CN1078248C - 分泌胰岛素的细胞系,生产方法及应用 - Google Patents
分泌胰岛素的细胞系,生产方法及应用 Download PDFInfo
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Abstract
本发明涉及生物学领域,特别是细胞生物学领域.本发明涉及一种新的葡萄糖敏感细胞系,称为β细胞系(INS-Ⅰ),其在可与正常β细胞相比的水平表达葡萄糖激酶和葡萄糖载体Glut-2,而另一方面由于基因操作其不能进行依赖于IGFⅡ表达的增殖.本发明也涉及生产所说新的细胞系的方法,其呈假胰岛形式的聚集体,以生物相容性水凝胶固定化并以硬化溶液将其硬化。应用于分泌胰岛素的β细胞移植物中。
Description
本发明涉及生物学领域,特别是涉及细胞生物学领域。
其主题特别地是一种新的细胞系,其能被移植入人的器官以便使其表达该新的细胞系在培养物中正常表达的生物制品。
本发明的主题特别地是一种新的、称为β细胞系(INS-I)的葡萄糖敏感的细胞系,其主要性质是对葡萄糖敏感。因此这些细胞的特征为具有高含量的胰岛素,在可与那些正常β细胞相比的水平上表达葡糖激酶和葡萄糖载体Glut2的可能性,除此之外,通过基因工程方法使这些细胞不增殖。
因此,这一细胞系能被移植入胰岛素依赖的受治疗者的器官,并能在胰岛素依赖的糖尿病情况中提供对糖血症的“生理”控制。
器官移植或组织移植成为部分治疗工具用于许多疾病中。最近,基因工程技术已指出了为治疗至今还不能治愈的疾病所设想的其它可能性。
因此提出一项研究方案,结合使用移植和基因疗法来治疗糖尿病。实际上,申请人已建立了一种β细胞系,即INS-I系。此细胞系根据葡萄糖的生理浓度分泌胰岛素,并且在基因工程操作之后能被用于移植及胰岛素依赖的糖尿病中糖血症的“生理”控制。
虽然在过去的十年间已做出医疗上的努力,但对糖尿病的治疗仍是很不令人满意。这就是为什么要开发新的疗法的原因;胰腺或胰岛移植是其中的一部分(Hellerstrom C,Andersson A,Groth C-G,Sandler s,Jansson L,Korsgren O,Swenne I,PeterssonB,Tollemar J,Tyden G-Diabetes Care 11(Suppl.1)45-531988)(Pipelleers DG,Pipelleers-Marichal M,Hannaert J-C,Berghmans M,In’t Veld PA,Rozin J,Van de Winkel M,Gepts W-Diabetes 40:908-919 1991)。世界范围内已进行了一千多例整个胰腺移植和几十例胰岛移植。虽然前景光明,但这些麻烦的技术主要带来两种类型的问题:一些是免疫学上的问题,其它的是来源的问题。
除了对每种移植所固有的耐受性问题之外,应该意识到,糖尿病是一种自身免疫病,接受者即糖尿病患者保持有破坏移植的β细胞的倾向因此存在一种绝对需要,需将免疫抑制疗法与移植技术结合使用。为避免这种免疫抑制,一些作者提出包囊的胰岛移植(Lacy P,Hegre D,Gerasimidi-Vazeo A,Gentile FT,DionneKE-Science 254:1782-1784 1991)(Chicheportich D,Reach G-Diabetologia 31:54-57 1988)。保护作用特别是包囊化的必备优点是保护移植的组织不受免疫***的攻击。然而,胰岛的低获得性使得制备足够数量的用于治疗目的的材料变得很困难。因此,在过去的十年间已付出巨大的努力建立β细胞系作为研究胰岛素分泌和糖尿病的模型。这些细胞系的多数已失去了其必备的β细胞功能,也就是说通过胰岛素分泌应答葡萄糖,这种丢失发生于传代培养和连续的细胞循环。因此,本申请已能够建立一种高度分化的β细胞系(INS-I),其特征非常相似于正常β细胞的特征(Asfari M,Janjic D,Meda P,Li G,Halban PA,WollheimCB-Endocrinology 130:167-178 1992)并且这一点构成了本发明的精华。
在这些细胞的突出的性质中,可特别提到的是可与正常β细胞相比的高胰岛素含量、葡糖激酶的表达、葡萄糖载体Glut2。最后,对分化或生长因子如生长激素、催乳素、IGF-I和IGF-II在生理浓度〔sic〕上的应答。这些细胞所必备的优点最基本的是其对葡萄糖的敏感性。申请人先前已能够表明,在使cAMP胞内水平提高的物质存在时,葡萄糖浓度从3提高至20mM使INS-I细胞的胰岛素分泌提高了4倍。
最近,已进一步表明这些细胞与生理浓度的葡萄糖(48h,5mM)一起保温使其对葡萄糖浓度的变化更加敏感。这些分化特征使这些细胞成为最适于糖尿病患者的生物相容性胶囊中异种移植。
然而,移植前,有必要使这些细胞不增殖。事实上,如果需要避免不受控制的生长和保护性胶囊的可能破裂,必须抑制增殖。这一点是通过损坏细胞增殖中(直接或非直接)涉及的基因而达到的。最近已可能表明IGF-II在这些细胞中被充分表达。IGF-II是一种生长因子,其主要以自泌方式调节细胞生长。因此,根据随后描述的近其结果可以认为,在这些细胞的不受控制的生长中涉及IGF-II,并且抑制IGF-II基因表达(通过破坏基因)能够抑制或大大减少细胞增殖。通过这种基因工程手段,INS-I细胞能够适于包囊和移植,方法是可能避免制备和移植胰岛的任何复杂性及困难。
最后,当在一种结合蛋白IGFBP-3存在条件下培养细胞时,以基因工程手段修饰的并在无血清条件下培养的β细胞的增殖大大减少(Gopinath R,Walton PE,Etherton ED-Endocrinol120:231-236 1989)。在申请人开发的IGFBP-3模型中分离IGF-II,其生物活性降低。IGFBP的加入抑制INS-I细胞增殖。这一观察结果说明了IGF-II在INS-I细胞增殖中起重要作用,因为结合蛋白(BP)特别是BP3已知的唯一功能是与IGFs结合。
大量间接证据支持所获得的结果:IGF-II在肿瘤细胞系中以自泌-旁泌方式刺激细胞增殖(El-Bardy OH,Romanus JAI,Helman LH,Cooper MH,Rrcher MM,Israel MA-J.Clin.Invest.84:829 1989)。这一点在转化细胞上(Park JHY,McCusker RH,Vanderhoof JA,Mohammadpour H,Harty RF,MacDonald RG-Endocrinology 131:1359-1368 1992)和在初级β胰岛培养物上(Rabinovith A,Quigley C,Russel T,Patel Y,Mintz DH-Diabetes 31:160-164 1982)(Hill DJ,Hogg J-E.M.Spencer(ed),Elsvier Science Publishing CO.INC 1991 235-240)也已经得到证明。
本发明主题也是细胞系INS-I的建立,以将其植入体内为目的对其进行基因修饰和包囊。
初步结果
这些结果主要涉及IGF-II、培养基中葡萄糖浓度和细胞增殖的关系。
5mM葡萄糖时,细胞增殖未表现出被改变。从5mM至20mM,在培养达72h的过程中观察到增殖的提高。同时观察到,葡萄糖浓度从10增至20mM时IGF-II信使RNAs有提高。
以前即使长时间暴露于生长激素后在这些细胞中不可能检测到IGF-I蛋白(放射免疫分析)(Binoux M,Seurin D,LassarreC,Gourmelen M-J.Clin.Endocrinol.Metab.59:453-4621984),或其信使RNA。
本方法原理
应分两步完成该工作:
-建立携带非活性IGF-II基因的细胞系,在保持其对葡萄糖的敏感性的同时使细胞不增殖。
-包囊这些细胞并移植入糖尿病动物,目的是随后按同样的步骤移植入人体。
实验步骤
第I部分:
通过同源重组技术完成IGF-II基因的永久性灭活(RieteH,Maandag ER,Clarke A,Hooper M,Berns A-Nature 384:649-651 1990)(Pennington S,Wilson JH-Proc.Natl.Acad.Sci.88:9498-9502 1991)。申请人具有可获得的质粒pUC119中的克隆化IGF-II基因(Dr HOLTHUIZEN)(Holthuizen P,Van der Lee FM,Ikejiri K,Yamamoto M,Sussenbach JS-BiochemBiophys,Acta 1087:341-343 1990)。在编码IGF-II的四种不同类型的信使RNAs中,3.6kb的信使RNA主要在INS-I细胞中被表达。使用P3启动子转录该信使(Matsuguchi T,Takahashi K,Ikejiri K,Ueno T,Endo H,Yamamoto M-BiochemBiophys Acta 1048:165-170 1990)。
提议在CMV早期基因启动子的控制下,***编码新霉素(neo)抗性基因的盒放置于IGF-II基因的外显子4相中。在这一构建物下游,将***编码单纯疱疹病毒胸苷激酶的盒。此构建物使得通过同源重组而不是随机***来选择具有IGF-II-neo构建物的稀有克隆成为可能。事实上,同源重组的克隆对新霉素和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤(gancyclovir)以均具有抗性,而随机重组的克隆将对新霉素具有抗性而对9-(1,3-二羟-2-丙氧甲基)鸟嘌呤敏感。
转染后,细胞于新霉素、9-(1,3-二羟-2-丙氧甲基)鸟嘌呤和IGF-II存在条件下进行培养。测试抗性克隆在内源IGF-II不存在但外源IGF-II存在时的增殖能力,并且用Southern印迹技术进行研究,用PCR进行分析以检查整合是否正确。将在信使RNAs和蛋白质水平分别以Northern和Western印迹技术研究IGF-II的表达。因为已证实只有IGF-II基因的父系等位基因是有活性的(De Chaiara TM,Robertson EJ,Efstratiadis A-Cell 64:849-859 1991),且因为同源重组在转录活性的DNA片断水平是更为有效的(Nickoloff JC-MEB12:5311-5318 1992),所以可设想通过单一转染/选择步骤得到不再表达IGF-II的INS-I细胞。然而,如果不是这种情况,可破坏IGF-II基因的第二次复制(Riete H,Maandag ER,Clarke A,Hooper M,Berns A-Nature 384:649-651 1990)。在该工作的每一步,应检查葡萄糖诱导胰岛素分泌的能力。
第II部分:
如果成功地破坏了(剔除了)IGF-II基因,最终会得到INSI细胞系,其生长完全或很大程度地依赖于IGF-I的外源给予。理论上,这一新的细胞系以与父系同样的方式对葡萄糖敏感。因此,在这种情况下这些细胞可用于包囊和移植。
1.INS-I细胞系的包囊
a)为充分地包囊,细胞应制成“假胰岛”形式的聚集体。这可通过在非粘附性培养皿中培养细胞来完成。聚集体(=“假胰岛”)的大小可通过培养时细胞密度来测定。另一方面,在对收集的“假胰岛”(PI)进行胰蛋白酶作用之后,可检查聚集体中细胞数目。
b)将一已知数目的PIs(含有数量相当于500-1,000个成熟鼠的胰岛(2-4×108个细胞)中发现的胰岛素)悬浮于藻酸钠溶液中(Lacy P,Hegre OD,Gerasimidi-Vazeo A,Gentile FT,Dionne KE-Science 254:1782-1784 1991)(Lanza RP,Butler DH,Borland KM,Staruk JE,Faustman DL,Solomon BA,Muller TE,Rupp RG,Maki T,Monaco AP,Chick WL-Proc.Natl.Acad.Sci.88:11100-11104 1991)并通过使用钙盐水溶液的包囊法将其固定于生物相容性的水凝胶中。然后将包囊化PIs放置于管形丙烯酸膜中,该膜对分子量低于50,000-80,000的分子是可渗透的(Lacy P,Hegre OD,Gerasimidi-Vazeo A,Gentile FT,Dionne KE-Science 254:1782-1784 1991)(LanzaRP,Butler DH,Borland KM,Staruk JE,Faustman DL,SolomonBA,Muller TE,Rupp RG,Maki T,Monaco AP,Chick WL-Proc.Natl.Acad.Sci.88:11100-11104 1991)。
c)通过灌流在体外研究包囊化PIs,以测定引起响应于不同葡萄糖浓度的胰岛素分泌变化的密度和速度。当这部分实验表明令人满意时,包含于纤维中的PIs将用于移植。
2.移植
包囊化PIs以皮下或腹膜内方式移植。这两种方法足以在人胰岛素依赖的糖尿病动物模型中保持血糖量正常。自发发生糖尿病的生物繁殖(biobreeding)(BB)鼠和非肥胖糖尿病(NOD)鼠相关于特定的组织相容性基因型,并且从免疫角度看,呈现与糖尿病受治疗者同样的缺陷(细胞毒性T淋巴细胞的存在),受治疗者即已进行移植的糖尿病动物(Lacy P,Hegre OD,Gerasimidi-Vazeo A,Gentile FT,Dionne KE-Science 254:1782-1784 1991)(Lanza RP,Butler DH,Borland KM,Staruk JE,Faustman DL,Solomon BA,Muller TE,Rupp RG,Maki T,Monaco AP,Chick WL-Proc.Natl.Acad.Sci.88:11100-111041991)。通过腹膜内途径进行的移植似乎更加有利于为移植细胞保证一良好的生物环境。
该技术可以同样方式用于胰岛素依赖的受治疗者。为达到有效的胰岛素浓度,应植入50,000至200,000PIs。
Claims (11)
1.新的高度分化的分泌胰岛素的β细胞系(INS-I),其表达特性非常近似于正常β细胞的表达特性,并且其IGF-II表达基因以至少是显著和永久的方式被抑制。
2.新的高度分化的分泌胰岛素的β细胞系(INS-I),其表达特性为正常β细胞的表达特性,其IGF II-表达基因被完全抑制。
3.根据权利要求1或2的新的分泌胰岛素的β细胞系,其以称为“假胰岛”的聚集体形式存在。
4.生产根据权利要求1至3之一的细胞系的方法,它包括,在质粒pUC119的克隆化IGF II基因中***编码新霉素抗性基因的盒,该盒在IGF II基因外显子4中的相中,在该构建物的下游***编码病毒胸苷激酶的盒,以获得同源重组的具有IGF II-新霉素构建物的克隆,通过转染将新霉素抗性的和9-(1,3-二羟-2-丙氧甲基)鸟嘌呤敏感的克隆导入细胞中,它们在新霉素、9-(1,3-二羟-2-丙氧甲基)鸟嘌呤和IGF II存在条件下培养,在转录活性DNA片断水平有抗性的克隆被收集,以得到完全或主要依赖于IGF II外源给予的INS-I细胞系。
5.根据权利要求4的方法,其中通过在非粘附性培养皿中对INS-I系的细胞进行培养将细胞制成假胰岛形式的聚集体。
6.根据权利要求4或权利要求5的方法,其中假胰岛存在于胶凝剂悬浮液中并通过加入固定化剂被固定化于生物相容性水凝胶中。
7.根据权利要求6的方法,其中胶凝剂是一种藻酸钠。
8.根据权利要求6的方法,其中固定化剂是一种钙盐的水溶液。
9.根据权利要求4至5之一的方法,其中通过包囊法固定化于生物相容性水凝胶中并通过加入一种钙盐水溶液被硬化的假胰岛随后被置于管形丙烯酸膜中,该膜对分子量低于50,000-80,000的分子是可渗透的。
10.根据权利要求4至5的方法,其中被固定化和硬化的假胰岛掺入可移植的纤维中。
11.根据权利要求4至5的方法,其中被附集和固定化的假胰岛含有数量相当于成熟鼠2-4×108个胰岛细胞中所发现的胰岛素。
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FR93/11687 | 1993-09-30 | ||
FR9311687A FR2710654B1 (fr) | 1993-09-30 | 1993-09-30 | Nouvelles lignées de cellules sécrétrices d'insuline, leurs procédés d'obtention par voie de manipulation génétique et leur utilisation chez des sujets diabétiques sous forme protégée. |
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CN1078248C true CN1078248C (zh) | 2002-01-23 |
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EP (1) | EP0721500B1 (zh) |
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CN (1) | CN1078248C (zh) |
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ES (1) | ES2243929T3 (zh) |
FI (1) | FI961440A (zh) |
FR (1) | FR2710654B1 (zh) |
HU (1) | HU219826B (zh) |
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US5427940A (en) * | 1991-06-03 | 1995-06-27 | Board Of Regents, The University Of Texas System | Engineered cells producing insulin in response to glucose |
US5744327A (en) * | 1990-02-20 | 1998-04-28 | Board Of Regents, The University Of Texas System | Methods for producing insulin in response to non-glucose secretagogues |
US5792656A (en) * | 1991-06-03 | 1998-08-11 | Board Of Regents, The University Of Texas System | Methods of preparing genetically engineered cells that produce insulin in response to glucose |
US6642003B2 (en) | 2001-08-02 | 2003-11-04 | Cedars-Sinai Medical Center | Human glucose-dependent insulin-secreting cell line |
CA2692325C (en) * | 2001-12-07 | 2015-10-20 | Geron Corporation | Islet cells from human embryonic stem cells |
US7141240B2 (en) * | 2002-03-12 | 2006-11-28 | Cedars-Sinai Medical Center | Glucose-dependent insulin-secreting cells transfected with a nucleotide sequence encoding GLP-1 |
WO2007149182A2 (en) | 2006-06-19 | 2007-12-27 | Geron Corporation | Differentiation and enrichment of islet-like cells from human pluripotent stem cells |
US10767164B2 (en) | 2017-03-30 | 2020-09-08 | The Research Foundation For The State University Of New York | Microenvironments for self-assembly of islet organoids from stem cells differentiation |
CN109957539A (zh) * | 2017-12-14 | 2019-07-02 | 深圳先进技术研究院 | 一种人造胰岛组织及其制备和应用 |
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DE4229526A1 (de) * | 1992-09-07 | 1994-03-10 | Juergen Dr Schrezenmeir | Vorrichtung zur Aufnahme und zum Einbringen von biologisch aktiven Geweben oder Zellen in den menschlichen Körper |
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Also Published As
Publication number | Publication date |
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FI961440A0 (fi) | 1996-03-29 |
FR2710654A1 (fr) | 1995-04-07 |
JP2005185293A (ja) | 2005-07-14 |
HU219826B (hu) | 2001-08-28 |
CA2172997A1 (en) | 1995-04-06 |
FI961440A (fi) | 1996-03-29 |
US5902577A (en) | 1999-05-11 |
AU7815794A (en) | 1995-04-18 |
HUT75985A (en) | 1997-05-28 |
HU9600789D0 (en) | 1996-05-28 |
NO961268L (no) | 1996-03-29 |
FR2710654B1 (fr) | 1995-12-22 |
KR20040017357A (ko) | 2004-02-26 |
NO961268D0 (no) | 1996-03-29 |
KR100463366B1 (ko) | 2004-12-29 |
DE69434433D1 (de) | 2005-08-25 |
ES2243929T3 (es) | 2005-12-01 |
AU688383B2 (en) | 1998-03-12 |
BR9407682A (pt) | 1997-02-04 |
DK0721500T3 (da) | 2005-10-17 |
EP0721500A1 (fr) | 1996-07-17 |
KR100432851B1 (ko) | 2004-11-06 |
CN1134724A (zh) | 1996-10-30 |
EP0721500B1 (fr) | 2005-07-20 |
WO1995009231A1 (fr) | 1995-04-06 |
NO317236B1 (no) | 2004-09-27 |
DE69434433T2 (de) | 2006-04-20 |
JPH09503662A (ja) | 1997-04-15 |
ATE299930T1 (de) | 2005-08-15 |
KR960705028A (ko) | 1996-10-09 |
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