CN116515759A - 改造间充质干细胞外泌体装载miR-34c-5p靶向清除白血病干细胞 - Google Patents
改造间充质干细胞外泌体装载miR-34c-5p靶向清除白血病干细胞 Download PDFInfo
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Abstract
本发明涉及一种间充质干细胞外泌体的改造方法及应用,属于生物医药领域。首先构建并转染Lamp2b‑IL3‑eGFP慢病毒至间充质干细胞内,使其外泌体膜表达白血病干细胞(leukemia stem cells,LSCs)表面特异性分子CD123受体(IL3),而后通过岩藻糖基化体系处理使外泌体膜表面CD44分子转化成骨髓定向归巢能力更强的HCELL分子,最后,通过电转染技术将miR‑34c‑5p导入到改造后的MSC外泌体,最终实现靶向清除骨髓白血病干细胞。本发明通过改造间充质干细胞外泌体装载miR‑34c‑5p治疗急性髓系白血病,克服miR‑34c‑5p在体内循环中易被降解、难以到达骨髓小龛作用白血病干细胞及其可能导致正常体细胞衰老等不足。
Description
技术领域
本发明属于生物医药领域,更具体地,涉及一种间充质干细胞外泌体的改造方法及应用,尤其通过改造间充质干细胞外泌体使其具有定向归巢到骨髓,并靶向结合骨髓中的白血病干细胞的能力,然后装载miR-34c-5p靶向诱导白血病干细胞衰老与清除,促进急性髓系白血病的治疗。
背景技术
白血病干细胞(leukemia stem cells,LSCs)是急性髓系白血病(acute myeloidleukemia,AML)复发和耐药的根源,多项研究提示,LSCs存在衰老抵抗是导致AML复发与耐药的重要原因之一。Ablain等人发现,维甲酸治愈急性早幼粒细胞白血病(Acutepromyelocytic leukemia,APL)机制是通过诱导APL始动细胞衰老来实现的。我们的研究发现,miR-34c-5p作为诱导细胞衰老的中心调控分子,在LSCs内低表达,上调miR-34c-5p水平可诱导LSCs衰老并且在一定程度上延长AML模型小鼠的生存期。尽管miR-34c-5p在治疗AML上可能具有较好的应用前景,然而,将miR-34c-5p应用到临床仍然面临很多挑战。首先,miR-34c-5p作为一种小分子RNA,在血液循环运输中极易被RNA酶降解,难以到达骨髓小龛作用于LSCs;其次,miR-34c-5p可能非选择性的诱导人正常体细胞衰老,因此,本项目拟探索一种理想的载体使miR-34c-5p高效运输至骨髓靶向作用于LSCs。
现有的药物载体主要包括:胶束、微乳液、凝胶、液晶囊泡等,对胞膜具有良好的渗透性,这些材料是目前药物载体的主要研究对象。与之相比,间充质干细胞作为药物载体有其独特的优势,它是一类来源广泛且具有多向分化潜能的多能干细胞。MSC具备的许多优势有助于临床转化,这些特性包括易于从多个组织中分离出来、体外扩增能力强、具有多分化潜能和免疫调节功能、能被操纵或基因修饰等等。此外,MSC与生俱来地倾向于向恶性位点迁移,这些特性使得它们能够有效传递抗肿瘤药物,也被认为是癌症靶向治疗的优良载体。然而,MSC不是简单的递送载体,它具有积极的生理过程,会产生和分泌大量的生长因子、细胞因子和趋化因子等等。因此,采用MSC运输药物时还需要解决一些关键问题,例如,如何准确地将抗癌药物递送至目标组织而不会递送至非目标组织,是否存在潜在毒性等。近年来有研究表明MSC因为体积大,大部分都在肝、脾和肺内被截留,被单核巨噬细胞吞噬,最终到达作用部位的数量小于1%,且在到达靶组织的MSC中,大部分在几天后消失,只有少量的MSC长期停留在靶点部位。此外,还需要考虑所运载的药物对MSC本身产生的不利影响,利用MSC运载miR-34c-5p可能诱导其自身衰老从而影响正常功能,这些不利因素都会大大的限制MSC临床应用价值。
近年来研究表明,MSC能够通过分泌的外泌体发挥对肿瘤细胞的调节作用。MSC被认为是分泌外泌体能力最强的细胞,MSC来源的外泌体是其在生理或者病理状态下分泌的40-100nm双层膜结构的囊泡,内含多种蛋白和RNA,MSC分泌的外泌体具有与MSC相似的生物学功能,如低免疫原性、免疫调节能力、向炎症或肿瘤部位归巢并释放活性物质而发挥作用等,是介导细胞间信息交流及传递的重要因素。外泌体本身的一些特性也使之成为一种理想的载体:如(1)外泌体将分子包裹在膜内,可以保护RNA免于降解;(2)外泌体相比于脂质体,对亲水性物质如核酸类分子具有更高的包装效果;(3)相比于病毒来源以及合成的纳米载体,外泌体具有更低的免疫原性;(4)相比于MSC,外泌体体积更小、复杂性更低,易于生产和存储,没有细胞的活性,并且不构成形成肿瘤的风险等;(5)此外,研究人员发现,MSC外排的囊泡高表达CD47(整合素相关蛋白),在AML及多种肿瘤细胞上均有表达,是信号调控蛋白SIRPa的配体,CD47与巨噬细胞表面的SIRPa结合,产生一系列级联反应抑制巨噬细胞吞噬作用。因此MSC外泌体表面表达CD47分子可防止MSC外泌体被血循环中单核细胞和巨噬细胞所消化。基于以上这些特点,使得MSC来源的外泌体成为一种理想的运输载体
尽管如此,MSC来源的外泌体仍不能直接作为miR-34c-5p的运输载体靶向清除LSCs,原因如下:(1)目前尚无利用MSC来源的外泌体作为载体传递药物或目标分子治疗AML的研究;(2)如何使MSC来源的外泌体特异性结合LSCs而不影响正常细胞;(3)LSCs大多存在于骨髓壁龛内,如何使MSC来源的外泌体更好的归巢至骨髓并发挥作用;(4)如何使miR-34c-5p高效装载至MSC外泌体内。解决好这几个问题是实现MSC外泌体作为载体运输miR-34c-5p靶向治疗AML关键。
发明内容
本发明首先构建并转染Lamp2b-IL3-eGFP慢病毒至间充质干细胞(Mesenchymalstem cell,MSC)内,使其外泌体膜表达LSCs表面特异性分子CD123受体(IL3),而后通过岩藻糖基化体系处理使外泌体膜表面CD44分子转化成骨髓定向归巢能力更强的HCELL分子,通过转染技术将miR-34c-5p导入到改造后的MSC外泌体最终实现靶向清除骨髓白血病干细胞。本发明通过改造间充质干细胞外泌体的装载miR-34c-5p治疗急性髓系白血病,克服miR-34c-5p在体内循环中易被降解、难以到达骨髓小龛作用白血病干细胞及其可能导致正常体细胞衰老等不足。
根据本发明第一方面,提供了一种间充质干细胞外泌体的改造方法,包括以下步骤:
(1)构建带有外泌体膜蛋白lamp2b基因、白血病干细胞膜表面特异性分子CD123受体IL3基因及增强型绿色荧光蛋白eGFP基因的融合质粒,将该融合质粒包装成慢病毒后转导至间充质干细胞中,使间充质干细胞分泌的外泌体膜表达IL3分子;
(2)收集步骤(1)得到的外泌体,并进行岩藻糖基化,使所述外泌体的膜表面CD44蛋白进行岩藻糖基化修饰,即实现间充质干细胞外泌体的改造。
优选地,步骤(2)中,所述岩藻糖基化为将步骤(1)得到的外泌体与岩藻糖基化酶促反应体系进行反应。
优选地,所述岩藻糖基化酶促反应体系含有a-1,3-岩藻糖转移酶。
优选地,所述a-1,3-岩藻糖转移酶为a-1,3-岩藻糖转移酶VII。
优选地,步骤(1)中,所述融合质粒的序列为SEQ ID NO:1所示。
优选地,步骤(1)中,所述间充质干细胞为脐带经体外培养获得的第3-8代传代细胞。
根据本发明另一方面,提供了任意一项方法得到的改造后的间充质干细胞外泌体。
根据本发明另一方面,提供了所述的改造后的间充质干细胞外泌体用于制备靶向清除骨髓白血病干细胞药物载体的应用。
优选地,所述应用具体为:将miR-34c-5p小分子转染入所述改造后的间充质干细胞外泌体中。
优选地,所述转染为电转染。
总体而言,通过本发明所构思的以上技术方案与现有技术相比,主要具备以下的技术优点:
(1)本发明选择间充质干细胞的外泌体作为载体,并利用基因工程改造使其分泌的外泌体膜高表达IL3,进而靶向结合白血病干细胞表面的特异性CD123分子。
在第一步改造的基础上,进一步通过岩藻糖基化反应体系,使外泌体膜表面的CD44分子转化为归巢能力更强的HCELL,使外泌体归巢骨髓的能力加强,现有的技术改造对象多为细胞,外泌体糖基化改造本研究为首例,优势在于盐藻糖基化改造的酶促反应体系对细胞本身会有影响,但外泌体却不受此影响,此外,MSC来源的外泌体继承了MSC本身的优势的同时又有其独特的优势,如体积小,不容易被肝肺巨噬细胞吞噬影响生物利用度,因此,相比MSC来说更适合作为药物载体。
(2)本发明首次通过两步法改造以实现MSC外泌体作为载体的靶向性和归巢能力同时增强;本研究两步法先后顺序必须为先进行靶向性改造,再进行归巢能力的改造。
(3)本发明新型改造的间充质干细胞外泌体载体,通过装载miR-34c-5p归巢至骨髓靶向清除白血病干细胞,靶向治疗AML小鼠,有望实现临床转化。
附图说明
图1:本发明的外泌体改造及应用的示意图。
图2:脐带来源的间充质干细胞的鉴定及过表达IL3的MSC的构建。其中A为脐带培养后得到的MSC在光学显微镜下的形态;B为MSC的成脂分化和成骨分化;C为MSC的表面分子的鉴定;以上结果证实本体系下成功培养并鉴定了MSC。D为转染Lamp2b-IL3-eGFP慢病毒后转染效果的流式鉴定及E为PCR鉴定结果,说明本体系下成功构建了过表达IL3的MSC。
图3:为过表达IL3的MSC外泌体靶向性的鉴定。其中A和B分别为利用慢病毒改造前后的MSC外泌体与KG1a细胞共培养后荧光显微镜检测外泌体进入KG1a的比率及流式检测的KG1a细胞的Dil的平均荧光强度,提示改造后的MSC外泌体与白血病干细胞的结合水平更高。
图4:岩藻糖基化改造外泌体促进骨髓归巢能力。其中:A为分别用FTV和FTVII岩藻糖基化处理MSC后流式鉴定CD162的表达情况;说明与FTV处理组相比,FTVII具有更好的糖基化效果;B为动物实验验证岩藻糖基化改造后的外泌体的骨髓归巢能力增强。以上结果证实利用FTVII酶促反应体系下成功改造IL3过表达的MSC外泌体,使其骨髓归巢能力增强。
图5:动物实验鉴定改造后的MSC外泌体装载miR-34c-5p靶向清除骨髓白血病干细胞。其中:A为动物实验流程图;B为验证NC组、miR-34c-5p组、改造前的MSC外泌体装载miR-34c-5p的作用组与改造后的MSC外泌体装载miR-34c-5p的作用组,比较四个不同处理组下AML小鼠的生存期;C为四个不同组AML小鼠脾脏的大小;D为比较四组不同处理的AML小鼠一次移植和二次移植后骨髓白血病干细胞的负荷。
图6为过表达载体GM-9646示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
(1)改造MSC使其来源的外泌体膜表达白血病干细胞表面特异性分子CD123的受体(IL3)
与脐带血及骨髓液相比,选择脐带作为间充质干细胞的来源最佳,因为脐带获取途径方便,涉及的伦理问题更少,扩增能力更强,更适合大规模的生产。因此,本课题通过收集脐带,处理成组织小块后进行培养,大量扩增得到P3-P8代的MSC,通过构建带有增强型绿色荧光蛋白(eGFP)、外泌体膜蛋白lamp2b及白血病干细胞膜表面特异性分子CD123受体(IL3)这三种基因的融合质粒,包装成慢病毒后转导至MSC中,使MSC分泌的外泌体膜表达IL3分子。
(2)岩藻糖基化修饰使eGFP-lamp2b-IL3携带MSC来源的外泌体表面CD44分子转化为HCELL分子以促进其骨髓归巢
以eGFP-lamp2b-IL3的MSC来源的外泌体为研究对象,分别利用α-1,3-岩藻糖基转移酶(V、VII)以及相应底物的酶促体系将CD44分子转化成HCELL,比较糖基化处理组与未处理组的外泌体表面CD162表达并通过动物实验验证骨髓归巢能力。
(3)利用改造后的外泌体装载miR-34c-5p靶向清除白血病干细胞
收集改造后的MSC外泌体,通过电转染途径将miR-34c-5p转染至改造后的MSC外泌体内,动物实验验证改造后的外泌体装载miR-34c-5p对AML模型小鼠生存期及小鼠体内白血病细胞干细胞的影响。
图1为本发明外泌体改造及应用的示意图,由图1可知,通过构建含Lamp2b-IL3-GFP融合基因的慢病毒,转染至MSC,使其分泌的外泌体表达IL3,可特异性靶向定位于白血病干细胞表面的CD123分子;在IL3阳性外泌体的基础上,进一步通过岩藻糖计划处理外泌体表面CD44分子,使其转换为归巢能力更强的HCELL分子,最后通过电转染将miR-34c-5p装载至改造后的外泌体使其归巢至骨髓靶向作用于白血病干细胞。
本发明改造间充质干细胞来源的外泌体增强其向骨髓归巢并靶向白血病干细胞的能力,具体涉及到的关键技术如下:
(1)第一步改造:获取脐带并处理成组织小块进行体外培养得到P3代的MSC,为提高MSC外泌体的靶向性,选择LSC特异性标志CD123作为靶标,CD123的受体为IL3,而Lamp2b是外泌体膜表面表达的特异性标记。构建Lamp2b-IL3-eGFP慢病毒并转染至MSC内,抗性筛选得到纯化的Lamp2b-IL3-eGFP阳性的MSC,收集培养上清分选外泌体并鉴定IL3在外泌体膜上的表达情况。
其中,Lamp2b-IL3-eGFP质粒构建的引物序列为:
其中,Lamp2b-IL3-eGFP过表达载体的序列为SEQ ID NO:1所示。
(2)第二步改造:在第一步改造的外泌体的基础上,进行进一步修饰以增强其骨髓归巢能力。CD44分子高表达于MSC外泌体膜上,通过a-1,3-岩藻糖转移酶VII体系处理权利1中改造的外泌体使CD44转化为骨髓定向归巢能力更强的HCELL分子。
其中岩藻糖基化反应选择a-1,3-岩藻糖转移酶VII。
(3)第三步改造:将miR-34c-5p装载到第二步改造的外泌体中,通过电转染的途径,转染程序为X-01。
实施例1:MSC分离、体外扩增及鉴定
(1)人脐带间充质干细胞分离及体外扩增(组织块贴壁法)
①准备一支装有适量含2%FBS+双抗的无菌PBS溶液的50ml离心管,无菌操作剪取健康的足月剖宫产新生儿脐带(近胎儿端),快速浸泡于含2%FBS+双的无菌PBS溶液中;
②转移至无菌超净台中,取出脐带标本,浸泡于装有2%FBS双抗PBS溶液的培养平皿中,用手术剪将脐带剪成约2cm长的小段,并用器械挤压出脐动/静脉内残留的脐血,反复冲洗,直至无血液及其它杂质;
③纵向剖开脐带组织块,剥离3条血管,之后用眼科剪将脐带组织剪成肉糜(约1mm3的组织小块,剪碎过程注意适当加含15%FBS+DMEM/F12+双抗的培养基保持湿润,但完全培养基不能太多,否则不易剪碎)。
④将肉糜状的脐带组织小块均匀铺与10cm培养皿底中,间隔1cm,倒置培养皿,放入37.0℃、5%CO2培养箱中;
⑤30min后,见组织块已经粘附在培养皿底部,将培养皿反转过来,从一侧轻轻加入适量(约6ml)的DMEM/F12完全培养基,要让培养基逐渐蔓延整个培养体系,刚刚浸没组织块一半即可,置于37.0℃,5%CO2的饱和湿度培养箱内静置培养;
⑥3天后观察有无污染,并适当补加培养基;1周后换液,去除漂浮组织块,其后每3天换液一次,观察组织小块周围梭形细胞的爬出和汇合情况;
⑦待贴壁细胞生长至80%-90%汇合后,加入1.5mL胰酶(10cm培养平皿)消化1min后,在倒置显微镜下观察,见细胞变形呈圆形后,立即加入完全培养基3mL终止消化。用移液管轻轻吹打培养瓶底部,镜下观察见细胞基本脱落后,吸出液体,70um滤网过滤去除组织块后,于室温下1000rpm/min离心5min,取沉淀,即为原代细胞。
⑧细胞传代:吸弃培养基,PBS缓冲液漂洗1遍,胰酶消化1min后(1.5ml胰酶→10cm培养平皿),加入2倍于胰酶的完全并于室温下1000rpm/min离心5min,弃上清,所得沉淀即细胞,将细胞再悬浮于培养液中,按5×104/ml再次接种传代至新的10cm培养平皿中,以后每3-4天换液一次(1:5传);第3-8代细胞用于后续实验。
(2)MSC鉴定
1)形态学观察:使用倒置显微镜观察脐带来源的MSCs的生长及形态变化。细胞呈长梭形,漩涡状生长。
2)流式检测MSCs表面标志:
收培养到P3代的MSC细胞,标记流式抗体(FITC-CD105、BV421-CD73、APC-CD90、PE-cy7 CD34、PE-CD45)进行流式细胞仪检测。
3)鉴定MSC成骨分化
成骨培养基:DMEM/F12、10mmol/Lβ-磷酸甘油钠、0.1umol/L地塞米松、50ug/mL维生素C、10% FBS
①消化收集P3/4代脐带MSCs,以4x104/孔接种于6孔板内。待细胞长到60%时,弃去培养液,用无菌PBS洗3遍;
②加入成骨培养基继续培养,并设置加完全培养基组为对照组,每隔三天进行半量换液;
③培养一段时间(14-21天),镜下观察有明显钙盐形成时,吸走培养液,加入PBS漂洗3次;再以4%多聚甲醛固定30min,用ddH2O润洗细胞3次,每次5min;加入茜素红染色20min,吸去染色液,ddH2O润洗3次后,加入0.1mlddH2O,显微镜下观察并采集图像。
4)鉴定MSC成脂分化
成脂培养基:DMEM/F12、0.5mmol/L IBMX(3-异丁基-1-甲基黄嘌呤)、10% FBS、0.2mmol/L吲哚美辛、10mg/L胰岛素、1umol/L地塞米松1%青-链霉素;油红O染液:称取0.21g油红O,溶于120ml异丙醇,室温静置过夜,过滤后加入90ml蒸馏水充分混匀,过滤两次后室温保存;
①消化收集P3/4代BMMSCs,细胞计数,以4×104/孔接种于6孔板内,使用MSC培养基(含10%FBS的DMEM/F12完全培养基);
②待细胞融合到80%后,弃去培养液。用无菌PBS清洗培养皿3次,加入成脂诱导培养基,每3天换液一次;
③成脂诱导第14天吸弃诱导培养液,PBS冲洗2-3次,4%甲醛固定30min;
④ddH2O清洗3次,加入0.2%油红O染液,37℃染色30分钟。
⑤弃去染色液,70%异丙醇数秒脱色,PBS洗3次。显微镜观察并拍照。
图2为脐带来源的间充质干细胞的鉴定及过表达IL3的MSC的构建。取健康足月剖宫产儿的脐带,组织块贴壁培养10天(A左)可见梭形贴壁细胞从组织块周围“爬出”,培养20-22天,长梭形细胞融合度达80%~90%,呈旋涡状生长(A右);体外诱导P3代hUC-MSCs细胞向成脂分化(B左)和成骨分化(B右)的能力;流式细胞术检测P3代hUC-MSCs细胞的免疫表型(C)。hUC-MSCs细胞转染慢病毒空载体及表达Lamp2b-IL3慢病毒载体,72h后收集细胞进行流式分析GFP阳性细胞的百分比(D),qPCR检测hUC-MSCs细胞内IL3 mRNA的表达水平(E)。p<0.05表示两组间差异有统计学意义,****p<0.0001
图3为过表达IL3的MSC外泌体靶向性的鉴定。由图3可知,浓度15ug/ml CTRL-MSC细胞来源的外泌体(CTRL-exo)和IL3OE-MSC细胞来源的外泌体(IL3OE-exo)与KG1a细胞共培养,并设置PBS作为对照,激光共聚焦显微镜观察外泌体(红色)被KG1a细胞(蓝色)摄取情况;红色荧光为Dil染料标记的外泌体,蓝色荧光为Hoechest标记的细胞核;(A)流式细胞术检测KG1a细胞内Dil的平均荧光强度;及对不同组KG1a细胞内Dil荧光的MFI统计分析(B)。
实施例2:Lamp2b-IL3-eGFP慢病毒的构建
(1)总流程为:设计引物将目的基因片段从原质粒上扩增下来,再通过引物两端所含无缝克隆识别位点将其重组到酶切后的过表达载体上;将连接产物转入制备好的细菌感受态细胞,对长出的单克隆菌落进行测序鉴定,比对正确的克隆即为构建成功的过表达质粒。
(2)实验步骤如下:
1)所用载体如图6所示。构建含嘌呤霉素抗性、绿色荧光蛋白的慢病毒空载体和表达融合蛋白Lamp2b-IL3的重组慢病毒载体,其中载体信息为(PGMLV-CMV-MCS-6×His-EF1-ZsGreen1-T2A-Puro)。
2)设计PCR扩增片段引物,并在引物5’端引入线性化克隆载体末端的同源序列,使得扩增产物5’和3’最末端序列分别和线性化克隆载体两末端序列完全一致。
3)将含载体质粒的菌液过夜培养,并取新鲜菌液3-5ml提取质粒。具体方法参考QIAGEN质粒小抽说明书。
4)取1μg新鲜质粒,用相应的限制性内切酶进行双酶切,37℃酶切3h。酶切体系如下:
载体 | 1μg |
green Buffer | 3μL |
XhoI | 1.5μL |
MluI | 1.5μL |
ddH2O | 补足30μL |
5)将酶切产物进行琼脂糖凝胶电泳,电泳结束后,进行胶回收,步骤如下:在紫外灯下,切下包含目的片段的胶条。用天平称量总重量并减去空管的重量算出凝胶的重量,按100mg=100μL来计算凝胶的体积,并加入1倍凝胶体积的Binging Solution置于65℃水浴锅内将凝胶彻底融化。期间适当摇晃EP管,加快凝胶的溶解。
6)将上述液体全部转移到滤柱内,13000转离心30S(可重复一次)。然后弃掉管内液体,向柱内加入500μL的WA Solution,13000转离心30S。弃掉管内液体,再向柱内加入500μL的Wash Solution,13000转离心30S(可重复一次)。然后空离3min。将滤柱放置在一个新的1.5mL EP管中,室温晾干。最后在柱子内加入35μL的ddH2O,静置5min,13000转离心1.5min。为了提高回收率,可将溶解的DNA再次加入柱子内离心一分钟。弃掉柱子,即为回收的载体片段,并测定浓度。
7)目的片段的扩增
将合成的引物稀释成终浓度为10μmol/L的储藏液。利用稀释的引物及模板进行PCR扩增。体系如下:
片段一
模板 | 1-2μg |
Primer-F | 2μL |
Primer-R | 2μL |
PCR mix | 25μL |
ddH2O | 补足50μL |
片段二
模板 | 1-2μg |
Primer-F | 2μL |
Primer-R | 2μL |
PCR mix | 25μL |
ddH2O | 补足50μL |
将上述材料加入薄壁管内混匀并点离后放入PCR仪内,选择好合适的退火温度和延伸温度,即可开始PCR扩增。PCR结束后进行琼脂糖凝胶电泳,并回收目的基因。
8)过表达载体与目的片段的连接(无缝克隆)
①测定回收载体和目的片段的浓度
②HieffCloneTM重组反应体系最适克隆载体使用量为0.03pmol;最适克隆载体与插入片段摩尔比为1∶2,即最适插入片段使用量为0.06pmol。这些摩尔数对应的DNA质量可由以下公式计算获得:最适克隆载体使用量=[0.02×克隆载体碱基对数]ng(0.03pmol)最适插入片段使用量=[0.04×插入片段碱基对数]ng(0.06pmol)
③过表达载体与目的片段的连接。连接体系如下:
于50℃作用20min;
9)转化
①将感受态细胞置于冰上(4℃)待其自然解冻后,取10μl连接产物加入感受态细胞中于冰上(4℃)放置30min。
②之后于42℃水浴中热击90S。然后迅速置于冰上(4℃)放置2-3min。
③加入500μL不含抗生素的SOC培养基于37℃,225rpm振荡培养45min。3000转离心2min,弃掉900μL的上清液,将管底的菌液吹打散开,加入到含有载体上对应抗性(氨苄或卡那等)的培养平板中,用灭菌的涂布器涂匀(涂布器的温度不能太高,以免烫死菌体),倒置于37℃恒温培养箱内过夜培养。
10)测序和比对
每个克隆挑选两个送测序;经过比对,重组克隆中插入片段序列与目的片段序列完全一致,因此质粒构建成功。
11)过表达载体的验证:用构建好的过表达载体转染工具细胞293,利用WesternBlot验证过表达效果。
12)慢病毒包装
抽提高纯度、无内毒素的慢病毒载体及其辅助包装原件载体质粒,使用HGtransgene reagent将构建好的慢病毒载体及其辅助包装原件载体质粒共转染进293T细胞,转染10~12h后加Enhancing buffer,接着8h后更换新鲜培养基,继续培养48h后,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。在一定滴度范围内的慢病毒颗粒可以满足大部分体内、体外实验需求。
实施例3:慢病毒转染及稳转株构建
①取P3代脐带MSC,待其融合度达80%-90%后换液,保证细胞处于最佳状态,并于次日消化,用完全培养基制备密度为2×104个/ml的细胞悬液,取2ml/孔种板于6孔板中(10cm培养皿→10ml,T25→5ml)
②吸取适量病毒原液如下所示,分别加到对应组的MSC细胞中,并标记;
(病毒使用原浓度;Polybrene稀释浓度为1ug/ul,终浓度5ug/ml;)
③感染48h后(感染后4h、8h、12h对细胞进行观察,如细胞形态发生变化,则立即进行细胞换液,注意保持细胞正常生长),荧光显微镜下观察绿色荧光表达情况,并更换为含有2ug/ml嘌呤霉素的完全培养基,进行筛选。
④每3天更换一次含purocymin的培养液,直至未感染病毒的对照组细胞全部被puromycin杀光,而感染病毒组的再无细胞出现死亡。
⑤此后用新鲜培养基取代含有嘌呤霉素的培养基,继续培养(或将puromycin浓度减至维持浓度,筛选浓度的1/2或1/4,继续培养);
⑥待细胞长起来并达到最佳状态后,流式细胞术检测GFP阳性细胞百分比(GFP+%)。
⑦继续对感染后的细胞进行扩增,同时收集细胞进行qPCR或WB鉴定,并将鉴定结果正常的细胞冻存保种。
实施例4:改造的MSC来源的外泌体提取
①无外泌体血清制备:无菌条件下,将血清分装至环氧乙烷消毒后的超离管内逐一配平,精确至小数点后两位,4℃,120000g,超离过夜;将超离后的血清转移至洁净50ml离心管内,避免吸入管壁的外泌体沉淀,全程应注意无菌操作,储存于4℃冰箱备用。
②5%无外泌体FBS-DMEM/F12细胞培养基:无菌条件下,将体积比为5%的无外泌体FBS和终浓度为100U/ml的青/链霉素加入相应体积的DMEM/F12中,混匀后密封,4℃保存。
③消化传代,取P3代,常规培养,在细胞融合度达70%后,吸弃培养基,并用PBS洗涤2遍,更换为5%无外泌体FBS-DMEM/F12细胞培养基;
④48h后收集细胞上清;(细胞上清可以保存在-80℃备用,提取外泌体前一天放4℃冰箱溶解),将上清液移入超速离心管中,用PBS补齐配平;
⑤4℃,2000g/min离心20min,弃沉淀(去除死细胞及碎片);
⑥将上述分离得到的上清用PBS补齐配平,4℃,10000g/min离心,30min,弃沉淀;再用0.22um滤器过滤进一步去除细胞碎片及微泡;
⑦将上述分离所得的上清用PBS补齐配平,4℃,110000g/min离心70min,弃上清;沉淀用PBS洗一遍,收集沉淀即为外泌体,用适量PBS重悬进行后续实验(或分装放﹣80℃冻存);
⑧透射电镜下观察外泌体形态(送公司检测):透射电镜下可见一群大小具有较明显异质性,直径介于40-150nm,呈圆形或椭圆形的膜性小囊泡。
⑨纳米流式检测(送公司检测):明确外泌体粒径。
⑩提取外泌体蛋白步骤如下:取50μL外泌体样本加入50μL的蛋白裂解液(RIPA:PMSF=100ul:1ul);同时制备一份细胞蛋白裂解液作为对照。充分混匀后冰上裂解40min,每10min涡旋震荡3次;4℃下12,000g/min离心25min;吸取蛋白上清至新的EP管中,吸取适量样品进行BCA法检测蛋白浓度及进行后续实验,或直接冻于-80℃冰箱保存。
实施例5:岩藻糖基化IL3-MSCs及其来源的外泌体
(1)储存溶剂配置:
①α-1.3-岩藻糖基化转移酶(FT)V、VII:浓度为0.9ug/ul,共25ul(使用浓度50ug/ml)
②GDP-岩藻糖(1mg,分子量633.31):将2mg试剂溶于200ul HBSS中,即可获得浓度16mmol/L,分装每支8ul;(工作浓度为1mmol/L)
③HSA(500mg):将500mgHSA溶于10ml HBSS中,即可获得5%浓度,分装成0.5ml/支,50ul/支;(工作浓度为1%)
④BSA:已配置浓度2%;工作浓度为0.2%;
⑤HEPES(238.3g/mol):溶液为1mol/L;
(2)MSC岩藻糖基化反应
1)准备岩藻糖基化反应体系:将α-1,3-岩藻糖转移酶V、VII以50ug/ml溶于200ul含有20mM HEPES、1%HSA及1mM GDP-岩藻酸的HBSS,置于37℃,5% CO2培养箱中预热待用。
2)将融合至90%的IL3OE-MSCs用0.25%胰酶消化,并转入15ml离心管中。
3)4℃以1000rpm离心5分钟,弃上清,加入HBSS清洗2次。
4)离心后后取沉淀,重悬于提前准备好的岩藻糖基化反应体系,置入37℃,5%CO2培育箱中反应60min,每15min轻柔混合一次。
5)用含100mM HEPES、2% BSA的HBSS溶液按上述方法再次清洗。
6)4℃以1000rpm离心5分钟后取沉淀,重悬于2% FBS/PBS中备用。
7)岩藻糖基化的验证:流式检测及Western blot验证CD162的表达
(3)岩藻糖基化糖基化改造外泌体膜表面CD44分子(反应体系100ul):
1)准备岩藻糖基化反应体系(100ul):将α-1,3-岩藻糖基转移酶VII(FTVII)以50ug/ml溶于200ul含有20mM HEPES、1% HSA及1mM GDP-岩藻酸的HBSS,置于37℃,5%CO2培养基中预热待用。
2)将300ml IL3OE-MSC细胞培养上清提取好的外泌体重悬于岩藻糖基化反应体系,置入37℃培养箱中反应60min,每隔15min轻柔混匀一次;
3)反应结束后,先加含100mM HEPES、2%BSA的HBSS溶液终止反应10min;再用PBS补齐;
4)4℃,110 000g/min,70min,取沉淀,重悬于200ulPBS中备用;
(4)收集岩藻糖基化和未岩藻糖基化的外泌体,Western blot验证唾液酸化Lewisx蛋白CD162)的表达;
(5)流式检测外泌体表面CD162的表达
取30ug外泌体与2.5ul微珠在100ul体系中室温孵育15min;用PBS补至1ml,于摇床上室温孵育2h;加入终浓度100mM的甘氨酸,室温孵育30min以终止反应;用2%的BSA的溶液清洗吸附外泌体的微球2遍,3000g,10min,沉淀微球;用PBS重悬吸附的微球,与PE-CD162抗体于4℃共孵育30min;PBS清洗,3000g,10min,两遍;流式细胞术检测。
HCELL是CD44蛋白的唾液岩藻糖苷糖型,HCELL和CD44之间的区别在于前者有sLeX基团,sLeX与选择素结合在介导骨髓归巢中起决定性作用。
实施例6:动物实验验证改造后外泌体骨髓归巢能力增强
①选择6周雌性NCG小鼠,于隔离包适应性培养2周,给予1.16g/L新霉素(Biosharp,China)水喂养;
②移植前一天给KG1a细胞换液,移植当天收集细胞,调整细胞浓度为4×10^7/ml,用EP管分装,放置于冰上准备移植(每只小鼠注射8×10^6个Kg1a细胞即200ul细胞悬液);无菌条件下将小鼠转移出隔离器,照射剂量200cGy,照射后24小时内尽快进行移植实验。照射条件如下:SAD:100cm;D=200/min;FS:35cm*35cm;d:1.5cm;GA:0,180°,对穿;D=200cGry;跳数:85,85(正反各85跳);
③照射结束后,尾静脉将细胞悬液一次性打入小鼠体内,每只小鼠移植8×10^6细胞数;分为PBS组,CTRL-exo组,IL3OE-exo组,HCELL-IL3OE-exo组;给予1.16g/L新霉素水继续喂养3周方可换成无抗生素的水喂养,注意密切观察一次小鼠状态(精神、毛色、食欲及小鼠重量等);
④移植后第3周,尾静脉取血检测hCD45%的比例以明确白血病模型是否构建成功;白血病模型构建成功后,根据分组开始尾静脉注射同等量的Dil染色的外泌体(150ugexosome/只),24h后处死小鼠,取骨髓并分离出单个核细胞进行流式检测hCD45+CD34+细胞群体的Dil的平均荧光强度。
图4为岩藻糖基化改造外泌体促进骨髓归巢能力结果图。由图4可知,分别用两种不同的a-1,3-岩藻糖基化转移酶(FT V和VII)处理过表达Lamp2b-IL3的hUC-MSC外泌体进行岩藻糖基化反应后,流式检测CD162阳性细胞的比例及平均荧光强度(A),经过FT VII改造后,外泌体表面CD162表达显著提高;构建小鼠AML模型,每只小鼠根据不同分组注射150ug Dil标记的外泌体,24h后取骨髓细胞,流式分析hCD45+CD34+中Dil阳性细胞比例及MFI,并进行统计学分析(B),说明FT VII改造后的外泌体骨髓归巢能力明显增强
实施例7:动物实验验证改造后的外泌体装载miR-34c-5p靶向清除白血病干细胞
(1)外泌体电转
1)配置电转混合物:exosomes:siRNA=1:1(wt/wt),电转缓冲液使用PBS。
在适量电转缓冲液中加入电转混合物,外泌体终浓度不超过0.5ug/ul。
2)电穿孔前将电穿孔比色皿在冰上预冷30min。
3)取2支无菌EP管,并做好标miR-34c-5p+exo或miR-34c-5p+HCELL-IL3OE-exo。
4)充分混匀外泌体悬液,分别加入1640至100μL。(尽量减少气泡产生,以免影响电转效果)。
5)将混合物转至电转杯内,选择程序X-01,电转完成后,室温静置10min。
6)用无菌微型吸管将液体吸出,并加PBS至4ml;使用超速离心法回收外泌体。
miR-34c-5p小分子的序列具体为:AAUCACUAACCACACGGCCAGG
(2)动物试验部分
①同上方法进行AML模型小鼠构建;
②移植结束后第4周,根据分组开始尾静脉注射同等量的外泌体,每周2次,连续2周;
③移植后第3W开始尾静脉取血,流式检测小鼠外周血hCD45+%,每周尾静脉取血检测人源细胞嵌合率;移植第7周(根据小鼠具体情况来定)处死小鼠,取外周血、骨髓、肝、脾细胞,一部分用于流式检测hCD45+、hCD45+hCD33+、hCD45+hCD34+或hCD34+hCD38-细胞比例;另一部分用于二次移植的细胞;
④二次移植:
将一次移植的每组同等数量的CD34+细胞移植到照射后的小鼠体内;于移植第7-8周,处死小鼠,取小鼠外周血及骨髓进行检测,检测指标同一次移植实验。
图5为动物试验鉴定改造后的MSC外泌体装载miR-34c-5p靶向清除骨髓白血病干细胞结果图。为进一步证实改造后的外泌体装载miR-34c-5p靶向清除AML干细胞的能力,我们先构建AML小鼠模型,随后尾静脉注射改造或未改造的外泌体装载的miR-34c-5p,并进行二次移植,A为动物实验流程图;B图可见miR-34c-5p+HCELL-IL3OE-exo组小鼠较其它组生存期明显延长;同时miR-34c-5p+HCELL-IL3OE-exo组小鼠相对其他组脾脏明显缩小(C);miR-34c-5p+HCELL-IL3OE-exo组一次移植小鼠骨髓hCD45+CD34+及二次移植小鼠骨髓hCD34+CD38-细胞百分比较其他组显著下降,说明改造后的外泌体装载miR-34c-5p显著抑制小鼠AML的发生发展及促进小鼠白血病干细胞的清除。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种间充质干细胞外泌体的改造方法,其特征在于,包括以下步骤:
(1)构建带有外泌体膜蛋白lamp2b基因、白血病干细胞膜表面特异性分子CD123受体IL3基因及增强型绿色荧光蛋白eGFP基因的融合质粒,将该融合质粒包装成慢病毒后转导至间充质干细胞中,使间充质干细胞分泌的外泌体膜表达IL3分子;
(2)收集步骤(1)得到的外泌体,并进行岩藻糖基化,使所述外泌体的膜表面CD44蛋白进行岩藻糖基化修饰,即实现间充质干细胞外泌体的改造。
2.如权利要求1所述间充质干细胞外泌体的改造方法,其特征在于,步骤(2)中,所述岩藻糖基化为将步骤(1)得到的外泌体与岩藻糖基化酶促反应体系进行反应。
3.如权利要求2所述间充质干细胞外泌体的改造方法,其特征在于,所述岩藻糖基化酶促反应体系含有a-1,3-岩藻糖转移酶。
4.如权利要求3所述间充质干细胞外泌体的改造方法,其特征在于,所述a-1,3-岩藻糖转移酶为a-1,3-岩藻糖转移酶VII。
5.如权利要求1所述间充质干细胞外泌体的改造方法,其特征在于,步骤(1)中,所述融合质粒的序列为SEQ ID NO:1所示。
6.如权利要求1所述间充质干细胞外泌体的改造方法,其特征在于,步骤(1)中,所述间充质干细胞为脐带经体外培养获得的第3-8代传代细胞。
7.如权利要求1-6任意一项方法得到的改造后的间充质干细胞外泌体。
8.如权利要求7所述的改造后的间充质干细胞外泌体用于制备靶向清除骨髓白血病干细胞药物载体的应用。
9.如权利要求8所述的应用,其特征在于,所述应用具体为:将miR-34c-5p小分子转染入所述改造后的间充质干细胞外泌体中。
10.如权利要求9所述的应用,其特征在于,所述转染为电转染。
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