CN110628719B - 一种诱导细胞快速产生囊泡的方法及其应用 - Google Patents
一种诱导细胞快速产生囊泡的方法及其应用 Download PDFInfo
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Abstract
本发明提供一种诱导细胞快速产生囊泡的方法及其应用,本发明通过使用一种新型纳米颗粒2I‑Bodipy‑5C‑UiO‑Hf,与有核细胞共同培养,通过光照的方法获得囊泡。同时在囊泡中获得了目的蛋白的高效表达,而且在各型有核细胞中获得广泛应用。在探索蛋白间相互作用,细胞膜蛋白的分子功能和免疫治疗等方面,提供了一种通用、高效、易行的方法,具有良好的实际应用之价值。
Description
技术领域
本发明属于医药生物技术领域,具体涉及一种诱导细胞快速产生囊泡的方法及其应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
细胞外囊泡是目前医药卫生行业中的研究热点,目前研究认为:根据细胞外囊泡的大小可以将细胞外囊泡分为凋亡小体,外泌体(60-100nm),小囊泡(100-1000nm),大囊泡(大于1μM)。
囊泡发挥重要作用:
1,囊泡的治疗作用。用囊泡装载药物,将具有严重副作用的药物运输到靶细胞,将减少治疗期间患者的痛苦和不便。在SCID鼠中,装载Cisplatin的小囊泡显著抑制卵巢癌细胞的增长。在肝癌H22荷瘤鼠中,装载MTX的小囊泡显著提高小鼠的生存率。在晚期肺癌恶性胸腔积液患者的临床治疗中,胸腔灌注甲氨喋呤囊泡能靶向杀伤肿瘤细胞,有效治疗复发性恶性胸腔积液,并展示出了良好的安全性。如果来源于异源细胞的囊泡作为治疗物质,由于MHC的不同,患者将出现免疫排斥;若应用来源于自体细胞的囊泡,患者将不会产生免疫反应,并且可以和免疫抑制剂联合应用。此外,囊泡内无细胞核,因此可以避免肿瘤细胞内核物质的转化作用,因此在临床上具有广阔的应用前景。
2,囊泡在生物膜蛋白分子功能的研究。质膜蛋白占所有已知药物靶点的70%,包括离子通道蛋白,G蛋白偶联受体,转运蛋白,免疫球蛋白,黏附分子等,质膜蛋白质组对于阐明其蛋白生物学功能,深入理解生理过程,免疫应答,疾病机制和发现新的药物靶点具有非常重要的意义。
3,囊泡在医学领域中的其他应用。肿瘤细胞产生的囊泡中含有大量含肿瘤相关抗原物质,经抗原提呈细胞处理后,可以作为肿瘤疫苗。在囊泡中装载生物活性物质如引物,探针,造影剂,反义核酸,荧光分子或抗体后,可用于疾病的诊断。在囊泡中装载生物活性物质如Cas9蛋白和gRNA后,产生可具有基因编辑功能的囊泡。
目前,囊泡生成剂的研究有很多,有化学试剂法(巯基封闭剂法),细胞毒素法(如细胞松弛素或蜂毒素)等,但是发明人发现,囊泡生成的效率,囊泡生成所需时间,收集囊泡所需后续处理却有所缺陷,目前诱导细胞产生囊泡的过程,仍存在周期较长,产量偏低,流程复杂等问题,都制约着囊泡的应用研究。
发明内容
基于上述现有技术的不足,本发明提供一种诱导细胞快速产生囊泡的方法及其应用,本发明通过使用一种新型纳米颗粒2I-Bodipy-5C-UiO-Hf,与有核细胞共同培养,通过光照的方法获得囊泡。同时还可以在囊泡中获得目的蛋白的高效表达,并且在各型有核细胞中获得广泛应用,因此具有良好的实际应用之价值。
本发明的一个方面,提供一种诱导细胞快速产生囊泡的方法,所述方法包括:纳米颗粒与细胞共孵育培养,在光照的作用下,迅速、高效的诱导细胞产生囊泡。
其中,所述纳米颗粒为2I-Bodipy-5C-UiO-Hf。
本发明的第二个方面,提供上述方法产生的囊泡。采用上述方法制备得到的囊泡产生后其直径为不小于100nm,进一步优选为直径在100-10000nm的液性囊泡。需要说明的是,本发明上述产生囊泡的方法,并不是通过诱导细胞凋亡而产生,因此囊泡内不含凋亡小体。
本发明的第三个方面,提供上述方法和/或上述囊泡在如下任一种或多种中的应用:
1)研究蛋白间相互作用;
2)研究膜蛋白的分子功能;
3)药物递送;
4)疾病诊断或制备疾病诊断药物和/或试剂;
5)疾病治疗或制备疾病治疗药物和/或试剂。
进一步的,所述疾病为癌症,所述癌症包括但不限于白血病、软组织肿瘤、恶性黑色素瘤、骨肿瘤、皮肤癌、肺癌、甲状腺癌、肝癌、子宫内膜癌、***癌、结肠癌、食管癌、胃癌和乳腺癌。
采用本发明的技术方案可以高效地诱导细胞产生囊泡,与现有技术相比,本发明技术方案具有以下有益效果:
1、本发明无需昂贵的仪器设备,耗费时间短,效率高,可在1小时内获得大量囊泡;
2、通过转基因技术,在囊泡内可以获得细胞质内表达的EGFP蛋白,也可在囊泡中高效表达膜蛋白;
3、利用本方法可获得小鼠,大鼠,人不同种属,不同组织来源的有核细胞囊泡,因此本发明具有普遍适用性。从而在比较不同细胞系中蛋白质组,比较目的细胞中的蛋白表达谱及蛋白相互作用,以及临床上肿瘤疫苗及药物的研究领域提供更优化的技术方案,因此本发明具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为纳米颗粒2I-Bodipy-5C-UiO-Hf联合光照诱导MCF-7细胞生成囊泡的步骤图;其中,
A:待细胞处于对数生长期时,在细胞培养皿中加入纳米颗粒2I-Bodipy-5C-UiO-Hf,使其终浓度为1μM;
B:避光,在37度,5%CO2条件下,共孵育培养30min;
C:用PBS洗涤细胞2次,以去除培养基中残余的纳米颗粒;
D:绿色激发光照射2-3min,诱导的MCF-7细胞产生囊泡,通过玛瑙球晃动,使囊泡脱落并游离在上清中;
E:将细胞上清溶液中加入2M/L蔗糖溶液,1500转/分离心20min,使细胞上清分层,细胞碎片沉积在蔗糖层中,分泌的囊泡存在囊泡层中。
图2为荧光显微镜检测纳米颗粒2I-Bodipy-5C-UiO-Hf在共孵育30min时,被MCF-7细胞吞噬,经绿色光照射2min后试验结果图;其中,
图2A为MCF-7细胞在绿光照射后,无囊泡产生图;
图2B为MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,无囊泡产生图;
图2C为MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生囊泡图;
图2D为MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生的脱落的囊泡图;
图3为纳米颗粒2I-Bodipy-5C-UiO-Hf诱导MCF-7细胞产生包裹目的蛋白的囊泡相关图;其中,
图3A为MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生囊泡图;
图3B为MCF-7-EGFP稳转细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生囊泡图;
图3C为荧光显微镜下,MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生的囊泡无绿色荧光图;
图3D为MCF-7-EGFP稳转细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生的囊泡显示绿色荧光图。
图4为流式细胞术检测纳米颗粒2I-Bodipy-5C-UiO-Hf在30min内被MCF-7细胞吞噬,及光照处理后产生囊泡图;其中,
图4A为MCF-7细胞作为阴性对照流式图;
图4B为MCF-7细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生无绿色荧光的囊泡流式图,可见在SSC-,FITC+处无明显分群;
图4C为MCF-7-EGFP稳转细胞与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育后,在绿光照射下,产生绿色囊泡流式图,可见在SSC-,FITC+的位置显著分群,数量占总细胞数量的5%。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
如前所述,目前囊泡生成剂的研究有很多,有化学试剂法(巯基封闭剂法),细胞毒素法(如细胞松弛素或蜂毒素)等,但是发明人发现,囊泡生成的效率,囊泡生成所需时间,收集囊泡所需后续处理却有所缺陷,所有诱导细胞产生囊泡的过程,仍存在周期较长,产量偏低,流程复杂等问题。
有鉴于此,本发明的一个具体实施方式中,提供一种诱导细胞快速产生囊泡的方法,所述方法包括:纳米颗粒与细胞共孵育培养,在光照的作用下,迅速、高效的诱导细胞产生囊泡。
其中,所述纳米颗粒为2I-Bodipy-5C-UiO-Hf。
所述纳米颗粒使用浓度为1-2μM(按BODIPY计)。
本发明的又一具体实施方式中,所述纳米载体与细胞共孵育培养的时间为30-60min,光照时间为1-30min。
本发明的又一具体实施方式中,所述光照的波长分布于1-580nm,优选分布于450-570nm;通过控制光照波长,可以有效提高囊泡产生效率。
本发明的又一具体实施方式中,所述细胞为有核细胞,所述有核细胞来源种属来源和组织来源不受限制,只要能产生囊泡的细胞皆在本发明的保护范围之内,如各种人源肿瘤细胞(包括人类乳腺癌细胞MCF-7)等。
同时产生的囊泡中可以装载有以下至少一种物质:目标蛋白,微囊泡,核酸,药物和纳米载体;进一步的,其中所述装载的物质为两种或两种以上不同物质,如cas9蛋白与核酸gRNA。
本发明的又一具体实施方式中,所述目标蛋白,至少包含以下任意一种蛋白:细胞因子,抗体,靶向蛋白,生物活性肽,毒素和融合蛋白。
本发明的又一具体实施方式中,所述目标蛋白制备方法,包括:通过构建质粒,利用脂质体,慢病毒载体,腺病毒载体等转基因技术,获得瞬转或稳转细胞系,使目的蛋白在细胞质中或细胞膜上有效表达,从而在产生囊泡的过程中被包裹在囊泡内。
所述核酸为反义核酸,DNA,mRNA,lncRNA,circRNA,PNA和gRNA中的任意一种或多种。
本发明的又一具体实施方式中,所述方法还包括收集囊泡的步骤,所述步骤包括使用密度梯度离心法收集囊泡。
本发明的又一具体实施方式中,提供上述方法产生的囊泡。采用上述方法制备得到的囊泡产生后其直径为不小于100nm,进一步优选为直径在100-10000nm的液性囊泡。需要说明的是,本发明上述产生囊泡的方法,并不是通过诱导细胞凋亡而产生,因此囊泡内不含凋亡小体。
本发明的又一具体实施方式中,提供上述方法和/或上述囊泡在如下任一种或多种中的应用:
1)研究蛋白间相互作用;
2)研究膜蛋白的分子功能;
3)药物递送;
4)疾病诊断或制备疾病诊断药物和/或试剂;
5)疾病治疗或制备疾病治疗药物和/或试剂。
本发明的又一具体实施方式中,所述疾病为癌症,所述癌症包括但不限于白血病、软组织肿瘤、恶性黑色素瘤、骨肿瘤、皮肤癌、肺癌、甲状腺癌、肝癌、子宫内膜癌、***癌、结肠癌、食管癌、胃癌和乳腺癌。
以下通过实施例对本发明做进一步解释说明,但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1纳米颗粒2I-Bodipy-5C-UiO-Hf的合成
1、UiO-66-OH(Hf)(1)的合成
将HfCl4(336mg,1.05mmol),2-羟基对苯二甲酸(182mg,1.0mmol)和乙酸(4.0mL)置于水中(6.0mL)加热至100℃维持48h;通过离心分离(12000rpm,30min),并使用乙醇清洗,在40℃条件下干燥即得纳米UiO-66-OH(Hf)颗粒。FT-IR(ATR,cm-1):3253(b),2977(m),1699(w),1583(s),1500(s),1424(vs),1245(m),1158(w),1045(w),963(w),798(w),768(m),680(m),578(w),475(w).
2、合成BODIPYC5Br
在氮气氛围下,6-溴己酰氯(5.6g,26mmol)和2,4-二甲基-1H-吡咯(5.0g,52.5mmol)在二氯甲烷(200mL)中回流处理1h,冷却至室温,然后向其中加入N,N-二异丙基乙胺(15mL)加热回流处理1h。然后加入BF3·Et2O(15mL)再加热回流处理3h。搅拌过夜,过中性氧化铝柱(洗脱液为二氯甲烷)纯化即得BODIPYC5Br.产率:4.7g(46%).1H NMR(400MHz,Chloroform-d)δ6.06(s,2H),3.44(t,J=5.1Hz,2H),2.99-2.94(m,2H),2.52(s,6H),2.42(s,6H),1.95-1.89(m,2H),1.69-1.63(m,4H).13C NMR(101MHz,Chloroform-d)δ153.90,145.90,140.25,131.36,121.66,33.38,32.18,30.88,28.55,28.19,16.39,14.44.MALDI-TOF MS,Calcd.For[M],396.118,Found,396.386.UV-vis(EtOH),497nm.FT-IR(ATR,cm-1):2927(m),2868(w),1550(vs),1509(vs),1475(s),1440(m),1409(m),1372(m),1340(w),1307(m),1270(m),1252(w),1224(m),1200(vs),1157(m),1107(m),1080(s),1070(s),1028(m),986(s),837(w),819(w),798(w),716(w),644(vw),626(w),583(w),554(vw),482(w).Anal.Calcd.For C18H24BBrF2N2(%):C,54.44;H,6.09;N,7.05,Found:C,54.14;H,5.91;N,7.32.
3、合成2I-BODIPYC5Br(2)
将BODIPYC5Br(1.0g,2.5mmol)和碘(1.6g,6.25mmol),and碘酸水溶液(1.0mL,0.9g/mL)加入乙醇(400mL)中回流处理30min。冷却至室温,过中性氧化铝柱(洗脱液,二氯甲烷/正己烷,v/v=1:1)纯化即得2I-BODIPYC5Br。产率:1.1g(69%).1H NMR(400MHz,Chloroform-d)δ3.44(t,J=6.5Hz,2H),3.07-2.99(m,2H),2.62(s,6H),2.49(s,6H),1.93(p,J=6.5Hz,2H),1.73-1.60(m,4H).13C NMR(101MHz,Chloroform-d)δ155.41,145.54,142.15,131.31,86.56,33.21,32.07,30.64,29.03,28.43,19.00,16.13.MALDI-TOF MS,Calcd.For[M],647.912,Found,647.557.UV-vis(EtOH),527nm.FT-IR(ATR,cm-1):2925(m),2859(w),1538(vs),1462(m),1394(m),1347(m),1306(w),1272(w),1252(w),1207(w),1186(s),1146(m),1077(m),994(m),753(w),719(w),587(w),527(w).Anal.Calcd.ForC18H22BBrF2I2N2(%):C,33.32;H,3.42;N,4.32,Found:C,33.48;H,3.21;N,4.55.
4、合成2I-Bodipy-5C-UiO-Hf
将UiO-66-OH(Hf)(5.0mg)、2I-BODIPYC5Br(5.0mg,7.7μmol)和KI(1.0mg,6.0μmol)溶于DMF(5.0mL)在室温下搅拌处理72h。通过离心分离(12000rpm,30min),并使用DMF和水依次清洗即得2I-Bodipy-5C-UiO-Hf。FT-IR(ATR,cm-1):3233(b),2976(m),1700(w),1584(s),1500(s),1424(vs),1245(m),1158(w),1099(w),1044(w),963(w),833(w),798(w),768(m),592(w),576(w),527(vw),475(w).
实施例2
1,囊泡生成(图1)。
传代培养人类乳腺癌细胞MCF-7,调整MCF-7细胞的浓度,使细胞处于对数生长期,在培养基中加入终浓度为1μM(按BODIPY计)的纳米颗粒2I-Bodipy-5C-UiO-Hf,与MCF-7细胞共孵育培养30min,共孵育结束后,通过荧光显微镜观察细胞形态,吞噬了纳米颗粒2I-Bodipy-5C-UiO-Hf的细胞在荧光显微镜下呈现绿色荧光。用PBS漂洗细胞2次以去除纳米颗粒,继而以绿色激发光照射细胞,光照时间为2-3min,诱导细胞生成囊泡。倒置显微镜下可以看到细胞产生无色的囊泡,随光照时间的延长,囊泡的体积增大,细胞处于半贴壁状态,囊泡可脱落至上清溶液中(图2)。
2,囊泡的收集:
动态观察细胞形成囊泡的情况,待约50%的细胞生成囊泡后,加入玛瑙球,轻轻摇动细胞培养瓶或培养皿,使囊泡脱落并游离在上清中。通过密度梯度离心法收集囊泡,小心轻柔的将囊泡生成液加到2M的蔗糖溶液中,1500转/分钟,离心10分钟,囊泡富集于上层,细胞碎片则富集于下层蔗糖层。
3,在MCF-7细胞囊泡中装载绿色荧光蛋白:
提取质粒pLVX-EGFP-IRES-PURO,与pSPAX2和pMD2.G,通过脂质体共同转染293T细胞,包装产生EGFP慢病毒,感染乳腺癌细胞MCF-7,观察细胞中绿色荧光蛋白的表达,通过嘌呤霉素稳定筛选得到MCF-7-EGFP稳转细胞系。通过与纳米颗粒2I-Bodipy-5C-UiO-Hf共孵育培养,经光照后,快速诱导细胞产生囊泡。与对照MCF-7细胞诱导产生无色囊泡相比,MCF-7-EGFP稳转细胞生成的囊泡为含有EGFP蛋白的绿色囊泡(图3),说明囊泡中装载有细胞质中稳定表达的EGFP蛋白。通过流式细胞术检测细胞生成囊泡的各项参数显示,绿色囊泡集中位于SSC-,FITC+的位置,此群囊泡产生的数量约占总细胞数量的5%(图4)。
最后应该说明的是,以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
Claims (4)
1.一种诱导细胞快速产生囊泡的方法,其特征在于,所述方法包括:纳米颗粒与细胞共孵育培养,在光照的作用下,诱导细胞产生囊泡;所述纳米颗粒与细胞共孵育培养的时间为30-60min,光照时间为1-30min;所述光照的波长分布于450-570nm;
2.如权利要求1所述方法,其特征在于,所述方法还包括收集囊泡的步骤,所述步骤包括使用密度梯度离心法收集囊泡。
3.一种通过权利要求1或2所述方法在囊泡中装载目标蛋白的应用,所述囊泡装载目标蛋白的方法包括:利用脂质体、慢病毒载体或腺病毒载体,获得瞬转或稳转细胞系,使目标蛋白在细胞质中或细胞膜上有效表达,并在如权利要求1所述产生囊泡的方法中被包裹在囊泡内。
4.如权利要求3所述应用,进一步包括研究蛋白间相互作用或膜蛋白的分子功能。
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