CN114632161B - 肿瘤坏死因子-α作为一种核酸基因药物体内递送载体的应用 - Google Patents
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Abstract
本发明公开了属于核酸药物研究领域中一种人体内源蛋白质分子作为新型核酸基因药物体内递送载体的发现与应用。本发明通过RNA免疫沉淀结合深度测序技术、RT‑PCR技术证明肿瘤坏死因子‑α(TNF‑α)能特异性结合microRNA。通过活细胞内吞实验、激光共聚焦、流式细胞术证明TNF‑α蛋白质本身可以将结合的外源microRNA递送进入细胞内。细胞增殖实验证明经TNF‑α携带进入细胞内的外源microRNA可以影响细胞的生长能力,证明TNF‑α蛋白质本身可以作为新型载体递送核酸基因药物进入细胞并影响细胞功能。
Description
技术领域
本发明属于医药学领域,具体涉及一种新型核酸基因药物递送载体的发现和应用。
背景技术
核酸基因药物是生物医药发展的前沿领域,包括反义核酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、小激活RNA(saRNA)、信使RNA(mRNA)、适配体(aptamer)、核酶(ribozyme)、抗体核酸偶联药物(ARC)等,是基因治疗的一种形式,也是继小分子药物、蛋白药物、抗体药物之后的新一代制药技术。核酸基因药物能够以化学药物或抗体药物无法靶向(例如mRNA和miRNA)的分子作为作用靶点,有望对传统药物疗效不佳的疾病产生突破性的进展,特别是难以治疗的遗传疾病、癌症以及某些病毒感染(例如SARS-CoV-2)。考虑到DNA、mRNA、siRNA、microRNA等核酸类生物活性物质分子量大,不能独自进入细胞内,因而这些核酸基因类药物进入细胞成为此类药物发挥作用的技术瓶颈之一,新型核酸基因药物递送载体的研究与发现是核酸药物发展的重中之重。
目前核酸基因药物载体主要分为病毒类和非病毒类载体,前者包括腺相关病毒(adenovirus-associated virus,AAV)、慢病毒(lentivirus)、腺病毒(adenovirus)和逆转录病毒(Retrovirus)等,后者包括脂质体、纳米颗粒、微囊泡、外泌体等。从已上市药物情况来看,病毒载体、脂质体在DNA基因药物递送较为成熟,小核酸基因药物则更多采用脂质体、ASO及GalNAc等载体或技术平台。无论是何种递送方式,其递送效率、稳定性、免疫原性、细胞毒性等都存在一些问题。因此,寻找一种新的、天然的、高效的、无毒的递送载体迫在眉睫。
肿瘤坏死因子α(TNF-α)是一个典型的促炎性细胞因子,在肿瘤发生中具有双重作用,其主要来源于单核细胞和巨噬细胞,非免疫细胞如内皮细胞、神经细胞也可分泌。TNF-α有两种存在形式,分子量为26kD的膜结合型与分子量为17kD的可溶性形式,可溶性TNF-α为其主要功能形式。膜结合型TNF-α经过TNF-α转化酶(TACE,即ADAM17)剪切,形成可溶性TNF-α(主要功能形式),与细胞表面TNF-α受体(TNFR1与TNFR2)结合,发挥抗感染、促进组织修复、破坏肿瘤血管系统、诱导细胞凋亡等多种抗肿瘤作用。在慢性炎症中,TNF-α的促肿瘤作用也已被证实。研究表明,健康人血清中TNF-α含量在10pg/mL以下,而当体内形成炎症病征时,血清中TNF-α含量可以提高至少5-10倍,病灶处浓度更高。TNF-α大量产生和释放会破坏机体的免疫平衡,造成病理损伤,导致多种疾病发生。大量研究表明,在今年爆发的新型冠状病毒感染的肺炎患者中,中度和重度感染病人的TNF-α显著上调。
发明人通过前期研究基础发现TNF-α具有RNA结合蛋白的生物学功能,即重组可溶性性人源TNF-α能够与随机68nt RNA结合,在体外具有RNA伴侣分子的活性,然而TNF-α是否是miRNA结合蛋白目前尚无文献报道。发明人通过研究发现,可溶性TNF-α可以结合microRNA并将其带入到细胞内,影响细胞增殖能力,说明TNF-α作为一种天然的、新型核酸递送载体的可能性。同时发明人通过序列分析结合仪gator确定了TNF-α结合microRNA的特异碱基,发现连接poly U的任意microRNA与TNF-α的结合均有明显增强,这说明任意核酸分子连接poly U都可以实现通过TNF-α递送到细胞内,实现任意核酸的天然、高效的递送。
发明内容
本发明的目的在于发现一种天然的核酸药物递送载体,即可溶性TNF-α蛋白。
本发明的目的还在于提供上述核酸基因药物递送载体的鉴定方法。
本发明的目的还在于提供上述核酸基因药物递送载体在核酸基因药物递送中的应用。
本发明的目的还在于提供以结合可溶性TNF-α蛋白的核酸基因药物的应用,包括但不限于microRNA、siRNA、反义RNA、核酸适配体、核酶、mRNA及其类似物。
上述核酸基因药物递送载体的鉴定方法,其特征在于,TNF-α在体外具有RNA伴侣分子的活性,能够与随机68nt的RNA结合。EMSA、生物大分子相互作用仪gator可以确定TNF-α与microRNA结合能力,激光共聚焦、流式细胞检测确定TNF-α转运外源microRNA到细胞内。细胞增殖、细胞迁移实验验证了进入细胞内的外源microRNA具有促进细胞生长和迁移的功能。
上述核酸基因药物递送载体在核酸基因药物递送中的应用。
本发明的有益效果:本发明利用EMSA、生物大分子相互作用仪gator等方法证实,可溶性TNF-α蛋白是可以与microRNA结合的。以结直肠癌细胞株HCT116细胞为模型,利用激光共聚焦显微镜、流式细胞仪证实可溶性TNF-α蛋白可以转运外源microRNA进入细胞。利用细胞增殖和细胞迁移实验,证实可溶性TNF-α蛋白可以结合并转运microRNA进入细胞,促进细胞增殖和细胞迁移。
附图说明
图1为TNF-α与miRNA体外结合实验的EMSA结果。
图2为Gator测定TNF-α与不同的miRNA的结合情况。
图3为Gator测定TNF-α与miR-146a结合亲和力测定结果。
图4为Gator测定TNF-α与加poly U的miRNA的结合情况。
图5为TNF-α的细胞内化实验Western blot结果。
图6为细胞流式检测TNF-α转运外源miRNA进入结直肠癌细胞结果。
其中,左图为TNF-α转运miR-146a的情况,右图为TNF-α转运let-7c的情况;TNF-α为绿色荧光标记,miRNA为红色荧光标记。
图7为激光共聚焦检测TNF-α转运外源mi-146a进入结直肠癌细胞的结果。
其中,TNF-α为AF488标记,显示为绿色;miRNA为Cy3标记,显示为红色;细胞核为DAPI染色,显示为蓝色。
图8为激光共聚焦检测TNF-α转运带polyU的外源miRNA进入结直肠癌细胞的结果。
其中,TNF-α为AF488标记,显示为绿色;miRNA为Cy3标记,显示为红色;细胞核为DAPI染色,显示为蓝色。上排miR-21为阴性对照,TNF-α无法结合并转运其进入细胞;下排为带polyU后,TNF-α可以结合并转运其进入细胞。
图9为细胞增殖和细胞迁移实验检测TNF-α转运miRNA进入细胞促进结直肠癌增殖和迁移;
其中,上图为细胞增殖实验,下图为细胞迁移实验。
具体实施方式
下面结合附图和具体实施方式对本发明做进一步说明。以下实施例并不限定本发明。
实施例1
利用凝胶阻滞实验(EMSA)确定TNF-α与miRNA的结合。
1、结合实验,10μL体系见下表,于37℃孵育30min。
2、配制6%的PAGE天然胶,见下表,放入含有0.5×TBE的电泳槽中,80V预电泳15min。
3、孵育体系加入上样缓冲液,全部加到上样孔中,80V电泳至溴酚蓝迁移至距底部1/4处,停止电泳。
4、小心取出胶,把胶上的TNF-α蛋白和miRNA湿转至尼龙膜上,湿转用0.25×TBE缓冲液,于70V转膜1h。
5、取出尼龙膜,标记正反面,将正面置于紫外交联仪1.2×105μJ/cm2交联2min。
6、将膜置于含60μg/mL蛋白酶K和10mM CaCl2的50mM Tris-HCl中,pH值为7.5,37℃消化过夜。
7、膜用封闭液室温封闭1h。
8、封闭液稀释HRP-抗生物素抗体,室温孵育膜1h。
9、1×洗脱液洗膜4次,每次5min,再用平衡液室温孵育5min。
10、膜用ECL显色液于化学发光成像系统中显影。
注:7-10采用Thermo公司化学发光核酸检测试剂盒。
实施例2
激光共聚焦检测TNF-α转运miRNA进入细胞的情况。
1、细胞铺板,共聚焦小皿种HCT116细胞,细胞汇合度在30%左右。
2、将TNF-α(AF488绿色荧光标记)终浓度10μg/mL,与microRNA(Cy3红色荧光标记)终浓度10nM,用100μL含1%血清的培养基进行稀释混匀,37度孵育30min。
3、将细胞换成含1%血清的培养基,加入孵育好的TNF-α和microRNA混合物,放入37℃、5%二氧化碳孵箱继续培养5h。
4、吸干净培养基,PBS漂洗2次,加入Hocheset染色30min。
5、PBS继续漂洗3-5次,立即进行共聚焦拍摄。
实施例3
生物大分子相互作用仪gator确定TNF-α与microRNA结合能力。
1、相互作用条件:TNF-α蛋白浓度为25μg/mL,microRNA为100nM;Kd值测定条件:TNF-α蛋白浓度为50、25、12.5、6.25μg/mL,microRNA为100nM;其中microRNA为生物素标记。
2、探针为SA偶联的探针,生物素标记的microRNA为固定相,经过基线、上样、结合、解离等步骤后得到相应结果。
可以与TNF-α结合的microRNA是(结合能力从高到低):let-7c、miR-146a、miR-155、miR-146b;不结合的是:miR-146a-antisense、miR-21。
实施例4
连接poly U的microRNA增强了与TNF-α的结合能力。
1、选择与TNF-α结合能力强的let-7c、miR-146a、miR-155;不结合的miR-21进行poly U连接(由公司合成,进行生物素或者荧光Cy3标记);
2、生物素标记的microRNA进行生物大分子相互作用仪gator检测,作用条件与实施例3相同,确定其与TNF-α结合能力的变化,发现所有microRNA的结合能力均有增强,不结合的miR-21也可以有较高的结合能力;
3、荧光标记的microRNA进行激光共聚焦检测,孵育时间、孵育浓度与实施例2相同,发现TNF-α与miR-21孵育后不能检测到有荧光的microRNA,说明没有外源的microRNA进入细胞;而TNF-α与带poly U的miR-21孵育后,可以检测到有荧光的microRNA,说明带polyU的microRNA被TNF-α转运进入了细胞。
实验结果:
为了确定TNF-α与miR-146a是否具有直接相互作用,我们进行了非放射性EMSA实验。TNF-α和生物素标记的miR-146a体外孵育,然后进行凝胶电泳,化学发光系统检测。结果显示,游离miR-146a上方出现一条阻滞带,且随着miR-146a加入量的增多阻滞带越明显(图1);利用生物大分子相互作用仪Gator测定TNF-α与不同的microRNA的结合能力以及对miR-146a进行KD值测定,TNF-α与miR-146a结合的KD值在nM级水平(图2、图3)。同时发现,TNF-α结合不同microRNA存在一定的差异,其中,对let-7c、miR-155、miR-146a的结合力较高,对miR-146家族的miR-146b的结合力较弱,而对miR-21不结合。这引起我们的注意。miR-146a和miR-146b只有3’端1个碱基的不同,这提示我们U可能是与TNF-α结合一个关键碱基。为了验证这个猜想,我们将结合与TNF-α强结合的let-7c、较强结合的miR-146a、不结合的miR-21分别在3’端加上U,我们发现,所有的microRNA加上U之后与TNF-α的结合能力大大提高,包括之前不结合的miR-21(图4)。这提示我们,U可以作为任意一个microRNA与TNF-α结合的“抓手”,利用U就可以将任意microRNA通过TNF-α带入到细胞内。
TNF-α可以通过受体进入细胞发挥其生物学作用已经被广泛研究,我们也对此进行验证。结果显示,TNF-α能通过细胞表面的TNF-α受体内化到细胞(HepG2、HeLa、未活化U937),Western blot结果显示,未检测到TNF-α在这几种细胞中的表达;rhTNF-α与细胞孵育后,细胞中都能检测到17kD的TNF-α,并且随着rhTNF-α加入量和刺激时间的增加而增加(图5)。说明TNF-α-microRNA结合复合物可以通过TNF-α受体进入到细胞内发挥生物学功能,同时TNF-α更有望成为一种天然的microRNA递送载体。
为确定体外TNF-α-microRNA结合复合物是否能够进入细胞内,我们分别将TNF-α和microRNA标记了绿色荧光和红色荧光,进行细胞流式检测和共聚焦分析。流式结果显示,加入TNF-α比不加TNF-α(绿色荧光),microRNA(红色荧光)的荧光比例明显增加(图6)。激光共聚焦结果显示,当不加TNF-α的时候,细胞内几乎没有红色荧光(microRNA),加入TNF-α之后,细胞内红色荧光明显增加,且红色荧光和绿色荧光存在部分共定位,共定位系数约为30%,说明TNF-α结合microRNA进入细胞是结合-解离的动态过程,结合态和解离态同时存在(图7)。同时我们也检测了带poly U的microRNA是否可以被TNF-α带入到细胞内。激光共聚焦实验发现TNF-α与miR-21孵育后不能检测到有荧光的microRNA,说明没有外源的microRNA进入细胞;而TNF-α与带poly U的miR-21孵育后,可以检测到有荧光的microRNA,说明带poly U的microRNA被TNF-α转运进入了细胞(图8)。
我们通过体外实验确定了TNF-α可以结合microRNA并找到了结合的关键碱基,进一步我们想确定被TNF-α带入到细胞内的microRNA是否可以发挥其生物学功能,影响细胞表型。于是我们利用细胞增殖实验、细胞迁移实验验证我们的猜想。我们发现,当TNF-α单独存在时,对细胞增殖和迁移的影响很小,microRNA单独存在几乎没有影响,而当两者共同孵育之后加入到细胞中,细胞的增殖和迁移能力明显增强,高于单独加TNF-α或microRNA(图9),说明TNF-α结合microRNA进入细胞之后,microRNA发挥了其促进细胞增殖和迁移的生物学功能,说明TNF-α作为microRNA递送载体的有效性。
Claims (4)
1.一种递送microRNA进入细胞的方法,其特征在于,利用可溶性TNF-α作为递送载体递送microRNA进入细胞的方法,所述方法为非治疗或诊断目的,所述microRNA为let-7c、miR-155、miR-146a、miR-146b,所述microRNA带poly U,所述microRNA在连接poly U后与可溶性TNF-α的结合有明显增强,递送效率增加。
2.根据权利要求1所述方法,其特征在于,可溶性TNF-α蛋白体外直接与microRNA孵育后,将microRNA递送至细胞内。
3.根据权利要求1所述方法,其特征在于,使用的可溶性TNF-α是通过天然提取,或基因工程重组获得。
4.根据权利要求1所述方法,其特征在于,所述方法在非治疗或诊断目的的药物研发中的应用。
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