Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (2): 363-368.doi: 10.3969/j.issn.2095-4344.2013.02.032
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Li Shan-yi1, 2, Chen Jian-su1, 2, 3
Received:
2012-04-08
Revised:
2012-07-14
Online:
2013-01-08
Published:
2013-01-08
Contact:
Chen Jian-su, Doctoral supervisor, Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou 510632, Guangdong Province, China; Institute of Ophthalmology, Medical College, Jinan University, Guangzhou 510632, Guangdong Province, China; Ophthalmology Department, First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong Province, China chenjiansu2000@163.com
About author:
Li Shan-yi★, Studying for master’s degree, Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou 510632, Guangdong Province, China; Institute of Ophthalmology, Medical College, Jinan University, Guangzhou 510632, Guangdong Province, China Gm77338@126.com
Supported by:
Supported by: the National Natural Science Foundation of China, No. 30973244*; the Project 211 at Jinan University, the Fundamental Research Funds for the Central Universities of China, No. 21609407
CLC Number:
Li Shan-yi, Chen Jian-su. Transplantation of tissue-engineered corneal endothelium[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(2): 363-368.
2.1 角膜内皮种子细胞的来源 2.1.1 分离培养的角膜内皮细胞 这类细胞主要来源于人[1-2]、猴[3-4]、兔[5]、猫[6]、牛和猪等哺乳类动物。人来源的角膜内皮细胞能够直接反应出临床情况,但是人的角膜组织来源有限,从而限制了广泛利用此类细胞。猴是最接近人类的高等动物,但由于数量有限且价格较高,也不能大量使用。兔、猫、猪和牛等眼球容易得到,价格也相对便宜,可以广泛的用于前期的基础研究。 2.1.2 角膜内皮细胞系 由于人的原代角膜细胞不易获得且不稳定,有学者开始建立能够长期传代培养的永生化角膜内皮细胞系,但由于其具有潜在致瘤性,对于这种细胞系的临床安全性有待进一步考察,故临床应用受到限制,只能用于体外的实验研究。Gotze等[7]利用永生化人角膜内皮细胞系体外进行细胞片研究。Kim等[8]建立了永生化角膜内皮细胞系并检测了在冻干人羊膜上角膜内皮特异性蛋白的表达。 2.1.3 干细胞 干细胞是一类能够自我更新并可分化为多种组织细胞的能力,在修复和重建方面成为疾病治疗研究的热点。骨髓间充质干细胞是一种成体干细胞,存在于骨髓基质,分化程度低,增殖能力强,经定向诱导后可分化为角膜内皮样细胞。2010年,Shao等[9]在体外把人骨髓间充质干细胞分化成角膜内皮样细胞,再接种在脱细胞猪角膜基质表面并移植到内皮及后弹力层损伤的猫模型中,发现可使角膜逐渐变透明。Joyce等[10]根据角膜内皮细胞的发育特点,用晶状体上皮细胞产生的条件培养液培养脐血间充质干细胞,结果显示脐血间充质干细胞分化成角膜内皮样细胞。 2.2 角膜内皮构建方式 2.2.1 利用载体构建内皮层 以各种材料为载体进行角膜内皮细胞体外培养与移植的实验研究广泛开展,是目前角膜构建的研究热点并获得了初步成功。 载体类:这类载体材料主要有羊膜[11-12]、羊膜基底膜[13]、明胶水凝胶薄膜[14-16]、晶状体前囊[17],以及最新应用的丝素蛋白膜[18],这些载体膜片透明度好,但操作很困难,不易插入并恰好放置在基质受体面上且不损坏重建的内皮层。角膜后弹力层由内皮细胞分泌形成,含有Ⅳ型胶原和层粘连蛋白,是内皮细胞的天然底物。人的后弹力层来源有限,异种的后弹力层受到学者的青睐并以猪的为首选。但由于其免疫原性,仍然达不到临床移植的要求。 Wencan等[13]利用羊膜基底膜为载体,体外培养内皮细胞融合后单层密度达到(3 486±53)个/mm2、大多数具有六角形、铺路石状且大小均匀;在体内移植后,细胞密度达到平均(2 837±57)个/mm2,并能保持稳定。Yoeruek 等[17]用消化酶处理过的晶状体前囊为载体,人的角膜内皮细胞为种子细胞,结果显示细胞密度、形态和功能标志物的表达都与体内特征相似。此外,可降解的水凝胶膜[19]、胶原-硫酸软骨素-戊二醛等构成的复合物[20]、Ⅰ型胶原[3]、vitrigel胶原膜等是刚性的载体,它们在没有缝合的情况下较难整合到宿主角膜后弹力膜[3, 21]。 新鲜天然角膜基质膜:新鲜天然角膜基质膜有很多的优点,如透明度好、一个生理的曲度、生物相容性和机械稳定性等。由于这些优点,也有研究人员使用新鲜天然的眼角膜基质作为载体,用于体内和体外研究。然而在新鲜的基质膜上培养内皮细胞,可能会污染上皮细胞和基质细胞。此外,移植有活性的载体可显著提高免疫排斥的风险。因此,这类基质膜并不被广泛研究。 失活的角膜基质膜:失活的天然角膜基质膜是替代使用新鲜原生组织的一种选择。失活的天然基质载体在组织工程内皮重建中富有好的黏附性,而且在全层移植中也很好的被接受。这类载体首先是消除活细胞,即消除载体上内皮细胞、上皮细胞和基质细胞,同时也是为了降低载体的免疫原性[22-23]。这种失活的天然基质膜可以选择人、猪、猫等动物的角膜基质用于研究。Proulx等[24]和Choi等[25]研究来源于人的失活的天然角膜基质作为载体支架。Shao等[9]用猪的失活角膜基质为载体,在猫的角膜中进行了移植。Amano等[26]研究认为,猪的角膜基质有望替代来源匮乏的人角膜基质,成为体外构建组织工程角膜的支架材料。 在脱细胞方面也取得了很大的进步。Amano 等[27]发现用液氮脱掉角膜基质上的细胞而不会影响移植。研究表明用化学试剂氢氧化铵等清除细胞也可以达到相同的结果[25, 28]。另外,基质中胶原纤维的完整性对于保持角膜透明起着至关重要的作用。Proulx等[6, 24]研究发现经过3次的冷冻和解冻后能够有效的使细胞失活且不改变基质的完整性。 2.2.2 培养角膜内皮细胞片 培养的角膜内皮细胞片可以直接用于移植。聚N-异丙基丙烯酰胺凝胶是一种温度敏感性聚合物,随着温度的变化表现出亲水性和疏水性。用这种材料包被培养皿可制成温度响应性培养皿,当细胞在温度响应性智能水凝胶培养皿中达到融合的单层之后,通过降低温度可以得到完整的单层细胞片[14, 29-31]。Ide等[30]的研究显示在温度敏感培养皿上培养出单层的人角膜内皮细胞片,与正常人角膜内皮细胞相似,呈六边形,有丰富的微绒毛和细胞器,并表达细胞间的紧密连接蛋白ZO-1。Hitani等[2]使用EDTA收获已经融合的单层细胞片。目前,有研究显示这些融合的单层细胞可以黏附到宿主的基质上[2, 29],或是可以依附在明胶水凝胶处理的细胞培养皿[14]。 角膜内皮细胞片进行体内移植的方式也会影响移植的成功率。目前,研究者常用的体内移植方式主要有:①在体外让角膜内皮细胞片贴附在去除内皮层和后弹力层的角膜基质层,再把这种角膜片缝合到受体动物模型[2]。②机械性的刮伤试验动物角膜内皮层,再将培养的细胞片通过角巩膜缘切口送入前房,在前房内注入气泡将其托起后缝合切口[3-4]。③去除内皮层后,将角膜内皮细胞片贴附在明胶凝胶载体,植入眼内后,利用载体的膨胀作用使细胞片恰好贴壁在后弹力层[32]。 2.2.3 前房注射法移植角膜内皮细胞 一些学者研究了注射法移植角膜内皮细胞到前房的可行性。这种移植方法,首先要除去后弹力层上病态的内皮细胞。已有报道,在动物模型中用冷冻的方法铲除内皮层细胞[33]。Mimura等[5, 34]和Patel等[35]利用铁离子或者超顺磁性微球在磁场下具有良好的控制性能来定位移植的内皮细胞到后弹力层上。体内和体外的研究显示,标记铁离子的内皮细胞很容易整合到受体的后弹力层表面。但是这种方法的长期效果仍需要进一步的实验验证。另一些研究结果表明Rho激酶抑制剂Y-27632可促进培养的猴角膜内皮细胞贴壁、抑制凋亡和促进增生的作用[36]。研究也显示这种激酶抑制剂有利于兔角膜内皮创伤的愈合[37],作者认为结合Y-27632进行注射移植角膜内皮细胞到前房的方式将会提高移植的成功率。 2.3 实验动物模型 一个好的角膜内皮实验动物模型应具有以下特点:①再现性好,能够再现所要研究的人类角膜内皮疾病,动物疾病表现与人类疾病相似。②动物模型背景资料完整,生命周期满足实验需要。③复制率高。④专一性好,即一种方法只能复制出一种疾病模型。 2.3.1 猴模型 猴是动物界最高等的类群,有几种品系的猴也是在结构和生理上最接近人类的动物模型。成年猕猴的角膜直径大约为10.6 mm,中心厚度约为470 μm,细胞密度约为4 000个/mm2,高于人类的角膜内皮密度[38-39]。目前已有文献报道过使用此模型用作组织工程角膜内皮的实验研究[3-4]。然而,在北美和欧洲的一些国家限制使用这类动物模型,并且这种模型相对也比较昂贵,使这种模型的广泛使用受到限制。 2.3.2 猫模型 由于猫不易被驯服、反抗性强等特点导致实验不易顺利进行,以至于也不能广泛使用。目前也有文献报道使用这类模型用于组织工程角膜内皮的移植试验[6, 40]。成年猫的角膜直径为15.5- 18 mm,中心厚度为545-650 μm[41-42]。与人类一样,猫的角膜内皮在体内也不能增殖,平均大约在 2 300-2 900个/mm2[41-43]。Shao等[9]将骨髓来源的间充质干细胞经过共培养诱导成角膜内皮样细胞,并利用猫模型在体内检测角膜内皮样细胞的功能。 2.3.3 兔模型 由于低成本、体积小、容易操作而且购买和饲养方便,许多研究者使用兔子模型进行体内功能评价。但其不能真实的反应人体的生理状态,所以只能在前期的基础移植实验中广泛的使用。Ishino等[11]用羊膜作为载体,培养人的角膜内皮细胞,再移植到兔体内进行体内角膜内皮功能检测。Mimura 等[44]在胶原上培养人的角膜内皮细胞,并将融合的细胞片移植到兔体内进行体内功能检测。Okumura等[37]用兔内皮层创伤模型检测Rho激酶抑制剂的滴眼液在体内作用。 2.3.4 其他模型 其他的一些动物,如猪模型、鼠模型等,也有用于角膜内皮试验。Mimura等[1]利用裸鼠进行体内实验模型,但它们的角膜很小,不容易操作,因此很少被用于角膜内皮的体内功能检测。"
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