CN109846904B - 间充质干细胞外泌体在制备促进线粒体自噬制剂中的应用 - Google Patents
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Abstract
本发明提供间充质干细胞外泌体在制备促进线粒体自噬制剂中的应用。通过制备脂肪间充质干细胞来源的外泌体,在细胞水平通过Western Blot,流式等实验明确AMSC‑Exo对实质细胞和免疫细胞线粒体自噬的调控作用;同时通过体内实验明确AMSC‑Exo对肝组织中线粒体自噬通路的影响;并进一步制备促线粒体自噬的生物制剂或药物。同时通过对AMSC‑Exo的修饰,以进一步提高其促线粒体自噬效能。本发明能促进线粒体自噬,纠正线粒体功能障碍;对实质细胞和免疫细胞均具有促线粒体自噬的作用;可用于制备治疗线粒体功能障碍相关疾病的制剂或药物;经特定修饰后,可进一步提高其促线粒体自噬能力。
Description
技术领域
本发明属于生物技术领域,涉及间充质干细胞来源外泌体的新应用,具体涉及一种脂肪来源间充质干细胞(AMSC)外泌体在制备促进线粒体自噬制剂中的应用。
背景技术
线粒体是细胞的能量工厂,健康的线粒体就像是一个动力工厂,通过三羧酸循环和氧化磷酸化来实现能量转换。线粒体功能可决定细胞的生死存亡,一方面其通过氧化磷酸化产生ATP提供细胞的能量需求,并通过钙离子通道将细胞质中的钙离子转运至线粒体储存,使细胞能够维持正常的功能;另一方面线粒体胞内活性氧(ROS)的异常累积以及产生凋亡相关蛋白,如细胞色素C和凋亡诱导因子,会导致细胞受损并诱导细胞死亡。此外,线粒体拥有单独的基因编码系统,因老化或者受损等因素引起的线粒体DNA突变由于线粒体基因构成的特殊性而不易修复。考虑到这些关键因素,损伤的线粒体会对细胞造成伤害,功能障碍的线粒体的积累会导致多种类型疾病,包括心脏衰竭,阿尔茨海默病,帕金森病和癌症。
线粒体自噬(mitophagy)是细胞选择性清除损伤或衰老的线粒体,并循环利用其组成元素的过程。线粒体自噬可使细胞内线粒体的质量和数量保持平衡,因而对于维持细胞稳态及细胞生存具有重要意义。研究表明,线粒体自噬在不同致病因素、不同细胞中都具有明显保护作用,其缺陷及功能障碍与衰老、神经退行性疾病和癌症等诸多生理病理过程密切相关。近来,越来越多的研究显示线粒体自噬也参与了多种肝脏疾病的发生发展,在非酒精性脂肪肝、肝纤维化/肝硬化、肝癌和肝缺血再灌注损伤中均存在线粒体自噬障碍。
PINK1和PARKIN是线粒体自噬的重要调控分子。Parkin可选择性聚集在线粒体基因突变的线粒体上,并将其清除。PINK1和PARKIN与维持线粒体网络的完整和细胞功能的正常具有密切联系,其编码基因的突变会导致受损线粒体的聚集,进而导致细胞损伤或恶变。因此,上调PINK1和PARKIN介导的线粒体自噬通路可作为去除受损线粒体的潜在治疗策略,有望为多种因素所致的肝脏损伤,以及线粒体自噬障碍相关的疾病治疗提供新的思路和选择。
发明内容
本发明的目的是要提供一种间充质干细胞(AMSC)外泌体在制备促进线粒体自噬制剂中的应用,所述间充质干细胞(AMSC)外泌体来源于脂肪,通过以下步骤实现:
1.脂肪来源间充质干细胞(AMSC)的制备:
小鼠AMSC(mAMSC)和成人AMSC(hAMSC)按常规方法分离,mAMSC用含5%胎牛血清(MSC适宜胎牛血清)的低糖DMEM培养基(Gibco)培养,hAMSC采用含2%MSC培养无血清替代物(EliteCell,EPA-050)的MSC培养基进行培养。AMSCs增殖接近80%融合时,进行消化、传代,3~8代细胞用于后续实验;
2.AMSC-Exo分离:
上述细胞在无血清MSC培养基中培养48小时,收集上清并离心(4℃,300g,10分钟);取上清后再次离心(4℃,2000g,10分钟);再次取上清进行离心(4℃,10,000g,30分钟),弃沉淀,将上清于100,000g离心70分钟,收集沉淀用PBS重悬后,再次离心(100,000g,70分钟),所得沉淀即为脂肪来源间充质干细胞外泌体(AMSC-Exo);
3.AMSC-Exo鉴定:
通过NTA法(Nanoparticle Tracking Analysis)分析其粒径,范围在64.5-143.8nm。并采用Western Blot或磁珠法(流式细胞术)检测其表面的外泌体标志物CD63、CD9和CD81。
本发明提供的AMSC-Exo制备的促线粒体自噬的生物制剂,该制剂含有有效剂量的AMSC-Exo,将该制剂施用于作用对象可有效促进线粒体自噬,纠正线粒体功能障碍。可用于线粒体功能障碍相关疾病的治疗。
本发明所述线粒体自噬,涉及的相关疾病包括但不限于急慢性肝损伤、肝炎(包括病毒性肝炎、药物性肝炎、脂肪性肝炎、酒精性肝炎和自身免疫性肝炎)、肝衰竭、肝纤维化/肝硬化、肝硬化等肝脏疾病。所述方法可以是体内的。也可以体外的,例如仅用于科学研究。
本发明的另一个目的是提供提高AMSC-Exo促线粒体自噬效能的制备方法(一种是通过在AMSC-Exo的基础上直接电转法荷载特定核酸;一种是通过基因修饰AMSC,使其分泌的AMSC-Exo带有特定核酸),通过如下步骤实现:
1.构建及制备SEQ:NO.1荷载的AMSC-Exo:
(1)采用前述制备AMSC-Exo的方法制备AMSC-Exo(包括AMSC制备,AMSC-Exo分离);
(2)采用电转法导入SEQ:NO.1核酸片段:
SEQ:NO.1:GCAGGCGCCCAAUUAAUGUCUGUUGAUGAUU
将AMSC-Exo和SEQ:NO.1核酸在4℃的电穿孔缓冲液中轻轻混合。以0.4cm的电穿孔试管,在350V和150μF的电压下采用基因脉冲II电转仪(Bio-Rad,USA)进行电穿孔。将混合物在37℃下放置30分钟,以确保AMSC-Exo膜完全恢复。荷载后的AMSC-Exo以预冷的PBS清洗2次(120,000×g超速离心90分钟)以去除未载入的多余核酸序列。
2.构建及制备XIST(X非活性特异性转录物)片段修饰的AMSC-Exo:
(1)构建XIST修饰的AMSC:
XIST慢病毒表达质粒pLVX-IRES-Zsgreen1-XIST感染AMSC。表达质粒包含部分XIST目的片段序列如SEQ:NO.2所示,其特征为对特定ceRNA功能区进行选择性拼接后的片段。
SEQ:NO.2
ggatgacctctagtcaagacctttgcactaggatagttaatgtgaaccatggcaactgatcacaacaatgtctttcagatcagatccattttatcctccttgttttacagcaagggatattaattacctatgttacctttccctgggactatgaatgtgcaaaattccaatgttcatggtctctccctttaaacctatattctaccccttttacattatagaaagggatgctggaaacccagagtccttctcttgggactcttaatgtgtatttctaattatccatgactcttaatgtgcctgtgtaatagacatgaaagcctcccctgccacaccccacctccaatcttcctttcccttccaccagggagtgtccactccatatacccttacatttggacaatcaaggtgcacaattgtaagtgagcataggcactcaccttggacatgaatgtgcataactgcacatggcccatcccatctgaataaggtcctactctcagaccctttttgcagtacagcaggggtgctgatcaccaaggccccttttcctggcctgttatgtgtgtgattatatttgttccagttcctgtgtaatagaaaacaaccaccacacgtcaagctcttcattgttcctatctgccaaatcattatacttcctacaagcagtgcagagagctgagtcttcagcaggtccaagaaatttgaacacactgaaggaagtcagccttcccacctgaagatcaacatgcctggcactctagcacttgaggatagctgaatgaatgtgtatttctttgtctctttctttcttgtctttgctctttgttctctatctaaagtgtgtcttacccatttccatgtttctcttgctaatttctttcgtgtgtgcctttgcctcattttctctttttgttcacaagagtggtctgtgtcttgtcttagacatatctctcatttctgctgttcatagcttaccaccccctgatgtattatttgttattcagagaaaatttctgaatactactagtttccttttctgtgcctgtccctgtgctaggcactaaaaatgcaatgattattgatatctaggtgacctgaaaaaaaatagtgaatgtgctttgtaaactgtaaagcacttgtattctactgtgataagcgttgtggatacaaagaaaggagcaagcataaaaaagtgctctttcaaaaggatatagtactatgcagacacaaggaattgtttgataaatgaataaatta。
AMSC细胞铺6孔板,完全培养基培养过夜,当融合度达到50%后备用。随后加入200μl OPTI-MEM培养基、50μl感染增强液和10μl XIST慢病毒(滴度为108TU/ml,MOI值=10),混匀后另加入1.75ml的完全培养基并一起加入到已贴壁的AMSCs,培养8-12h观察细胞状态,如果细胞状态出现不好则立即换液,如果未产生变化则继续培养。24h后换成含5μg/mlpolybrene(聚凝胺)的完全培养基筛选培养3-4天后荧光显微镜观察ZsGreen1荧光情况,当荧光率达到96%以上后则可撤除聚凝胺。所述感染增强液选用HitransG感染增强液。
(2)AMSC-Exo-XIST制备及鉴定:
上述细胞在无血清MSC培养基中培养48小时,收集上清并按上述超速离心法分离制备AMSC-Exo-XIST,并通过NTA法分析其粒径,采用Western Blot法检测其外泌体标志物。
(3)采用荧光定量PCR法鉴定其中XIST的表达:
采用ncRNA试剂盒抽提AMSC-Exo-XIST的RNA,然后进行逆转录。逆转录体系:RNA模板-1μg;200μM随机引物-1μl;25μM Oligo(dT)-1μl;5×逆转录缓冲液-2μl;逆转录酶混合液-2μl;无RNA酶水-补至10μl。逆转录反应程序为:42℃60min,70℃10min。
再进行荧光定量PCR检测,PCR体系为:2×SYBR Green Mix-10μl;XIST上游引物序列如见SEQ:NO.3所示:ggatgacctctagtcaagacctttgca)5μM-0.8μl;XIST下游引物序列如SEQ:NO.4所示:gctatgaacagcagaaatgagaga,5μM-0.8μl;模板-2ul;dd水-补至20μl。PCR条件为:95℃预变性10min,然后95℃5s,60℃30s,72℃30s,40个循环。PCR结果显示:经XIST修饰的AMSC,其XIST的表达水平明显高于对照组。
3.线粒体自噬调控作用比较:
相同剂量的AMSC-Exo-SEQ1、AMSC-Exo-XIST和AMSC-Exo分别作用于肝细胞系和巨噬细胞系,AMSC-Exo-SEQ1或AMSC-Exo-XIST较之AMSC-Exo可更为显著地影响细胞线粒体自噬水平,经特定修饰的AMSC-Exo的有效剂量可低至天然AMSC-Exo的数十倍。
本发明通过制备脂肪间充质干细胞来源的外泌体(AMSC-Exo),在细胞水平通过Western Blot,流式等实验明确AMSC-Exo对实质细胞和免疫细胞线粒体自噬的调控作用;同时通过体内实验明确AMSC-Exo对肝组织中线粒体自噬通路的影响;并进一步制备促线粒体自噬的生物制剂或药物。同时通过对AMSC-Exo的修饰,以进一步提高其促线粒体自噬效能。本发明还公开了AMSC-Exo在促进线粒体自噬,纠正线粒体功能以及治疗相关肝脏疾病中的作用,其具备以下作用中的任一项或多项:AMSC-Exo能(1)促进实质细胞(如肝细胞)线粒体自噬;(2)促进免疫细胞(如巨噬细胞)线粒体自噬;(3)抑制线粒体ROS水平;(4)促进损伤线粒体的清除;(5)纠正线粒体功能障碍;(6)治疗线粒体自噬相关肝脏疾病。
本发明的AMSC来源外泌体的优点有:(1)促进线粒体自噬,纠正线粒体功能障碍;(2)对实质细胞和免疫细胞均具有促线粒体自噬的作用;(3)可用于制备治疗线粒体功能障碍相关疾病的制剂或药物;(4)经特定修饰后,可进一步提高其促线粒体自噬能力。
附图说明
图1:AMSC-Exo上调肝细胞线粒体自噬水平:线粒体自噬相关分子PARKIN和PINK1的Western Blot条带灰度比值显示,AMSC-Exo能显著上调肝细胞系和原代肝细胞的线粒体自噬,并呈剂量依赖性,*P<0.05,**P<0.01,ns表示无统计学差异。
图2:AMSC-Exo上调巨噬细胞线粒体自噬水平:PARKIN和PINK1的Western Blot条带灰度比值显示,AMSC-Exo能显著上调巨噬细胞的线粒体自噬,并呈剂量依赖性,*P<0.05,**P<0.01,ns表示无统计学差异。
图3:AMSC-Exo对肝细胞线粒体损伤的保护作用:MitoSOX Red染色后流式细胞术检测显示AMSC-Exo能降低APAP所诱导的肝细胞线粒体ROS(mtROS)水平;JC-1探针染色后流式细胞术检测显示AMSC-Exo能纠正APAP所诱导的肝细胞线粒体膜电位异常;TOM20蛋白Western Blot条带灰度比值显示AMSC-Exo能清除APAP所导致的损伤线粒体。并且AMSC-Exo对肝细胞线粒体损伤的保护作用呈剂量依赖性,*P<0.05,**P<0.01,ns表示无统计学差异。
图4:AMSC-Exo对肝组织线粒体自噬的调控作用:在APAP诱导的药物性肝损伤小鼠模型中,线粒体自噬相关分子PARKIN和PINK1的Western Blot条带灰度比值显示,AMSC-Exo输注能显著上调肝组织的线粒体自噬水平;同时降低肝组织ROS水平(以DHE探针染色反应);并且AMSC-Exo的作用呈剂量依赖性,*P<0.05,**P<0.01,ns表示无统计学差异。
图5:AMSC-Exo-SEQ1和AMSC-Exo对线粒体自噬的作用比较:同剂量的AMSC-Exo-SEQ1较之AMSC-Exo,能更为显著地上调PARKIN和PINK1的表达;且AMSC-Exo-SEQ1在0.5μg/mL浓度时即有显著作用。*P<0.05,**P<0.01,ns—与Vehicle组相比无统计学差异,a—与Vehicle组相比有统计学差异。
图6:AMSC-Exo-XIST和AMSC-Exo对线粒体自噬的作用比较:同剂量的AMSC-Exo-XIST较之AMSC-Exo,能更为显著地上调PARKIN和PINK1的表达;且AMSC-Exo-XIST在0.5μg/mL浓度时即有显著作用。*P<0.05,**P<0.01,ns—与Vehicle组相比无统计学差异,a—与Vehicle组相比有统计学差异。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1 AMSC-Exo促进肝细胞线粒体自噬
人肝细胞系HL-7702中加入梯度浓度的hAMSC-Exo(5μg/mL、20μg/mL、80μg/mL)处理24h,然后收集细胞,进行细胞线粒体自噬相关分子的Western Blot检测。或小鼠原代肝细胞中加入梯度浓度的mAMSC-Exo(5μg/mL、20μg/mL、80μg/mL),处理24h后收集细胞,进行线粒体自噬相关分子的Western Blot检测。WB条带均采用ChemiScope Western BlotImaging System(Clinx Science Instruments Co.,Ltd)扫描后,再用Image J软件(RawakSoftware,Inc.Germany)进行灰度比值分析。如图1显示,AMSC-Exo能显著促进肝细胞系和原代肝细胞的线粒体自噬,具体表现为剂量依赖性地上调肝细胞中线粒体自噬相关分子PARKIN和PINK1水平。
实施例2 AMSC-Exo促进巨噬细胞线粒体自噬
巨噬细胞RAW264.7中分别加入梯度浓度的mAMSC-Exo(5μg/mL、20μg/mL、80μg/mL)处理24h,然后收集细胞,进行细胞线粒体自噬相关分子的Western Blot检测,WB条带采用ChemiScope Western Blot Imaging System(Clinx Science Instruments Co.,Ltd)扫描后,再用Image J软件(Rawak Software,Inc.Germany)进行灰度比值分析。如图2显示,mAMSC-Exo能显著促进巨噬细胞线粒体自噬,具体表现为剂量依赖性地上调线粒体自噬相关分子PARKIN和PINK1水平。
在经PMA诱导的THP-1或U937来源的巨噬细胞中加入hAMSC-Exo,同样也具有上述效应。
实施例3 AMSC-Exo可缓解药物诱导的肝细胞线粒体损伤
HL-7702细胞加入梯度浓度的AMSC-Exo(5μg/mL、20μg/mL、80μg/mL)培养12h后,再以5mM APAP作用12h诱导细胞线粒体损伤,然后采用MitoSOX Red染色结合流式细胞术分析线粒体ROS(mtROS)水平;采用JC-1探针染色结合流式细胞术检测线粒体膜电位;采用Western Blot检测TOM20蛋白水平来反应线粒体清除情况。
如图3显示,AMSC-Exo能有效降低APAP所诱导的肝细胞mtROS分泌;纠正APAP所诱导的肝细胞线粒体膜电位异常;清除APAP所导致的肝细胞中的损伤线粒体。
实施例4 AMSC-Exo在药肝小鼠模型中的作用
梯度剂量(8mg/kg、20mg/kg、50mg/kg)的AMSC-Exo分别尾静脉输注C57小鼠,并尾静脉注射对应体积的PBS作为对照组(Vehicle),隔天注射,共注射3次。第3次注射3h后小鼠腹腔注射APAP(500mg/kg)建立药物性肝损伤模型。造模24h后,处死小鼠并收集血清、肝组织等进行肝功能、肝脏病理、及肝脏线粒体自噬相关指标的检测。血清ALT、AST检测分别采用FUJI DRI-CHEM Slide GFP/ALT-PIII和GOT/AST-PIII试剂盒,用DRI-CHEM 4000ie(FUJIFILM)测定;肝脏病理采用HE检测;肝组织ROS水平采用二氢乙啶探针(dihydroethidium,DHE)染色分析;细胞线粒体自噬相关分子采用Western Blot检测,再行灰度比值分析。
AMSC-Exo输注可呈剂量依赖性地促进肝脏线粒体自噬,降低肝组织ROS水平(图4)。同时AMSC-Exo治疗组的小鼠血清ALT、AST水平,肝脏炎症,肝组织坏死灶等均显著低于对照组。显示AMSC-Exo可以通过促进肝脏线粒体自噬发挥对肝损伤的保护作用。
实施例5 SEQ:NO.1核酸荷载提高AMSC-Exo对肝细胞线粒体自噬的影响
人肝细胞系HL-7702中分别加入梯度浓度(0.5μg/mL、5μg/mL、50μg/mL)的hAMSC-Exo和hAMSC-Exo-SEQ1处理24h,或巨噬细胞RAW264.7中分别加入梯度浓度(0.5μg/mL、5μg/mL、50μg/mL)的mAMSC-Exo和mAMSC-Exo-SEQ1处理24h,然后收集细胞,进行细胞线粒体自噬相关分子的Western Blot检测,再行灰度比值分析。
如图5显示,相同浓度条件下,AMSC-Exo-SEQ1较之AMSC-Exo能更为显著地促进肝细胞线粒体自噬,具体表现为上调线粒体自噬相关分子PARKIN和PINK1水平。并且AMSC-Exo-SEQ1在0.5μg/mL浓度时即可显著上调PARKIN和PINK1水平,其效果与50μg/mL浓度的AMSC-Exo相当。在巨噬细胞中,AMSC-Exo-SEQ1亦较之AMSC-Exo能更为显著地促进线粒体自噬。
实施例6 XIST修饰提高AMSC-Exo对肝细胞线粒体自噬的影响
人肝细胞系HL-7702中分别加入梯度浓度(0.5μg/mL、5μg/mL、50μg/mL)的hAMSC-Exo和hAMSC-Exo-XIST处理24h,或巨噬细胞RAW264.7中分别加入梯度浓度(0.5μg/mL、5μg/mL、50μg/mL)的mAMSC-Exo和mAMSC-Exo-XIST处理24h,然后收集细胞,进行细胞线粒体自噬相关分子的Western Blot检测,再行灰度比值分析。
如图6显示,相同浓度条件下,AMSC-Exo-XIST较之AMSC-Exo能更为显著地促进肝细胞线粒体自噬,具体表现为上调线粒体自噬相关分子PARKIN和PINK1水平。并且AMSC-Exo-XIST在0.5μg/mL浓度时即可显著上调PARKIN和PINK1水平,其效果与50μg/mL浓度的AMSC-Exo相当。在巨噬细胞中,AMSC-Exo-XIST亦较之AMSC-Exo能更为显著地促进线粒体自噬。
序列表
<110> 浙江大学
<120> 间充质干细胞外泌体在制备促进线粒体自噬制剂中的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> RNA
<213> 人工序列(Unknow)
<400> 1
gcaggcgccc aauuaauguc uguugaugau u 31
<210> 2
<211> 1260
<212> DNA
<213> XIST片段(X-inactive specific transcript)
<400> 2
ggatgacctc tagtcaagac ctttgcacta ggatagttaa tgtgaaccat ggcaactgat 60
cacaacaatg tctttcagat cagatccatt ttatcctcct tgttttacag caagggatat 120
taattaccta tgttaccttt ccctgggact atgaatgtgc aaaattccaa tgttcatggt 180
ctctcccttt aaacctatat tctacccctt ttacattata gaaagggatg ctggaaaccc 240
agagtccttc tcttgggact cttaatgtgt atttctaatt atccatgact cttaatgtgc 300
ctgtgtaata gacatgaaag cctcccctgc cacaccccac ctccaatctt cctttccctt 360
ccaccaggga gtgtccactc catataccct tacatttgga caatcaaggt gcacaattgt 420
aagtgagcat aggcactcac cttggacatg aatgtgcata actgcacatg gcccatccca 480
tctgaataag gtcctactct cagacccttt ttgcagtaca gcaggggtgc tgatcaccaa 540
ggcccctttt cctggcctgt tatgtgtgtg attatatttg ttccagttcc tgtgtaatag 600
aaaacaacca ccacacgtca agctcttcat tgttcctatc tgccaaatca ttatacttcc 660
tacaagcagt gcagagagct gagtcttcag caggtccaag aaatttgaac acactgaagg 720
aagtcagcct tcccacctga agatcaacat gcctggcact ctagcacttg aggatagctg 780
aatgaatgtg tatttctttg tctctttctt tcttgtcttt gctctttgtt ctctatctaa 840
agtgtgtctt acccatttcc atgtttctct tgctaatttc tttcgtgtgt gcctttgcct 900
cattttctct ttttgttcac aagagtggtc tgtgtcttgt cttagacata tctctcattt 960
ctgctgttca tagcttacca ccccctgatg tattatttgt tattcagaga aaatttctga 1020
atactactag tttccttttc tgtgcctgtc cctgtgctag gcactaaaaa tgcaatgatt 1080
attgatatct aggtgacctg aaaaaaaata gtgaatgtgc tttgtaaact gtaaagcact 1140
tgtattctac tgtgataagc gttgtggata caaagaaagg agcaagcata aaaaagtgct 1200
ctttcaaaag gatatagtac tatgcagaca caaggaattg tttgataaat gaataaatta 1260
<210> 3
<211> 27
<212> DNA
<213> 人工序列(Unknow)
<400> 3
ggatgacctc tagtcaagac ctttgca 27
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Unknow)
<400> 4
gctatgaaca gcagaaatga gaga 24
Claims (1)
1.一种提高AMSC-Exo促线粒体自噬效能的制备方法,其特征在于,通过以下步骤实现:
(1)构建及制备SEQ:NO.1荷载的AMSC-Exo:
(a)制备AMSC-Exo:
1)脂肪来源间充质干细胞的制备:
小鼠AMSC和成人AMSC按常规方法分离,小鼠AMSC用含5%胎牛血清的低糖DMEM培养基培养,成人AMSC采用含2% MSC培养无血清替代物的MSC培养基进行培养,AMSCs增殖接近80%融合时,进行消化、传代,3~8代细胞用于后续实验;
2)AMSC-Exo分离:
步骤1)的AMSC细胞在无血清MSC培养基中培养48小时,收集上清并做第一次离心,取上清后第二次离心,再次取上清进行第三次离心,弃沉淀,将上清于100,000g离心70分钟,收集沉淀用PBS重悬后,再次离心100,000g,70分钟,所得沉淀即为脂肪来源间充质干细胞外泌体;所述第一次离心为4℃,300g,10分钟,第二次离心为4℃,2000g,10分钟,第三次离心为4℃,10,000g,30分钟;
3)脂肪来源间充质干细胞外泌体鉴定:
通过NTA法分析其粒径,范围在64.5-143.8nm,并采用Western Blot或磁珠法检测其表面的外泌体标志物CD63、CD9和CD81;
(b)采用电转法导入SEQ:NO.1的核酸片段:将AMSC-Exo和SEQ:NO.1所示的序列在4℃的电穿孔缓冲液中轻轻混合,以0.4 cm的电穿孔试管,在350 V和150μF的电压下采用基因脉冲II电转仪进行电穿孔,将混合物在37℃下放置30分钟,以确保AMSC-Exo膜完全恢复,荷载后的AMSC-Exo以预冷的PBS清洗2次以去除未载入的多余核酸序列;所述PBS清洗条件为120,000 × g超速离心90分钟;
(2)构建及制备X非活性特异性转录物片段修饰的AMSC-Exo:
(a)构建X非活性特异性转录物修饰的AMSC:
X非活性特异性转录物慢病毒表达质粒pLVX-IRES-Zsgreen1-XIST感染AMSC,表达质粒包含部分X非活性特异性转录物目的片段序列如SEQ:NO.2所示,所述SEQ:NO.2是对特定ceRNA功能区进行选择性拼接后的片段;
AMSC细胞铺6孔板,完全培养基培养过夜,当融合度达到50%后备用,随后加入200 μlOPTI-MEM培养基、50 μl感染增强液和10 μl XIST慢病毒,滴度为108TU/ml,MOI值=10,混匀后另加入1.75ml的完全培养基并一起加入到已贴壁的AMSCs,培养8-12h观察细胞状态,如果细胞状态出现不好则立即换液,如果未产生变化则继续培养,24h后换成含5 μg/ml聚凝胺的完全培养基筛选培养3-4天后荧光显微镜观察ZsGreen1荧光情况,当荧光率达到96%以上后则撤除聚凝胺,所述感染增强液选用HitransG感染增强液;
(b)AMSC-Exo-XIST制备及鉴定:
步骤(a)所述AMSC细胞在无血清MSC培养基中培养48小时,收集上清并按步骤2)中的离心法分离制备AMSC-Exo-XIST,并通过NTA法分析其粒径,采用Western Blot法检测其外泌体标志物;
(c)采用荧光定量PCR法鉴定其中XIST的表达:
采用ncRNA试剂盒抽提AMSC-Exo-XIST的RNA,然后进行逆转录,逆转录体系:RNA模板-1μg;200µM随机引物-1 μl;25µM Oligo (dT)-1 μl;5×逆转录缓冲液-2 μl;逆转录酶混合液-2 μl;无RNA酶水-补至10 μl;逆转录反应程序为:42℃ 60 min,70℃ 10 min;
再进行荧光定量PCR检测,PCR体系为:2×SYBR Green Mix-10 μl;XIST上游引物序列如见SEQ:NO.3所示,5 µM -0.8 μl;XIST下游引物序列如SEQ:NO.4所示,5 µM -0.8 μl;模板-2ul;dd水-补至20 μl;PCR条件为:95℃预变性10 min,然后95℃ 5s,60℃ 30s,72℃30s,40个循环,PCR结果显示:经XIST修饰的AMSC,其XIST的表达水平明显高于对照组;
(3)线粒体自噬调控作用比较:
相同剂量的AMSC-Exo-SEQ :NO.1、AMSC-Exo-XIST和AMSC-Exo分别作用于肝细胞系和巨噬细胞系,AMSC-Exo-SEQ:NO.1或AMSC-Exo-XIST较之AMSC-Exo更为显著地影响细胞线粒体自噬水平,经修饰的AMSC-Exo的有效剂量低于天然AMSC-Exo的有效剂量。
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