CN1166773C - 分离的人脂肪组织衍生的基质细胞 - Google Patents

分离的人脂肪组织衍生的基质细胞 Download PDF

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CN1166773C
CN1166773C CNB988117940A CN98811794A CN1166773C CN 1166773 C CN1166773 C CN 1166773C CN B988117940 A CNB988117940 A CN B988117940A CN 98811794 A CN98811794 A CN 98811794A CN 1166773 C CN1166773 C CN 1166773C
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Y-D��C��������ɭ
Y-D·C·哈尔沃森
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Abstract

本发明提供了使来源于脂肪组织的基质细胞分化为具有成骨细胞性质之细胞的方法和组合物，以及改善受治疗者骨骼结构的方法。这些方法包括在β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸中将来源于脂肪组织的基质细胞培养足够长时间以使所述细胞分化为成骨细胞。所述方法和组合物可应用于在外科手术和损伤部位产生自体移植入骨所需的成骨细胞。所述组合物包括脂肪基质细胞、能支持成纤维细胞生长的培养基以及一定量的足够所说的诱导基质细胞分化为成骨细胞的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸。本发明进一步提供了鉴定影响成骨细胞分化的化合物的方法。所述化合物可用于研究骨骼发育和治疗包括骨折和骨质疏松症在内的骨疾病的治疗。

Description

分离的人脂肪组织衍生的基质细胞
发明领域
本发明涉及使来自脂肪组织的基质细胞分化为成骨细胞的方法和组合物，及其应用。
发明背景
在美国每年有大约150万例的骨折归咎于骨质疏松症，其中大约30万是臀部骨折。臀部骨折50-75％的患者不能独立生活，导致看护费用的提高。骨质疏松症的特点是随着人们变老，骨质密度的降低超乎寻常。这种疾病在西方和亚洲发病率很高(＞30％的60岁以上妇女)，并且寿命越长患者越多。虽然还不知道导致这种骨骼修复紊乱的真正原因，但很明显骨骼重建的动态过程被一个以骨质形成(骨质形成细胞)活力降低而骨质退化(骨质退化细胞)活力增加为特点的过程所破坏(Parfitt(1992)三角形(Triangle)31：99-110；Parfitt(1992)骨骼，卷1，B.K.Hall编.Teleford Press and CRC Press，Boca Raton，FL，351-429页)。
使用骨髓移植物是矫形术、神经外科和牙科，以及塑造/重塑外科中的常规作法，并且在过去二十年中这种应用越来越频繁。除血液之外，骨骼是最经常被移植的组织，在美国，每年估计使用50万骨骼移植物。在普通矫形术中使用骨骼移植物包括处理不连合性和急性长骨骨折，关节重建以及在治疗多种脊髓异常中促进脊椎运动部分的融合(Lane(1987)Ortho Clin N Amer 18：213-225)。
目前临床上最能接受的移植材料是自体骨骼。所谓的自体移植材料通常是从第二个手术部位得到的。自体移植存在重要的问题。这些问题包括对于大的伤口和损伤而言缺乏足够的移植材料。患有骨质疏松症或骨质减少的年长者使用自体移植也成问题。与获取移植材料的手术相关的继发性发病率是很高的。这些并发症包括感染、骨盆不稳(通常在髂骨的顶端获取骨骼)、血肿和骨盆骨折(Laurie等(1984)Plas Rec Surg73：933-938；Summers等(1989)J Bone Joint Surg 71B：677-680；Younger等(1989)矫形术和外伤杂志3：192-195；Kurz等(1989)脊柱14：1324-1331)。另外，供体部位的慢性疼痛是第二种最经常报道的并发症(Turner等(1992)JAMA 268：907-911)。最后，由于骨骼材料的坚硬特性使自体移植物能配合损伤/伤口部位的形状的能力受到限制。
最近的研究集中在各种基质的使用上，所述基质或是无机材料如羟磷灰石(Flatley等(1983)Clin Orthop Rel Res 179：246-252；Shima等(1979)神经手术杂志51：533-538；Whitehill等(1985)脊柱10：32-41；Herron等(1989)脊柱14：496-500；Cook等(1986)脊柱10：305-309；各文章内容此处引作参考)或是有机材料如脱矿质的骨基质(DBM)(综述见Ashay等(1995)美国矫形术杂志24：752-761；该文内容此处引作参考)。这些基质被认为是骨质传导性的(促进骨质形成细胞在惰性基质中的入侵)或骨质诱导性的(诱导重新补充的前体细胞转化为成骨细胞)。食品和药品管理局批准的一些用于临床的产品在临床上已观察到许多成功的结果。尽管获得成功，使用这些基质仍存在很多问题。首先是受治疗者对DBM不同程度的反应。而且这些基质比自体骨骼移植需要更长的时间来形成重要的结构整体性和有效地承受负载。
移植物和使用单一基质的替代品是使用骨髓或骨髓基质细胞与DBM的混合物。较为理想的是细胞和DBM来源于同一受治疗者，尽管异源DBM也开始在临床上取得成功(Mulliken(1981)外科年鉴194：366-372；Kaban等(1982)J Oral Maxillofac Surg 40：623-626)。使用自体骨髓细胞与异源DBM的移植方法已经取得很好的结果(Connolly(1995)临床矫形术313：8-18)。但是，阻碍这些技术广泛使用的问题包括非自身物质污染的潜在可能、患者对捐献骨髓的可接受性，以及抽出骨髓后引起的潜在并发症和骨髓来源耗竭。
许多研究小组已经证明骨髓基质细胞和由此衍生的细胞系能够分化为在生化性质和形态上类似于成骨细胞的细胞(Dorheim等，(1993)细胞生理学杂志154：317-328；Grigoriadis等，(1988)细胞生物学杂志106：2139-2151；Benayahu等(1991)Calcif Tiss Int.49：202-207；文献内容引作参考)。在多数情况下，从人或动物的骨髓中分离类成纤维细胞并将这些细胞铺在标准的组织培养器上。一般来说，使用标准培养基配方，如添加10-20％胎牛血清和抗生素的Dulbecco改进的Eagle培养基(DMEM)来富集这些细胞(Ashton等(1980)临床矫形术151：294-307；Sonis等(1983)口腔医学杂志3：117-120)。然后通过将培养基换为含有5-20％胎牛血清、2-20mMβ-甘油磷酸和20-75μM抗坏血酸或抗坏血酸-2-磷酸的培养基来刺激细胞分化为成骨细胞(Asahina等(1996)实验细胞研究222：38-47；Yamaguchi等(1991)Calcif Tissue Int 49：221-225；文献内容此处引作参考)。培养14-21天后，许多细胞型和细胞系将矿化培养器中的基质，这由von Kossa染色为阳性可得到证实。成骨细胞谱系的其他表型指示包括分泌碱性磷酸酶活力的升高；培养基中存在分泌的骨钙蛋白；一些被认为是在成骨细胞中特异表达的基因的表达水平提高，包括骨钙蛋白，骨桥蛋白和骨涎蛋白(Stein等(1990)FASEB杂志4：3111-3123；Dorheim等(1993)细胞生理学杂志154：317-328；Asahina等(1996)实验细胞研究222：38-47；Yamaguchi等(1991)Calcif TissueInt 49：221-225)。
一些实验室所进行的许多详细研究证实被移植的骨髓基质细胞能够形成异位骨骼(Gundle等(1995)骨骼16：597-603；Haynesworth等(1992)骨骼13：81-89；Boynton等(1996)骨骼18：321-329)。例如，人和鼠的骨髓基质成纤维细胞已被移植到免疫缺陷的SCID小鼠中(Krebsbach等(1997)移植63：1059-1069；Kuzneysov等(1997)J BoneMin Res 12：1335-1347)。使用抗体和组织化学标记证明存在诱导性基质时，供体的骨髓基质细胞导致在异位骨骼形成的部位上发展出新的成骨细胞。有羟磷灰石/磷酸三钙颗粒(HA/TCP)、明胶、多聚-L-赖氨酸和胶原蛋白的情况下， 小鼠的(骨髓基质)细胞形成骨骼。与此不同的是，人的基质细胞只在有HA/TCP时才能有效地形成骨骼。在这些研究中不需要外源的BMP。
骨的形成并不限于骨架。例如，如果肌肉间、肾下囊或皮下等部位同时表达骨骼形态发生蛋白，在这些部位引入陶瓷的或脱矿化的骨基质将导致骨骼的形成。(Urist等(1996)科学150：893-899)。这些结果暗示在合适的环境条件下，这些组织的细胞具有某些形成骨骼前体细胞的能力。
在软组织如脂肪中的异位骨骼形成是一种罕见的病理状况，它发现于患有进行性纤维骨化—一种遗传疾病的患者中。虽然并不完全了解其病原学，该病部分是由于位于软组织损伤部位的淋巴细胞异常表达BMP所引起的(Kaplan等(1997)J Bone Min Res 12：855；Shafritz等(1996)N Eng J Med 335：555-561)。在脂瘤中也会偶尔发现骨骼形成(Katzer(1989)Path Res Pract 184：437-443)。
有证据表明分离自经胶原酶处理后的脂肪组织的基质—血管部分含有大量的前脂肪细胞，或倾向于分化为脂肪细胞的细胞(Hauner等(1989)临床研究杂志34：1663-1670)。这些细胞能以较低的频率自发地分化为脂肪细胞或能对产脂肪促进剂如四氢噻唑二酮产生应答从而提高分化频率(Halvorsen(1997)等方法11：58-60；Digby(1997)糖尿病4：138-141)。有证据表明在这些细胞系之间基质细胞呈现出一种互相分化的模式(Gimble等(1996)骨骼19：421-428；Bennett等(1991)细胞科学杂志99：131-139；Beresford等(1992)细胞科学杂志102：341-351)。具体来说，脂肪形成伴有骨生成可能性的降低，而骨生成伴随脂肪形成可能性的降低。
在某些条件下，骨髓基质细胞能分化为脂肪细胞。事实上，就这一能力，一些骨骼基质细胞系已经广泛地得到表征(Gimble等(1990)欧洲免疫学杂志20：379-387；Gimble等(1992)细胞生物化学杂志50：73-82；Gimble等(1996)骨骼19：421-428)。但是，在本发明之前，使从脂肪组织分离的基质细胞能分化为成骨细胞仍是未知的。
                         发明概述
本发明提供了使脂肪组织的基质细胞分化为具有成骨性质的细胞的方法和组合物，以及改善受治疗者骨骼结构的方法。所述方法包括将脂肪组织的基质细胞在β-甘油磷酸、抗坏血酸和抗坏血酸-2-磷酸中培养足够长时间以诱导所述细胞分化为成骨细胞。这种方法和组合物可用于产生在外科手术部位或伤口进行自体移植到骨骼中的成骨细胞。组合物中包含脂肪基质细胞，以及能支持成纤维细胞生长的培养基和一定量足够诱导分化的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸。
本发明进一步提供了鉴定影响成骨细胞分化的化合物的方法，所述化合物可用于研究骨骼发育和治疗骨疾病，其中包括骨折和骨质疏松症。
                        附图简述
图1显示了经成骨细胞分化培养基处理后，被诱导分化为成骨细胞的人脂肪基质细胞所发生的形态变化。
图2显示了经成骨细胞分化培养基或脂肪细胞分化培养基处理后的脂肪基质细胞和用油红O(图2A和图2C)或von Kossa方法(图2B和图2C)染色后的脂肪基质细胞。
图3显示了经基质细胞培养基(PA)、脂肪细胞培养基(AD)或成骨细胞培养基(DST)处理的脂肪基质细胞，分泌到培养基中的骨钙蛋白和leptin浓度随时间的变化。
图4显示了在基质细胞培养基(SC)、脂肪细胞分化培养基(AD)或成骨细胞分化培养基(OST)中培养21天后的脂肪基质细胞分泌的碱性磷酸酶的浓度。
                         发明详述
本发明提供了使脂肪基质细胞分化为成骨细胞的方法。由本发明所述方法产生的成骨细胞可用来提供用于研究或移植到受治疗者损伤或骨折部位的骨中的成骨细胞。因此，在一个方面，本发明提供了使脂肪基质细胞分化为成骨细胞的方法，其中包括：在一种组合物中培养所述细胞，该组合物包括能支持成骨细胞生长的培养基和一定量足够诱导分化的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸。
在另一方面，本发明提供了使脂肪基质细胞分化为成骨细胞的组合物。所述组合物包括：脂肪基质细胞，能支持成纤维细胞生长的培养基和足够量的β-甘油磷酸和抗坏血酸或抗坏血酸-2-磷酸以诱导所述基质细胞分化为成骨细胞。
“脂肪基质细胞”指来源于脂肪组织的基质细胞(也可称之为脂肪组织衍生的基质细胞)。“脂肪”意味着任何脂肪组织。脂肪组织可以是棕色或白色的脂肪组织。优选脂肪是皮下白色的脂肪组织。这些细胞可以包括原代细胞培养物或一个永生化的细胞系。脂肪组织可以来源于任何有脂肪组织的生物。优选哺乳动物的脂肪组织，最优选人的脂肪组织。人脂肪组织的一个方便来源是吸脂外科手术，但脂肪组织的来源或分离的方法对本发明并不重要。如果希望成骨细胞用于自体移植，应从受治疗者身上分离脂肪组织。
“一定量足够诱导分化的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸”指β-甘油磷酸和(抗坏血酸和/或抗坏血酸-2-磷酸)的浓度，当补充于能支持成纤维细胞(如NIH-3T3细胞、人类脂肪基质细胞以及类似细胞)生长的培养基中时，经5天到8周的培养，将诱导所述基质细胞分化为成骨细胞。处理的最佳浓度和时间将由操作者利用已知的分化成骨细胞的检测方法来确定。这样的检测方法包括，但不限于形态和生化特性的检测(如分泌的骨钙蛋白或其它成骨细胞特异性蛋白或RNA)。
抗坏血酸和/或抗坏血酸-2-磷酸的浓度指这些化合物的混合浓度，即所述浓度的总和。例如，“50μM的抗坏血酸和/或抗坏血酸-2-磷酸”的定义包括，但不限于这样的排列组合：50μM的抗坏血酸；50μM的抗坏血酸-2-磷酸；10μM的抗坏血酸和40μM的抗坏血酸-2-磷酸；或40μM的抗坏血酸和10μM的抗坏血酸-2-磷酸。
优选，培养基含有大约2-20mMβ-甘油磷酸和大约20-75μM的抗坏血酸和/或抗坏血酸-2-磷酸。更优选，培养基包含5-15mMβ-甘油磷酸和大约40-60μM的抗坏血酸和/或抗坏血酸-2-磷酸。最优选，培养基包含10mMβ甘油磷酸和大约50μM抗坏血酸和/或抗坏血酸-2-磷酸。
可以使用任何能支持成纤维细胞在细胞培养基中生长的培养基。支持成纤维细胞生长的培养基配方包括Dulbecco改进的Eagle培养基(DMEM)，α-改进的极限必需培养基(αMEM)和必需的基本培养基(BME)以及类似的培养基。通常，要向以上培养基中加入5-20％胎牛血清(FCS)以支持成纤维细胞的生长。但如果在FCS中对成纤维细胞生长所必须的成分已得到确定并补充到生长培养基中，也可以使用确定成分培养基。
可用于本发明所述方法的培养基可以含有一种或多种感兴趣的化合物，包括，但不限于，抗生素，骨诱导性、骨传导性或促进生长或分化的化合物，例如骨骼形态发生蛋白或其它生长因子。骨骼形态发生蛋白的例子包括，但不限于生骨蛋白-1，BMP-5，成骨素，骨骼诱导因子和骨骼形态发生蛋白-4(Asahina等(1996)实验细胞研究222：38-47；Takuwa(1991)生物化学和生物物理研究通讯174：96-101；Chen(1991)JBone Min Res 6：1387-1390；Sampath(1992)生物化学学报267：20352-20362；Wozney等(1988)科学242：1528-1534，文献此处引作参考)，以及类似的物质。
优选地，脂肪组织经过处理从而使基质细胞彼此解离并同其它类型的细胞分离，除去沉淀的血液成份。通常，可以用蛋白水解酶，比如胶原酶和/或胰蛋白酶以及能螯合钙离子的试剂来处理脂肪组织可以使其解离为单个可存活的细胞。然后通过各种本领域技术人员熟知的方法，如差速离心，荧光激发细胞分选术，亲和层析以及类似的方法可以部分或全部地纯化基质细胞。然后在经含有β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的培养基处理前，将部分或全部纯化的基质细胞在支持成纤维细胞生长的培养基中培养8个小时到5个细胞传代时间。
将基质细胞在含有β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的培养基中培养足够长的时间以诱导其分化为成骨细胞。为使得基质细胞分化为成骨细胞，用β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸处理时间的长短取决于多种因素。这些因素包括，但不限于，所用β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的浓度，所用培养基，脂肪组织或基质细胞的来源，铺板的起始密度，生长因子或骨骼形态发生蛋白质存在与否等。操作者可以优化β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的浓度以及其它条件和因子。通过测量已分化为成骨细胞的细胞百分比可以确定最佳的浓度和处理次数。通过本领域技术人员了解的形态学和生化检测和指数可以监测上述百分比。这样的检测和指数包括，但不限于对形态和生化特性检测，比如钙沉淀物或成骨细胞特异性蛋白质或RNA的存在、von Kossa染色、骨钙蛋白的分泌和碱性磷酸酶的分泌。
来源于脂肪组织基质细胞的成骨细胞可以导入人或动物受治疗者的外科手术或骨折部位。将成骨细胞导入骨骼可用于治疗骨折和骨疾病，包括骨质疏松症。因此，在另外一个方面，本发明着眼于改善受治疗者骨结构的方法，包括：
a)在含有能支持成纤维细胞生长的培养基和一定量足够诱导分化的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的组合物中培养来自脂肪组织的基质细胞。
b)将所述成骨细胞导入所述受治者的外科手术或骨折部位。
优选基质细胞分离自将接受分化的成骨细胞的受治疗者的脂肪组织，但也可以使用分离自与受治疗者同种或异种的生物的基质细胞。受治疗者可以是有骨组织的任何生物。优选的受治疗者是哺乳动物，最优选的受治疗者是人。
在移植到受治疗者的外科手术或骨折部位之前，可以用目的核酸来稳定或瞬时转化基质细胞或成骨细胞。目的核酸序列包括，但不限于能编码提高成骨细胞生长，分化和/或矿化的产物的基因。例如，为治疗没有愈合的骨折或骨质疏松症，可以将骨骼形态发生蛋白4的表达系统以稳定或瞬时的方式转化到前脂肪细胞中。转化基质细胞和成骨细胞的方法是本领域技术人员已知的，将成骨细胞移植到骨骼外科手术或骨折部位上的方法也同样为人所周知。
可以将成骨细胞单独或以它与有益于骨骼伤口和损伤的修复的组合物形成的混合物的形成导入。这些组合物包括，但不限于，骨骼形态发生蛋白，羟磷灰石/磷酸三钙颗粒(HA/TCP)，明胶，多聚-L-赖氨酸，和胶原。例如，可以将从脂肪基质细胞分化而来的成骨细胞与DMB或其它基质组合以使该复合材料具有骨质生成性(自身产生骨骼)和骨质诱导性。用自体骨髓细胞与同种异体的DMB组合的类似方法也产生了很好的结果(Connolly(1995)临床矫形术318：8-18)。
本发明另外一个目的是为鉴定和研究能促使基质细胞分化为成骨细胞的化合物提供方法。能提高成骨细胞的分化的化合物在各种骨疾病，包括骨折和骨质疏松症的治疗中可能有一定作用。另外，发现能诱导成骨细胞分化的化合物可应用于体外或体内使细胞分化。相反地，发现能阻碍成骨细胞分化的化合物或因子可应用于某些疾病状态，其中特应性骨产生，比如Paget’s病或成软骨组织变形，可以利用这些化合物进行治疗。因此，在另一个方面，本发明着眼于鉴定影响成骨细胞分化的化合物的方法，包括：
a)在存在或缺乏被检测其对成骨细胞分化之影响的化合物的情况下，在含有能支持成纤维细胞生长的培养基和一定量足够诱导分化的β-甘油磷酸和抗坏血酸和/或抗坏血酸-2-磷酸的组合物中培养脂肪基质细胞；以及
b)比较存在或缺乏所述化合物时培养的细胞的成骨细胞分化情况。
可以对任何化合物测试其影响基质细胞分化为成骨细胞的能力。与待测化合物相容的载体是本领域技术人员已知的，并且可以在《Remington’s制药科学》(此处引作参考)的现行版本中找到。
将检测结果与使用已知分化促进剂的结果进行比较，比如产骨蛋白1和骨质形态发生蛋白-4(Asahina等(1996)实验细胞研究222：38-47；Takawa(1991)supra；Chen(1991)supra；Sampath(1992)Supra；Wozney等(1988)科学242：1528-1534)，已知这些物质通过提高成骨细胞标记物(比如骨钙蛋白—一种成骨细胞功能的明确标记物)的表达来促进成骨细胞的分化(Celeste(1986)美国科学院学报87：9843-9872；Stein等(1990)FASEB杂志4：3111-3123)。并且也可以将这些检测的结果与已知的成骨细胞分化抑制剂(比如α-TNF)进行比较，这些抑制剂能全部或部分阻断基质细胞向成骨细胞的转变。
参考下面的实施例可以更清楚地理解本发明的特点和有利之处，但这些实施例并非用来限制本发明。
                      实施例
                      实施例1
              从人的脂肪组织中分离基质
根据Rodbell(1964，生物化学杂志239：275)和Hauner等(1989，临床研究杂志)描述的方法从人类脂肪组织中分离基质细胞。简要地说，通过吸脂术分离来自人皮下储存的脂肪组织。然后将脂肪组织从吸脂杯中转移到500ml无菌的烧杯中，并沉降大约10分钟。吸去沉淀的血。将125ml体积(或更少)的脂肪组织转移到250ml离心管中，然后在离心管中加入Krebs-Ringer缓冲液。组织和缓冲液放置沉降3分钟或直到产生清晰的分层，然后吸出缓冲液。再用Krebs-Ringer缓冲液将组织洗4-5次或直到组织为橙黄色，而缓冲液为浅褐色。
通过胶原酶处理来解离脂肪组织的细胞。简要地说，从脂肪组织中除去缓冲液，换上比例为1ml胶原酶溶液/ml组织的2mg胶原酶/ml KrebsBiffer溶液(Worthington，MA，USA，type 1)。离心管在37℃水浴中保温30-35分钟，并不时振荡。
经室温下500xg离心5分钟使得基质细胞与脂肪组织中其它组份分离。吸去油和脂肪细胞。剩余的基质-泡囊部分通过强烈旋转重新悬浮于大约100ml磷酸缓冲盐溶液(PBS)中，分到50ml的离心管并于500xg离心5分钟。小心吸去缓冲液，留下基质细胞。然后将基质细胞重新悬浮于基质细胞培养基(DMEM(Morton(1970)体外6：89-108；Dulbecco(1959)病毒学8：396)/Ham’s F-10培养基(Ham(1963)实验细胞研究29：515)(1∶1，v/v)；10％(v/v)胎牛血清；15mM HEPES，pH7.4；60U/ml青霉素；60U/ml链霉素；15μg/ml两性霉素B)，以合适的细胞密度下铺板并在5％CO2下37℃培养过夜。一旦附着在组织培养皿或培养瓶上，培养的基质细胞可以马上投入使用或者在被诱导而分化为成骨细胞(如以下实施例2所述)之前，在培养基中维持多达5个传代时间。
                       实施例2
             髓外脂肪基质细胞分化为成骨细胞
如实施例1所述分离脂肪基质细胞，然后如下处理以诱导分化为成骨细胞。基质细胞以大约22000个细胞/cm2的密度铺在24孔和/或6孔组织培养板中的基质细胞培养基(见上)上。24小时后，用成骨细胞分化培养基(补充有10％的胎牛血清(v/v)的DMEM；10mM β-甘油磷酸；50μg/ml抗坏血酸-2-磷酸；60U/ml青霉素；60U/ml链霉素，15μg/ml两性霉素)取代基质细胞培养基。三周内，每三天用新鲜的培养基置换成骨细胞分化培养基。换培养基时，收集1ml条件培养基并保存于-80℃，用于以后分析分泌的因子。另一种替代的方法是根据Hauner等人(1989临床研究杂志34：1663-1670)的方法，经脂肪细胞分化培养基处理，诱导分离自脂肪组织的基质细胞分化为脂肪细胞(即脂细胞细胞系)。
对如上所述经成骨细胞培养基处理的细胞进行显微观察显示与成骨细胞的外形相一致的形态学变化(图1)。延长培养(21-28天)后，在每个培养小孔内形成了一些多细胞结。成骨细胞(为非-脂细胞细胞系)培养物的颗粒状外型表明磷酸钙沉淀物和前骨结构的存在。因为分离自脂肪组织的基质细胞用脂肪细胞分化培养基处理时，也有可能分化为脂肪细胞，也应检查经成骨细胞培养基处理的细胞中是否存在脂肪细胞。细胞质中没有油滴以及缺乏具有特征性的圆形的脂肪细胞形态的细胞表明经成骨细胞培养基处理的培养物中没有明显的脂肪细胞。
经成骨细胞分化培养基处理的细胞用von Kossa的方法进行染色以鉴定基质细胞是否分化为成骨细胞。简要地说，通过用无血清的培养基置换80％的培养基数次，连续稀释出培养基中的胎牛血清。细胞在5％的甲醛中固定，然后用PBS洗涤数次以除去残余血清。固定化的细胞在100％的乙醇中4℃下保温大约10分钟。除去乙醇后，在波长为254nm的紫外下，固定化的细胞在0.5ml的5％硝酸银中保温10分钟。在蒸馏水中冲洗细胞2-3次，在5％硫代硫酸钠中温育5分钟，然后用水冲洗。染色的细胞可以在50％甘油中无限期地保存。图2B和图2C显示了von kossa染色的结果。只有得到成骨细胞培养基的细胞染色为阳性并变黑。
如下进行油红O染色：培养平板用PBS洗涤几次以除去培养基中的血清或牛血清白蛋白。细胞在甲醇中或溶于PBS的10％甲醛中固定15分钟到24小时。通过将6ml储存液(0.5油红O溶于100ml异丙醇)加入4ml蒸馏水中来制备油红O染色溶液。在经Whatman#1滤纸过滤之前，染色液在室温下放置一个小时。细胞以大约3ml/100mm平板或在6孔平板中以1ml/孔的比例在室温下染色1小时，然后用水洗涤几次。将残留的洗涤用水全部除去。加入150μl/孔的异丙醇，平板在室温下放置10分钟。将异丙醇吹吸几次以保证所用油红O溶解。在500nm处测量光密度。
接受脂肪细胞分化培养基的细胞用下述方法经油红O染色将呈现特征性的红色，表明脂质的积累(图2A)。在培养基中4周后，前脂肪细胞显示出积累的很小的油滴。细胞接受成骨细胞培养基显示了一些非特异性的油红O背景，但该染色与细胞无关(图2C)。这些结果表明分化为成骨细胞的基质细胞没有可检测到的脂肪细胞形态。缺乏这种中性脂质积累是失去脂肪细胞功能和谱系的标志。
还检测了指示成骨细胞分化的生化变化。骨钙蛋白是成骨细胞特异性分泌的蛋白，可由ELISA(完整人类骨钙蛋白K EIA试剂盒，货号BT-460，Biomedical Technologies Inc.，Stoughton，MA)来检测。检查成骨细胞、前脂肪细胞、脂肪细胞的培养基是否存在分泌的骨钙蛋白。简要地说，收集条件培养基(40000细胞培养72小时后的培养基2ml)，每种培养基取20μl进行检测。如图3所示，在成骨细胞培养基中骨钙蛋白增加，而脂肪基质细胞和脂肪细胞分泌的骨钙蛋白很少或没有。
用商业来源的ELISA试剂盒检测分泌的leptin肽。收集条件培养基(40000细胞条件培养72小时后的培养基2ml)，取100μl依照生产商建议的方案进行检测。正如预期的那样，在脂肪细胞分化期间，分泌到培养基中的leptin的量增加，而在成骨细胞分化中则否。在条件培养基中leptin--一种脂肪细胞特异性分泌蛋白的存在，是脂肪细胞活力明确的指示物。成骨细胞和基质细胞不能分泌可检测量的leptin到培养基中，这表示缺乏脂肪细胞谱系，而只有脂肪细胞分化两周后的细胞才分泌leptin。
成骨细胞谱系的另一个标记是分泌碱性磷酸酯酶的能力。按上述方法制备和培养细胞。使用商业来源的检测试剂盒(Sigma诊断公司，货号104-LS，St Louis，MO)检查条件培养基的碱性磷酸酯酶活性。如图4所示，前脂肪细胞有本底水平的分泌的碱性磷酸酯酶活力。经分化，脂肪细胞失去了本底分泌活力，而成骨细胞的碱性磷酸酯酶的水平则有提高。
                      实施例3
            鉴定影响成骨细胞分化的化合物
按照实施例1描述的方法，用分离自脂肪组织的基质细胞的培养物来研究目的化合物对成骨细胞分化的影响。如实施例2所述，在存在或缺乏目的化合物时，成骨细胞由脂肪基质细胞分化而来。通过分泌骨钙蛋白的检测方法来检测分化。使用本实施例的方法，发现促进成骨细胞分化的化合物包括骨形态发生蛋白4和骨桥蛋白-1(未显示数据)。这些结果与Wozney等，1988科学242：1528-1534；Asahina等，1996实验细胞研究222：38-47；Cook等，1996临床矫形术324：29-38以前的发现相符。如下讨论促进成骨细胞分化的化合物可用于增强受治疗者的骨结构。
                     实施例4
              骨修复中成骨细胞的应用
按照实施例1的方法，使用吸脂术从患有不能痊愈性骨折和骨质疏松症骨折的患者的脂肪组织中分离基质细胞。用实施例2的方法在体外诱导前脂肪细胞分化为成骨细胞。7-21天后，通过例如胰酶处理或从组织培养板中机械地剥离来收集已分化的成骨细胞，然后在无菌条件下4-20℃3000xg离心10分钟浓缩细胞。将收集到的细胞悬浮于胶原或MatrigelTM溶液中，然后使用20口径或更大孔径的针头直接注射到骨折或外科手术部位。另一个替代的方法是，在注射前，将细胞与DBM或比如ProOsteon 2000TM(Interpore Cross，Irvine，CA)或CollagraftTM(Zimmer，Warsaw，IN)的陶瓷基质相混合。所用细胞的数量取决于骨折的面积和性质。根据所需的骨折恢复速度可以进行多种治疗。结果是痊愈时间的缩短和骨折部位周围骨密度的增加。
                            实施例5
使用基因改变的(即经过基因修饰的)成骨细胞治疗骨折或骨质疏松症
如实施例1所述分离基质细胞，使用标准转染方法如氯化法(Maniatis等(1982)分子克隆实验指南，Cold Spring Harbor Press，Cold Spring Harbor，NY)将基因材料(例如编码有用基因产物，如骨骼形态发生蛋白的DNA，它可操纵地与启动子相连)转入基质细胞(即将核酸引入细胞)。使用Effectene试剂可改善该方法，并在这里描述。在1个微量离心管中，将0.1-2.0μg pCMV-βgal(Stratagene Inc.，LaJolla，CA)加入150μl Buffer EC(Qiagen EffecteneTM试剂盒，货号301425，Qiagen公司)。这样可以凝聚DNA。将8μl的促进剂(试剂盒)加入凝聚后的DNA。然后将含有凝聚DNA的离心管旋转1秒钟并在室温下放置2-5分钟。短暂离心以除去管顶端的液滴。加入10μl EffecteneTM，离心管旋转10秒，室温下放置5-10分钟。在5-10分钟的保温时间后，向DNA混合物中加入1ml培养基。
从细胞移去120μl日培养基并加入70μl新鲜培养基，然后将25μl DNA混合物移入每个孔。细胞于37℃培养约5小时。但是，Effectene没有毒性也可以一直保留在细胞上。
然后将细胞在感染72小时后用80μl新鲜培养基冲洗1次，并用Mantiatis等描述的方法(1982)检测细胞的β-半乳糖苷酶活力。由实施例1描述的方法可使这些细胞分化为成骨细胞。可替代地，或者DNA可以直接导入分化为成骨细胞的细胞中。
也可以使用其它将核酸序列导入细胞的方法。例如，通过与Becker等(1994，Meth Cell Biol 43：161-189)和Meunier-Durmont等(1996，欧洲生物化学237：660-667)描述的相类似的方法，可以使用腺病毒将DNA转入基质细胞。细胞经如以上实施例1描述的处理后分化为成骨细胞。或者，分化的成骨细胞能够用病毒颗粒感染。加入抗生素选择性标记使其能富集含有导入遗传材料的细胞。然后如以上实施例3所述，将得到的带有导入遗传材料的成骨细胞移植到骨折或骨质疏松症的骨髓中。
说明书中提到的所有出版物表明了本发明相关领域的技术人员的水平。所有出版物引入本文作为参考文献，正如每篇均是逐一并个别地全文引入作为参考文献一样。
虽然已参照特定的实施方案描述了本发明，可以想到很多变化、改善和实施方案都是可能的，并且可以相应地认为所有这些变化、改善和实施方案都在本发明的精神和范围内。

Claims (6)

1.一种分离的人脂肪组织衍生的基质细胞，所述细胞表现出至少一种非—脂细胞细胞系的特征，其中所述非—脂细胞细胞系是成骨细胞。
2.根据权利要求1的人脂肪组织衍生的基质细胞，其中所述细胞是基因修饰的。
3.根据权利要求2的人脂肪组织衍生的基质细胞，其中所述基因修饰是将核酸引入细胞。
4.与基质相结合的根据权利要求1的人脂肪组织衍生的基质细胞。
5.根据权利要求4的人脂肪组织衍生的基质细胞，其中所述基质选自羟磷灰石/磷酸三钙，明胶，多聚-L-赖氨酸，和胶原。
6.根据权利要求1的人脂肪组织衍生的基质细胞，其中所述细胞可以在培养基中维持多达5代。
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CN1584020A (zh) 2005-02-23
AU1615599A (en) 1999-06-16
WO1999028444A1 (en) 1999-06-10
ES2351082T3 (es) 2011-01-31
US20020119126A1 (en) 2002-08-29
JP4222726B2 (ja) 2009-02-12
CA2312847A1 (en) 1999-06-10
JP2001525165A (ja) 2001-12-11
US9200255B2 (en) 2015-12-01
JP2009279458A (ja) 2009-12-03
HK1075062A1 (en) 2005-12-02
US20160068813A1 (en) 2016-03-10
JP4394718B2 (ja) 2010-01-06
ATE478135T1 (de) 2010-09-15
CN100529063C (zh) 2009-08-19
EP1036163B1 (en) 2010-08-18
CN1280612A (zh) 2001-01-17
JP2008099708A (ja) 2008-05-01
US20070134211A1 (en) 2007-06-14
US6391297B1 (en) 2002-05-21
US7179649B2 (en) 2007-02-20
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DE69841850D1 (de) 2010-09-30

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