CN112941034A - 一种永生化人脐带间充质干细胞系的构建方法 - Google Patents
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Abstract
本发明公开了一种永生化人脐带间充质干细胞系的构建方法,是以人脐带间充质干细胞作为基础,转染携带SV40大T抗原的同源重组载体,将SV40大T抗原基因整合入人脐带间充质干细胞基因组AAVS1基因位点,经过连续传代筛选得到表达SV40大T抗原基因且具有多向分化潜能的细胞系,该细胞系在体外传代50代以上仍有较好的活力,保持较强的增殖能力,且保持间充质干细胞的特性,是一种永生化的细胞系。
Description
技术领域
本发明属于细胞工程技术领域,特别是涉及一种永生化人脐带间充质干细胞系的构建方法。
背景技术
人间充质干细胞(Human Mesenchymal Stem Cells,hMSCs)是来源于发育早期中胚层的具有自我更新和多向分化潜能的多能干细胞,具有广阔的临床应用前景。可用于抗衰老和治疗神经系统疾病、脊髓损伤、肝硬化、自身免疫疾病、糖尿病等疾病,具有广泛的临床应用前景。
脐带间充质干细胞具有比其他一些干细胞(如脐带造血干细胞、骨髓造血干细胞、骨髓间充质干细胞等)特有的优势,其材料来源广泛,细胞增殖能力强,免疫原性低,无伦理问题。但脐带间充质干细胞的寿命有限,只能在体外培养10-20代,其后衰老死亡。
发明内容
本发明主要解决的技术问题是提供一种永生化人脐带间充质干细胞系的构建方法,筛选出永生化的人脐带间充质干细胞,解决脐带间充质干细胞传代次数的限制,为基础研究提供充足稳定的间充质干细胞来源。
为解决上述技术问题,本发明采用的一个技术方案是:一种永生化人脐带间充质干细胞系的构建方法,包括以下步骤:
a、以AAVS1基因第一外显子序列,通过网站设计并筛选特异性靶向SV40基因的gRNA,引导Cas9蛋白进行定点切割;
b、构建了含有SV40大T抗原的同源重组载体AAVS1-SV40-T-NEO;
c、筛选出阳性的hMSC-SV40细胞系;
d、用实时定量PCR及常规PCR技术在DNA、mRNA水平检测了SV40大T抗原基因的明显表达;
e、用Western blot从蛋白水平检测了SV40大T抗原基因的表达;
f、用成骨试剂诱导hMSC-SV40细胞系;
g、用CCK8检测永生化细胞的增殖情况。
本发明为解决其技术问题所采用的进一步技术方案是:
进一步地说,所述步骤c的具体操作为:用AAVS1-SV40-T-NEO和AAVS1-Cas9-sgRNA共转染到分离后第二代人脐带间充质干细胞,在新霉素的筛选下,成功筛选出阳性的hMSC-SV40大T抗原细胞系,并连续50代而不衰老死亡。
进一步地说,AAVS1基因第一外显子序列如SEQ ID No:1所示。
进一步地说,SV40基因序列如SEQ ID No:2所示。
进一步地说,gRNA基因序列如SEQ ID No:3所示。
本发明的有益效果至少具有以下几点:
1、本发明使用了基因敲入技术将SV40大T抗原基因导入到人脐带hMSCs,整合入基因组中并能稳定表达,获得的永生化细胞为单克隆细胞,比较均一;
2、利用新霉素成功地筛选到了SV40阳性的细胞系hMSCs-SV40,在体外培养已达到50代,远超脐带间充质干细胞的体外增殖能力,证明成功地获得永生化的人脐带间充质干细胞系;
3、本发明选用的建系细胞为人脐带间充质干细胞,人脐带间充质干细胞来源于人脐带,容易获得;此外人脐带间充质干细胞具有自我更新和多向分化潜能的多能干细胞,在临床上应用前景十分广泛;
4、本发明是运用了先进的CRISPR/Cas9系统,将SV40大T抗原基因定点插入AAVS1位点,该位点是一个经过验证、能确保转入DNA片段预期功能的“安全港”位点,该位点是一个开放的染色体结构,能保证转入基因能被正常转录,另外很重要的一点是在该位点插入外源目的片段对细胞无已知的副作用;
5、本发明通过构建同源重组载体AAVS1-SV40-T-NEO,将SV40大T抗原整合入人脐带间充质干细胞基因组中,经过载体上的NeoR抗性基因筛选得到表达SV40大T抗原且具有多向项分化潜能的干细胞系,获得的永生化细胞系更加稳定,更加安全。
附图说明
图1是本发明PCR检测永生化人脐带间充质干细胞系SV40大T抗原表达的结果图;
图2是本发明Western blot检测永生化人脐带间充质干细胞在蛋白水平上SV40表达的结果图;
图3是本发明建立的人脐带间充质干细胞系的诱导分化成骨情况图;
图4是本发明永生化hMSC-SV40细胞的CCK8检测增殖图。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述,以使本发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
实施例1:AAVS1-SV40-T-NEO同源重组载体的构建
骨架载体AAVS1-HDR-donor由扬州大学表观遗传学实验室提供,SV40-polyA是从pIRES2-EF-1A-EGFP上扩增获得,T-antigen是从SV40-T-T上扩增获得,PGK是从pmirGLO上扩增获得,都是使用高保真酶扩增。
所用到的引物序列如下:
PGK:
上游引物:TCAGGGACACACCCAGAGATGGGGTTGGGGTTGCGCCTTTTCC(SEQ ID No:4)
下游引物:GTGGCGACCGGTGGATCCCCTGGGGAGAGAGGTCGGTGAT(SEQ ID No:5)
SV40-polyA:
上游引物:CACCATCACCATCACCATTGAACCTCCCACACCTCCCCCTG(SEQ ID No:6)
下游引物:AGACCCAGAGCAGTGTAGATTAAGATACATTGATGAGTTTGGACA(SEQ ID No:7)
T-antigen:
上游引物:GGGGATCCACCGGTCGCCACCGATGGATAAAGTTTTAAACAGAGAG(SEQ ID No:8)
下游引物:CAATGGTGATGGTGATGGTGTGTTTCAGGTTCAGGGGGAGG(SEQ ID No:9)
提取高纯度的骨架载体AAVS1-HDR-donor,使用ECORV进行单酶切,用凝胶回收试剂盒从AAVS1-HDR-donor质粒酶切产物中回收纯化线性化AAVS1-ECORV片段。用PCR纯化试剂盒纯化片段SV40-polyA、T-antigen、PGK,使用同源重组试剂,混合线性化载体和插入片段,5X CE buffer 4μl,Exnase 2μl,加ddH2O to 20μl。37℃反应30min后,转化DH5α大肠杆菌,接种于氨苄青霉素抗性的LB培养基,37℃培养过夜。挑菌检测,然后送公司测序。测序结果与标准序列比对正确。
实施例2:AAVS1-SV40-T-NEO和Cas9-gRNA共转染脐带间充质干细胞
用去内毒素质粒提取试剂盒提取高浓度的AAVS1-SV40-T-NEO和Cas9-gRNA质粒,并测定浓度。实验前一天,将P2代的脐带MSC按照4*104个细胞/cm2接种到6孔板中,细胞使用10%FBS的α-MEN培养基培养。实验当天,待细胞密度达到60-70%,将提取的高质量AAVS1-SV40-T-NEO 5μg和Cas9-gRNA 5ug及转染试剂Xfect 3μl,buffer 100μl混匀,室温孵育10min后滴加到6孔板中。37℃培养,4h后更换培养基。48h后按照1:3的传代比例传代,实验第四天,加含Puro(0.75μg/ml)的培养基持续培养。每隔三天更换抗性培养基。药筛一周后,细胞死亡大于90%,但镜下可以看到少部分细胞形成集落(阳性细胞),第9-18天维持抗生素的浓度,细胞少量死亡,集落增大。根据细胞的具体情况进行细胞传代,冻存。
实施例3:PCR鉴定hMSC-SV40LT细胞株
为了检测导入到hMSC细胞中的SV40大T抗原基因是否能够稳定表达,分别提取hMSCs、hMSC-SV40细胞的RNA,合成cDNA。设计引物,以cDNA为模板,进行PCR和荧光定量PCR分析,鉴定建立的hMSC-SV40细胞系中SV40大T抗原基因的整合情况。
在DNA水平,PCR分析结果显示(图1),在hMSC-SV40细胞系中,能够扩增出大约210bp和大T抗原基因的片段,而在野生型hMSCs中不能扩增出相应的片段,表明hMSC-SV40细胞中外源大T抗原基因已经成功整合到细胞染色体中。mRNA水平荧光定量PCR结果显示,永生化细胞SV40大T抗原的表达量超过普通MSC15倍。
实施例4:Western blot方法鉴定SV40大T抗原蛋白的表达情况
首先配制聚丙烯酰胺分离胶和浓缩胶,放置等待凝固。处理样品,将永生化人脐带间充质干细胞和野生型人脐带间充质干细胞消化,1000g离心,获得细胞沉淀。加入细胞裂解液,冰上放置10min,12000g离心5min,获得蛋白样品。加入上样缓冲液,煮沸5分钟,充分变性蛋白后直接上样,每孔上样15微升。剩下的孔用1Xloading buffer补齐。
电泳条件:通过上层浓缩胶时电压80V电泳1小时,然后电压升至100V,继续电泳1.5小时。当溴酚蓝到达胶的底端处停止电泳。根据胶的大小剪取膜和滤纸,放入转膜缓冲液中平衡5min,装配转移三明治:海绵一层,滤纸三层,胶,膜,滤纸三层,海绵一层,每层放好后,用试管赶去气泡,注意胶放在负极面将转移槽置于冰浴中,加转膜电泳液,插上电极,条件为90V2小时。转膜结束后,切断电源,取膜。
配置5%脱脂牛奶(TBST溶解),室温下封闭1.5小时,蛋白面朝上,摇床上摇动。倒掉牛奶后用TBST液洗3次(5min/次),加入合适稀释度的一抗SV40大T抗原抗体,4℃缓慢摇动过夜。TBST液洗3次(5min/次)后,加入二抗,室温缓慢摇动孵育1.5小时,最后TBST液洗3次(5min/次),结果如图2。
实施例5:诱导永生化hMSC-SV40细胞向成骨细胞分化
取第30代永生化的hMSC-SV40细胞置于培养箱中培养。当细胞融合度达到80%-90%时,用胰酶消化,将消化下来的细胞按照2×104cells/cm2的密度接种在事先包被0.1%明胶的六孔板中,每孔加入2ml完全培养基。当细胞融合度达到60%-70%时,小心将孔内完全培养基吸走,加入2ml成骨诱导分化培养基。每隔3天换液(使用前需预热37℃)。诱导2-4周后,视细胞的形态变化及生长情况,用茜素红染色。
成骨诱导分化结束后,吸走六孔板中的成骨诱导分化培养基,用PBS冲洗1-2次。每孔加入2ml 4%中性甲醛溶液,固定30min,用PBS冲洗3次后,用茜素红染液染3-5min。吸去茜素红染液,PBS冲洗2次后,用倒置显微镜观察。检测结果如图3所示,可以看到成骨细胞的染色标志,经茜素红染色,钙化结节明显。
实施例6:CCK-8试剂盒检测永生化hMSC-SV40-T的增殖情况
将细胞融合度达到80%-90%的永生化细胞和普通的间充质干细胞分别消化,进行细胞计数。按照5000个cells/孔,铺到96孔板中,做6个重复。24h后吸走培养基,加入cck-8 10ul每孔,完全培养基90ul每孔混匀,対照组为不加细胞只加cck-8和培养基的孔。置于培养箱培养2h,避光吸出培养基,酶标仪检测OD450和OD630。每隔24h重复操作,持续一周。
如图4,检测结果表明所构建的永生化的脐带间充质干细胞系是一种永生化的细胞系,能够传代至50次,而且具有定向分化潜能,同时能节省培养基成本,给脐带间充质干细胞随着传代次数的提高活力差,难以存活提供了帮助。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效结构变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
序列表
<110> 江苏易诺维生物医学研究院有限公司
<120> 一种永生化人脐带间充质干细胞系的构建方法
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 351
<212> DNA
<213> AAVS1第一外显子序列(人工序列)
<400> 1
agttcccggc ggcccccggg gcgggcgggc gggcgggtgg tggcggcggt tggggctcgg 60
cgctcgctcg ctcgctgggc gggcgggcgg tgcgatgtcc ggagaggatg gcccggcggc 120
tggcccgggg gcggcggcgg cggctgcccg ggagcggcga cgggagcagc tgcggcagtg 180
gggggcgcgg gcgggcgccg agcctggccc cggagagcgc cgcgcccgca ccgtccgctt 240
cgagcgcgcc gccgagttcc tggcggcctg tgcgggcggc gacctggacg aggcgcgtct 300
gatgctgcgc gccgccgacc ctggccccgg cgccgagctc gaccccgccg c 351
<210> 2
<211> 2294
<212> DNA
<213> SV40基因序列(人工序列)
<400> 2
ggggatccac cggtcgccac cgatggataa agttttaaac agagaggaat ctttgcagct 60
aatggacctt ctaggtcttg aaaggagtgc ctgggggaat attcctctga tgagaaaggc 120
atatttaaaa aaatgcaagg agtttcatcc tgataaagga ggagatgaag aaaaaatgaa 180
gaaaatgaat actctgtaca agaaaatgga agatggagta aaatatgctc atcaacctga 240
ctttggaggc ttctgggatg caactgaggt atttgcttct tccttaaatc ctggtaaggt 300
aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg tttgtgtatt 360
ttagattcca acctatggaa ctgatgaatg ggagcagtgg tggaacgcgt ttaatgagga 420
aaacctgttt tgctcagaag aaatgccctc gagtgatgat gaggctaccg cggactctca 480
acattctact cctccaaaaa agaagagaaa ggtagaagac cccaaggact ttccttcaga 540
attgctaagt tttttgagtc atgctgtgtt tagtaataga actcttgctt gctttgctat 600
ttacaccaca aaggaaaaag ctgcactgct atacaagaaa attatggaaa aatattctgt 660
aacctttata agtaggcata acagttataa tcataacata ctgttttttc ttactccaca 720
caggcataga gtgtctgcta ttaataacta tgctcaaaaa ttgtgtacct ttagcttttt 780
aatttgtaaa ggggttaata aggaatattt gatgtatagt gccttgacta gagatccatt 840
ttctgttatt gaggaaagtt tgccaggtgg gttaaaggag catgatttta atccagaaga 900
agcagaggaa actaaacaag tgtcctggaa gcttgtaaca gagtatgcaa tggaaacaaa 960
atgtgatgat gtgttgttat tgcttgggat gtacttggaa tttcagtaca gttttgaaat 1020
gtgtttaaaa tgtattaaaa aagaacagcc cagccactat aagtaccatg aaaagcatta 1080
tgcaaatgct gctatatttg ctgacagcaa aaaccaaaaa accatatgcc aacaggctgt 1140
tgatactgtt ttagctaaaa agcgggttga tagcctacaa ttaactagag aacaaatgtt 1200
aacaaacaga tttaatgatc ttttggatag gatggatata atgtttggtt ctacaggctc 1260
tgctgacata gaagaatgga tggctggagt tgcttggcta cactgtttgt tgcccaaaat 1320
ggattcagtg gtgtatgact ttttaaaatg catggtgtac aacattccta aaaaaagata 1380
ctggctgttt aaaggaccaa ttgatagtgg taaaactaca ttagcagctg ctttgcttga 1440
attatgtggg gggaaagctt taaatgttaa tttgcccttg gacaggctga actttgagct 1500
aggagtagct attgaccagt ttttagtagt ttttgaggat gtaaagggca ctggagggga 1560
gtccagagat ttgccttcag gtcagggaat taataacctg gacaatttaa gggattattt 1620
ggatggcagt gttaaggtaa acttagaaaa gaaacaccta aataaaagaa ctcaaatatt 1680
tccccctgga atagtcacca tgaatgagta cagtgtgcct aaaacactgc aggccagatt 1740
tgtaaaacaa atagatttta ggcccaaaga ttatttaaag cattgcctgg aacgcagtga 1800
gtttttgtta gaaaagagaa taattcaaag tggcattgct ttgcttctta tgttaatttg 1860
gtacagacct gtggctgagt ttgctcaaag tattcagagc agaattgtgg agtggaaaga 1920
gagattggac aaagagttta gtttgtcagt gtatcaaaaa atgaagttta atgtggctat 1980
gggaattgga gttttagatt ggctaagaaa cagtgatgat gatgatgaag acagccagga 2040
aaatgctgat aaaaatgaag atggtgggga gaagaacatg gaagactcag ggcatgaaac 2100
aggcattgat tcacagtccc aaggctcatt tcaggcccct cagtcctcac agtctgttca 2160
tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac 2220
acctccccct gaacctgaaa cacaccatca ccatcaccat tgaacctccc acacctcccc 2280
ctgaacctga aaca 2294
<210> 3
<211> 20
<212> DNA
<213> gRNA基因序列(人工序列)
<400> 3
ggggccacta gggacaggat 20
<210> 4
<211> 43
<212> DNA
<213> 扩增PGK的上游引物(人工序列)
<400> 4
tcagggacac acccagagat ggggttgggg ttgcgccttt tcc 43
<210> 5
<211> 40
<212> DNA
<213> 扩增PGK的下游引物(人工序列)
<400> 5
gtggcgaccg gtggatcccc tggggagaga ggtcggtgat 40
<210> 6
<211> 41
<212> DNA
<213> 扩增SV40-polyA的上游引物(人工序列)
<400> 6
caccatcacc atcaccattg aacctcccac acctccccct g 41
<210> 7
<211> 45
<212> DNA
<213> 扩增SV40-polyA的下游引物(人工序列)
<400> 7
agacccagag cagtgtagat taagatacat tgatgagttt ggaca 45
<210> 8
<211> 46
<212> DNA
<213> 扩增T-antigen的上游引物(人工序列)
<400> 8
ggggatccac cggtcgccac cgatggataa agttttaaac agagag 46
<210> 9
<211> 41
<212> DNA
<213> 扩增T-antigen的下游引物(人工序列)
<400> 9
caatggtgat ggtgatggtg tgtttcaggt tcagggggag g 41
Claims (5)
1.一种永生化人脐带间充质干细胞系的构建方法,其特征在于:包括以下步骤:
a、以AAVS1基因第一外显子序列,通过网站设计并筛选特异性靶向SV40基因的gRNA,引导Cas9蛋白进行定点切割;
b、构建了含有SV40大T抗原的同源重组载体AAVS1-SV40-T-NEO;
c、筛选出阳性的hMSC-SV40细胞系;
d、用实时定量PCR及常规PCR技术在DNA、mRNA水平检测了SV40大T抗原基因的明显表达;
e、用Western blot从蛋白水平检测了SV40大T抗原基因的表达;
f、用成骨试剂诱导hMSC-SV40细胞系;
g、用CCK8检测永生化细胞的增殖情况。
2.根据权利要求1所述的永生化人脐带间充质干细胞系的构建方法,其特征在于:所述步骤c的具体操作为:用AAVS1-SV40-T-NEO和AAVS1-Cas9-sgRNA共转染到分离后第二代人脐带间充质干细胞,在新霉素的筛选下,成功筛选出阳性的hMSC-SV40大T抗原细胞系,并连续50代而不衰老死亡。
3.根据权利要求1所述的永生化人脐带间充质干细胞系的构建方法,其特征在于:AAVS1基因第一外显子序列如SEQ ID No:1所示。
4.根据权利要求1所述的永生化人脐带间充质干细胞系的构建方法,其特征在于:SV40基因序列如SEQ ID No:2所示。
5.根据权利要求1所述的永生化人脐带间充质干细胞系的构建方法,其特征在于:gRNA基因序列如SEQ ID No:3所示。
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