CN114181907B - 一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡 - Google Patents
一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡 Download PDFInfo
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Abstract
一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡,其由转染目的质粒的工程细胞所表达分泌而获得,所述细胞微囊泡内包含由外泌信号肽引导的CRISPR/CasRx编辑酶和靶向炎症因子的sgRNA,CRISPR/CasRx在靶向炎症因子的导向RNA序列sgRNA的引导下,特异性结合并降解炎症因子的mRNA,降低靶细胞内的特定炎症因子的mRNA水平。该细胞微囊泡与宿主细胞融合性好,免疫原性低,用于治疗急性炎症因子风暴相关疾病,如急性呼吸窘迫综合征(ARDS)、细胞因子释放综合征(CRS)及败血症等。
Description
技术领域
本发明属于生物工程领域,具体涉及一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡。
背景技术
炎症因子风暴也称细胞因子风暴(cytokine storm),或系统性炎症反应综合征(system inflammatory response syndrome,SIRS),其机制是免疫细胞过度的活化,细胞内炎性因子包括白细胞介素、TNF-a、补体蛋白分子等大量释放,对感染源及被感染的细胞进行暴风雨般的自杀式攻击,造成自身组织细胞的旁观者损伤伴随血管通透性增加及循环障碍,最坏的结果是多器官功能衰竭(MOF)。
炎症本是一种保护性免疫反应,有利于清除感染源;但非可控的过度的炎症会带来自身免疫损伤,是一种过度的保护性反应。在重症监护室中,炎症因子风暴是导致脓毒症患者高死亡率的主要机制,因此如何预测或预防炎症因子风暴的形成一直是重症医学研究领域的难点。
众多参与新冠肺炎重症患者救治的专家都不约而同的关注到了细胞因子风暴现象,细胞因子风暴的发生与病毒感染重症化和致死有密切关联。在针对新型冠状病毒感染患者的治疗中,除依据国家卫健委正式公布认可的抗病毒治疗方案外,临床医生应密切关注细胞因子风暴带来的巨大冲击,这对于重症患者的治疗、降低病死率具有重大意义。
以冠状病毒肺炎为例,诱发炎症因子风暴的原因可能有以下几个途径:1)冠状病毒突然大量扩增或暴露,免疫细胞大量活化释放炎症因子,造成非可控炎性损伤;2)病毒感染后激发细菌感染,免疫细胞在应对细菌的过程中过度活化,造成非可控炎性损伤;3)病毒诱发肺、肾、肝等组织损伤后释放的大量胞内危害蛋白(可以作为DAMP)激活免疫细胞,引发炎症因子风暴,带来二次损伤。
这其中,比较重要的炎症因子有IL-6、TNFa、IL-1b、IL-18和IFNr等,这些炎症因子是炎症因子风暴的核心因素,也是基础研究、临床研究关注的重点指标。
目前在治疗炎症因子风暴方面,得到临床应用批准的只有少许药物。应用最广泛的为糖皮质激素类药物,其可以控制炎症反应,但副作用也是临床避免使用它的重要原因。单克隆抗体是近年来兴起的靶向敲低炎症因子的药物,如IL-6单抗,TNFa单抗等,其价格昂贵,且半衰期较短。
合成生物学是生物科学在二十一世纪刚刚出现的一个分支学科,近年来合成生物物质的研究进展很快。与基因工程把一个物种的基因延续、改变并转移至另一物种的作法不同,合成生物学的目的在于建立人工生物系统(artificial biosystem),让它们像电路一样运行。
CRISPR/CasRx是2018年Salk生物研究所和Arbor Biotechnologies公司(张锋是创始人之一)的研究人员通过常用的生物信息学筛选,在推定的效应核酸酶附近寻找CRISPR重复序列。他们鉴定出的Cas13d蛋白与之前发现的Cas13a-c同源物的序列相似度不高,但含有Cas13超家族特有的HEPN核酸结构域。与其他Cas13酶一样,这些Cas13d同源物能独立地将CRISPR序列加工成向导RNA。它们依赖crRNA来切割目标,而不依赖HEPN结构域。这些酶对目标的侧翼序列没有要求,因此可以靶向任何RNA序列。
细胞微囊泡(extracellular vesicle,EV)是由细胞释放的各种具有膜结构的囊泡结构统称,根据其直径不同,可分为4个亚群:外泌体、微粒、凋亡小体和癌小体(oncosomes)。外泌体(exosomes)直径为30~180nm;微粒也叫核外颗粒体(microvesicles/ectosomes),直径为100~1000nm;凋亡小体(apoptotic body/bleb)是细胞凋亡过程中产生的直径约为 50~5000nm的囊泡;癌小体是最新发现的类别,直径为1~10μm,已在癌细胞中观察到。目前研究热点是外泌体这个亚群,外泌体以及直径更大的囊泡已经被用来做药物递送以及基因编辑递送,其应用前景广阔。
现有技术中,利用细胞微囊泡治疗炎症性疾病的案例较多,大部分这些囊泡治疗全都是非生物人工合成的材料包裹相关药物,或者间充质干细胞等干细胞的微囊泡。也有针对IL-6、TNFa、IL-1b、IL-18和IFNr相关的炎症因子,但是作用机制及相关效果都不是非常明确。
CRISPR/CasRx已在多种遗传病和衰老相关疾病中显现出疗效,最常用的递送方式是AAV(腺相关病毒),但是没有使用细胞微囊泡包裹 CRISPR/CasRx编辑系统治疗急性炎症疾病的先例。
发明内容
本发明的目的在于提供一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡,包含CRISPR/CasRx编辑系统和靶向炎症因子的sgRNA,与宿主细胞融合性好,免疫原性低,可降低靶细胞内的特定炎症因子的mRNA水平,使得炎症因子表达降低,用于治疗急性炎症因子风暴相关疾病,如急性呼吸窘迫综合征(ARDS)、细胞因子释放综合征(CRS)及败血症等。
为了达到上述目的,本发明提供如下技术方案:
一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡,其由转染了目的质粒的工程细胞所表达而获得,所述细胞微囊泡内包含由外泌信号肽引导的CRISPR/CasRx编辑酶和靶向炎症因子的sgRNA。
进一步,所述目的质粒中包括CRIPPR/CasRx序列及其启动子、外泌信号肽、Tag序列、终止信号序列、靶向炎症因子的sgRNA序列及其启动子。
优选地,所述目的质粒中的外泌信号肽选自tPA、Human IgG2、Human Insulin、ARRDC1、ARRDC1+2xWW domain、Human OSM、Mouse Ig Kappa 或BM40;所述CRISPR/CasRx序列的启动子为CAG启动子;所述Tag 序列为HA-tag序列;所述终止信号为polyA终止信号序列;所述sgRNA序列的启动子为U6启动子序列;所述炎症因子为IL-6、TNFa、IL-18、IL-1b 和IFNr。
又,所述sgRNA序列的具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
又,所述工程细胞由普通细胞系或干细胞转染目的质粒得到。
一种用于制备工程细胞的目的质粒,其包括CRIPPR/Cas Rx序列及其启动子、外泌信号肽、Tag序列、终止信号序列、靶向炎症因子的sgRNA序列及其启动子。
优选地,所述炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述sgRNA序列的具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
一种靶向炎症因子的sgRNA序列的筛选方法,包括以下步骤:
1)将目的炎症因子的cDNA序列从小鼠或人的cDNA文库中拷贝下来,连接进VS005载体或DP375载体质粒,炎症因子片段后面依次接2A 序列和报告基因,获得的质粒命名为1号质粒;
2)构建携带CRISPR/CasRx、待筛选的sgRNA序列以及不同波段荧光报告基因的质粒,命名为2号质粒;
3)将所述1号质粒和2号质粒共同转染至自身不表达所述目的炎症因子的哺乳动物细胞,使得哺乳动物细胞先过表达1号质粒上相应的炎症因子,之后被2号质粒上的CRISPR/CasRx和待筛选的sgRNA敲低,最终通过1号质粒的荧光强度判断相应sgRNA的效率,筛选出合适的sgRNA序列。
进一步,所述的炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述报告基因为红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白或LacZ。
一种基于RNA编辑靶向炎症因子的细胞微囊泡的制备方法,
1)构建目的质粒
调取CAG启动子、外泌信号肽序列、CRISPR/CasRx序列、HA-tag 序列、polyA终止信号序列、U6启动子序列、靶向炎症因子的sgRNA序列,设计含所述各片段的上下游特异性PCR扩增引物,同时引入酶切位点,利用重叠延伸PCR进行扩增,将所述各元件依次连入cx850载体,获得目的质粒;
2)目的质粒转染至工具细胞
利用普通细胞系或干细胞制备工具细胞,将步骤1)获得的目的质粒转染至工具细胞,同时转染荧光报告基因,得到经所述目的质粒转染的工程细胞
3)更换培养基
所述工程细胞在适于工具细胞的完全培养基中培养16-30h后,将完全培养基更换为无血清培基,继续培养24-48h;
4)分离细胞微囊泡
收集工程细胞的上清,进行细胞外囊泡分离,得到所述细胞微囊泡。
本发明中,基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡由转染特定质粒的工程细胞所分泌,所述工程细胞可为293T、L929 等肿瘤细胞系,也可为间充质干细胞、神经干细胞等原代干细胞;特定质粒中的DNA序列在工程细胞中被转录翻译为功能蛋白,包括 CRISPR/CasRx、U6引导转录的靶向炎症因子的sgRNA,以及构成细胞微囊泡所必须的元件,如CD63、TSG101和CD81等蛋白。
本发明的细胞微囊泡为由转染目的质粒的工程细胞所表达而获得的外泌体,粒径大小为30-180nm,与未转染任何质粒的外泌体大小无明显区别;该细胞微囊泡免疫原性低,递送简单,不需要其它转染试剂,其递送方式为直接将该微囊泡悬液注入靶细胞或动物体内。递送的 CRISPR/CasRx为现成的蛋白,这意味着蛋白可以直接发挥作用,而AAV、Lentivirus等的递送方式递送的均为DNA序列,需要转录翻译成蛋白才能发挥作用;而现有技术中庸纳米脂质体LNP递送的CRRSPR/CasRx mRNA 需要经过翻译成蛋白才能发挥作用。
本发明的细胞囊泡内含有CRISPR/CasRx-RNA编辑酶和导向RNA序列(即sing-guide RNA,sgRNA),靶细胞可通过胞吞、融合等方式使工程化的细胞微囊泡进入其内部,细胞微囊泡中的CRISPR/CasRx-RNA编辑酶和靶向炎症因子的sgRNA随囊泡进入靶细胞或器官,CRISPR/CasRx在靶向炎症因子的导向RNA序列sgRNA的引导下,特异性结合炎症因子的mRNA,随后,CRISPR/CasRx将其切割,达到降低炎症因子表达的目的,可用于治疗急性炎症因子风暴相关疾病,如急性呼吸窘迫综合征(ARDS),细胞因子释放综合征(CRS),败血症等,包括慢性炎症性疾病的急性转变期,如慢性阻塞性肺疾病急性感染期等;还包括设计炎症因子调节的疾病,如脑卒中、脑外伤、骨折、外科手术、炎症性肠病及自身免疫性肝病等。
本发明中,CRISPR/CasRx蛋白是发挥作用的基本单元,在目的质粒中,外泌信号肽可以最大限度的将CRISPR/CasRx蛋白引入细胞微囊泡中,可以提高细胞微囊泡发挥作用的效能,CRISPR/CasRx蛋白表达量最高。
炎症因子主要由巨噬细胞、中性粒细胞等免疫相关细胞表达,这些免疫细胞及细胞系常规情况下炎症因子的表达量也比较低,只有在外部刺激如LPS等的刺激下,才会高表达炎症因子。但同时,这些免疫细胞及细胞系又比较难转染,做sgRNA的筛选的难度很大。本发明对靶向炎症因子的sgRNA序列筛选方法中,通过先过表达相关炎症因子,再敲低的方法,可以高效达到筛选sgRNA的目的。
与现有技术相比,本发明具有如下有益效果:
本发明的细胞微囊泡中,CRISPR/CasRx-RNA编辑酶和靶向炎症因子的sgRNA可以特异性切断靶细胞内对应的炎症因子mRNA,降低炎症因子的表达,可以敲低多种细胞内的炎症因子,包括巨噬细胞、中性粒细胞、内皮细胞等,最终可以达到敲低靶器官或全身炎症因子的作用。
本发明在对靶向炎症因子的sgRNA序列筛选中,采用使炎症因子先过表达再被敲低的方案,克服了免疫系统被激活所带来的影响,有效地筛选出能够降低炎症因子表达的sgRNA序列。
本发明的细胞微囊泡来源于生物细胞,属于膜性生物结构,其表面蛋白很少,可以通过胞吞、蛋白受体诱导的结合等方式与靶细胞结合,其内容物可以进入靶细胞,相比于AAV、lentivirus、LNP(纳米脂质体)等方式,细胞微囊泡的免疫原性大大降低。
附图说明
图1为本发明实施例2中基于RNA编辑靶向炎症因子的细胞微囊泡的制备流程。
图2为本发明实施例2中细胞微囊泡内部结构的分解图。
图3为本发明实施例2中细胞微囊泡的电镜图。
图4为本发明实施例2中细胞微囊泡中的CRISPR/CasRx电镜鉴定图。
图5为本发明实施例2中细胞微囊泡粒径分布图。
图6为本发明实施例2中不同外泌信号肽引导的细胞微囊泡中的标记蛋白以及CRISPR/CasRx蛋白Western blot鉴定图。
图7-10为本发明实施例2中激活原代巨噬细胞48h后,细胞上清中的各炎症因子含量水平,其中,LPS+Exo为加入本发明细胞微囊泡的实验组, LPS+ExoNaive为加入空白微囊泡的对照组,LPS+PBS为激活原代细胞后的初始状态组。
图11-13为本发明实施例2中细胞外囊泡治疗LPS激活的巨噬细胞后的流式图,其中,横坐标和纵坐标分别为PE-A CD86和APC-A CD206,图11为空白对照组:巨噬细胞加入LPS激活后只用PBS做治疗处理;图 12为阴性外泌体对照组:巨噬细胞加入LPS激活后加入只有 CRISPR/CasRx而没有sgRNA的外泌体;图13为实验组:巨噬细胞加入 LPS激活后加入CRISPR/CasRx以及靶向IL-6、TNFa、IL-1b炎症因子的 sgRNA的外泌体。
图14为本发明实施例2中小鼠ARDS模型细胞外囊泡治疗的肺部切片。
图15为本发明实施例2中小鼠ARDS模型细胞外囊泡治疗的肺部切片组化评价结果。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
本发明中出现的术语“基于RNA编辑靶向炎症因子的细胞微囊泡”简称细胞微囊泡;出现的“目的质粒”指包含CAG启动子、外泌信号肽序列、CRISPR/CasRx序列、HA-tag序列、polyA终止信号序列、U6启动子序列和靶向炎症因子的sgRNA序列的质粒;出现的术语“工程细胞”指通过转染目的质粒、慢病毒等进入真核细胞获得的细胞。
细胞因子风暴,也称为炎症风暴,是指机体感染微生物后引起体液中多种细胞因子,如TNF-α、IL-1、IL-6、IFN-γ等迅速大量产生的现象,是引起病毒性感染患者发生急性呼吸窘迫综合症和多器官衰竭的重要原因。
IL-6能够促进T细胞群体扩增和活化以及B细胞分化,调节急性期反应,可以影响血管疾病、脂质代谢、胰岛素抵抗、线粒体活动、神经内分泌系统和神经心理行为的激素样属性。此外,IL-6促进破骨细胞和血管生成的分化、角化细胞和肾小球膜细胞的增殖、以及骨髓瘤和浆细胞瘤细胞的生长。IL-6是机体受炎症刺激后由T细胞、B细胞、单核巨噬细胞及内皮细胞等分泌的细胞因子,升高的IL-6可能存在双重效应:一方面作为一个信号调动机体防御,对肿瘤坏死因子等引起的前炎症反应有分化和低调作用,另可诱导多形核白细胞的细胞凋亡,使炎症反应得以正常消失;另一方面,可导致免疫功能失调,促使巨噬细胞产生转化生长因子β (TGF-β),而TGF-β与损伤后免疫功能抑制有关。
肿瘤坏死因子(TNF)可能是最有名的和最强烈的促炎细胞因子,它在细胞因子风暴中起着重要作用。TNF被认为是急性病毒性疾病的中枢细胞因子,包括流感病毒、登革热病毒和埃博拉病毒引起的疾病。TNF由多种免疫细胞表达,TNF蛋白超家族扩大了TNF的多效性,过量的TNF产生与许多慢性炎症和自身免疫性疾病相关。TNF抑制剂已被批准用于治疗炎症性肠病、牛皮癣和类风湿性关节炎。
IL-1α和IL-1β是通过直接和间接机制介导宿主对感染的反应的促炎细胞因子。在它们的生物学功能中,这些细胞因子增加了急性期信号传导,免疫细胞向原发感染部位的运送,上皮细胞活化和继发性细胞因子产生。
人和小鼠IFN-γ基因分别定位于12号和10号染色体,在DNA水平上IFN-γ基因与IFN-α/β基因无同源性。人和小鼠IFN-γ在DNA水平上有 65%左右同源性,在氨基酸水平的同源性只有40%左右。小鼠成熟IFN-γ分子由133个氨基酸残基组成。人IFN-γ成熟分子由143个氨基酸组成,糖蛋白,以同源双体形式存在,分子量为40kDa,其生物学作用有严格的种属特异性。IFN-γ的生物学作用有较严格的种属特异性,人IFN-γ只作用于人或灵长类动物的细胞,其作用有:(1)诱导单核细胞、巨噬细胞、树突状细胞、皮肤成纤维细胞、血管内皮细胞、星状细胞等MHCⅡ类抗原的表达,使其参与抗原提呈和特异性免疫的识别过程。此外,IFN-γ可上调内皮细胞ICAM-1(CD54)表达,促进巨噬细胞FcγR表达,协同诱导TNF并促进巨噬细胞杀伤病原微生物;(2)促进LPS体外刺激小鼠B 细胞分泌和IgG2a,降低IgG1、IgG2b、IgG3和IgE的产生;抑制由IL-4 诱导小鼠B细胞增殖,IgG1和IgE产生以及FcεRⅡ表达;促进SAC诱导的人B细胞的增殖;(3)协同IL-2诱导LAK活性,促进T细胞IL-2R 表达;(4)诱导急性期蛋白合成,诱导髓样细胞分化。
IL-18属于IL-1超家族,主要由巨噬细胞产生,但也由其他细胞类型产生,刺激各种细胞类型并具有多效性功能。IL-18是促发1型反应的促炎细胞因子。它与IL-12一起,在被诸如脂多糖(LPS)的微生物产物感染后诱导细胞介导的免疫。IL-18与IL12联合作用于CD4,CD8 T细胞和 NK细胞以诱导IFNγ产生,II型干扰素在激活巨噬细胞或其他细胞中起重要作用。已证明此IL-18和IL-12的组合可抑制IL-4依赖性IgE和IgG1 的产生并增强B细胞中IgG2a的产生。重要的是,在没有IL-12或IL-15 的情况下,IL-18不会诱导IFNγ的产生,但在将幼稚T细胞分化为Th2 细胞并刺激肥大细胞和嗜碱性粒细胞产生IL-4时发挥重要作用,IL-13和化学介质,例如组胺。
这些炎症因子在炎症性疾病中都发挥至关重要的作用,本发明中,通过细胞微囊泡腹腔给药或肺部给药可以将这些炎症因子明显敲低,最终达到治疗相关炎症性疾病的目的。
实施例中的磷酸缓冲盐溶液(Phosphate buffer saline,PBS),pH7.4,购于Gibco公司,货号#10010049;DPBS,购于Gibco公司,货号#14190144;双蒸水,购于Sigma-Aldrich公司,货号#DENWAT3MSDS;4%多聚甲醛溶液,购于Biosharp公司,货号#BL539A;蔗糖,购于Sigma-Aldrich公司,货号#S9378MSDS;10-kDadextran-tetramethylrhodamine,购于Thermo Fisher公司,货号#D3312;AxyPrep质粒DNA小量试剂盒,购于Axygen 公司,货号#AP-MN-MS-GDNA-50;高纯质粒大提试剂盒,购于天根生化科技(北京)有限公司,货号#DP116;DMEM高糖培养基,购于Gibco公司,货号#11965092;超速离心机,购于Beckman公司,货号#Optima L-90k;26.3ml超速离心瓶,购于Beckman公司,货号#355654;EZ Trans细胞转染试剂,购于上海李记生物公司,货号#AC04L092;Opti MEM减血清培养基,购于Gibco公司,货号31985070;人工序列由铂尚生物技术(上海) 有限公司合成。
实施例1一种靶向炎症因子的sgRNA序列的筛选方法,包括以下步骤:
1)将目的炎症因子的cDNA序列从小鼠或人的cDNA文库中拷贝下来,连接进VS005载体或DP375载体质粒,炎症因子片段后面依次接2A 序列和报告基因,获得的质粒命名为1号质粒;
2)构建携带CRISPR/CasRx、待筛选的sgRNA序列以及不同波段荧光报告基因的质粒,命名为2号质粒;
3)将所述1号质粒和2号质粒共同转染至自身不表达所述目的炎症因子的哺乳动物细胞,使得哺乳动物细胞先过表达1号质粒上相应的炎症因子,之后被2号质粒上的CRISPR/CasRx和待筛选的sgRNA敲低,最终通过1号质粒的荧光强度判断相应sgRNA的效率,筛选出合适的sgRNA 序列,炎症因子IL-6、TNFa、IL-18、IL-1b和IFNr的sgRNA序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
实施例2
(一)构建用于治疗小鼠ARDS的细胞微囊泡
参见图1,在治疗小鼠ARDS中,基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡的制备,包括以下步骤:
1)构建目的质粒
调取CAG启动子,具体核苷酸序列参见SEQ ID NO.1、外泌信号肽tPA 序列,具体氨基酸序列参见SEQ ID NO.2、CRISPR/CasRx序列,具体氨基酸列参见SEQ ID NO.3,核苷酸序列如SEQ ID NO.4所示、HA-tag序列,具体氨基酸序列参见SEQ ID NO.5、polyA终止信号序列,具体核苷酸序列参见SEQ ID NO.6、U6启动子序列,具体核苷酸序列参见SEQ ID NO.7、靶向炎症因子的sgRNA序列,具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
设计上述各基因片段的上下游特异性扩增引物,同时引入酶切位点,采用高保真KOD酶或Phanta酶进行PCR扩增,3K内突变率为0%,从模板中(cDNA质粒或者文库)调取目的基因CDS,连入cx850载体,构建出含有CAG启动子、外泌信号肽序列、CRISPR/CasRx序列、HA-tag序列、polyA终止信号序列、U6启动子序列和靶向炎症因子的sgRNA序列的目的质粒。
其中,外泌信号肽tPA序列由商业公司合成;CAG启动子从VS005 质粒上经PCR获得,CRISPR/CasRx序列和HA-tag序列以及polyA序列和U6启动子序列从cx850质粒上PCR获得;靶向炎症因子的sgRNA序列由商业公司合成。
2)培养、扩增293T细胞
利用完全培养基在培养皿中将293T细胞培养至70%-80%密度,其中,所述完全培养基由DMEM、10%FBS、1%P/S和1%NEAA组成。
3)质粒转染
光镜下鉴定293T细胞长到合适密度,使用EZ trans细胞转染试剂进行转染,每盘15cm培养皿细胞加入50μg目的质粒和100μl转染试剂,具体步骤如下:
准备两个50ml离心管:A管和B管,两管中分别加入Opti MEM转染缓冲液,Opti MEM转染缓冲液体积为n(n=293T细胞盘数)×1ml。随后,在A管中加入n×50μg的质粒量,吹打混匀,其中,Opti MEM转染缓冲液为无血清培养基。
在B管中加入n×100μl的EZ trans细胞转染试剂,吹打混匀。两管静置 3min-5min后,将B管内液体加入到A管中,吹打混匀。再静置10min-15min 后,将混合液按2ml/盘细胞的量加入到293T细胞的完全培养基中进行转染,获得所述工程细胞。
4)更换培养基
转染24h之后,将293T细胞的完全培养基更换为无血清培养基,包括:DMEM、1%P/S和1%NEAA。
5)回收上清、离心分离外泌体
使用无血清培养基继续培养24-48h后,将293T细胞的培养基回收至 50ml离心管中,收集293T细胞上清,进行细胞外囊泡分离:
2000g离心10分钟,弃沉淀,上清转移至新的离心管。
10000g离心30分钟,弃沉淀,上清转移至超速离心管。
150000g离心70分钟,弃上清,加入DPBS,重悬管壁的细胞微囊泡。
150000g离心70分钟,弃上清,加入200μl DPBS,重悬管壁的细胞微囊泡,得到所述细胞微囊泡。
(二)细胞微囊泡的鉴定
图2为本发明实施例中细胞微囊泡内部结构的分解图,通过透射电子显微镜TEM检测可以检测到分泌出的细胞微囊泡(如图3所示),免疫电镜检测可以检测到CRISPR/CasRx蛋白,如图4所示,其中,箭头所示即为CRISPR/CasRx蛋白;nanoparticle trackinganalysis(NTA)数据如图 5所示,可见,本发明工程化细胞外囊泡粒径主要分布于80-220nm,与未转染任何质粒的外泌体的大小无明显区别。
图6为不同外泌信号肽引导的细胞微囊泡中的CRISPR/CasRx蛋白及标记蛋白的Western blot鉴定图,其中,1-6分别是转染了不同质粒的组,其中,这些质粒的不同就在于CRISPR/CasRx前方的引导序列的不同;第 1组转染的质粒CRISPR/CasRx前方没有引导序列,第2组为 ARRDC1+2xWW domain引导的CRISPR/CasRx的质粒组别,第3组为 ARRDC1引导的CRISPR/CasRx的质粒组别,第4组为tPA引导的 CRISPR/CasRx的质粒组别,第5组为HumanIgG2引导的CRISPR/CasRx 的质粒组别,第6组为人胰岛素引导系列(human insulin)引导的CRISPR/CasRx的质粒组别。
可见,不同外泌体引导下,目的蛋白的量存在明显区别,由于未转染任何质粒的空白组no plasmid组不可能有CasRx,因此图6中无no plasmid 组。
(三)细胞微囊泡的功能鉴定
1.细胞微囊泡治疗LPS激活的原代巨噬细胞
1)原代巨噬细胞的分离培养
小鼠取腿骨:小鼠麻醉后脱臼处死,将小鼠放置在盛有足量75%乙醇的烧杯中浸泡消毒5min,将浸泡好的动物用纸吸去多余的酒精。用剪刀在小鼠背部剪开一小口,用手直接撕开皮肤至小鼠小腿关节处,去除小鼠足关节以及皮肤。用剪刀沿着小鼠大腿根部大转子将后肢拆下来,去掉肌肉组织后放置在含有75%乙醇的培养皿内浸泡5min,更换新的75%乙醇的培养皿移入超净台中。
将乙醇浸泡的腿骨移入冷的PBS浸泡,洗去胫骨、股骨表面的乙醇,此过程可重复3次。将清洗好的股骨、胫骨分开,并用剪刀分别将股骨、胫骨两端剪断,使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出,反复吹洗3次,直至腿骨内看不到明显的红色为止。用5mL 移液枪将含有骨髓细胞的培养基反复吹打,使细胞团块分散,然后使用70 μm细胞滤器将细胞过筛,转移至15mL离心管内,1500rpm/min离心5min,弃上清,加入红细胞裂解液重悬静置5min后1500rpm/min离心5min,弃上清用冷的配置好的骨髓巨噬细胞诱导培养基重悬,铺板。
细胞培养期间不更换培养基,培养第三天后更换一半骨髓巨噬细胞诱导培养基(含20%的L929细胞系培养基),第五天更换全部培养基,第七天即可用于后续实验。
2)原代巨噬细胞的激活
原代巨噬细胞提取培养7天后,向培基内加入LPS,使LPS终浓度为 100ng/ml。激活48h后,收集细胞上清,通过ELISA检测细胞上清中的各个炎症因子水平,如图7-10所示。
细胞微囊泡敲低靶细胞炎症因子的功能实验验证:
使用1μg/ml的脂多糖LPS处理巨噬细胞,后在巨噬细胞培养基中加入细胞微囊泡,24h后收集巨噬细胞,提取RNA,逆转录为cDNA,通过特异性引物和qPCR验证靶向炎症因子敲低情况。48h后收集巨噬细胞上清,通过酶联免疫吸附反应炎症培养基中的炎症因子水平,结果参见图 7-10,可见,加入本发明细胞微囊泡后,炎症因子IL6、IL-1β、IL-18、TNF 的水平均明显降低。
通过流式检测,检测M1型巨噬细胞marker(CD86),和M2型巨噬细胞(CD206)表达情况(常规情况下,LPS会使巨噬细胞向M1型转化),如图11-13所示,其中,横坐标和纵坐标分别为PE-A CD86和APC-A CD206,图11为空白对照组:巨噬细胞加入LPS激活后只用PBS做治疗处理;图12为阴性细胞微囊泡对照组:巨噬细胞加入LPS激活后加入只有CRISPR/CasRx而没有sgRNA的细胞微囊泡;图13为实验组:巨噬细胞加入LPS激活后加入CRISPR/CasRx以及靶向IL-6、TNFa、IL-1b炎症因子的sgRNA的细胞微囊泡。
图11-13表明,加入工程化细胞微囊泡的组中,CD86+巨噬细胞明显减少,而加入阴性对照工程化细胞微囊泡和加入PBS的组中,CD86+巨噬细胞明显多于实验组,三组的CD206+细胞没有明显区别。
2.细胞微囊泡治疗ARDS模型小鼠
1)构建小鼠ARDS模型
腹腔注射戊巴比妥钠(0.4mg/10g)麻醉小鼠,将小鼠固定于小动物气管插管装置上。用生理盐水和LPS配好LPS脂多糖溶液,配置浓度 100μg/ml。按照剂量5mg/kg的剂量进行肺部给药,给药速度要快,将注射器内的脂多糖溶液在1-2秒内注射完毕。后拿起小鼠,使其保持垂直态,左右晃动10-20次,保证药物分布均匀。将小鼠挂在垂直架上,保持垂直态两分钟。后取下,保暖。
2)治疗小鼠ARDS模型
ARDS模型构建成功后1-2h内,小鼠肺部给药制备的细胞微囊泡,给药剂量单位为颗粒度,5×1012颗粒度/只小鼠。ARDS模型构建成功后12h 和24h再次给药。
3)验证治疗效果
将小鼠在ARDS模型构建成功后48h安乐死,取其肺组织进行切片染色。染色指标有苏木精-伊红染色法(HE染色),以及免疫组化染色(IHC 染色)。免疫组化染色标记物为F4/80(巨噬-单核细胞marker)和MPO (中性粒细胞marker)。比较实验组和对照组间的HE染色结果和IHC染色结果,参见图14,组化评价参见图15,其中,LPS+Exo为加入本发明细胞微囊泡的实验组,LPS+ExoNaive为加入空白微囊泡的对照组, LPS+PBS为单纯造模组。
由图14可见,本发明的细胞外囊泡可以有效使肺部的炎症细胞降低,肺部的损伤分数也明显降低,图15可见,加入本发明细胞微囊泡的实验组的组化评分明显降低,炎症细胞聚集明显减少。
序列表
<110> 李天文
朱剑虹
<120> 一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡
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c 241
Claims (6)
1.一种基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡,其由转染了目的质粒的工程细胞所表达而获得,所述细胞微囊泡内包含由外泌信号肽引导的CRISPR/CasRx编辑酶和靶向炎症因子的sgRNA;
其中,所述目的质粒中包括CRIPPR/CasRx序列及其启动子、外泌信号肽、Tag序列、终止信号序列、靶向炎症因子的sgRNA序列及其启动子;所述工程细胞由293T细胞转染目的质粒得到;所述炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述sgRNA序列的具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
2.根据权利要求1所述基于CRISPR/CasRx RNA编辑系统靶向炎症因子的细胞微囊泡,其特征在于,所述目的质粒中CRISPR/CasRx序列的启动子为CAG启动子;所述外泌信号肽选自tPA、Human IgG2、Human Insulin、ARRDC1、ARRDC1+2xWW domain、Human OSM、Mouse IgKappa或BM40;所述Tag序列为HA-tag序列;所述终止信号为polyA终止信号序列;所述sgRNA序列的启动子为U6启动子序列。
3.一种用于制备工程细胞的目的质粒,其包括CRIPPR/CasRx序列及其启动子、外泌信号肽、Tag序列、终止信号序列、靶向炎症因子的sgRNA序列及其启动子;所述炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述sgRNA序列的具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac。
4.根据权利要求1或2或3所述一种靶向炎症因子的sgRNA序列的筛选方法,包括以下步骤:
1)将目的炎症因子的cDNA序列从小鼠或人的cDNA文库中拷贝下来,连接进VS005载体或DP375载体质粒,炎症因子片段后面依次接2A序列和报告基因,获得的质粒命名为1号质粒;
2)构建携带CRISPR/CasRx序列、待筛选的sgRNA序列以及与1号质粒中报告基因不同波段的荧光报告基因的质粒,命名为2号质粒;
3)将所述1号质粒和2号质粒共同转染至自身不表达所述目的炎症因子的哺乳动物细胞,转染后使该哺乳动物细胞先过表达1号质粒上相应的炎症因子,之后被2号质粒上的CRISPR/CasRx和待筛选的sgRNA敲低,最终通过1号质粒的荧光强度判断相应sgRNA的效率,筛选出合适的sgRNA序列。
5.根据权利要求4所述靶向炎症因子的sgRNA序列的筛选方法,其特征在于,所述的炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述报告基因为红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白或LacZ。
6.一种基于RNA编辑靶向炎症因子的细胞微囊泡的制备方法,
1)构建目的质粒
调取CAG启动子、外泌信号肽序列、CRISPR/CasRx序列、HA-tag序列、polyA终止信号序列、U6启动子序列、靶向炎症因子的sgRNA序列,设计含所述各片段的上下游特异性PCR扩增引物,同时引入酶切位点,利用重叠延伸PCR进行扩增,将所述各元件依次连入cx850载体,获得目的质粒;
其中,所述目的质粒包括CRIPPR/CasRx序列及其启动子、外泌信号肽、Tag序列、终止信号序列、靶向炎症因子的sgRNA序列及其启动子;所述炎症因子为IL-6、TNFa、IL-18、IL-1b和IFNr;所述sgRNA序列的具体核苷酸序列如下:
IL-6 sgRNA:acaggtctgttgggagtggtatcctctgtg;
TNFa sgRNA:tttgctacgacgtgggctacaggcttgtc;
IL-18 sgRNA:ttcacagagagggtcacagccagtcctctt;
IL-1b sgRNA:aacgtcacacaccagcaggttatcatcatc;
IFNr sgRNA:actgtgccgtggcagtaacagccagaaac;
2)目的质粒转染至工具细胞
利用293T细胞制备工具细胞,将步骤1)获得的目的质粒转染至工具细胞,同时转染荧光报告基因,得到经所述目的质粒转染的工程细胞;
3)更换培养基
所述工程细胞在适于工具细胞的完全培养基中培养16-30h后,将完全培养基更换为无血清培基,继续培养24-48h;
4)分离细胞微囊泡
收集工程细胞的上清,进行细胞外囊泡分离,得到所述细胞微囊泡。
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