CN100366735C - 脂肪细胞,脂肪细胞系及其应用 - Google Patents
脂肪细胞,脂肪细胞系及其应用 Download PDFInfo
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- CN100366735C CN100366735C CNB998165387A CN99816538A CN100366735C CN 100366735 C CN100366735 C CN 100366735C CN B998165387 A CNB998165387 A CN B998165387A CN 99816538 A CN99816538 A CN 99816538A CN 100366735 C CN100366735 C CN 100366735C
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Abstract
本发明描述了脂肪细胞。本发明尤其涉及具有可连续传代培养许多代这一性质的皮脂腺细胞和皮脂腺细胞系。所述脂肪细胞很适合各种应用。
Description
描述
本发明涉及含脂肪或脂并产生皮脂的皮肤和粘膜细胞,亦称脂肪细胞。本发明特别涉及皮脂腺细胞和一个能通过大规模的传代培养而连续培养的皮脂腺细胞系。脂肪细胞尤其适合有价值的应用,如人和动物的皮脂腺的生理和病理研究,研究痤疮、皮脂溢或其他疾病的发生,测定不同的物质和药物的有效性,开发基于二维或三维的细胞装配和器官样结构构建的细胞培养系统,及来自于这些细胞的产物的生产。
背景技术
越来越多的迹象表明脂肪细胞在病理生理过程和皮脂腺/毛发复合体疾病尤其是痤疮中扮演了重要角色(Gollnick等。《皮肤病学杂志》1991;18:489-499;Brown和Shalita,《柳叶刀》杂志,1998;351:1871-1876;Cunliffe,《皮肤病学》1998;196:182-184)。我们对于皮脂腺的生理和病理生理的了解主要来自于实验动物模型(Pochi《皮肤病中的模型》第二卷,Lowe N和Maibach H编辑,巴塞尔,1985;70-75)。然而,目前发现这些动物模型不适合对用于人的抗痤疮药物的疗效价值进行理性的预测(Geiger,皮肤病学1995;1991:305-310)。痤疮只在人体发生,而且皮脂腺分泌活性有很强的种属特异性(Nikkari,《皮肤病学调查杂志》。1974;257-267)这些事实,引导了人模型的研究。为了消除这些不利,早期的研究采用人皮肤样本,它们可以体外培养(Hsia等。Proc.Soc.Exp.Biol.Med.1970;135:285-291;Cooper等。《英国皮肤病学杂志》1976;94:156-172;Sharp等,《内分泌学杂志》,1976;70:491-499),或移植至裸鼠(Petersen等,《临床消化学杂志》,1984;74:1358-1365)。最近十年在皮脂腺分离(Kealex等。《英国皮肤病学杂志》,1986,114:181-188)和体外人脂肪细胞培养模型建立(Xia等,《皮肤病学调查杂志》1989,93:315-321)时,才开始人脂肪细胞活性的基础研究。
依靠Xia等培养技术的改进,最近几年脂肪细胞的体外培养的可重复性已经有提高。因此,Ziyboulis等(《皮肤药理学》1991;4:74-83)在培养基中省略了皮质醇,加入了人血清。Lee(上皮:细胞生理学和细胞培养进展;C.J.Jones,编辑:Kluwer,Dordrecht,1990;333-350)在有丰富添加物的无血清培养基中培养皮脂腺之前利用胶原酶处理它们。还有,原代脂肪细胞培养物通过去掉作为粘附基质层的3T3成纤维细胞层而获得(Akamatsu等。《皮肤病学调查杂志》1992;99:509-511)。传代培养物维持在添加无脂血清的培养基中(Zouboulis等,《皮肤病学调查杂志》。1993;101:628-633),及在无血清的含角质形成细胞但没有添加物的基本培养基中(Akamazu等。《皮肤病学调查杂志》1992;99:509-511)。并且,研究表明角质形成细胞生长因子明显增加人脂肪细胞的产量和增殖(Chen等。《皮肤病学调查杂志》1998;110:84-89)。
尽管有这些技术的进步,未来的进展非常受限于一个状况:来自于分离的人皮脂腺的脂肪细胞的大量培养非常困难。尤其是在培养时长期保存细胞材料非常困难。基于以上的原因,可以设想脂肪细胞通过细胞膜破裂趋于分化并且死亡,随后释放内容物。最好的结果是Fujie等获得的(《皮肤病研究档案》1996;288:703-708),他以Xia等(1989)的技术为基础分离了皮脂腺,利用分散细胞培养的方法,在无血清无细胞粘附层的角质形成细胞生长因子培养基中通过六次传代培养培养了脂肪细胞。
发明摘要
本发明的目的是提供可通过较高代次的传代培养而稳定培养的脂肪细胞。也就是说,从形态学、表型和功能特征的表象上来说,提供的脂肪细胞应接近活的正常的人脂肪细胞,至少是在这种程度上:即它们适合作为用于生理、病理生理和制药评价和研究的细胞材料或含脂、产皮脂的细胞,尤其脂肪细胞的细胞培养模型。
本发明目的通过提供永生化的脂肪细胞而实现。合适地,本发明的细胞来自于人,因为这是实际应用最感兴趣的。这种脂肪细胞存在于人皮脂腺细胞系SZ95中,其在德意志微生物保藏中心保藏,保藏号为DSM ACC2383。
优选的实施方案描述
本发明以下参考附图进行详细描述。图1和图2表明本发明提供的永生化的SZ95脂肪细胞保持了正常的原代脂肪细胞(本例中来自于人)的表皮的、多态的表观。而且,提供的永生化的脂肪细胞和其克隆(一个克隆意味着来自于单个细胞)表达典型的94kD大小的SV-40大T抗原,其用编码DNA序列进行了转染,在传代培养物中也是如此。图1表明(a)正常,第二次传代培养的脂肪细胞,本发明提供的永生化的脂肪细胞来源于此,(b)由初次传代培养来源的永生化的脂肪细胞提供的粘附脂肪细胞培养物,(c)提供的永生化的脂肪细胞(克隆的第50代传代培养物)。所有的细胞表现了相似的表皮的多态结构。图2显示了下述细胞的细胞离心样品,(a)提供的永生化的脂肪细胞,(b)作为阳性对照细胞的内皮细胞培养物细胞HMEC-1,两者均用SV-40大T抗原的单克隆抗体标记。两种样品均被阳性标记,且显示人SV-40大T抗原尤为集中在细胞核内表达,部分在细胞浆中表达。在(c)中,Western印迹分析说明了人SV-40大T抗原在提供的永生化的脂肪细胞中的表达。尽管人SV-40大T抗原在未转染的、正常的人脂肪细胞(第1道)和正常的人表皮角质形成细胞(第2道)中位检测到,但是特征性94kD大的蛋白质在第34代传代培养的永生化的脂肪细胞的蛋白提取物中检测到(第3道),在三个分离的克隆中也检测到(第4、5、6道)。
本发明的永生化的脂肪细胞是人来源的,术语“脂肪细胞”的含义具有很广泛的意义,即,涉及所有的或多或少含有脂肪或脂质以及产生皮脂的细胞。皮脂纯粹由不同的脂肪或脂质物质组成。由此,细胞的脂肪或脂质含量可在脂质物质成分和脂质物质成分的含量方面有所变化。通常但非必需地,细胞的脂肪或脂质物质含量包括游离的脂肪酸、甘油三酯、蜡、鲨烯、游离胆固醇、胆固醇酯、二羟基胆固醇和其他的类固醇及烃。那些来源于人皮脂腺细胞的永生化的脂肪细胞是特别优选的。假如脂肪细胞来自于人面部皮脂腺,就非常的适合医学用途。
本发明的脂肪细胞的基本特征是它们的永生化。本发明的永生化基本上意味着在多次传代培养中保持了细胞的重要特征。本发明的永生化脂肪细胞SZ95可保持培养4.5年、超过50次传代,而正常的人脂肪细胞仅生长3-6代便死亡。
本发明的永生化的脂肪细胞可利用与增殖抑制基因形成稳定的非活性复合物的DNA,转染人源的尤其是人皮脂腺的正常脂肪细胞而获得。本发明的一个非常成功的永生化是通过用包含编码SV-40大T抗原的DNA序列的DNA转染正常人脂肪细胞、特别是衍生自面部皮脂腺的脂肪细胞而实现。SV-40大T抗原(蛋白)的永生化效果和DNA编码序列转染人细胞的相应应用是已知的。这样,通过用编码大T抗原的DNA转染例如表皮来源的其它细胞(见Tohyama等,《实验医学杂志》1997;182:75-82;Bae等,《前列腺》1998;34:275-282),及内皮来源的细胞(见Ades等,《皮肤病学调查杂志》)从而获得永生化的细胞系。
利用阳离子脂(Lipofectin试剂,其是含阳离子脂DOTMA(1.2-二油酰氧基丙基-3-三甲基氯化铵)和DOPE[二油基磷脂酰乙醇胺]于膜过滤的水中[1mg/ml]的1∶1(w/w)脂质体配制品),通过细胞的胞吞作用经阳离子/DNA复合物摄入外源DNA(见Wang等,《体外细胞发育生物学》1991;27A:63-74;Staedel等,《皮肤病学调查杂志》,1994;102:768-772)的基因转移方法转染脂肪细胞后发现,永生化得到了很好的结果,而转染混合物中优选含另外0.25到2.0%(体积)、尤其是2.0%(体积)的Lipofectin试剂及0.05-0.5%(重量)、特别是0.5%(重量)的于合适的转染缓冲液中的外源DNA。外源DNA,如编码SV-40大T抗原蛋白的DNA,典型地插入一个合适的表达载体中,由此优选通过启动子和增强子序列而使蛋白质的表达水平增强。如果在优选的实施方案中,正常的人脂肪细胞用编码SV-40的大T抗原的DNA转染,则可以设预期脂肪细胞在成功的转染和永生化后会表达SV-40的大T抗原。利用针对SV-40大T抗原的单克隆抗体进行的免疫细胞化学和Western印迹分析确证了本发明提供的永生化脂肪细胞中大T抗原的表达。
这样得到的永生化的脂肪细胞优选地以细胞系的状态存在,这样可达到理想的应用目的。
本发明的永生化的脂肪细胞在适应无血清培养基后,其生长比未转染的、正常人脂肪细胞状态好,它们保持合成皮脂腺细胞特征性脂质的能力,这与在无血清培养中的未转染的正常的人脂肪细胞相反。根据本发明的永生化的脂肪细胞可作为持续更新和繁殖的细胞系并可在一定的培养基中生长。
根据本发明的永生化的脂肪细胞的特殊价值是它们具有未转染的、正常的和分化的脂肪细胞的形态、表型和功能方面的特征。这样,本发明的永生化的脂肪细胞可作为生理学、病理生理学和药理学研究的优秀模型。同时,人源的常规培养的脂肪细胞的有限的生存能力的不利因素也消除。因此,本发明提供的永生化的脂肪细胞能基本保持正常脂肪细胞的表型,在功能方面与未转染的正常人面部脂肪细胞相似。
根据本发明的永生化的脂肪细胞或脂肪细胞系表现出多态、表皮表观,与未转染的正常人脂肪细胞相似。细胞培养时,细胞大小与细胞内结构多样,后者表明了细胞成熟的不同阶段。因此,观察到不同大小的细胞,是融合生长的平均5-6倍,这与未转染的正常人脂肪细胞在体外的程序性分化(平均4-5.5倍大小差异)相符合。而且,根据本发明,永生化的脂肪细胞在细胞浆中富含脂肪物质或脂质颗粒,与未转染的正常人脂肪细胞相似。特征性皮脂腺的脂质鲨烯和蜡酯(正常的人脂肪细胞产生)的合成在本发明的实验中被证实。而且,本发明的永生化的脂肪细胞合成游离脂肪酸,同样与未转染的正常人脂肪细胞的体外发现相符合,即使多次传代培养也是如此。
而且,确证脂肪细胞来源,提示能存活的分化的表达标记也被证实是本发明的永生化的脂肪细胞及细胞系的脂肪细胞的典型标记。因此,永生化的脂肪细胞或脂肪细胞系表达人多形态表皮粘液蛋白团,如皮脂腺抗原、人奶脂球蛋白1和2、人表皮唾液粘蛋白、Thomsen-Friedenreich抗原、粘蛋白样致癌物相关抗原和表皮膜抗原。以上在本发明过程中利用免疫细胞化学和Western印迹的方法得到了确证。另外,本发明的永生化的脂肪细胞或脂肪细胞系表达角蛋白抗原,它在未转染的正常人脂肪细胞如亚类7、13和19很典型。该抗原表型说明了脂肪细胞来源和脂肪细胞的分化。
在功能方面,本发明的永生化的脂肪细胞或脂肪细胞系与未转染的正常人脂肪细胞相似。这样,本发明的永生化的脂肪细胞对雄激素如5α-双氢睾丸酮产生应答,体外增殖加强。而且,本发明的永生化的脂肪细胞或脂肪细胞系能通过类维生素A尤其是非芳香族类型的类维生素A(如13-顺式视黄酸、全反式视黄酸)改变它们的增殖。
本发明的优选的实施方案是永生化的优选地为人的脂肪细胞得到克隆。这是一个优势,因为永生化的脂肪细胞或由此产生的脂肪细胞系通过其独特的基因组基础很好地定义并特异地表征。克隆的永生化人脂肪细胞系可通过在足够长的培养管中逐渐稀释永生化的脂肪细胞直到每个培养管中仅从一个细胞重新开始细胞分裂而得到。可通过显微镜来观察和控制这一过程。
相应地,本发明中发现,与未转染的正常人脂肪细胞相比,本发明得到的永生化的人脂肪细胞或因此产生的脂肪细胞系基本保留了脂肪细胞的特征。这一点从特征鉴定和功能测定中证实。
具有本发明以上阐述的优点的一个脂肪细胞系,命名为SZ95,是皮脂腺细胞系,其保藏于DSMZ,保藏号为ACC2383。
因此,本发明的永生化的脂肪细胞或脂肪细胞系提供了有价值应用的非常好的可能性。一般地,本发明的脂肪细胞或脂肪细胞系可用于诊断、治疗和化妆品应用。特别是,上述的脂肪细胞或脂肪细胞系可以用于开发和研究含脂质的产生皮脂的细胞,尤其是人或动物皮脂腺细胞的生理学或病理生理学,和它们在皮肤的病理生理过程和皮肤病如痤疮疾病中的作用。在本发明的帮助下,可以研究痤疮和/或皮脂溢和/或其他疾病,尤其是在皮脂腺功能起作用或可能起作用的皮肤疾病的产生。本发明的产品将来可作为检测和评价抗痤疮化合物和/或抗皮脂溢化合物或治疗剂的优异模型,还可作为抗病、特别是皮脂腺功能相关的皮肤病的治疗剂。尤其是临床研究,如体外研究药物的药理特性研究中发挥作用。
本发明的脂肪细胞或脂肪细胞系的优点还包括可建立进一步的细胞培养系统。这包括开发简单或复杂的细胞培养系统。简单的细胞培养系统是二维单层或多层粘附培养物或非粘附培养物,其例如通过将前述的脂肪细胞加入到其他细胞中,或通过半透膜或非透膜培养而形成(Schartz等,《外科研究杂志》1998;76:79-85;Nackman等,《外科》1998;124:353-361)。复杂的细胞培养系统是三维的单层或多层培养物,其例如通过培养细胞形成球状体,在球状体上,在胶原或其他的胶状物材料或人工皮肤样结构上培养细胞(Korff和Augustin,《细胞生物学杂志》1998;143:1341-1352;Hamamoto等;《生物化学杂志》(东京)1998;24:972-979;Desoize等,《抗癌研究》1998;18:4147-4158;Hamilton《癌症通讯》1998;131:29-34;Niemann等,《细胞生物学杂志》1998;143:533-545;Awata等,《胃肠病学肝病学杂志》1998;13增刊:S55-61);Voura等,《显微研究技术》1998;43:265-275;Pipili-Synetos等,《不列颠药理杂志》1998;125:1252-1257;Vasile等,《组化和细胞化学杂志》1999;47:159-168;Michalopoulos等。《肝脏病学》1999;29:90-100;Trent和Kirsner,《国际临床医师杂志》1998;52:408-413;Fransson等《不列颠皮肤病学杂志》1998;139:598-604;Konstantinova等,《皮肤病学研究档案》1998;290:610-614;Black等,FASEB杂志,1998;12:1331-1340;Zhao等,《生物化学与生物物理研究通讯》1999;254:49-53)。
一个特别有利的应用涉及三维细胞集群的产生或者基于本发明脂肪细胞或脂肪细胞系的器官样结构的建立或重建。为此,单独使用脂肪细胞,但优选地也加入其它产生皮肤的细胞,尤其是角质形成细胞、成纤维细胞、黑素细胞、内皮细胞、朗罕氏细胞和/或来自毛囊的细胞。为产生三维细胞集群或器官样结构的建立或重建,首先提供一个含有胶原或其它胶状材料和/或部分非活性组织的支架,然后前面提到的细胞填充或敷于支架上。这个方法对于本领域熟练技术人员是基本知识(Trent和Kirsner,《国际临床医师杂志》1998;52:408-413;Fransson等《不列颠皮肤病学杂志》1998;139:598-604;Konstantinova等,《皮肤病学研究档案》1998;290:610-614;Black等,FASEB杂志,1998;12:1331-1340;Zhao等,《生物化学与生物物理研究通讯》1999;254:49-53)。由此产生一种“人工皮肤”或皮肤替代物,其为移植医学、对于受损伤皮肤如烧伤的重建或皮肤病损的治疗提供了很好的可能。借助于本发明,当本发明的脂肪细胞掺入其中时,这样的“人工皮肤”可合成足量的脂质/皮脂。
更进一步的应用领域涉及生产来自于本发明的脂肪细胞或脂肪细胞系的产品。这包括细胞物质如脂质、蛋白质、DNA和/或RNA的分离和纯化。由于细胞是永生化的,它们可以不断提供这些细胞物质。可从这些细胞中获得的特别合适的例子包括,局部药用的皮肤脂质、下面实施例3提到的与脂肪细胞表型特征关联的抗原蛋白、更进一步的质粒DNA或载体DNA的产生。质粒DNA或载体DNA是通过遗传工程产生的,这对于本领域熟练的技术人员是知道的。因此,可以得到诱导脂质产生的基因。尤其是用这样合适的质粒和载体,也包括病毒载体,其他的细胞或生物体可被改变或转染。
本发明参考如下非限制的实施例更详尽地解释。
实施例
如下描述的实施例,可应用如下具体的材料和方法。实施例不应当被理解为是限制性的。
细胞培养
如不另外说明,所有的细胞均于由改良的DMEM/HAM F12培养基(1∶1)(Biochrom公司,柏林,德国)组成的培养基中粘附培养,该培养基含2mM的N-乙酰-L-丙氨酰-L-谷氨酸,并加入10%热灭活的胎牛血清(FCS;Biochrom)和50μm/ml庆大霉素(Gibco公司,卡尔斯鲁厄,德国)。在含5%CO2的湿度气氛下于37℃培养。每2-3天换液。
正常人脂肪细胞的分离和培养
正常的脂肪细胞是从一个87岁的外科手术女性病人面部皮肤上分离的,Xia曾报道(皮肤病学观察杂志1989;93:315-321)。分离的皮脂腺在无滋养层的补加9ng/ml的表皮生长因子,9ng/ml的角质形成细胞生长因子(均得自Boehringer Mannheim,德国),0.4μg/ml氢化可的松(得自Sigma,Deisenhofen,德国),10-9M霍乱毒素(得自Calbiochem,Bad Soden,德国)的标准培养基中培养。原代正常脂肪细胞培养物作为皮脂腺小叶周围的生长晕而获得。
免疫细胞化学测定
次融合的正常脂肪细胞培养物的分散细胞经细胞离心吸附到玻璃片上。样品在空气中晾干后冰丙酮固定10分钟。样品随后在室温与相应的单克隆抗体或对照抗体孵育30分钟。加入1∶100稀释的兔抗鼠IgG(重链+轻链)和碱性磷酸酶/抗碱性磷酸酶复合物(Dianova,汉堡,德国)室温30分钟,检测结合的抗体。一抗和二抗在含10%RPMI-1640和10%胎牛血清(pH7.4)的溶液中稀释。用不含钙离子和镁离子的PBS缓冲液(得自Biochrom)洗涤三次。样品用Neufuchsin作为粘附试剂,萘酚盐作为偶联试剂,在缓冲液中染色30分钟,用Mayer’s Haemalum(Merck,达姆施塔特,德国)复染,覆盖并用光显微镜观察。
分离和定量蛋白
细胞培养物用PBS洗两次,在培养瓶中用冰冷的溶液(50mMHEPES,1%Nonidet P-40(ICN,Aurora,美国),150mM NaCl,蛋白酶抑制剂(CompleteTM Mini,宝灵曼公司))裂解,随后刮下并收集细胞转移至离心管分离细胞蛋白。得到的材料利用超声破碎搅均,离心后收集上清置于冰上。加入双辛可宁酸(BCA-蛋白分析,Pierce,Rochford,伊利诺伊斯州,美国)可见总蛋白,蛋白浓度通过测定550nm的吸收值来定量。
Western印迹分析
分离的蛋白等份(20μg)加热至95℃15分钟。一维SDS-PAGE电泳,使用7.4%浓度的胶。然后,利用标准的转印系统(伯乐公司,Munchen,德国)将蛋白转印到膜(PVDF Immobilon-P,Millipore公司,Eschbom,德国)上。印迹与第一抗体在室温孵育60分钟,然后分别和0.2μg/ml稀释的辣根过氧化酶缀合的羊抗鼠单抗和羊抗兔单抗(Oncogene Science)在室温孵育60分钟。彻底洗涤后,信号通过化学发光法用标准分析(ECL,Amersham公司,Braunschweig)显影在X光片(XAR 5,柯达公司,Rochester,纽约州,美国)上,其中不同的发光间隔可调。
油红和尼罗红染色
细胞生长在钳片中与0.6%的油红或60%的异丙醇孵育15-120分钟,或用1μg/ml的尼罗红(柯达公司)在室温染色15分钟,如Xia报道(1989)。培养物随后在光学显微镜下观察(油红染色后)或利用450-500nm波段发光>580nm的激发滤光片在荧光显微镜下观察。
流式细胞方法
分散的未标记的细胞利用常规的分选仪依据细胞大小判断,而标记尼罗红染料(柯达公司)的细胞利用流式细胞仪根据荧光来判断脂质含量。每个样品约测定10.000个细胞。
标记和脂质提取
细胞培养物在培养基中培养2天,然后通过[2-14C]醋酸钠(45-60mCi/mmol;杜邦公司,波士顿,美国)进行放射性脉冲,其在PRMI-1640中的浓度是0.5μCi/ml,培养基中含2mM的L-谷氨酸,10%热灭活的胎牛血清和100IU/ml的青霉素和100μg/ml的链霉素。孵育进一步持续24小时,脂质从培养的细胞和培养上清中分离,并分为中性脂质、脂肪酸和磷脂(见Seifert等,《皮肤病学调查杂志》1997;108:375)。
中性脂质和脂肪酸的大小分离组分可通过在20×10cm2硅胶覆盖的玻璃板(Merck公司,达姆施塔特,德国)上进行的高效薄层层析法(HPTLC)观察。玻璃板用正己烷预处理,晾干24小时。样品利用自动的脂质加样器(Linomat IV;Camag,柏林,德国)加样。中性脂质层析谱在正己烷-二乙醚溶液(9∶1)中显色在9厘米,晾干,在氯仿/二乙醚,乙酸乙酯(80∶4∶16)的溶液中显色后在4.5厘米。利用发光片(TR 2040S,富士公司,东京,日本)发光,用图象分析仪(BAS 1000生物成像分析仪,富士)扫描。脂质标准作为对照样品。
生长行为测定
细胞接种到96孔板,浓度为0.5-4×103/孔。细胞增殖利用4-甲基umbelliferyl庚酸酯荧光分析自动测定(Zouboulis等,《黑素瘤研究》1991;1:91-95)。
至此,在测定时,倒掉培养基,细胞用PBS洗两次,每孔加入100μl的稀释于PBS的100μg/ml 4-甲基umbelliferyl庚酸酯溶液(Sigma,Deisenhofen,德国)。培养板在37℃放置30分钟,利用合适的荧光测量仪器(Titertec-Fluoroscan II;Flow,Meckenheim,德国)测定发射的荧光。在355nm激发和460nm发射滤片获得荧光单位。
用5α-二氢睾丸酮和类维生素A处理
将5α-二氢睾丸酮(Sigma)溶于二甲基亚砜,然后溶于无血清无酚的含2mM的N-乙酰-L-丙氨酰-L-谷氨酸的改良的DMED/Ham’s F12培养基(1∶1)(Gibco-BRL)中,该培养基补加5ng/ml的EGF,50μg/ml的牛垂体提取物,1mg/ml无脂肪酸的牛血清白蛋白(宝灵曼)和50μg/ml庆大霉素,从而5α-二氢睾丸酮终浓度为10-6M和二甲基亚砜为0.1%。用0.1%二甲基亚砜作为对照组。细胞(0.5-2.0×103/孔)用5α-二氢睾丸酮处理18天。
对于类维生素A处理,将全反式视黄酸、13-顺式视黄酸和acitretin溶于DMSO,随后置于无血清的含2nM N-乙酰-L-丙氨酰-L-谷氨酸的改良DMEM/Ham’s F12培养基(1∶1)中,该培养基补加5ng/ml的EGF,50μg/ml的牛垂体提取物,1mg/ml无脂肪酸的牛血清白蛋白(宝灵曼)和50μg/ml庆大霉素,从而类维生素A终浓度为10-7M和二甲基亚砜为0.1%。用0.1%二甲基亚砜作为对照组。类维生素A在暗琥珀色光下操作。细胞(0.5-1×103/孔)用类维生素A处理9天。
统计学分析
生长研究在96孔板上做6次重复。所有的其他实验做3次。
实施例1
正常人脂肪细胞转染
转染正常人脂肪细胞所用载体为pSVT,其是以pBR322为基础构建的,其包含编码SV-40大T抗原的序列,蛋白表达由劳斯肉瘤病毒长末端重复序列所控制(见Dutt,《癌基因》,1990;5:195-200;Wang等,《体外细胞发育生物学》,1991;27A:63-74)。第二次传代培养的人脂肪细胞培养物在35mm的培养皿(Becton Dickinson,Plymouth,UK)中长到50%融合时用于转染。转染以阳离子脂质体基因转染法为基础。为此,使用LIPOFECTIN试剂,其含有阳离子脂质DOTMA(1,2-二油酰氧基丙基-3-三甲基氯化铵)和DOPE[二油基磷脂酰乙醇胺]于膜过滤的水中[1mg/ml]中的1∶1(w/w)脂质体配制品)。至此,弃掉培养基,培养的细胞用无血清培养基(Opti-MEM,来自Gibco-BRL)洗2次,在此培养基中孵育4小时。然后培养基以转染混合物代替,所述混合物由不含抗生素量的Opti-MEM培养基(1.5ml)和适量的LIPOFECTIN试剂(Gibco-BRL;5-30μl,最优选1.5%(体积))及适量的溶于0.5ml PBS的pSVT DNA(1-10μg,DNA终浓度最优选为0.5%(wt))组成。培养物在含5%CO2的湿度环境于37℃孵育24小时。培养物用培养基洗2次,然后在上述的脂肪细胞培养基中培养。
转染处理后,在4周过程中观察到pSVT处理的脂肪细胞的存活性急剧降低。但是用最佳量的LIPOFECTIN试剂和pSVT DNA,增殖的脂肪细胞集落出现。这些细胞(SZ95)能够传代至今超过50次。它们在4.5年的观察期间始终存活。
实施例2
永生化的人脂肪细胞克隆
将如此的永生化的人脂肪细胞SZ95系列稀释接种到96孔板中,细胞数由1×102为第一组,直到最后一组为0个细胞(Zouboulis等,“皮肤恶性黑素瘤”,CE Orfanos和C.Garbe编辑,Muckschwerdt,Munchen,德国,1990;158-168)。细胞在加入5mg/ml EGF和3ng/mlKGF的标准培养基中培养。显微镜下观察,每孔由单个细胞长成的细胞认为是克隆。这样,得到了SZ95克隆细胞。
实施例3
永生化的脂肪细胞的鉴定
检测SV-40大T抗原
永生化的脂肪细胞中,利用免疫细胞化学和Western印迹法,采用鼠血清来源的抗大T抗原的单克隆抗体(Oncogene Science,剑桥,美国)检测了SV-40大T抗原的表达,所述单克隆抗体1∶1000稀释用于免疫细胞化学分析,1∶100稀释用于Western印迹分析。人正常表皮角质形成细胞,皮肤成纤维细胞和作为阳性对照的内皮细胞HMEC-1(被SV-40大T抗原永生化,见WO-A-992/175 69)作为参比。
根据实施例1,利用大T抗原的单抗进行的永生化的脂肪细胞的免疫细胞化学实验的结果发现大多在细胞核,部分在细胞浆有明显的染色(见图2a)。正常的角质形成细胞和成纤维细胞一致为大T抗原阴性,HMEC-1细胞作为阳性对照则在细胞核,部分细胞浆中有SV-40大T抗原的染色(图2b)。
图2c显示了在未转染的正常人SV-40大T抗原的Western印迹的分析结果(第1道),及正常人表皮角质形成细胞(第2道)和本发明的永生化的脂肪细胞(第34代传代培养,第3道),及本发明的不同克隆脂肪细胞(4、5、6道)。94kD带被确认是永生化的脂肪细胞和它的克隆中表达的SV-40大T抗原(见Harlow等,《病毒学杂志》,1981;39:861-869)。
本发明的永生化的脂肪细胞的表型鉴定
根据实施例1,永生化的脂肪细胞SZ95形态学是表皮的,表现出多形态性,细胞大小不同,在细胞浆中有很多小滴(见图1b和1c)。
用相关的抗体进行的本发明永生化脂肪细胞的免疫细胞化学实验结果显示,与正常表皮角质形成细胞没有染色相比,检测到皮脂腺抗原的染色和表达(见图3)。图3显示免疫细胞化学结果,(a)本发明的永生化的脂肪细胞,(b)正常人表皮角质形成细胞。制备物用抗皮脂腺抗原的单克隆抗体染色。本发明的永生化的脂肪细胞显示胞浆染色呈阳性,正常人表皮角质形成细胞没有染色。
另外,利用Western印迹检测到永生化的脂肪细胞中,角蛋白7、13和19和人多态表皮粘蛋白组的各种蛋白的表达,而人角质形成细胞只表达角蛋白13(见图4)。根据图4,在Western印迹分析中,本发明的永生化的脂肪细胞(第34代培养物,第1道),本发明的各种克隆的永生化的脂肪细胞(第2、3、4道)和正常人表皮角质形成细胞(第5道)的总蛋白提取物用来检测人表皮唾液粘蛋白(>300kD),人乳脂球蛋白(HMFG-1)(400kD),人乳脂球蛋白(HMFG-2)(80-400kD),粘蛋白型癌关联蛋白抗原(MCA)(350kD),表皮膜蛋白(EMA)(250-400kD),Thomsen-Friedenreich抗原(TF抗原)(155kD),角蛋白7(54kD),角蛋白13(54kD),角蛋白19(40kD),5α-还原酶I型(21-27kD)。本发明的永生化的细胞系和它的克隆均表达所测的蛋白,而角质形成细胞只表达角蛋白13和5α-还原酶I型。
脂质合成
用尼罗红染色和荧光显微镜观察表明细胞浆内有脂质存在。本发明的永生化的脂肪细胞SZ95(图5)用尼罗红荧光染色,中性脂质着色,结果单个或成团脂质小团随机分散在脂肪细胞的胞浆中,尼罗红染色荧光细胞计数实验结果表明,本发明的永生化的脂肪细胞的内容物由有血清培养基中每细胞510荧光单位(中间值)下降至无血清培养中的每个细胞429个荧光单位。本发明的永生化的脂肪细胞(实施例1)合成多种中性脂质部分,包括典型的皮脂腺脂质鲨烯、蜡酯、甘油三酯、胆固醇、胆固醇酯、甘油二酯、羊毛甾醇和游离脂肪酸。25-40传代培养物均观察到这一点。图6示出了结果,其利用放射性测量成像评价永生的和克隆的脂肪细胞培养物(见第3、4道和5道);放射性标记醋酸钠后脉冲记录测定了HPLC分离的脂质。第3、4、5道显示,细胞合成多种中性脂质部分,包括鲨烯、蜡酯、甘油三酯、胆固醇、胆固醇酯、甘油二酯、羊毛甾醇和游离脂肪酸。在细胞外上清中所有的中性脂质被发现,但含量较少(见6道)。为进行比较,采用1道脂质标准,2道人皮脂、4道游离脂肪酸。作为进一步比较,7道和8道表明角质形成细胞的结果。7道表明细胞中胆固醇和甘油三酯为主,而上清(8道)主要是胆甾醇。
增殖研究
本发明的永生化的脂肪细胞对数生长曲线在正常培养状态下观察到,细胞群倍增时间是52.4+1.6,而与最初的细胞培养物密度无关。图7显示一个永生化的克隆脂肪细胞系SZ95在18天内在脂肪细胞培养基中的增殖情况。
永生化的脂肪细胞在加入无血清培养基后增殖下降,但加入5α-DHT后,增殖恢复正常。图8显示了这样一个脂肪细胞克隆的例子,在无血清培养基(对照)以及加入10-6M的5α-DHT无血清培养基中观察细胞增殖(接种2000/孔)18天。在第8天后,5α-DHT明显增加细胞增殖,所测定的细胞群倍增时间是136小时(对照)和53.7小时(5α-DHT处理的细胞,*,p<0.05;**,p<0.01)。
类维生素A对永生化细胞的影响显示出增殖行为的分化应答。尽管一些克隆表现为类维生素A的增殖抑制(典型的顺序是13-顺式视黄酸>全反式视黄酸>>acitretin),但其他的克隆增殖被促进(如被全反式视黄酸和13-顺式视黄酸促进),与正常人表皮角质形成细胞增殖应答相应。图9展示了这一点(*,<0.05;**,p<0.01;***,p<0.001)。
Claims (11)
1.人脂肪细胞系DSM ACC2383。
2.权利要求1的人脂肪细胞系在制备诊断、治疗或化妆品制剂中的应用。
3.权利要求1的人脂肪细胞系在制备检查人或动物皮脂腺的生理或病理生理学的药物或制剂中的应用。
4.权利要求1的人脂肪细胞系在制备检查痤疮和/或皮脂溢性皮炎和/或其它皮肤疾病的起因的药物或制剂中的应用,其中其它皮肤疾病是其中皮脂腺功能是起作用的因素的皮肤病。
5.权利要求1的人脂肪细胞系在检测抗痤疮和/或抗皮脂溢的化合物或药剂中的应用。
6.权利要求1的人脂肪细胞系在检测抗疾病的化合物或药剂中的应用。
7.权利要求6的应用,其中所述疾病是其中皮脂腺功能是起作用的因素的皮肤病。
8.权利要求1的人脂肪细胞系在开发简单的或复杂的细胞培养系统中的应用。
9.权利要求1的人脂肪细胞系在形成或用于三维细胞集群或器官型结构的构建中的应用。
10.权利要求1的人脂肪细胞系在制备来自细胞的产品中的应用,其中所述细胞为来源于人脂肪细胞系的细胞。
11.权利要求10的应用,所述来自细胞的产品为脂质、质粒、载体、细胞表达的蛋白质和/或所述蛋白质的DNA或RNA序列。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992017569A1 (en) * | 1991-04-04 | 1992-10-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immortalization of endothelial cells |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992017569A1 (en) * | 1991-04-04 | 1992-10-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immortalization of endothelial cells |
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US20160237406A1 (en) | 2016-08-18 |
DK1151082T3 (da) | 2006-04-10 |
WO2000046353A1 (de) | 2000-08-10 |
HUP0200048A2 (hu) | 2002-05-29 |
US20140120569A1 (en) | 2014-05-01 |
EP1151082B1 (de) | 2006-03-08 |
DE19903920B4 (de) | 2005-08-25 |
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