CN116875556A - 一种miR-204-5p过表达细胞系、高表达miR-204的外泌体及其制备方法与应用 - Google Patents
一种miR-204-5p过表达细胞系、高表达miR-204的外泌体及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种过表达miR‑204的细胞系、富含高表达miR‑204的外泌体及其制备方法与应用,所述高表达miR‑204的外泌体为miR‑204‑5p过表达细胞系生成的miR‑204。所述miR‑204‑5p过表达细胞系的制备方法为:将miR‑204基因克隆到pLVX‑IRES‑ZsGreenl载体上,获得重组质粒;将所述重组质粒转染进HEK293T细胞进行培养,得到包装有稳定表达miR‑204基因的慢病毒;用所述慢病毒感染MCF‑10A细胞,后通过细胞分选得到所述细胞系。本发明的高表达miR‑204的外泌体能够高效靶向脂肪细胞,并促进其分解代谢和减少机体的脂质储存,作为治疗药物与其他减肥药或保健药相比,具有更高的安全性。
Description
技术领域
本发明涉及细胞生物学技术领域,特别涉及一种miR-204-5p过表达细胞系、高表达miR-204的外泌体及其制备方法与应用。
背景技术
在经济全球化的潮流下,人们工作的生活节奏不断加快、饮食结构也发生了改变,肥胖症逐渐成为一种十分常见的疾病,其患病率呈逐年上升趋势,成为全球卫生健康的重大负担。肥胖症是机体能量摄入长期超过消耗的结果,常伴随高血压、糖尿病、血脂异常、心脏病等并发症,是一种代谢紊乱性疾病。尽管减肥降脂药物或保健品种类繁多,但减肥降脂药物的毒副作用及反弹效应大大降低了患者服药的依从性;各种保健品因功效成分不详、功效不稳定也逐渐失去了患者的信任。因此近年来,成分明确的生物来源的减肥方法越来越被减重代谢领域的研究者重视。
当前应用较多的肥胖疗法包括食疗、体育锻炼、行为疗法、药物疗法、综合治疗手术(胃大部切除术)等等。食疗、体育锻炼、行为疗法等非医疗技术治疗方法存在很大的局限性,首先这些疗法需要患者的高度配合以及自律性,往往患者会因为工作、生活忙碌及心情等多种原因无法坚持进行下去,其次,一旦中途停止治疗,患者的体重会迅速反弹导致前功尽弃。曾经在市面上售卖的减肥药种类很多,但是由于副作用较大,已禁止使用。例如曾经风靡一时的西布曲明。西布曲明本是作为抗抑郁症药物进行使用的,后来发现其具有抑制食欲和增强代谢的双重作用,就被用于饮食控制和运动不能控制的肥胖症的治疗。由于使用药物西布曲明可能增加严重心血管风险,减肥治疗的风险大于效益。而综合治疗手术(胃大部切除术)等则是一种创伤性较大治疗手段,通过减少胃的容量抑制患者的进食量,但此方法会带来很多不可预知的并发症,首先手术本身的风险较大,其次研究报道胃除了基本的消化功能以外还具有特殊的免疫功能,切除胃可能会某种程度上影响患者的免疫健全导致疾病的发生。
因此,有必要开发一种避免免疫排斥、安全性高的减肥降脂药物。
发明内容
本发明目的是提供一种miR-204-5p过表达细胞系、高表达miR-204的外泌体及其制备方法与应用,本发明的高表达miR-204的外泌体能够高效靶向脂肪细胞促进分解代谢减少机体的脂质储存,作为治疗药物与其他减肥药或保健药相比,具有更高的安全性。
为了实现上述目的,本发明采用如下技术方案:
在本发明的第一方面,提供了一种miR-204-5p过表达细胞系,所述miR-204-5p过表达细胞系为MCF-10A-miR-204细胞系;所述MCF-10A-miR-204细胞系是通过以下制备方法制备得到的:
将miR-204基因克隆到pLVX-IRES-ZsGreenl载体上,获得重组质粒
pLVX-IRES-ZsGreen1-miR-204;
用含有pMD2.G、psPAX2和pLVX-IRES-ZsGreen1-miR-204三种质粒的第一混合液转染进HEK293T细胞进行培养,后收集培养液并处理,得到包装有miR-204基因的慢病毒;
用所述包装有miR-204基因的慢病毒加入聚凝胺的第二混合液感染MCF-10A细胞进行培养,后通过细胞分选,得到miR-204-MCF-10A细胞系。
进一步地,所述将miR-204基因克隆到pLVX-IRES-ZsGreenl载体上,包括:
以SEQ ID NO.1-SEQ ID NO.2所示的引物对以MCF-10A细胞cDNA为模版进行PCR,获得PCR产物;
将所述PCR产物和pLVX-IRES-ZsGreenl质粒经XhoI和BamHI双酶切、酶连、转化、筛选和测序鉴定,获得重组质粒pLVX-IRES-ZsGreen1-miR-204。
进一步地,所述pMD2.G、psPAX2和pLVX-IRES-ZsGreen1-miR-204三种质粒的质量比为1.8~2.2:2.8~3.2:3.8~4.2。优选为2:3:4。
进一步地,所述聚凝胺的浓度为4.5~5.5μg/ml,优选为5μg/ml。
本发明的第二方面,提供了一种高表达miR-204的外泌体,所述高表达miR-204的外泌体为所述miR-204-5p过表达细胞系生成的miR-204mRNA。
在本发明的第三方面,提供了所述高表达miR-204的外泌体的制备方法,所述方法包括:
将所述miR-204-5p过表达细胞系进行细胞培养,获得培养液;
将所述培养液进行分离提纯,获得外泌体。
进一步地,所述细胞培养包括:
将所述miR-204-5p过表达细胞系培养至密度70%~90%,用PBS清洗后换成无血清的DMEM培养基。优选地,同时给予EGF生长因子20ng/ml、胰岛素10ug/ml第一天添加以促细胞增殖成分培养。
进一步地,所述分离提纯包括:
将所述培养液10000~25000rpm高速离心后弃去上清,收集沉淀即为外泌体。
在本发明的第四方面,提供了所述的高表达miR-204的外泌体在制备减肥降脂药物中的应用。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明提供的高表达miR-204的外泌体为基因工程化人乳腺上皮细胞外泌体,与人类的免疫反应较低,同时此种过表达高表达miR-204的外泌体可以工业化量产,在临床治疗上方式简单且易操作,除静脉注射外也可根据实际情况局部注射肥胖部位,是未来非常有前景的肥胖症治疗方法。与现有的减肥降脂药物相比,高表达miR-204的外泌体具有如下优点:
(1)首次提出应用外泌体天然介质包裹的miR-204达到减肥治疗肥胖症的疗效。
(2)效果好:外泌体可以避免其他治疗方法的一些缺陷(如免疫排斥,伦理等),并可直接与靶细胞发生融合高效发挥生物学效应。
(3)性质更稳定:外泌体具有脂质双分子层在运输活性调控因子时可保护其免于降解,从而防止活性成分在机体内转移时被快速降解。
(4)治疗方式更多样:外泌体作为药物治疗载体可以通过静脉注射、局部皮下注射、雾化吸入等多种途径进行治疗。
(5)保存运输更方便:外泌体在-20℃下储存6个月,-80℃长期保存,同时不会损失其生化活性。
(6)可量产:可通过规模化细胞培养收集培养基大量富集所需要使用的外泌体。
(7)可控制:外泌体功能可通过改变细胞环境来改变,同时也可以通过基因工程技术改造外泌体以达到控制外泌体效应,并且没有移植细胞带来的免疫排斥反应和肿瘤形成的风险。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图做简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为过表达高表达miR-204的外泌体尾静脉注射小鼠后脂肪组织形态图例。MCF-10AEV为阴性对照组尾静脉注射人乳腺上皮细胞外泌体,10A/miR-204EV为实验组尾静脉注射基因克隆过表达miR-204的人乳腺上皮细胞外泌体。
图2为高表达高表达miR-204的外泌体处理小鼠后小鼠脂肪组织;图中eWAT为内脏脂肪,iWAT为皮下脂肪;PBS为尾静脉注射PBS对照组;MCF-10A EV为阴性对照组尾静脉注射人乳腺上皮细胞外泌体,MDA-MB-231EV为实验组尾静脉注射miR-204高表达的乳腺癌细胞外泌体,Tumor Free为未种瘤空白对照组小鼠,Tumor Bearing为4T1细胞种瘤产生高表达miR-204组小鼠,4T1/Rab27a KO为抑制外泌体分泌的4T1细胞种瘤组小鼠;
图3为脂肪组织放置小动物活体成像系统检测明显可见GFP信号,说明经尾静脉注射的外泌体高效被脂肪组织吸收;
图4为过表达高表达miR-204的外泌体尾静脉注射小鼠体内皮下及内脏脂肪油红染色脂肪形态。
图5为规模化细胞培养上清中外泌体的提取流程及外泌体尾静脉注射小鼠模型的构建。
图6为超速离心法技术提取细胞培养基中高纯度高表达高表达miR-204的外泌体的结果。MCF-10A EV为阴性对照组尾静脉注射人乳腺上皮细胞外泌体,MDA-MB-231EV为实验组尾静脉注射miR-204高表达的乳腺癌细胞外泌体,10A/miR-204EV为实验组尾静脉注射基因克隆过表达miR-204的人乳腺上皮细胞外泌体;
图7为miR-204与体重之间的相关性结果。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买获得或者可通过现有方法获得。
本发明的“miR-204-5p过表达细胞系”又称“miR-204-5p过表达细胞系”或“MCF-10A-miR-204细胞系”;“高表达miR-204的外泌体”又称“高表达miR-204的外泌体”。
本发明实施例提供一种高表达miR-204的外泌体,总体思路如下:
首先,制备得到了miR-204-5p过表达细胞系,所述miR-204-MCF-10A细胞系是通过以下制备方法制备得到的:
步骤S101、将miR-204基因克隆到pLVX-IRES-ZsGreenl载体上,获得重组质粒pLVX-IRES-ZsGreen1-miR-204;
步骤S102、用含有pMD2.G、psPAX2和pLVX-IRES-ZsGreen1-miR-204三种质粒的第一混合液转染进HEK293T细胞进行培养,后收集培养液并处理,得到包装有miR-204基因的慢病毒;
步骤S103、用所述包装有miR-204基因的慢病毒加入聚凝胺的第二混合液感染MCF-10A细胞进行培养,后通过细胞分选,得到miR-204-MCF-10A细胞系。
接着,将所述miR-204-5p过表达细胞系进行培养,获得高表达miR-204的外泌体:
步骤S201、miR-204-5p过表达细胞系进行细胞培养,获得培养液;
步骤S202、将所述培养液进行分离提纯,获得外泌体。
然后,将所述外泌体处理细胞或尾静脉注射小鼠体内(每周2次,持续5周),测验是否具有减肥效果:
使用无外泌体血清配制的完全培养基培养MCF-10A、MDA-MB-231细胞,提取细胞外泌体,尾静脉注射NSG小鼠,每只小鼠每次注射10ug,每周注射两次;使用2×105MDA-MB-231及231/Rab27a KD细胞注射NSG小鼠第四对乳腺位置,构建荷瘤小鼠。每两天测量小鼠体重,进食量。
小鼠模型构建第6周后处死小鼠,解剖分离小鼠内脏脂肪、皮下脂肪组织,送公司RNA-Seq高通量测序。
结合生物信息学及miRNA测序结果,鉴定miR-204可以引起脂解的增加。小鼠尾静脉注射MCF-10A/miR-204OE外泌体6周后,全身内脏脂肪、皮下脂肪体积分布及重量均明显减少。
在肿瘤恶液质和肿瘤非恶液质病人血清外泌体的检测中,我们也证实了miR-204与体重之间的相关性。
下面将结合实施例及实验数据对本申请的一种高表达miR-204的外泌体及其制备方法与应用进行详细说明。
实施例1、一种高表达miR-204的外泌体的制备方法
一、构建miR-204-5p过表达细胞系
步骤S1、将miR-204基因克隆到pLVX-IRES-ZsGreenl载体(Takara,Cat#632187)上,获得重组质粒pLVX-IRES-ZsGreen1-miR-204;具体地,miR-204由下列引物PCR扩增所得
Forward primer(5'-3'):TTACTCGAGTCCAGGGGAATCTTTGCTT(SEQ ID NO.1);
Reverse primer(5'-3'):CGCGGATCCAGGAAGAAATACAACAGGGTGC(SEQ ID NO.2);
PCR产物(SEQ ID NO.3)和pLVX-IRES-ZsGreenl质粒经酶切、酶连、转化、筛选、测序鉴定后获取重组质粒pLVX-IRES-ZsGreen1-miR-204。
步骤S2,用含有pMD2.G(Takara,Cat#12260)、psPAX2(Takara,Cat#12259)和上述步骤所得的pLVX-IRES-ZsGreen1-miR-204三种质粒的混合液转染进HEK293T细胞进行培养,后收集培养液并处理,得到包装有miR-204基因的慢病毒;
步骤S3、用所述包装有miR-204基因的慢病毒加入聚凝胺的混合液感染MCF-10A细胞进行培养,后通过细胞分选,得到miR-204-MCF-10A细胞系。
二、细胞培养
将所述miR-204-5p过表达细胞系进行细胞培养,获得培养液;培养方法包括:
(1)提前将细胞培养基、高压灭菌的PBS溶液等置于37℃水浴锅预热,将生物安全柜的紫外灯打开,照射30min。
(2)酒精擦拭双手生物安全柜台面及培养箱把手,从培养箱中取出细胞培养皿。
(3)吸去培养基,1×PBS清洗细胞,弃去PBS后加入适量胰酶(10cm细胞培养皿1ml;15cm细胞培养皿2ml),以平铺培养瓶底部为宜,放置在细胞培养箱中消化。
(4)根据细胞类型调整消化时间(MDA-MB-231细胞2-3min;MCF-10A细胞及MCF-10A/miR-204OE 15min),消化结束后,轻轻拍打培养瓶侧边,可观察到细胞细沙样从皿底滑落。加入与胰酶等体积的新鲜培养基,轻轻摇晃培养皿混匀,终止消化,移液枪反复吹打,冲洗皿底的细胞使之脱落,收集细胞悬液于离心管中。
②1400rpm离心5分钟,使细胞沉淀,在生物安全柜中,弃去上清。
③加入1mL新鲜培养基轻轻吹打,使细胞分散混匀,1:2或1:3传代细胞,将细胞加入含有新鲜培养基的细胞培养皿中,十字法水平晃动培养皿,使细胞分散均匀。
④倒置显微镜下观察传代后的细胞密度,后放入细胞培养箱(37℃,5%CO2)中培养。
三、外泌体的提取
将所述培养液进行分离提纯,获得外泌体。具体操作如下:
(1)外泌体提取
①清洗高速离心瓶、超离管等,离心瓶高压灭菌并烘干,与超离管一同在细胞间生物安全柜紫外照射灭菌30min。
②在细胞间生物安全柜中将细胞培养液上清收集至离心瓶中,500g,15min,离心后将上清转移至500ml高速离心瓶中。
③日立高速离心机10000r,20min,4℃,离心后将上清分装至超速离心管中,每管35ml。
④贝克曼超速冷冻离心机25000r,70min,4℃,离心后弃去上清,沉淀即为外泌体。
⑤PBS重悬外泌体(每15cm细胞培养皿细胞分泌的外泌体30ul PBS的比例)
超速离心法技术提取细胞培养基中高纯度高表达高表达miR-204的外泌体,与对照组野生型细胞相比miR-204高表达约20倍(如图6)。
实施例2、高表达miR-204的外泌体能够高效靶向脂肪细胞促进分解代谢减少机体的脂质储存
1、构建荷瘤小鼠
使用无外泌体血清配制的完全培养基培养MCF-10A、MDA-MB-231细胞,提取细胞外泌体,尾静脉注射NSG小鼠,每只小鼠每次注射10ug,每周注射两次;使用2×105MDA-MB-231及231/Rab27a KD细胞注射NSG小鼠第四对乳腺位置,构建荷瘤小鼠。每两天测量小鼠体重,进食量。
小鼠模型构建第6周后处死小鼠,解剖分离小鼠内脏脂肪、皮下脂肪组织,送公司RNA-Seq高通量测序。
RNA-seq数据GSEA分析在MDA-MB-231EV组及TB组小鼠的内脏脂肪组织与对照组相比低氧信号通路VHL-HIF1A显著富集。
2、外泌体处理细胞方法
①细胞密度在80-90%时加外泌体重悬液,2μg的外泌体可用于处理2×105个受体细胞。
②24小时后再处理一次。
3、小鼠尾静脉注射外泌体方法
①收集细胞培养基,三轮离心后离心管中沉淀用灭菌的PBS重悬再离心一次富集,沉淀称重。
②每只小鼠每次注射10μg外泌体,每周注射2次,共6周。
4、如图1-2,外泌体处理6周后小鼠置于micro-CT拍摄,分析发现小鼠体内性腺白色脂肪组织和皮下白色脂肪与对照组相比明显减少,解剖小鼠收取脂肪组织拍照可见外泌体处理组形态大小与对照组相比明显减少。
5、如图3,脂肪组织放置小动物活体成像系统检测明显可见GFP信号,说明经尾静脉注射的外泌体高效被脂肪组织吸收。
6、脂肪组织OCT冰冻切片制备,冰冻切片机切片后蒸馏水充分洗涤,油红稀释液染10-15分钟,避光,密封,60%乙醇镜下分化至间隙清楚,甘油明胶封片。镜下观察可见实验组脂肪大小明显减少。由图4可知,小鼠尾静脉注射MCF-10A/miR-204OE外泌体6周后,全身内脏脂肪、皮下脂肪体积分布及重量均明显减少。
7、在肿瘤恶液质和肿瘤非恶液质病人血清外泌体的检测:准备500ul病人血清,第一步300g低速离心10分钟去除死细胞、大的细胞碎片,第二步10000g高速离心30分钟去除小的细胞碎片、大囊泡等;最后100000g超速离心70分钟得到外泌体。结果如图7所示,证实了miR-204与体重之间的相关性。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种miR-204-5p过表达细胞系,其特征在于,所述miR-204-5p过表达细胞系为MCF-10A-miR-204细胞系;所述MCF-10A-miR-204细胞系是通过以下制备方法制备得到:
将miR-204基因克隆到pLVX-IRES-ZsGreenl载体上,获得重组质粒pLVX-IRES-ZsGreen1-miR-204;
用含有pMD2.G、psPAX2和pLVX-IRES-ZsGreen1-miR-204三种质粒的混合液转染进HEK293T细胞进行培养,后收集培养液并处理,得到包装有miR-204基因的慢病毒;
用所述包装有miR-204基因的慢病毒加入聚凝胺的混合液感染MCF-10A细胞进行培养,后通过细胞分选,得到miR-204-5p过表达细胞系。
2.根据权利要求1所述的一种miR-204-5p过表达细胞系,其特征在于,将miR-204基因克隆到pLVX-IRES-ZsGreenl载体上,包括:
以SEQ ID NO.1-SEQ ID NO.2所示的引物对,以MCF-10A细胞cDNA为模版进行PCR,获得PCR产物;
将所述PCR产物和pLVX-IRES-ZsGreenl质粒经酶切XhoI和BamHI双酶切、酶连、转化、筛选和测序鉴定,获得重组质粒pLVX-IRES-ZsGreen1-miR-204。
3.根据权利要求1所述的一种miR-204-5p过表达细胞系,其特征在于,所述pMD2.G、psPAX2和pLVX-IRES-ZsGreen1-miR-204三种质粒的质量比为1.8~2.2:2.8~3.2:3.8~4.2。
4.根据权利要求1所述的一种miR-204-5p过表达细胞系,其特征在于,所述聚凝胺的浓度为4.5~5.5μg/ml。
5.一种高表达miR-204的外泌体,其特征在于,所述高表达miR-204的外泌体为权利要求1-4任一项所述miR-204-5p过表达细胞系生成的非编码RNA-miR-204。
6.一种权利要求5所述高表达miR-204的外泌体制备方法,其特征在于,所述方法包括:
将权利要求1-4任一项所述miR-204-5p过表达细胞系进行细胞培养,获得培养液;
将所述培养液进行分离提纯,获得外泌体。
7.根据权利要求6所述的制备方法,其特征在于,所述细胞培养包括:
将所述miR-204-5p过表达细胞系培养至密度70%~90%,用PBS清洗后换成无血清的DMEM培养基。
8.根据权利要求6所述的制备方法,其特征在于,所述分离提纯包括:
将所述培养液10,000~25,000rpm高速离心后弃去上清,收集沉淀即为外泌体。
9.权利要求1-4任一项所述miR-204-5p过表达细胞系miR-204-5p过表达细胞系在制备减肥降脂药物中的应用。
10.权利要求5所述的高表达miR-204的外泌体在制备减肥降脂药物中的应用。
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