CN109336982B - 基因改造的干细胞及其应用 - Google Patents
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Abstract
本发明涉及基因工程技术领域,尤其涉及基因改造的干细胞及其应用。本发明提供了基因改造的干细胞,该干细胞能够表达融合蛋白Pdx‑1‑Linker‑IGF‑1,能够高效分化成为胰岛样细胞团。实验表明,诱导5天转化率可达90%,胰岛素分泌量可达125±16pg/mL。与其他各组相比,经基因转化后的干细胞具有更强的胰岛细胞转化效果。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及基因改造的干细胞及其应用。
背景技术
糖尿病(Diabetes Mellitus,DM)是一组以高血糖为特征的代谢性疾病。高血糖则是由于胰岛素分泌缺陷或其生物作用受损,或两者兼有引起。糖尿病时长期存在的高血糖,导致各种组织,特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍。
据世界卫生组织推荐的分型,可将糖尿病分为以胰岛素绝对不足为主的I型糖尿病和以胰岛素相对不足且胰岛素抵抗为主的II型糖尿病两种类型。其中,约10%的患者属于I型糖尿病,遗传因素在该型糖尿病中的重要性高达50%,发病常见于儿童和青少年人群。另外,90%的患者属于II型糖尿病,是一种多基因遗传因素、环境因素联合作用引起的代谢性疾病,具有很高的遗传倾向,多见于40岁以上的成年患者。
目前在糖尿病的临床治疗上,通过注射胰岛素来控制血糖虽然是一种有效的缓解措施,但并不能彻底治愈糖尿病,还有可能造成低血糖等风。真正根治糖尿病将是恢复机体内功能性胰岛β细胞的数量和消除糖尿病引发的各种并发症。其中,进行胰岛移植是治疗I型糖尿病和部分II型糖尿病最有效的方法之一。但是,由于供体来源严重不足以及器官移植将面临终身免疫抑制治疗等问题,极大地限制了胰岛移植在临床治疗上的广泛应用。
近年来,由干细胞体外定向诱导分化成胰腺祖细胞和功能性胰岛来进行移植,为我们提供了一个全新的细胞替代疗法。自体诱导分泌胰岛素细胞微球可以有效的缓解病人的痛苦,同时该技术是以自体间充质干细胞为基础,能够从根本上避免免疫排斥问题以及其他传染性疾病的困扰,并且利用自体细胞可以更有效的发挥其修复作用,使移植细胞更好的与胰岛相结合,并最终替代损伤的胰岛细胞,进而达到修复其功能的作用。但是,目前的技术对干细胞的诱导效率较低,尚无法满足临床的应用。因此,应进一步研究,提高干细胞体外定向诱导分化成为分泌胰岛素的细胞的效率。
发明内容
有鉴于此,本发明要解决的技术问题在于提供基因改造的干细胞及其应用,该干细胞体外定向分化为分泌胰岛素的细胞的效率较高。
由于骨髓干细胞本身具有可诱导成型的特点可将其改造成胰岛素分泌性细胞,但是普通诱导方法效果并不是很好,因此采用慢病毒转基因加后期诱导的方法,对干细胞进行改造。
本发明提供了一种融合蛋白Pdx-1-Linker-IGF-1,其氨基酸序列如SEQ ID NO:1所示。
Pdx1基因是胰十二指肠同源盒基因-1,是控制胰腺细胞正常发育和维持胰岛素正常分泌的基因。Pdx-1基因最终转录为Pdx-1蛋白,其具有283个氨基酸,分子量约为31kDa。主要利用氨基端和胰岛素基因启动子区域结合,进而调控胰岛素基因的转录以及表达,同时引导胰岛素mRNA从细胞核中转移到核外。IGF-1胰岛素样生长因子是具有促进生长的多肽结构的分泌性激素,多功能细胞调控因子,对细胞的生长,促进组织的发育和细胞转化,以及生理代谢等起到了极强的作用。
本发明充分利用Pdx-1和IGF-1的特性,设计了以G3S(即GGGS,G:甘氨酸,S:丝氨酸)为柔性链接的表达Pdx-1和IGF-1的融合蛋白。通过基因表达慢病毒载体,将Pdx-1于IGF-1的融合基因嵌入到干细胞基因组中,促使干细胞最终能表达Pdx-1-Linker-IGF-1融合蛋白。通过G3S柔性链连接到一起的融合蛋白同时具有Pdx-1和IGF-1两个蛋白的性质,既可以促进干细胞分泌胰岛素同时又具有定向促进细胞的生长,并且能够很好的促进干细胞像类胰岛细胞及组织进行转化,从而达到修复胰岛组织的作用。
本发明还提供了表达融合蛋白Pdx-1-Linker-IGF-1的DNA分子。
所述表达融合蛋白Pdx-1-Linker-IGF-1的DNA分子的核苷酸序列如SEQ ID NO:2所示。
本发明还提供了包含所述DNA分子的质粒载体。
本发明实施例中,其骨架载体为pRRLSIN.cPPT.PGK-WPRE载体(图谱如图1)。
本发明中,所述质粒载体中,表达融合蛋白Pdx-1-Linker-IGF-1的DNA分子的插入位点为BamH I,Sal I。
本发明还提供了一种慢病毒,其由本发明所述的质粒载体与慢病毒包装质粒共转染293T细胞构建获得。
所述慢病毒包装质粒为pMD2G(表达VSV G包膜蛋白)和pCMVR8.74(表达HIV-1Gag,Pol,Tat and Rev蛋白)。所述共转染采用CaCl2转染。
本发明利用pMD2G和pCMVR8.74来表达HIV-1病毒的基本组件,将目标基因Pdx-1-Linker-IGF-1嵌入到pRRL质粒载体上,制备出含有目标基因的慢病毒载体,并将目标基因有效的整合到宿主细胞(干细胞)的染色体上,并最终实现表达目的序列的效果。
所述慢病毒的制备方法为:将293T细胞与本发明所述的质粒载体和pMD2G、pCMVR8.74质粒混合,以含有CaCl2的HeBS缓冲液,37℃,5%CO2培养后收集上清液,经过滤、离心收集病毒。
表达融合蛋白Pdx-1-Linker-IGF-1的干细胞。
本发明所述的干细胞为骨髓干细胞。
一些实施例中,所述骨髓干细胞为自体骨髓干细胞。
本发明所述干细胞的制备方法,其以本发明所述的慢病毒感染干细胞获得。
具体的,以本发明所述的慢病毒感染骨髓干细胞制得。
所述感染为:将病毒悬液和完全培养基混合后加入含有干细胞的培养皿中,37℃孵育。4小时后,再加入一定量的完全培养基,37℃孵育,继续培养24小时,用新鲜培养基替换含有病毒的培养基,继续培养获得所述干细胞。
本发明所述干细胞在制备治疗糖尿病的制剂中的应用。
本发明还提供了一种治疗糖尿病的制剂,其包括本发明所述干细胞。
本发明所述制剂的制备方法,其将本发明所述干细胞以含有30mmol/L葡萄糖和10mmol/L C肽的完全培养基诱导分化成为胰岛样细胞团,添加辅料制成所述制剂。
本发明提供了基因改造的干细胞,该干细胞能够表达融合蛋白Pdx-1-Linker-IGF-1,能够高效分化成为胰岛样细胞团。实验表明,诱导5天转化率可达90%,胰岛素分泌量可达125±16pg/mL。与其他各组相比,经基因转化后的干细胞具有更强的胰岛细胞转化效果。
附图说明
图1示质粒图谱;其中图1-a示pRRLSIN.cPPT.PGK-WPRE的质粒图谱;图1-b示pRRLSIN.cPPT.PGK-Pdx-1-Linker-IGF-1-WPRE的质粒图谱;
图2示显微镜下观察诱导分化干细胞的检测结果,图2-a为未进行双硫腙染色的细胞,图2-b为经过双硫腙染色后胰岛样细胞团被染成铁红色;
图3示胰岛素分泌趋势。
具体实施方式
本发明提供了基因改造的干细胞及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1
人工合成SEQ ID NO:2所示的DNA片段,以BamH I,Sal I,酶切位点BamH I:G^GATCC,4021,Sal I:G^TCGAC,4049位点连接入载体pRRLSIN.cPPT.PGK-WPRE(图1-a),构建获得表达融合蛋白Pdx-1-Linker-IGF-1的质粒载体(图1-b,全序列如SEQ ID NO:3所示)。
实施例2
实验材料:
病毒生产所需物品:293T细胞为病毒包装细胞;Dulbecco’s modified Eaglemedium(DMEM培养基);FBS(胎牛血清);1×penicillin-streptomycin抗生素;3质粒;2.5MCaCl2;2×HeBS缓冲液。
病毒生产流程:
A:293T细胞使用含10%FBS,+penicillin-streptomycin抗生素的DMEM培养基(完全培养基)进行培养,按15ml培养皿为例,加入8×106细胞,22.5ml完全培养基,37℃,5%CO2进行培养。
B:过夜培养后,更换为无血清培养基DMEM。
C:按一定比例将3个质粒(pMD2G、pCMVR8.74、pRRLSIN.cPPT.PGK-WPRE-Pdx-1-Linker-IGF-1)进行混合,缓冲液为HeBS。
D:转染方法采用CaCl2转染方案,将CaCl2按一定比例和含有3质粒的HeBS缓冲液混合,充分混匀后,加入到293T培养皿中,37℃,5%CO2进行培养。
E:过夜培养后,将培养基吸出,重新更换完全培养基继续培养,37℃,5%CO2进行培养。
F:培养24小时后,轻轻的吸尘细胞培养基,4℃保存,再更换新鲜完全培养基。
G:培养24小时后,轻轻的吸出细胞培养基,4℃保存。
H:将两次收集到的细胞培养上清,通过0.45um滤膜进行过滤,去除杂质,4℃保存。
I:将过滤后的培养上清,移入离心管中,25000转,4℃,离心120分钟,离心后去除上清,沉淀即为病毒,用PBS重悬,-80℃保存。
实施例3
离心提取骨髓组织的MNC(单核细胞),PBS清洗后,将细胞悬浮在8-10ml生理盐水(2.2~3.8×108cells)。使用完全培养基,配方如下:
含10%FBS(胎牛血清)的干细胞培养基(北京达科为生物技术有限公司:Mesenchymal Stem Cell Basal Medium(MSCBM),人间充质干细胞基础培养基),加庆大霉素和青霉素,37℃,5%CO2的条件下进行贴壁培养,扩增传代2次后,细胞扩增效率平稳并且活率达到98%时,用于细胞转染。
将培养好的干细胞重新贴壁培养,加完全培养基,过夜培养,第二天将培养基吸出,将一定量的病毒悬液和完全培养基混合后加入培养皿中,37℃孵育。4小时后,再加入一定量的完全培养基,37℃孵育,继续培养24小时,用新鲜培养基替换含有病毒的培养基,继续培养3天,获得基因改造干细胞。
实施例4
通过定量PCR技术对实施例3构建获得的干细胞中的目的基因进行检测,评估转染效率,本检测采用提取经转染和未经转染的细胞进行对比性检测。分别提取1×107经转染和未经转染的细胞,采用15cm玩去培养皿培养,加入15ml完全培养基,37℃,5%CO2进行培养24小时。培养结束后,经离心,去除培养基,经离心沉淀并提取全部细胞,然后提取细胞总RNA(Qiagen,RNA Neasy Plus Kits提取试剂盒),采用Roche公司的LightCycler96荧光定量PCR检测,针对Pdx-1-Linker-IGF-1表达效果进行检测。一般情况下,未经转染细胞mRNA表达量为0copie,转染后效率达到100copies/细胞,即可认定细胞转染效率达到90%,此时细胞可用作后期诱导实验。经检测,实施例3构建获得的干细胞中,mRNA表达量为1×1019copies/mL。
实施例5
对实施例3制备的干细胞的细胞分化效果进行鉴定,采用双硫腙染色法和ELISA(酶联免疫吸附测定)确定最终转化效果,实验分为未转化组和基因改良组:
未转化组为没有经过基因改良的原始干细胞;
基因改良组为经慢病毒转染可表达Pdx-1-Linker-IGF-1融合蛋白的干细胞(实施例3)。
每组细胞分三个平行试验组:
组1,使用干细胞完全培养基
组2,使用干细胞完全培养基,加入到浓度为3mmol/L葡萄糖
组3,使用干细胞完全培养基,加入到浓度为25mmol/L葡萄糖
实验分组如表1:
表1实验分组
进行测试中,3组细胞初始量一致:1×107细胞,采用15cm直径培养皿培养,加入10-15ml干细胞完全培养基,分别在培养的第1天至第5天,提取培养基上清1ml进行检测,主要对培养基中分泌的胰岛素进行ELISA定量检测(采用人胰岛素ELISA检测试剂盒),同时提取一定量的细胞进行双硫腙染色法检测。配制出质量体积比为10%双硫腙溶液,取适量的溶剂与一定量的细胞混合,室温放置1h后,利用显微镜和细胞计数板对细胞进行计数,同时经双硫腙染色后,胰岛细胞染色后成铁红色,未染成红色的为阴性(图2),取1ml,离心提取细胞,利用细胞计数板对细胞进行细胞计数,通过不同的颜色分辨阴性(未染色)和阳性(铁红色)细胞,利用公式:
胰岛样细胞团%=阳性细胞数/(阳性细胞数+阴性细胞数)
测算出胰岛样细胞成团效果,测试结果如表2和图2:
表2测试结果
编号 | 转化率 | 胰岛素分泌量 | 胰岛样细胞团 |
MSC-A | 0 | 0 | 无 |
MSC-B | 30% | 15±15pg/ml | 20% |
MSC-C | 50% | 55±22pg/ml | 30% |
MSCT-A | 20% | 35±10pg/ml | 20% |
MSCT-B | 50% | 65±7pg/ml | 50% |
MSCT-C | 90% | 125±16pg/ml | 75% |
MSC-A组正常情况下无胰岛素蛋白表达,MSC-B组表达量变可达到15±15pg/ml,MSC-C组表达量可达到55±22pg/ml,但是诱导后的转化率和胰岛样细胞团相对较低。MSCT-A组在没有葡萄糖诱导下也可以分泌胰岛素并能够形成胰岛样细胞团,同时在葡萄糖的诱导下MSCT-3组能够达到90%的转化效果,经过对比,经基因转化后的干细胞具有更强的胰岛细胞转化效果。
对经转染后的干细胞,传代一次后,重新进行贴壁培养,更换完全培养基,同时中加入高剂量葡萄糖(达到30mmol/L)。继续培养10天(期间每隔两天传代一次),同时抽取培养基检测胰岛素分泌情况,建立胰岛素分泌曲线(图3)。
对比例
Pdx-1-Linker-IGF-1融合蛋白表达
利用Pet32表达系统,采用大肠杆菌BL21进行Pdx-1-Linker-IGF-1融合蛋白表达,并利用分子筛进行纯化,纯化后的蛋白在-80度冷冻保存,用于后期实验。
将大肠杆菌表达外源Pdx-1-Linker-IGF-1融合蛋白(浓度为10pg/mL)加入高浓度葡萄糖(25mmol/L)对正常骨髓干细胞进行诱导;诱导7天,检测结果表明胰岛素分泌量在51±12pg/ml,双硫腙结果显示转化效率50%±5%,成团现象45±5%。与在相同条件下诱导的基因改造骨髓干细胞相比,该效果不论在胰岛素分泌量或是转化率、成团现象方面,存在显著差异,p<0.05。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 长春万成生物电子工程有限公司
<120> 基因改造的干细胞及其应用
<130> MP1806681
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gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 480
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 540
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 600
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 660
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 720
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 780
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 840
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 900
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 960
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1020
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1080
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1140
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1200
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1260
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1320
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1380
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1440
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 1500
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1560
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1620
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1680
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1740
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1800
cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 1860
gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 1920
aattttatcg atcacgagac tagcctcgag aagcttgata tcgaattcca cggggttggg 1980
gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 2040
ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag 2100
cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 2160
cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 2220
acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 2280
gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga 2340
aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg 2400
gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg 2460
aatcaccgac ctctctcccc agggggatcc accggtcgcc accatgaacg gcgaggagca 2520
gtactacgcg gccacgcagc tttacaagga cccatgcgcg ttccagcgag gcccggcgcc 2580
ggagttcagc gccagccccc ctgcgtgcct gtacatgggc cgccagcccc cgccgccgcc 2640
gccgcacccg ttccctggcg ccctgggcgc gctggagcag ggcagccccc cggacatctc 2700
cccgtacgag gtgccccccc tcgccgacga ccccgcggtg gcgcaccttc accaccacct 2760
cccggctcag ctcgcgctcc cccacccgcc cgccgggccc ttcccggagg gagccgagcc 2820
gggcgtcctg gaggagccca accgcgtcca gctgcctttc ccatggatga agtctaccaa 2880
agctcacgcg tggaaaggcc agtgggcagg cggcgcctac gctgcggagc cggaggagaa 2940
caagcggacg cgcacggcct acacgcgcgc acagctgcta gagctggaga aggagttcct 3000
attcaacaag tacatctcac ggccgcgccg ggtggagctg gctgtcatgt tgaacttgac 3060
cgagagacac atcaagatct ggttccaaaa ccgccgcatg aagtggaaaa aggaggagga 3120
caagaagcgc ggcggcggga cagctgtcgg gggtggcggg gtcgcggagc ctgagcagga 3180
ctgcgccgtg acctccggcg aggagcttct ggcgctgccg ccgccgccgc cccccggagg 3240
tgctgtgccg cccgctgccc ccgttgccgc ccgagagggc cgcctgccgc ctggccttag 3300
cgcgtcgcca cagccctcca gcgtcgcgcc tcggcggccg caggaaccac gaggtggcgg 3360
agggagtggg ggtggaggct ctatgtgtca gtcccctgag agtcatgtgg aaaaaaaaaa 3420
aaagaaaaaa ttcaaggtcc aggttatttc caccactcct gggaaaccag gcctggagag 3480
ctctctaggg aaagaggtga agatgcacac catgtcctcc tcgcatctct tctacctggc 3540
gctgtgcctg ctcaccttca ccagctctgc cacggctgga ccggagacgc tctgcggggc 3600
tgagctggtg gatgctcttc agttcgtgtg tggagacagg ggcttttatt tcaacaagcc 3660
cacagggtat ggctccagca gtcggagggc gcctcagaca ggcatcgtgg atgagtgctg 3720
cttccggagc tgtgatctaa ggaggctgga gatgtattgc gcacccctca agcctgccaa 3780
gtcagctcgc tctgtccgtg cccagcgcca caccgacatg cccaagaccc agaaggaagt 3840
acatttgaag aacgcaagta gagggagtgc aggaaacaag aactacagga tgtagagcgg 3900
ccgcgtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 3960
ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 4020
tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 4080
tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 4140
aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 4200
ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 4260
ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc 4320
atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 4380
ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 4440
tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc 4500
tggaattcga gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc 4560
cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat 4620
ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 4680
ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 4740
gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 4800
gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt 4860
gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac 4920
aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4980
tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc 5040
taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 5100
gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 5160
agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc 5220
gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 5280
tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 5340
ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc 5400
ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 5460
tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 5520
cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 5580
acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 5640
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 5700
gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 5760
tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 5820
ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga 5880
acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 5940
ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 6000
gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 6060
ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 6120
gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 6180
agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 6240
caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 6300
gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 6360
agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 6420
gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 6480
aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 6540
ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 6600
tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 6660
tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 6720
gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 6780
atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 6840
ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 6900
aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 6960
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 7020
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 7080
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 7140
cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 7200
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 7260
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 7320
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 7380
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 7440
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 7500
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 7560
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 7620
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 7680
gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 7740
acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg 7800
cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg 7860
aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag 7920
gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt 7980
cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg 8040
gaacaaaagc tggagctgca 8060
Claims (12)
1.融合蛋白Pdx-1-Linker-IGF-1,其氨基酸序列如SEQ ID NO:1所示。
2.表达权利要求1所述融合蛋白Pdx-1-Linker-IGF-1的DNA分子。
3.根据权利要求2所述的DNA分子,其特征在于,其核苷酸序列如SEQ ID NO:2所示。
4.含有权利要求2~3任一项所述DNA分子的质粒载体。
5.根据权利要求4所述的质粒载体,其特征在于,其骨架载体为pRRLSIN.cPPT.PGK-WPRE载体。
6.一种慢病毒,其特征在于,由权利要求4~5任一项所述的质粒载体与慢病毒包装质粒共转染293T细胞构建获得。
7.表达权利要求1所述融合蛋白Pdx-1-Linker-IGF-1的干细胞。
8.根据权利要求7所述的干细胞,其特征在于,其为骨髓干细胞。
9.权利要求7~8任一项所述干细胞的制备方法,其特征在于,以权利要求6所述的慢病毒感染干细胞获得。
10.权利要求7~8任一项所述干细胞在制备治疗糖尿病的制剂中的应用。
11.一种治疗糖尿病的制剂,其特征在于,其原料包括权利要求7~8任一项所述干细胞。
12.权利要求11所述的制剂的制备方法,其特征在于,将权利要求7~8任一项所述干细胞以含有30mmol/L葡萄糖和10mmol/L C肽的完全培养基诱导分化成为胰岛样细胞团,添加辅料制成所述制剂。
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