的一种或多种。
SEQ ID NO.1~SEQ ID NO.1411所示的miRNA在制备抗肿瘤制剂中的应用。
进一步地,针对肿瘤的类型包括但不限于:急性淋巴细胞白血病,急性髓系白血病,肾上腺皮质癌,艾滋病相关的癌症,肛门癌,星形细胞瘤,非典型畸胎/横纹肌样瘤,中枢神经系统瘤,基底细胞癌-皮肤癌(非黑色素瘤),胆管癌,膀胱癌,骨肿瘤,骨肉瘤,恶性纤维组织细胞瘤,脑干胶质瘤,脑肿瘤,乳腺癌,支气管肿瘤,中枢神经系统肿瘤,子宫颈癌,脊索瘤,慢性淋巴细胞白血病(CLL),慢性粒细胞性白血病(CML),慢性骨髓增生性疾病,结肠癌,大肠癌,颅咽管瘤,皮肤T细胞淋巴瘤-蕈样肉芽肿,乳腺导管原位癌,胚胎性肿瘤,中枢神经系统瘤,子宫内膜癌,室管膜瘤,食管癌,嗅神经母细胞,尤因肉瘤家族肿瘤,颅外生殖细胞瘤,性腺外生殖细胞肿瘤,肝外胆管肿瘤,眼癌,骨纤维组织细胞瘤,骨肉瘤,胆囊癌,胃癌,胃肠道类癌,胃肠道间质瘤-成人软组织肉瘤,生殖细胞瘤,妊娠滋养细胞肿瘤,神经胶质瘤,毛细胞白血病,头颈部癌症,心脏肿瘤,肝癌,组织细胞增多症,朗格汉斯细胞,霍奇金淋巴瘤,下咽癌,眼内黑色素瘤,胰岛细胞瘤(内分泌胰腺),卡波西氏肉瘤,肾癌,朗格汉斯细胞组织细胞增生症,喉癌,白血病,唇及口腔癌,肝癌,小叶原位癌,肺癌,淋巴瘤,巨球蛋白血症,男性乳腺癌,骨恶性纤维组织细胞瘤,骨肉瘤,髓母细胞瘤,髓上皮瘤,黑色素瘤,恶性间皮瘤,转移性鳞癌颈部肿瘤,口腔癌,多发性内分泌瘤综合征,多发性骨髓瘤/浆细胞肿瘤,蕈样肉芽肿,骨髓增生异常综合征,骨髓增生异常/骨髓增殖性肿瘤,慢性粒细胞性白血病,髓细胞白血病,多发性骨髓瘤,骨髓增生性疾病,鼻腔鼻窦肿瘤,鼻咽癌,神经母细胞瘤,非霍奇金淋巴瘤,非小细胞肺癌,口腔癌,口腔癌,唇口咽癌,骨肉瘤,恶性纤维组织细胞瘤骨,卵巢癌胰腺癌,乳头状瘤,副神经节瘤,鼻窦和鼻腔癌,甲状旁腺肿瘤,阴茎癌,咽癌,嗜铬细胞瘤,松果体实质肿瘤,垂体瘤,浆细胞肿瘤/多发性骨髓瘤,胸膜肺母细胞瘤,原发性中枢神经系统(CNS)淋巴瘤,前列腺癌,直肠癌,肾细胞(肾)癌症,肾盂和输尿管,移行细胞癌,呼吸道癌症,视网膜母细胞瘤,横纹肌肉瘤,涎腺肿瘤,皮肤癌,小细胞肺癌,小肠肿瘤,软组织肉瘤,鳞癌颈部肿瘤,原始神经外胚层肿瘤,T细胞淋巴瘤,睾丸癌,咽喉癌,胸腺瘤和胸腺癌,甲状腺癌,滋养细胞肿瘤,输尿管及肾盂移行细胞癌,尿道癌,子宫癌,子宫肉瘤,阴道癌,外阴癌,瓦尔登斯特巨球蛋白血症和肾母细胞瘤。
本发明的有益效果在于:本发明提出了一种区别于抗生素抗菌、抗肿瘤的手段,将aNK细胞分泌的外泌体及其相关的miRNA用于抗菌和抗肿瘤,在增强治疗效果的同时,极大的降低了副作用;通过大量的实验验证,这种外泌体及其相关的miRNA对细菌和肿瘤均有抑制效果,这种抑制作用来源于外泌体或相关的miRNA对于细菌或肿瘤细胞共有的信号通路的抑制。此外,外泌体可低温储存,miRNA可以人工合成,成本低,可以实现大规模的生产;而且,这种外泌体和aNK细胞的联合使用将进一步增强抗菌效果。
附图说明
图1经中空纤维切向过滤系统提取的aNK外泌体的粒径分布图;
图2Werstern blot检测aNK外泌体的蛋白表达情况图;
图3透射电镜观察aNK外泌体的形态和大小图;
图4aNK中miRNA韦恩分析图;
图5aNK外泌体抑制多种细菌生长图;
图6aNK外泌体抑制柠檬酸杆菌的生长图;
图7aNK外泌体联合NK细胞对小鼠肠道细菌移位的影响;
图8hsa-miR-330-2-3p抑制柠檬酸杆菌的生长图;
图9hsa-miR-181-5p抑制柠檬酸杆菌的生长图;
图10hsa-30a-5p、hsa-106a-5p、hsa-296-3p对细菌生长的影响;
图11hsa-134-5p、hsa-4463、hsa-145-5p对细菌生长的影响。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1:aNK外泌体的获取及其结构特征
利用现有的方法,采用跨膜IL-21滋养细胞扩增NK体系:将来源于志愿者的外周静脉血分离获得PBMC,加入辐照后表达有跨膜IL-21的滋养细胞和IL-2,期间加入完全培养液培养18天。
收集培养18天的aNK细胞培养液,流式细胞仪检测aNK细胞的纯度达到95%以上,并且培养液无细菌和支原体污染。
1.1aNK细胞培养液通过台式低速离心机400g离心5min去除细胞沉淀,收集上清4度保存待用。
1.2采用中空纤维切向过滤系统(Spectrum Laboratories KrosFlo Research IITFF System)对培养液中的外泌体进行纯化。首先,利用0.45μm mPES中空纤维过滤柱(P-S02-E45U-10-N)去除细胞培养液中的细胞碎片;滤出液进一步通过截留分子量在300-kDa的mPES中空纤维过滤柱(S02-E300-05-N)进行浓缩,获得外泌体粗制品;为了进一步缩小体积并且去除残余的培养基和盐离子,用3倍体积的PBS对外泌体粗制品进行稀释,并采用截留分子量在300-kDa的mPES中空纤维过滤柱(D02-E300-05-N)进行浓缩,得到纯度很高的外泌体。
1.3将获取的外泌体用PBS稀释100倍,利用Zetasizer Nano ZSE系统检测外泌体的粒径分布,并用Dispersion Technology Software对检测结果进行分析。如图1所示,大部分aNK外泌体分布在50-150nm,峰值分布在90nm。
1.4用BCA蛋白定量试剂盒对外泌体进行蛋白定量,并用裂解液释放外泌体中的蛋白,采用Werstern blot方法对外泌体Marker蛋白CD63、ALIX,aNK外泌体特有的granzymeA、granzyme B和perforin进行检测。如图2所示,与293T的外泌体相比,aNK外泌体含有NK细胞特有的颗粒酶A、颗粒酶B和穿孔素。
1.5将获取的外泌体用去离子水重悬,取少量外泌体置于有碳涂层的铜网,去除多余的水分,并用2%乙酸铀酰进行染色,自然干燥后利用透射电镜观察外泌体的形态和大小。如图3所示,TEM结果显示分离的aNK外泌体具有典型的外泌体结构,呈现内部半透明、椭圆、大小不一的封闭膜结构。
实施例2:aNK外泌体miRNA测序
根据实施例1获得的aNK外泌体,委托南京世和基因生物技术有限公司对aNK外泌体中miRNA进行测序。采用NEBNext Multiplex Small RNA Library Prep Set forIllumina(NEB,USA)进行建库,并对待测序样本添加测序接头。采用Illumina X-ten PE150平台对样本进行测序,每个样本至少300M reads。为了减少假阳性,信噪比大于100的独立序列纳入miRDeep log-odds score的计算,并设置miRDeep log-odds score的范围为-10~10.为了发掘aNK外泌体中全新的miRNA,以miRDeep score=0作为阈值,所有超过阈值的miRNA作为全新的miRNA,如图4所示,在测序中发现了732个已知的miRNA(SEQ ID NO.1412~SEQ ID NO.2144)。除此之外,全新序列的miRNA有1411个(SEQ ID NO.1~SEQ IDNO.1411)。
实施例3:aNK外泌体抗菌性能以及aNK外泌体联合NK细胞的抗菌性能
为了确定外泌体是否可以抑制细菌生长,选取柠檬酸杆菌(C.R.),大肠杆菌,金黄色葡萄球菌,伤寒沙门氏菌(CT18),金黄色葡萄球菌(LAC),鲍曼不动杆菌(XH386)作为检测对象。将实施例1获得的aNK外泌体按照不同比例和上述6中细菌混合,通过测量光密度(OD)量化细菌的数量。结果如图5所示,与没有外泌体的阴性对照相比,随着外泌体浓度的增加,大肠杆菌,金黄色葡萄球菌,伤寒沙门氏菌(CT18)和金黄色葡萄球菌(LAC)的生长呈现浓度依赖性的显著抑制。同时,通过检测柠檬酸杆菌中荧光素酶活性量化细菌的数量,随着aNK外泌体浓度的增加,生物荧光的强度随之降低,说明aNK外泌体可以浓度依赖性的抑制柠檬酸杆菌(图6)。
进一步的结果表明,aNK外泌体联合NK细胞相对于aNK细胞的抗菌作用有进一步的增强(图7)。
实施例4:已知序列的miRNA的抗菌性能
将实施例2筛选得到的已知序列的miRNA随机分成8组,每一组中随机选出30个样本进行抗菌测试,结果表明,均具有抗菌性能。下面以这240个样本中的hsa-miR-330-5p(SEQ ID NO.1571)、hsa-miR-181a-2-3p(SEQ ID NO.1657)、hsa-miR-30a-5p(SEQ IDNO.1503)、hsa-miR-106a-5p(SEQ ID NO.1603)、hsa-miR-296-3p(SEQ ID NO.1731)、hsa-miR-134-5p(SEQ ID NO.1531)、hsa-miR-4463(SEQ ID NO.2048)、hsa-miR-145-5p(SEQ IDNO.1586)为例,人工合成这些miRNA类似物,进行抗菌试验如下:
(1)将hsa-miR-330-5p、hsa-miR-181-2-3p类似物按照1.25μM、2.5μM、5μM和柠檬酸杆菌共孵育,通过检测OD600量化细菌的数量。测试结果如图8和图9所示。
(2)将hsa-miR-30a-5p、hsa-miR-106a-5p、hsa-miR-296-3p、hsa-miR-134-5p、hsa-miR-4463、hsa-miR-145-5p类似物以1.25μM浓度和柠檬酸杆菌共孵育,通过检测OD600量化细菌的数量。测试结果如图10-11所示。
通过与阴性对照PBS组和NC组对比,可以证明,这随机挑选的hsa-miR-330-5p、hsa-miR-181-2-3p、hsa-miR-30a-5p、hsa-miR-106a-5p、hsa-miR-296-3p、hsa-miR-134-5p、hsa-miR-4463、hsa-miR-145-5p均能显著的抑制细菌的生长,并且hsa-miR-330-5p抑制柠檬酸杆菌生长的效果最为明显。
实施5:全新序列的miRNA的抗菌性能
将实施例2筛选得到的全新序列的miRNA随机分成10组,每一组中随机选出30个样本进行抗柠檬酸杆菌测试,结果表明,均具有抗菌性能。下面以这300个样本中的hsa-miR-9502(chr7_17512,SEQ ID NO.878)、hsa-miR-9543(chr1_354,SEQ ID NO.531)、hsa-miR-9550(chr11_26139,SEQ ID NO.510)、hsa-miR-9555(chr10_23533,SEQ ID NO.448)、hsa-miR-9564(chrX_44483,SEQ ID NO.497)、hsa-miR-9609(chr16_37097,SEQ ID NO.520)、hsa-miR-9684(chr2_4515,SEQ ID NO.515)、hsa-miR-9700(chr19_37125,SEQ IDNO.290)、hsa-miR-9719(chr3_8592,SEQ ID NO.997)、hsa-miR-9773(chr12_29879,SEQ IDNO.1142)、hsa-miR-9773(chr22_40092,SEQ ID NO.284)、hsa-miR-9825(chr10_22446,SEQID NO.47)、hsa-miR-9832(chr22_39917,SEQ ID NO.144)、hsa-miR-9857(chr8_19943,SEQID NO.1186)为例,人工合成这些miRNA类似物,进行抗菌试验如下:
(1)将hsa-miR-9502、hsa-miR-9543、hsa-miR-9550、hsa-miR-9555、hsa-miR-9564、hsa-miR-9609、hsa-miR-9684、hsa-miR-9700、hsa-miR-9719、hsa-miR-9773、hsa-miR-9773、hsa-miR-9825、hsa-miR-9832、hsa-miR-9857类似物按照1.25μM、2.5μM、5μM和柠檬酸杆菌共孵育,通过检测OD600量化细菌的数量。测试结果如表1所示。
(2)将hsa-miR-9502、hsa-miR-9550、hsa-miR-9609类似物以5μM浓度和柠檬酸杆菌共孵育,通过检测OD600量化细菌的数量。
通过与阴性对照PBS组和NC组对比,可以证明,挑选的hsa-miR-9502、hsa-miR-9550、hsa-miR-9609均能显著的抑制细菌的生长,并且hsa-miR-9609抑制柠檬酸杆菌生长的效果最为明显。
表1
实施5:全新序列的miRNA的抗肿瘤性能
将实施例2筛选得到的全新序列的miRNA随机分成10组,每一组中随机选出30个样本进行抗肿瘤细胞测试,我们用神经母细胞CHLA-255作为靶细胞。结果表明,均具有抗肿瘤细胞性能。下面以这300个样本中的hsa-miR-9507(chr3_7489,SEQ ID NO.600)、hsa-miR-9514(chr1_354,SEQ ID NO.531)、hsa-miR-9758(chr1_131,SEQ ID NO.862)、hsa-miR-9796(chr2_5702,SEQ ID NO.177)、hsa-miR-10029(chr12_27203,SEQ ID NO.49)hsa-miR-10157(chr8_19314,SEQ ID NO.412)、hsa-miR-10315(chr5_12999,SEQ ID NO.983)、hsa-miR-10367(chr17_35006,SEQ ID NO.660)、hsa-miR-10434(chr7_19106,SEQ ID NO.769)、hsa-miR-10469(chr7_17145,SEQ ID NO.680)为例,人工合成这些miRNA类似物,进行抗肿瘤试验如下:
将hsa-miR-9507、hsa-miR-9514、hsa-miR-9758、hsa-miR-9796、hsa-miR-10029、hsa-miR-10157、hsa-miR-10229、hsa-miR-10315、hsa-miR-10367、hsa-miR-10434、hsa-miR-10469类似物按照1.25μM、2.5μM、5μM和aNK外泌体共孵育12小时,将miRNA和外泌体混合物加入到荧光化酶标记的神经母细胞瘤细胞中,共培养48小时。通过检测肿瘤细胞对荧光底物水平的改变,量化肿瘤细胞的存活率。测试结果如表2所示,可以证明,其中挑选的hsa-miR-10029、hsa-miR-10367、hsa-miR-10434等miRNA均能显著的抑制肿瘤细胞的生长,并且hsa-miR-10029抑制生长的效果最为明显。
表2
序列表
<110> 浙江大学
上海博慷生物科技有限公司
<120> NK细胞外泌体hsa-miR-330-5p在抗菌中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 未知(Unknown)
<400> 1
ucucugggcc ugugucuuag gc 22
Claims (2)
1.一种经跨膜IL-21激活的NK细胞分泌的外泌体在制备抗柠檬酸杆菌制剂中的应用,所述外泌体的miRNA为hsa-miR-330-5p,序列为ucucugggcc ugugucuuag gc。
2.根据权利要求1所述的应用,其特征在于,将所述外泌体单独用于抗菌,或将所述外泌体与经跨膜IL-21激活的NK细胞共同用于抗菌。
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CN110951729A (zh) | 2020-04-03 |
CN110613727A (zh) | 2019-12-27 |
CN110613728B (zh) | 2022-06-21 |
CN110613728A (zh) | 2019-12-27 |
CN110613735B (zh) | 2022-07-01 |
CN110951729B (zh) | 2021-01-12 |
CN110684770B (zh) | 2021-01-12 |
CN110613729B (zh) | 2022-06-21 |
CN110684770A (zh) | 2020-01-14 |
CN110623981A (zh) | 2019-12-31 |
CN110664843A (zh) | 2020-01-10 |
CN107998149B (zh) | 2020-05-19 |
CN110669764B (zh) | 2020-12-11 |
CN110613727B (zh) | 2022-06-21 |
CN110613735A (zh) | 2019-12-27 |
CN110669764A (zh) | 2020-01-10 |
CN107998149A (zh) | 2018-05-08 |
CN110664843B (zh) | 2022-07-01 |
CN110613729A (zh) | 2019-12-27 |
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