CN1296102C - 上皮干细胞的扩展方法 - Google Patents
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Abstract
上皮干细胞移植术，在特别处理过的羊膜上来自体内地培养上皮干细胞，同羊膜一起回收具有扩展的上皮干细胞的外科移植物。制备这种移植物的方法以及移植物本身提供了简单而有效的方法以重建损坏的组织，一个优选的实施例是角膜组织。上皮干细胞的来源可以是来自健康的自体和同种异体组织的生物活组织检测的一小块外植体。处理羊膜杀死细胞但使细胞外基质保存下来。
Description
技术领域
本发明涉及上皮干细胞和角膜缘干细胞缺损以及，更具体的说，涉及这种上皮干细胞缺损问题中的治疗或移植方法，例如重建角膜表面。
背景技术
关于眼睛，正常的眼睛表面是由角膜，角膜缘和结膜上皮覆盖的。它们不同的细胞类型同稳定的眼前泪膜一起保持了眼睛表面的完整性。严重的角膜缘上皮损坏是由于：化学或热灼伤。Stevens-Johnson综合症，眼瘢痕类天疱疮，角膜缘的多次手术和冷冻疗法，佩戴隐形眼镜以及严重的微生物感染能够导致角膜缘和上皮干细胞缺损。角膜缘上皮干细胞缺损通常显示出结膜炎，血管化，慢性炎症以及角膜表面的纤维状内生和角膜浑浊的症状。
当角膜缘缺损是单侧或非对称性的双侧时，建议施用自体角膜缘组织移植术。对自体角膜缘移植方法的主要的担忧是一个或两个角膜缘的移植物-不得不取自健康的其它眼睛的角膜要跨越2到3个小时的范围。已经有报道描述了对供体眼睛的潜在的并发症。对兔子的实验也说明如果先移走角膜缘接着从供体眼中随后移走中央上皮角膜，就会发生角膜缘缺损。Pellegrini等，柳叶刀1997；349：990-993，报道了为了给两个角膜缘完全缺损的患者重建角膜表面，在3T3成纤维细胞饲养层上扩展移植的角膜上皮细胞层。
最近，Kim和Tseng，Cornea 1995；15：473-84已经说明了用羊膜移植作为底物替代在重建角膜缘干细胞完全缺损的兔子角膜表面中是有效的。
发明内容
本发明涉及以下方面：
1.一种施用到受试部位的外科移植物，所述的移植物的特征在于含有：包含羊膜细胞和保有完整性的细胞外基质的羊膜；和在所述羊膜上扩展的上皮干细胞。
2.根据第1方面的外科移植物，其中所述的羊膜具有一个基膜侧；其特征在于，所述的上皮干细胞在所述的基膜侧上扩展。
3.根据第1方面的外科移植物，其特征在于，在健康眼睛上进行的角膜缘生物活组织检测得到的角膜缘组织是所述上皮干细胞的来源。
4.根据第1方面的外科移植物，其中所述的羊膜具有一个基膜侧；其特征在于，所述的上皮干细胞源自在培养基中处理过的组织，其接着转移到所述羊膜上；并且在所述羊膜的所述基膜侧上离体培养所述的上皮干细胞。
5.根据第1方面的外科移植物，其特征在于，所述的上皮干细胞取自与受试部位相应的生物和组织相容性的健康部位的生物活组织。
6.一种用于损坏的受试部位的外科移植物的制备方法，所述的方法的特征在于，包括的步骤是：a)将生物活组织作为外植体放置于羊膜上，所述羊膜为包含羊膜细胞和保有完整性的细胞外基质的羊膜；以及b)使上皮干细胞在所述的羊膜上扩展。
7.根据第6方面的方法，其中所述的羊膜具有一个基膜侧；其特征在于，所述的放置于羊膜上的步骤包括将所述的外植体载置于所述羊膜的基膜侧。
8.根据第7方面的方法，其特征在于，所述的载置使得所述扩展的上皮细胞朝上定位于被损坏的受试部位。
9.根据第6方面的方法，其特征在于，使所述的上皮干细胞在所述的羊膜上扩展的步骤包括：在所述放置于羊膜上的步骤前在培养基中培养所述的外植体。
10.根据第6方面的方法，其中使上皮干细胞在所述的羊膜上扩展的步骤的特征在于，培养所述带有外植体的羊膜一段足够的时间以使所述上皮干细胞扩展到直径为2到3厘米的范围。
11.根据第6方面的方法，其特征在于，在将所述移植物用外科手术固定在受试部位之后从所述的羊膜去除所述的外植体。
优选的实施方案
本发明的一个实施例中，从健康眼取的一小块角膜缘生物活组织的角膜缘干细胞和上皮细胞，在培养基中在一个特殊处理过的羊膜上扩展。用这种羊膜作为底物有助于修复没有炎症的角膜缘基质并扩展角膜缘干细胞群和上皮干细胞群。所得到的“产品”可以作为移植物被移植到剥落的角膜表面，接着进行表面切除术以去除维管组织内生长。对于除了眼睛以外的被破坏的部分，健康上皮细胞的生物活组织来自具有相同或相近的生物/组织特性-即同损坏区具有组织相容性的邻近组织区域。
角膜缘活组织检测在健康眼上进行，它可能是患者的另外一只眼睛或者来自于另一个生命个体的眼睛。用Betadine(聚维酮碘)对眼睑消毒。在灭菌条件下，用No.66 Beaver刀片(Becton Dickinson，Franklin Lakes，NJ)通过片状角膜切除术从角膜缘分离并从表面角膜基质中切除含有上皮细胞和部分角膜基质组织的1到2mm2角膜缘组织。将该组织放置在一个含有1.5ml培养基的35mm的培养皿上，每毫升培养基含有：DMEM(Dulbecco′s改进的Eagle′s培养基)以及Ham′s F12(比率1∶1)，添加0.5％DMSO(二甲基亚砜)，2ug小鼠EGF(上皮生长因子)，1ug牛胰岛素，0.1ug霍乱毒素以及5％胎牛血清并迅速将其放置在实验室的灭菌的，层流罩超静台中培养。
根据Tseng SCG在Am.J.Opthalmol 1997；124：765-774中的报道获得、处理并保存用作培养系统的羊膜(购自Bio Tissue，Miami，F1.)，文中的教导在此引入。使用前，将基膜朝上的羊膜平滑地附着到培养平皿上并于37℃和5％CO2以及95％空气中，在潮湿的恒温箱中过夜。根据前面的描述加上一些改进，进行角膜缘外植体培养(Tsai和Tseng，InvestOpthalmol Vis Sci 1988；29：97-108；和Tsai等.，Invest Opthalmol VisSci 1994；35：3865-2875)。带有上皮干细胞的角膜外植体被种植/转移到含有1ml上述培养基的35mm平皿中的羊膜的基膜侧上，以替代这些参考中所教授的转移到塑料基质上。为了重建角膜，每两天更换一次培养基，并将培养物保持2到3星期以使上皮干细胞生长并且扩展形成直径约为2到3厘米的覆盖范围。对其它组织的修复，对需要，可以或多或少可以使用上皮干细胞层区。
继续角膜修复的实施例，接着角膜缘的外周切割术，去除眼前结膜下疤痕和发炎的组织使巩膜裸露出来。利用No.57和66 Beaver刀片通过片状角膜切除术去除维管组织，手术方式和为同种移植角膜缘移植术所描述的(Tsai和Tseng Cornea 1994；13：389-400)相似。对于那些有部分完全角膜缘损坏但是具有正常的中央角膜的患者，则用培养的带有羊膜的角膜缘干细胞作为切割角膜缘移植物，或者角膜缘等价物，根据受试者眼睛的尺寸进行精加工，并移植到相应的受试角膜缘区(从90度到360度)。对于那些角膜缘和角膜表面完全损坏的患者，这种新的移植物被用作一个完整的片状角膜组织，或者角膜缘角膜等价物，并用片状角膜成形术移植以覆盖整个区域。
在处理过的羊膜基质上培养的合适大小的上皮干细胞层的上皮侧向上附着并覆盖到整个缺损上，这可以通过荧光染色或者松弛附着的原始外植体容易地鉴定。然后将移植物固定在损坏部位。对于损坏的角膜，可以通过角膜侧面上间断的10-0尼龙缝线固定，以及用间断的8-0 Vicryl缝线固定对有巩膜支持的周围结膜边缘。在整个过程中，要通过覆上透明质酸钠(透明质酸盐)Healon(Pharmacia & Upjohn AB，Uppsala，瑞典)来防止培养的上皮干细胞层暴露，干燥以及擦伤。在手术的最后，可以从羊膜去除原始的外植体组织。如果该移植物在角膜之上，就将眼睛压贴过夜，第二天放上一个治疗用接触透镜，过一个星期；第一周每天四次局部施用1％氢强的松醋酸盐溶液，后两周每天两次，接着根据手术部位周围结膜发炎的严重程度每天施用两次0.1％氟米龙2到3个月。
如上所述，在特别处理的羊膜的基膜侧上培养外植体。两到三周后，上皮干细胞在羊膜上生长形成了2到3cm2大小的薄层。平板封固制备物显示干细胞层对PAS和Alcian蓝染色显示阴性；并且裸露的羊膜被染成紫色。组织学检测说明上皮层是由其边缘的4到5层干细胞层和在边缘和原始外植体组织区域之间1到4个细胞层组成的。超微结构检测显示存在有广泛且松散的细胞间隙和基质膜结构，在基底细胞-羊膜结合处有电子密集基质的聚集。
关于本发明治疗眼睛的一个具体的实施方案，根据Snellen视力表显示在随后的平均(±SD)14±1.9个月期间有不同程度的视力改善。在2到4天内所有的眼睛显示出了完全的上皮再生，平均周期(±SD)为2.7±0.8天。重建的角膜表面在1到2周内显示出炎症和血管化的消退。手术一个月后，角膜的澄清度改善并且光滑而湿润。
根据角膜缘的损坏面积，培养的上皮干细胞在经特殊制备的羊膜基质上扩展，可以用其作为角膜缘的等价物或角膜缘-角膜等价物。
为了移植而去除了一片相当大的角膜缘而造成的供体眼睛的角膜缘缺损已经在兔子中有报道。因此，本发明的这种新方法和所得到的移植物实质上减少了供体眼潜在的并发症，这是因为仅仅去除了一小片角膜缘。而且，这种方法可以用于非对称性的两侧角膜缘缺损。在特别预处理的羊膜上来自体内(ex vivo)的扩展的自体上皮干细胞提供了在2到3周，内用于移植手术的充足的上皮干细胞。对于双侧角膜缘完全缺损的患者，应当按照本发明考虑使用相匹配的来源，另一个生命体，以及亲属个体的角膜缘。
将以独特的羊膜作为基底的自体上皮干细胞用于移植也提供了所有羊膜移植术所固有的有益效果，包括有利于上皮形成，减少炎症和疤痕，以及当下面的基质组织被破坏时可替换基质。最重要的是，按照Tseng预处理了的羊膜为要保存和扩展的上皮干细胞提供了天然基质，形成了角膜重建所必需的自体细胞群。此外，因为仅仅是自体细胞被移植，在移植手术后不需要免疫抑制。对于以这种方式进行的同种异体干细胞移植，排斥率应当减少，这是由于仅仅移植了上皮干细胞而没有其它类型的细胞。
正如上面阐明的，这种独特的移植和其方法的形成，是通过利用特殊处理的羊膜，扩展其上来自外植体的上皮干细胞，这在眼科手术以外也有用途，例如，皮肤烧伤部位的修复；尤其是当必须使供体部位的外植体较小时。同样，从小的外植体得到的生物活组织不一定是角膜缘组织。外植体将具有健康组织，含有上皮干细胞并且同要移植的受试部位有组织相容性。如果生物活组织不能来自同受试部位相同的身体部分，可以选择相应的类似的身体部分作为外植体；正如在优选的实施例中的情况，损坏的眼睛是受试部位而另外的眼睛-健康的眼睛-提供供体外植体。
应当认为一种独特的并且有创造性的外科移植物及其外科移植手术方法已经被充分公开，并且对于本领域技术人员来说不需要充分的试验过程就可以实施，对于在本发明权利要求所定义的精神和范围内进行的改进也是如此。
Claims (11)
1.一种施用到受试部位的外科移植物，所述的移植物的特征在于含有：包含羊膜细胞和保有完整性的细胞外基质的羊膜；和在所述羊膜上扩展的上皮干细胞。
2.根据权利要求1的外科移植物，其中所述的羊膜具有一个基膜侧；其特征在于，所述的上皮干细胞在所述的基膜侧上扩展。
3.根据权利要求1的外科移植物，其特征在于，在健康眼睛上进行的角膜缘生物活组织检测得到的角膜缘组织是所述上皮干细胞的来源。
4.根据权利要求1的外科移植物，其中所述的羊膜具有一个基膜侧；其特征在于，在所述羊膜的基膜侧上培养有移至其上的带有上皮干细胞的角膜外植体。
5.根据权利要求1的外科移植物，其特征在于，所述的上皮干细胞取自与受试部位相应的生物和组织相容性的健康部位的生物活组织。
6.一种用于损坏的受试部位的外科移植物的制备方法，所述的方法的特征在于，包括的步骤是：a)将生物活组织作为外植体放置于羊膜上，所述羊膜为包含羊膜细胞和保有完整性的细胞外基质的羊膜；以及b)使上皮干细胞在所述的羊膜上扩展。
7.根据权利要求6的方法，其中所述的羊膜具有一个基膜侧；其特征在于，所述的放置于羊膜上的步骤包括将所述的外植体载置于所述羊膜的基膜侧。
8.根据权利要求7的方法，其特征在于，所述的载置使得所述扩展的上皮细胞朝上定位于被损坏的受试部位。
9.根据权利要求6-8任意一项的方法，其特征在于，使所述的上皮干细胞在所述的羊膜上扩展的步骤包括：在所述放置于羊膜上的步骤前在培养基中培养所述的外植体。
10.根据权利要求9的方法，其中使上皮干细胞在所述的羊膜上扩展的步骤的特征在于，培养所述带有外植体的羊膜一段足够的时间以使所述上皮干细胞扩展到直径为2到3厘米的范围。
11.根据权利要求6-8任意一项的方法，其中使上皮干细胞在所述的羊膜上扩展的步骤的特征在于，培养所述带有外植体的羊膜一段足够的时间以使所述上皮干细胞扩展到直径为2到3厘米的范围。
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| DE2780078T9 (de) * | 2001-11-19 | 2005-05-19 | Amniotec Inc | Ektokornea-ähnliches blatt und herstellungsverfahren dafür |
| US8642338B2 (en) | 2003-02-06 | 2014-02-04 | Cellseed Inc. | Anterior ocular segment related cell sheets, three-dimensional structures, and processes for producing the same |
| US20060234377A1 (en) | 2003-02-06 | 2006-10-19 | Teruo Okano | Cell sheets for ectocornea formation, method of producing the same and method of using the same |
| JPWO2005087286A1 (ja) * | 2004-03-11 | 2008-01-24 | アルブラスト株式会社 | 生体組織シート及びその作製方法、並びに同シートを用いる移植方法 |
| JP4576545B2 (ja) * | 2004-05-27 | 2010-11-10 | 独立行政法人理化学研究所 | 羊膜由来因子による胚性幹細胞の培養方法 |
| CN100336567C (zh) * | 2004-10-19 | 2007-09-12 | 北京科宇联合干细胞生物技术有限公司 | 医用角膜贴片 |
| CN101102800A (zh) * | 2005-01-14 | 2008-01-09 | 阿如布勒斯特有限公司 | 使用羊膜的片状组合物及其制作方法 |
| JP4863655B2 (ja) * | 2005-06-17 | 2012-01-25 | 国立大学法人 東京医科歯科大学 | 細胞含有シート |
| TWI407967B (zh) * | 2006-04-28 | 2013-09-11 | Ray Jui Fang Tsai | 人類角膜內皮幹細胞之擴增方法 |
| CN102166375B (zh) * | 2011-02-16 | 2013-04-10 | 中国海洋大学 | 一种组织工程人角膜上皮的重建方法 |
| CN115671398B (zh) * | 2022-11-22 | 2024-03-01 | 首都医科大学附属北京同仁医院 | 一种3d打印仿生角膜缘移植物及其制备方法和用途 |
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