CN113234759A - 一种nampt基因修饰的人脐带间充质干细胞外泌体的制备方法 - Google Patents
一种nampt基因修饰的人脐带间充质干细胞外泌体的制备方法 Download PDFInfo
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Abstract
本发明涉及一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，包括以下步骤：1）、针对人AAVS1位点设计sgRNA并构建sgRNA‑CAG‑hCas9载体；2）、将sgRNA‑CAG‑hCas9载体转染至293T细胞验证sgRNA切割效率；3）、构建含NAMPT基因、抗性基因neo、转录终止序列BGH polyA、启动子EF‑1A重组载体；4）、将步骤2）中切割效率好的sgRNA‑CAG‑hCas9载体与步骤3）中构建的重组载体共转染间充质干细胞，G418筛选得到稳转细胞系；5）、收集高表达NAMPT稳转细胞系培养基，利用外泌体试剂盒提取，得到高表达NAMPT基因的人脐带间充质干细胞外泌体。通过本发明，较于慢病毒构建方法更加安全。
Description
技术领域
本发明涉及一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，具体的涉及一种利用CRISPR Cas9技术构建NAMPT基因敲入间充质干细胞的实验方法，属于生物技术领域。
背景技术
衰老是机体细胞、组织和器官等的结构和功能随着年龄的增长所发生的自然退化过程，是生命过程中的一种自然现象，同时也是引发代谢性疾病、神经退行性疾病、心血管等系统重大疾病的发生和发展的重要因素。因此，如何有效的延缓衰老已成为生命科学领域研究热点之一，也是当今社会迫切需要解决的问题。
外泌体是一类直径在30-150nm之间的纳米级囊泡，可由许多类型的细胞分泌产生，其内容物包含蛋白质、DNA、miRNA、lncRNA等，参与细胞间的物质交换和信息交流[3]。外泌体作为新型的药物载体具有良好的生物相容性、低免疫原性、来源广泛、稳定性好、递送效率高、膜修饰方式多样等优势，有望成为优良蛋白质药物递送的载体。
烟酰胺磷酸核糖转移酶（NAMPT）是NAD+生物补救合途径的主要限速酶，在细胞周期、细胞代谢、细胞衰老及DNA损伤修复等生理、病理活动中发挥着重要作用[8]。NAMPT在哺乳动物体内有两种存在形式：细胞内和细胞外NAMPT（iNAMPT和eNAMPT）。iNAMPT作为关键NAD+生物合成酶的功能已得到充分确立，而eNAMPT的生理相关性和功能长期以来一直存在争议。eNAMPT以前被鉴定为B前细胞集落增强因子（PBEF）和内脂肪素(visfatin)，至今均未得到证实。但已有研究证实细胞外囊泡内eNAMPT具有延缓衰老和延长小鼠寿命的作用。本研究利用CRISPR Cas9技术对人脐带间充质干细胞系（hUCMSC）进行NAMPT基因修饰以达到对外泌体进行基因修饰的目的，为抗衰老后续研究提供依据。
发明内容
本发明的目的就是针对上述问题，提供一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，可以有效替代间充质干细胞成为具有抗衰老作用的新型药物。
本发明的目的是通过以下技术方案实现的，一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，包括以下步骤：
1）、针对人AAVS1位点设计sgRNA并构建sgRNA-CAG-hCas9载体；
2）、将sgRNA-CAG-hCas9载体转染至293T细胞验证sgRNA切割效率；
3）、构建含NAMPT基因、抗性基因neo、转录终止序列BGH polyA、启动子EF-1A重组载体；
4）、将步骤2）中切割效率好的sgRNA-CAG-hCas9载体与步骤3）中构建的重组载体共转染间充质干细胞，新霉素药物G418筛选得到稳转细胞系；
5）、收集高表达NAMPT稳转细胞系培养基，利用外泌体试剂盒提取，得到高表达NAMPT基因的人脐带间充质干细胞外泌体。
所述NAMPT基因的核苷酸序列如SEQ ID NO:1所示。
步骤1）中，基因敲入位点为人AAVS1位点，该位点是能确保插入靶基因正常转录且不影响邻近基因表达的“安全港”位点。
步骤2）中，切割效率高的sgRNA具体序列为SEQ ID NO:2所示。、
步骤3）中，构建的重组载体骨架为T-easy-vector-multi-5改造而来。
步骤4）中，切割效率好的sgRNA-CAG-hCas9载体与步骤3）中构建的重组载体共转染间充质干细胞的比例为1：1。
步骤4）中，新霉素药物G418筛选浓度为400µg/ml。
所述间充质干细胞为永生化人脐带间充质干细胞。
本发明方法先进科学，通过本发明，包括如下步骤：1）在人的AAVS1位点设计sgRNA并构建sgRNA/cas9质粒；2）转染至293T细胞内验证切割效率；3）构建含AAVS1位点的左同源臂LR、右同源臂RR、NAMPT基因、EF-1A启动子、neo、polyA等元件的载体质粒；4）将sgRNA/cas9质粒与含NAMPT基因的载体质粒共转染至间充质干细胞；5）G-418筛选得到单克隆间充质干细胞株。6）收集上清，提取外泌体。AAVS1位点是一个能确保插入靶基因正常转录且不影响邻近基因表达的“安全港”位点。通过本发明，较于慢病毒构建方法更加安全。
附图说明
图1为T7内切酶验证sgRNA切割效率结果；
图2为NAMPT基因敲入后间充质干细胞NAMPT基因mRNA表达水平上调；
图3为NAMPT基因敲入后间充质干细胞基因组鉴定；
图4为NAMPT基因敲入后间充质干细胞外泌体内NAMPT蛋白表达量升高。
具体实施方式
实验仪器与试剂：
RCP仪、离心机、37℃恒温培养箱、恒温摇床、倒置显微镜、CO2培养箱、超速离心机、凝胶成像仪、酶标仪；切胶回收试剂盒、PCR产物纯化试剂盒、质粒提取试剂盒、xfect转染试剂、外泌体提取试剂盒、MEM培养基、FBS、PBEF抗体、CD81抗体、GAPDH抗体、RIPA裂解液、鲑精蛋白、BCA试剂盒、CCK-8试剂盒。
下面结合具体实施例子，对本发明的技术方案详细描述。
实施例1 、AAVS1位点sgRNA切割效率验证；
1.sgRNA-CAG-hCas9载体构建；
利用https://chopchop.rc.fas.harvard.edu/网址在线设计sgRNA并合成正反义寡核苷酸序列，且将其进行退火反应后与Bbs1酶切后的sgRNA-CAG-hCas9质粒连接，鉴定，挑取阳性菌落。
2.T7内切酶验证切割效率；
将上述sgRNA-CAG-hCas9参考xfect转染试剂盒说明书转染至293T细胞内，72h收集细胞，提取基因组DNA，利用AAVS1-after-cut-F和AAVS1-after-cut-R引物进行PCR扩增，得到的PCR产物纯化并进行退火处理，用T7内切酶酶切后进行琼脂糖凝胶电泳。
实施例2 、AAVS1-NAMPT-NEO同源重组载体构建；
骨架载体AAVS1-T-HDR-donor由扬州大学表观遗传学实验室提供，其上含有IRES、bGH polyA、LOXP，左同源臂LR，右同源臂RR从人的基因组模板上扩增得到的，将AAVS1-T-HDR-donor、左同源臂LR、右同源臂RR及neo用ClonExpress MultiS One Step Cloning Kit试剂盒进行连接，送测。将上述序列正确的质粒用EcoRV进行单酶切和启动子EF-1A、PolyA及3xFLAG-NAMPT-6His进行连接，送测，得到AAVS1-NAMPT-NEO同源重组载体。
实施例3、 获得AAVS1-NAMPT-NEO稳转细胞系及鉴定；
将切割效率好的sgRNA-CAG-hCas9载体与AAVS1-NAMPT-NEO同源重组载体按照xfect说明书共转染间充质干细胞，400ug/mlG418筛选10天得到稳转细胞系。一部分细胞提取基因组，PCR扩增验证。一部分细胞用于提取总RNA并反转录合成cDNA，再用荧光定量PCR法检测细胞内NAMPT mRNA的相对水平。
实施例4、外泌体提取及NAMPT基因修饰外泌体的鉴定；
1.提取外泌体；
取对数生长期的NAMPT稳定高表达的间充质干细胞铺于100mm皿中，待其贴壁后，换成MT无血清培养基培养72h，收集细胞上清。细胞上清用0.22µm滤器过滤，随后16000g离心30min，将上清转移到新的离心管内，加入1/2体积的EXO-SpinTM Buffer（EXO-SpinTMkit，CELL guidance systems），颠倒混匀后4℃过夜孵育，次日，16000g离心1h，弃上清，用500µlPBS重悬沉淀，得到NAMPT高表达的间充质干细胞外泌体。
2.NAMPT基因修饰外泌体的鉴定；
用蛋白质裂解液（RIPA）裂解外泌体，采用BCA试剂盒测蛋白含量。蛋白质免疫印迹检测NAMPT高表达间充质干细胞外泌体的表面标记及NAMPT高表达间充质干细胞外泌体内NAMPT的表达量。
SEQ ID NO:1
atgaatcctgcggcagaagccgagttcaacatcctcctggccaccgactcctacaaggttactcactataaacaatatccacccaacacaagcaaagtttattcctactttgaatgccgtgaaaagaagacagaaaactccaaattaaggaaggtgaaatatgaggaaacagtattttatgggttgcagtacattcttaataagtacttaaaaggtaaagtagtaaccaaagagaaaatccaggaagccaaagatgtctacaaagaacatttccaagatgatgtctttaatgaaaagggatggaactacattcttgagaagtatgatgggcatcttccaatagaaataaaagctgttcctgagggctttgtcattcccagaggaaatgttctcttcacggtggaaaacacagatccagagtgttactggcttacaaattggattgagactattcttgttcagtcctggtatccaatcacagtggccacaaattctagagagcagaagaaaatattggccaaatatttgttagaaacttctggtaacttagatggtctggaatacaagttacatgattttggctacagaggagtctcttcccaagagactgctggcataggagcatctgctcacttggttaacttcaaaggaacagatacagtagcaggacttgctctaattaaaaaatattatggaacgaaagatcctgttccaggctattctgttccagcagcagaacacagtaccataacagcttgggggaaagaccatgaaaaagatgcttttgaacatattgtaacacagttttcatcagtgcctgtatctgtggtcagcgatagctatgacatttataatgcgtgtgagaaaatatggggtgaagatctaagacatttaatagtatcaagaagtacacaggcaccactaataatcagacctgattctggaaaccctcttgacactgtgttaaaggttttggagattttaggtaagaagtttcctgttactgagaactcaaagggttacaagttgctgccaccttatcttagagttattcaaggggatggagtagatattaataccttacaagagattgtagaaggcatgaaacaaaaaatgtggagtattgaaaatattgccttcggttctggtggaggtttgctacagaagttgacaagagatctcttgaattgttccttcaagtgtagctatgttgtaactaatggccttgggattaacgtcttcaaggacccagttgctgatcccaacaaaaggtccaaaaagggccgattatctttacataggacgccagcagggaattttgttacactggaggaaggaaaaggagaccttgaggaatatggtcaggatcttctccatactgtcttcaagaatggcaaggtgacaaaaagctattcatttgatgaaataagaaaaaatgcacagctgaatattgaactggaagcagcacatcat
SEQ ID NO:2
sgRNA具体序列：
F:CACCGTAAGGAATCTGCCTAACAGG
R: AAACCCTGTTAGGCAGATTCCTTAC
序列表
<110> 扬州大学
<120> 一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1473
<212> RNA
<213> 人 (Homo sapiens)
<400> 1
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cat 1473
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
caccgtaagg aatctgccta acagg 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaccctgtt aggcagattc cttac 25
Claims (8)
1.一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，包括以下步骤：
1）、针对人AAVS1位点设计sgRNA并构建sgRNA-CAG-hCas9载体；
2）、将sgRNA-CAG-hCas9载体转染至293T细胞验证sgRNA切割效率；
3）、构建含NAMPT基因、抗性基因neo、转录终止序列BGH polyA、启动子EF-1A重组载体；
4）、将步骤2）中切割效率好的sgRNA-CAG-hCas9载体与步骤3）中构建的重组载体共转染间充质干细胞，新霉素药物G418筛选得到稳转细胞系；
5）、收集高表达NAMPT稳转细胞系培养基，利用外泌体试剂盒提取，得到高表达NAMPT基因的人脐带间充质干细胞外泌体。
2. 根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，所述NAMPT基因的核苷酸序列如SEQ ID NO:1所示：
atgaatcctgcggcagaagccgagttcaacatcctcctggccaccgactcctacaaggttactcactataaacaatatccacccaacacaagcaaagtttattcctactttgaatgccgtgaaaagaagacagaaaactccaaattaaggaaggtgaaatatgaggaaacagtattttatgggttgcagtacattcttaataagtacttaaaaggtaaagtagtaaccaaagagaaaatccaggaagccaaagatgtctacaaagaacatttccaagatgatgtctttaatgaaaagggatggaactacattcttgagaagtatgatgggcatcttccaatagaaataaaagctgttcctgagggctttgtcattcccagaggaaatgttctcttcacggtggaaaacacagatccagagtgttactggcttacaaattggattgagactattcttgttcagtcctggtatccaatcacagtggccacaaattctagagagcagaagaaaatattggccaaatatttgttagaaacttctggtaacttagatggtctggaatacaagttacatgattttggctacagaggagtctcttcccaagagactgctggcataggagcatctgctcacttggttaacttcaaaggaacagatacagtagcaggacttgctctaattaaaaaatattatggaacgaaagatcctgttccaggctattctgttccagcagcagaacacagtaccataacagcttgggggaaagaccatgaaaaagatgcttttgaacatattgtaacacagttttcatcagtgcctgtatctgtggtcagcgatagctatgacatttataatgcgtgtgagaaaatatggggtgaagatctaagacatttaatagtatcaagaagtacacaggcaccactaataatcagacctgattctggaaaccctcttgacactgtgttaaaggttttggagattttaggtaagaagtttcctgttactgagaactcaaagggttacaagttgctgccaccttatcttagagttattcaaggggatggagtagatattaataccttacaagagattgtagaaggcatgaaacaaaaaatgtggagtattgaaaatattgccttcggttctggtggaggtttgctacagaagttgacaagagatctcttgaattgttccttcaagtgtagctatgttgtaactaatggccttgggattaacgtcttcaaggacccagttgctgatcccaacaaaaggtccaaaaagggccgattatctttacataggacgccagcagggaattttgttacactggaggaaggaaaaggagaccttgaggaatatggtcaggatcttctccatactgtcttcaagaatggcaaggtgacaaaaagctattcatttgatgaaataagaaaaaatgcacagctgaatattgaactggaagcagcacatcat。
3.根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，步骤1）中，基因敲入位点为人AAVS1位点，该位点是能确保插入靶基因正常转录且不影响邻近基因表达的“安全港”位点。
4. 根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，步骤2）中，切割效率高的sgRNA具体序列为SEQ ID NO:2所示:
F:CACCGTAAGGAATCTGCCTAACAGG
R: AAACCCTGTTAGGCAGATTCCTTAC。
5.根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，步骤3）中，构建的重组载体骨架为T-easy-vector-multi-5改造而来。
6.根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，步骤4）中，切割效率好的sgRNA-CAG-hCas9载体与步骤3）中构建的重组载体共转染间充质干细胞的比例为1：1。
7.根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，步骤4）中，新霉素药物G418筛选浓度为400µg/ml。
8.根据权利要求1所述的一种NAMPT基因修饰的人脐带间充质干细胞外泌体的制备方法，其特征是，所述间充质干细胞为永生化人脐带间充质干细胞。
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