CN112410376A - 一种高效装载特异性miRNA的功能性外泌体构建方法 - Google Patents

一种高效装载特异性miRNA的功能性外泌体构建方法 Download PDF

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CN112410376A
CN112410376A CN202011382967.2A CN202011382967A CN112410376A CN 112410376 A CN112410376 A CN 112410376A CN 202011382967 A CN202011382967 A CN 202011382967A CN 112410376 A CN112410376 A CN 112410376A
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李倩坤
胡文治
张翠萍
付小兵
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Chinese PLA General Hospital
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Abstract

本发明公开了一种高效装载特异性miRNA的功能性外泌体构建方法，为了使外泌体能够更加高效地携带具有特定调控功能的miRNA，以更加精准高效地发挥靶向调控作用，利用MS2噬菌体衣壳蛋白，编辑构建特定miRNA分子的捕捉元件，重编程胎盘间充质干细胞，使其分泌的外泌体高效装载目的miRNA分子，从而递送至组织细胞发挥有效调控作用，为将来实现特异性精准治疗提供一种新的策略。

Description

一种高效装载特异性miRNA的功能性外泌体构建方法
技术领域
本发明涉及生物医用材料和细胞分子治疗领域，具体涉及一种高效装载特异性miRNA的功能性外泌体构建方法。
背景技术
随着细胞分子治疗的快速发展，外泌体治疗成为生物医疗领域研究的热点，并受到广泛的关注。外泌体是细胞外分泌囊泡中的一员，它可以作为运输载体，携带细胞内特定的蛋白和RNA等成分，通过融合到靶细胞膜或者靶细胞胞吞作用摄入，将RNA等分子直接递送到靶细胞内部，对受体细胞组织发挥有效调控作用。相比于干细胞治疗，干细胞来源的外泌体，不仅含有干细胞分泌的多种有效成分，而且可以避免异种来源细胞表面抗原带来的一过性排异反应，在临床治疗应用上更加安全。
MicroRNA(miRNA)是一类内生的、长度约为20-24个核苷酸的小RNA，其在细胞内具有多种重要的调节作用。据推测，miRNA调节着人类三分之一的基因，在细胞分化、生物发育及疾病发生发展过程中发挥巨大作用。然而，单独的miRNA分子在体外稳定性差，并且需要进入细胞内部才能发挥调控作用，而外泌体恰好可以作为miRNA的载体在细胞间起到有效的递送作用。由于外泌体自身携带的细胞内蛋白和RNA数量有限、成分复杂，特异性作用效率不高。以往的方法单纯通过脂质体转染或电转染等方式使细胞过表达miRNA，仍是依靠外泌体自组装能力携带miRNA分子，装载效率依然有限。
发明内容
针对现有技术的不足，本发明旨在提供一种高效装载特异性miRNA的功能性外泌体构建方法。
为了实现上述目的，本发明采用如下技术方案：
一种高效装载特异性miRNA的功能性外泌体构建方法，包括如下步骤：
S1、利用MS2噬菌体衣壳蛋白，将MS2蛋白编码基因与外泌体Lactadherin蛋白中的C1C2结构域相连接，构建C1C2-MS2(CM)慢病毒质粒；
S2、将用于与MS2铆合的位点pac蛋白与目的miRNA相连，构建pac-miRNA-pac(p-miRNA-p)慢病毒质粒，其两端均具有与MS2结合的pac位点；
S3、将步骤S1和步骤S2获得的两种质粒包装为慢病毒感染间充质干细胞，通过抗性药物筛选获得确定的稳转系，留取干细胞上清液，利用超速离心法提取外泌体。
进一步地，步骤S3的具体过程：
S3.1、将C1C2-MS2慢病毒质粒和pac-miRNA-pac慢病毒质粒分别利用三质粒慢病毒包装系统，通过转染293T细胞，包装得到CM慢病毒和p-miRNA-p慢病毒；
S3.2、无菌条件下，将胎盘绒毛膜来源的间充质干细胞PMSCs加入含有干细胞培养基中，置于37℃、CO2体积分数为5％的培养箱内孵育；所述干细胞培养基中含有质量百分比10％的胎牛血清；
S3.3、通过CM慢病毒感染PMSCs，48小时后利用含有1.0ug/mL puromycin的培养基进行药物筛选，10-14天后获取稳定表达MS2的细胞系CM-PMSCs；
S3.4、将CM-PMSCs进一步感染p-miRNA-p慢病毒，48h后利用含有600μg/mL G418的培养基进行药物筛选，2周后获取稳定表达目的miRNA的细胞系CM-miRNA-PMSCs；
S3.5、利用含有质量百分比10％的无外泌体血清的干细胞培养基孵育CM-miRNA-PMSCs，24-48h收集细胞上清液，超滤浓缩后，通过超速离心法与外泌体提取试剂盒，获得高效装载目的miRNA的外泌体CM-miRNA-Exo。
进一步地，步骤S3.2中，所述干细胞培养基由高糖DMEM与DMEM-F12以体积比1：1比例配制而成，并含有100U/mL青链霉素、10ng/mL成纤维细胞生长因子和10ng/mL表皮生长因子。
本发明的有益效果在于：本发明为了使外泌体能够更加高效地携带具有特定调控功能的miRNA，以更加精准高效地发挥靶向调控作用，利用MS2噬菌体衣壳蛋白，编辑构建特定miRNA分子的捕捉元件，重编程胎盘间充质干细胞，使其分泌的外泌体高效装载目的miRNA分子，从而递送至组织细胞发挥有效调控作用，为将来实现特异性精准治疗提供一种新的策略。
附图说明
图1为本发明实施例2中高效表达miR-146a工程化外泌体的构建示意图；
图2为本发明实施例2中CM-miR146a-Exo的装载效率及功能验证结果示意图。
图3为本发明实施例2中CM-miR146a-Exo对糖尿病小鼠创面愈合作用的结果示意图。
图4为本发明实施例2中CM-miR146a-Exo作用于糖尿病小鼠创面组织转录组测序的炎性相关蛋白网络分析示意图。
具体实施方式
以下将结合附图对本发明作进一步的描述，需要说明的是，本实施例以本技术方案为前提，给出了详细的实施方式和具体的操作过程，但本发明的保护范围并不限于本实施例。
实施例1
一种高效装载特异性miRNA的功能性外泌体构建方法，包括如下步骤：
S1、利用MS2噬菌体衣壳蛋白，将MS2蛋白编码基因与外泌体Lactadherin蛋白中的C1C2结构域相连接，构建C1C2-MS2(CM)慢病毒质粒；
S2、将用于与MS2铆合的位点pac蛋白与目的miRNA相连，构建pac-miRNA-pac(p-miRNA-p)慢病毒质粒，其两端均具有与MS2结合的pac位点；
S3、将步骤S1和步骤S2获得的两种质粒包装为慢病毒感染间充质干细胞，通过抗性药物筛选获得确定的稳转系，留取干细胞上清液，利用超速离心法提取外泌体。
进一步地，步骤S3的具体过程为：
S3.1、将CM慢病毒质粒和p-miRNA-p慢病毒质粒利用三质粒慢病毒包装系统，通过转染293T细胞，包装得到CM慢病毒和p-miRNA-p慢病毒；
S3.2、无菌条件下，将胎盘绒毛膜来源的间充质干细胞(PMSCs)加入含有10％胎牛血清的干细胞培养基中，置于37℃、5％CO2孵育箱内培养；
S3.3、通过CM慢病毒感染PMSCs，48小时后利用含有1.0ug/mL puromycin的培养基进行药物筛选，10-14天后获取稳定表达MS2的细胞系CM-PMSCs；
S3.4、将CM-PMSCs进一步感染p-miRNA-p慢病毒，48h后利用含有600μg/mL G418的培养基进行药物筛选，2周后获取稳定表达目的miRNA的细胞系CM-miRNA-PMSCs；
S3.5、利用10％无外泌体血清的干细胞培养基孵育CM-miRNA-PMSCs，24-48h收集细胞上清液，超滤浓缩后，通过超速离心法与外泌体提取试剂盒，获得高效装载目的miRNA的外泌体CM-miRNA-Exo。
进一步地，步骤S3.2中，所述干细胞培养基由高糖DMEM与DMEM-F12以体积比1：1比例配制而成，并含有100U/mL青链霉素、10ng/mL成纤维细胞生长因子和10ng/mL表皮生长因子。
实施例2
本实施例基于实施例1所述方法，提供一种构建高效携带miRNA-146a的具有抗炎作用的功能性外泌体的方法。
大量研究表明，miRNA-146a在炎症反应中具有重要的调控作用。IRAK是miR-146a经典的下游靶基因，也是NF-κB通路的关键调控因子，miR-146a可以通过调控IRAK1抑制NF-κB通路的激活。因此，本实施例通过工程化外泌体作用于表皮细胞，检测表皮细胞中IRAK1的表达量，对CM-miR146a功能性外泌体(CM-miR146a-Exo)的抗炎功效进行验证，并观察CM-miR146a-Exo对糖尿病小鼠炎性创面的愈合作用，通过创面组织转录组测序分析差异性基因表达及相关炎性蛋白与信号通路。
具体过程如下：
1.构建pac-pre-miR-146a-pac慢病毒质粒：查询miR-146a前体的基因序列，构建miRNA-146a的前体pre-miR-146a慢病毒质粒，两侧各带有一组与MS2结合的pac锚定位点，形成pac-pre-miR-146a-pac(p-miR146a-p)慢病毒质粒，如图1A所示。图1A为构建工程化外泌体的示意图：通过CM和p-miR146a-p慢病毒质粒基因重编程，使干细胞来源的外泌体高效携带miR-146a靶向调控分子。
2.培养293T细胞以包装慢病毒：通过胰酶消化收集293T细胞，用适当的完全培养基平铺细胞于35mm培养皿上，使细胞融合面积达到培养皿总面积的80％以上。将细胞置于含5％CO2的37℃培养箱中孵育8-24h，当细胞贴壁完全后即可开始转染，转染前2h换液。
3.包装p-miR146a-p慢病毒：利用三质粒系统，将p-miR146a-p慢病毒质粒与病毒包装质粒Mix:1μg/μl(Mix＝pMDL:VSV-G:REV＝5:3:2)经lipo3000转染至293T细胞。6h后去除质粒转染培养基，加入2.5mL 37℃预热的完全培养基，继续将细胞放置培养箱孵育。
4.收集p-miR146a-p慢病毒上清：48h后可以收集含慢病毒的上清，1500rpm离心5分钟(min)，一般可收集到2mL慢病毒上清，可直接用于外泌体分泌细胞的感染，或者分装后置于－80℃保存。
5.p-miR146a-p慢病毒感染：将CM-PMSCs(按实施例1方法获得)平铺于6孔板，最佳密度为30％－70％。加入收集的p-miR146a-p慢病毒上清感染CM-PMSCs，12h后更换为正常干细胞培养基，48h后加入含有600μg/mL G418的培养基对细胞进行药物筛选，2w后获取稳定表达miR-146a的细胞系(CM-miR146a-PMSCs)。
6.提取高效表达miR-146a的外泌体：利用10％无外泌体血清的干细胞培养基孵育CM-miR146a-PMSCs，24-48h收集细胞上清液，超滤浓缩后通过超速离心法与外泌体提取试剂盒，获得高效表达miR-146a的外泌体CM-miR146a-Exo。通过SEM观察胎盘间充质干细胞来源的工程化外泌体形态，并对外泌体进行粒径分析(如图1B、1C所示)。从图1B可见透射电子显微镜观察胎盘间充质干细胞来源的工程化外泌体呈盘状囊泡。从图1C可见工程化外泌体的粒径检测直径为70-120nm。从图1D可见Western blot鉴定外泌体表面特有的标记蛋白CD63、CD9、TSG101表达阳性，且细胞内质网特异性分子Calnexin表达阴性。
7.检测工程化外泌体装载miR-146a的效率：通过QRT-pcr检测miR-146a和CM慢病毒感染PMSCs后外泌体中miR-146a的表达量。结果显示，CM-miR146a-Exo组中的miR-146a的相对表达量显著增加，高于单纯过表达miR-146a的外泌体miR146a-Exo组近十倍(如图2A所示)。从图2可见，CM-miR146a-Exo组中的miR-146a的相对表达量显著增加，与单纯过表达miR-146a组相比，miR-146a的相对表达量增加了近十倍。
8.CM-miR146a功能性外泌体靶向抗炎功能的验证：miR-146a对下游靶基因IRAK1具有抑制调控作用。IRAK1是NF-κB通路的关键调控因子，miR146a可通过调控IRAK1抑制NF-κB通路的激活。通过工程化外泌体作用于表皮细胞，利用双荧光素酶实验检测miR-146a对下游靶基因IRAK1的抑制水平，从而验证外泌体的抗炎功能。结果显示，单纯过表达miR-146a的外泌体作用表皮细胞后，IRAK1相对荧光值下降38％，有一定抑制作用，CM-miR146a-Exo作用的细胞中IRAK1的相对荧光值显著减少81％，表明CM-miR146a-Exo显著抑制下游炎性因子IRAK1的表达(如图2B所示)。从图2B可见，双荧光素酶实验显示，CM-miR146a-Exo作用的表皮细胞IRAK1的相对荧光值显著减少，表明CM-miR146a-Exo对IRAK1的抑制作用最为显著。
9.CM-miR146a功能性外泌体对糖尿病小鼠炎性创面的作用：在糖尿病小鼠背部构建直径为1cm的全层皮肤创面，空白对照和不同外泌体组分别作用创面，观察各组创面愈合情况，并对各组残余创面面积和创面闭合率进行统计分析。结果如图3A、B、C所示。从图3A可见糖尿病创面第14天和第21天的大体愈合情况，CM-miR146a-Exo组与其他对照组相比，创面愈合速度显著增加，具有促进创面修复的作用。图3B通过各组残余创面面积的统计分析，发现CM-miR146a-Exo组的创面残余面积显著小于同期对照组创面。从图3C可见，与糖尿病创面对照组相比，CM-miR146a-Exo组作用的创面愈合速度更快，创面愈合率＝(原创面面积-残余创面面积)/原创面面积×100％。
10.CM-miR146a功能性外泌体作用于糖尿病创面组织转录组测序的炎性相关蛋白分析：通过创面组织的转录组测序，分析差异性基因表达和参与调控的相关炎性蛋白及信号通路。如图4所示，结果显示，经CM-miR146a-Exo作用的皮肤创面组织中，炎性相关蛋白IL-1a、IL-1b、IL-11、TNF、NF-κB1、Rela等表达量减少，相关炎性调控通路受到抑制。表明CM-miR146a功能性外泌体具有显著的抗炎作用。
对于本领域的技术人员来说，可以根据以上的技术方案和构思，给出各种相应的改变和变形，而所有的这些改变和变形，都应该包括在本发明权利要求的保护范围之内。

Claims (3)

1.一种高效装载特异性miRNA的功能性外泌体构建方法，其特征在于，包括如下步骤：
S1、利用MS2噬菌体衣壳蛋白，将MS2蛋白编码基因与外泌体Lactadherin蛋白中的C1C2结构域相连接，构建C1C2-MS2慢病毒质粒；
S2、将用于与MS2铆合的位点pac蛋白与目的miRNA相连，构建pac-miRNA-pac慢病毒质粒，其两端均具有与MS2结合的pac位点；
S3、将步骤S1和步骤S2获得的两种质粒包装为慢病毒感染间充质干细胞，通过抗性药物筛选获得确定的稳转系，留取干细胞上清液，利用超速离心法提取外泌体。
2.根据权利要求1所述的方法，其特征在于，步骤S3的具体过程：
S3.1、将C1C2-MS2慢病毒质粒和pac-miRNA-pac慢病毒质粒分别利用三质粒慢病毒包装系统，通过转染293T细胞，包装得到CM慢病毒和p-miRNA-p慢病毒；
S3.2、无菌条件下，将胎盘绒毛膜来源的间充质干细胞PMSCs加入含有干细胞培养基中，置于37℃、CO2体积分数为5％的培养箱内孵育；所述干细胞培养基中含有质量百分比10％的胎牛血清；
S3.3、通过CM慢病毒感染PMSCs，48小时后利用含有1.0ug/mL puromycin的培养基进行药物筛选，10-14天后获取稳定表达MS2的细胞系CM-PMSCs；
S3.4、将CM-PMSCs进一步感染p-miRNA-p慢病毒，48h后利用含有600μg/mL G418的培养基进行药物筛选，2周后获取稳定表达目的miRNA的细胞系CM-miRNA-PMSCs；
S3.5、利用含有质量百分比10％的无外泌体血清的干细胞培养基孵育CM-miRNA-PMSCs，24-48h收集细胞上清液，超滤浓缩后，通过超速离心法与外泌体提取试剂盒，获得高效装载目的miRNA的外泌体CM-miRNA-Exo。
3.根据权利要求2所述的方法，其特征在于，步骤S3.2中，所述干细胞培养基由高糖DMEM与DMEM-F12以体积比1：1比例配制而成，并含有100U/mL青链霉素、10ng/mL成纤维细胞生长因子和10ng/mL表皮生长因子。
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