体外培养角膜缘上皮细胞重建眼表的实验研究
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摘要
角膜感染、外伤、热及化学性烧伤、先天性异常、反复复发的翼状胬肉和肿瘤等疾病严重损伤眼表组织，造成视力下降，甚至致盲。角膜盲的患者在盲病人中占相当比例，由于眼表疾病以及角膜缘干细胞缺失所导致的角膜盲欠缺有效治疗手段，故对其治疗一直是步履蹒跚。粘膜或结膜移植眼表重建手术的临床效果较差，因为粘膜和结膜与角膜上皮表型不同而不能恢复眼表正常的形态与功能，最终导致新生血管化。同种异体板层角膜移植供体来源匮乏，冷冻保存的植片由于角膜缘干细胞的失活不能改善重建正常的眼表。
随着对角膜缘干细胞在维持眼表中的重要作用的不断认识，角膜缘干细胞移植成为目前的研究热点。1998年12月美国科学家在《science》上报导，成功地在体外培养和扩增了人体胚胎干细胞，这一研究为利用干细胞治疗疾病提供了理论依据。一年之后，美国科学家发现小鼠肌肉组织的成体干细胞可以“横向分化”为血液细胞，这一发现陆续被世界各国科学家所证实。随着研究的深入，人们还发现人类的成体干细胞同样具有“横向分化”的功能，这也表明人类有望利用患者自身健康组织的干细胞，定向诱导分化成有功能的细胞用于治疗疾病。随着干细胞研究的深入，组织工程学也应运而生，组织工程技术的兴起为解决眼表重建的难题带来了希望。组织工程技术的基本方法是取少量自体健康角膜缘组织，体外培养扩增后种植于一种生物相容性良好的载体上，组成复合角膜上皮植片后移植到患侧眼表。其优点在于：取材量少、健眼损伤小、解决了供体来源问题、不存在免疫排斥反应。角膜缘干细胞经体外培养后其抗原性会有所降低，这就更有利于异体移植。因此，角膜缘干细胞培养后移植解决了干细胞来源不足的问题，对异体角膜缘干细胞的体外培养是目前研究的重点，是治疗眼表疾病最有前途的治疗方法。
目前角膜缘干细胞理想的培养体系还处于探索研究阶段，正确获取培养用角膜缘组织是体外成功培养角膜缘干细胞的首要因素，但由于角膜缘干细胞缺乏明确的阳性标志，对其的研究一直在进行中。还有角膜缘干细胞体外培养时，细胞增殖、分化等过程的调节受许多因素制约，其中许多环节人们还知之甚少。如能在这些方面有所突破，有望产生巨大的社会效益和经济效益，并为数以十万计的角膜盲患者带来福音。
基于上述，本实验研究利用组织工程学、细胞生物学和干细胞与干细胞工程学的基本原理与方法，试图在体外成功培养角膜缘干细胞、构建组织工程化角膜上皮，并将组织工程化角膜上皮移植于动物模型，以达到重建眼表、恢复角膜表面的光滑和透明，恢复眼表面较为正常的解剖结构和功能，为进一步组织工程化眼表重建奠定理论和实验基础。
在本研究中应用体外组织培养方法，对兔角膜上皮前体细胞或角膜缘干细胞(祖细胞)存在的确切部位和生理特性、在完整羊膜和去上皮层羊膜为载体培养的生物学特性和以去上皮层羊膜为载体培养后自体及异体移植治疗全角膜缘干细胞缺损以及临床应用等情况进行了实验，主要方法和结果概括如下：
一．应用体外组织培养方法，以兔角结膜分界线(Cornea-Conjuctiva Line CCL)为基准，分成8个定位区带，并分别以12点、3点、6点、9点为中心，取材进行体外培养。逐日观察细胞成膜状态，测量成膜面积并进行统计学分析。结果显示：1．角膜缘不同区域的上皮组织在细胞生长时间、成膜时间、成膜速率以及成膜类型方面具有明显差异(P＜0.01)，角膜缘附近的角膜上皮细胞生长最快、出膜最早、膜状态最好。2．CCL内各区组培养后均无成纤维细胞出现；而CCL外各区组培养后可见成纤维细胞，距CCL线越远成纤维细胞数量越多。3．水平方位与垂直方位即12点位、6点位、3点位和9点位的角膜上皮前体细胞成膜形态与成膜规律的比较，发现二者不存在明显差异(p＞0.05)。4．角膜缘附近的角膜上皮细胞体外培养形成的膜都是完整、均质、透明的，当膜在其增长过程中，可将扩展范围内的成纤维细胞化解。提示：1．角膜缘不同定位区组织细胞形成膜的特性不同，含有干细胞的角膜缘附近的角膜上皮细胞生长最快、出膜最早、膜状态最好。2．角膜缘部栅栏组织的丰富程度与角膜上皮前体细胞的数量及功能未必存在一致关系。3．角膜缘干细胞形成的膜有保持其透明性及排它性的生理特性。4．进行角膜缘干细胞体外培养，从CCL附近取材较为适宜。
二．对兔角巩膜缘不同定位区的角结膜上皮细胞进行体外组织培养，探讨了角膜缘干细胞的生理特性。结果显示：1．当角膜缘上皮细胞形成的膜片相互融合时，他们之间是兼容连接，最终形成的膜片之间无连接的痕迹。2．当角膜缘上皮细胞形成的膜片与结膜上皮细胞形成的成纤维细胞融合时出现相互竞争现象。3．角膜缘存在有角膜缘干细胞，角膜缘上皮细胞形成的膜有保持其透明性及排它性的生理表现。提示：1．角膜缘上皮细胞和结膜上皮细胞存在相互竞争机制，为角膜缘干细胞缺乏症引起的角膜结膜化提供了必要的实验基础。2．角膜缘存在有角膜缘干细胞，角膜缘干细胞对维持角膜的透明性和正常生理功能有重要意义。
三．采用组织块培养法，将角膜缘及角膜中心组织块分别种植于完整羊膜和去上皮层羊膜上进行培养，观察并比较在完整羊膜、去上皮层羊膜为载体培养兔角膜上皮细胞的生物学特性。结果显示：1．角膜上皮细胞在去上皮层羊膜上培养成膜速率与成膜面积方面均快于在完整羊膜上培养，去上皮层羊膜更利于角膜上皮细胞的生长。2．角膜上皮细胞在完整羊膜上培养，当膜在其增长过程中，需将扩展范围内的羊膜上皮细胞推开，膜才能继续增长。提示：1．不同性质羊膜为载体对角膜上皮细胞的生物学特性有一定的影响，去上皮层羊膜更利于角膜上皮细胞的生长，可作为一个良好的载体用于移植。2．角膜上皮细胞具有排它性的生理特性，对维持角膜的透明性和正常生理功能有重要意义。
四．制作兔眼(以右眼为实验眼)角膜缘干细胞完全缺损模型3个月，将实验动物随机分为两组即实验组和对照组。实验组取对侧眼角膜缘组织以去除上皮细胞的羊膜基底膜为载体制成组织工程角膜上皮，约培养后12天行角膜缘干细胞羊膜移植术；对照组行单纯羊膜移植术(仅将刮除上皮的羊膜)，术后观察3个月。以角膜上皮染色，角膜浑浊和新生血管三项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果显示：1．体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增殖。2．自体角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS(+)，而移植后角膜上皮细胞PAS(-)；组织病理学显示：移植前角膜上皮大部分缺损，移植后角膜上皮为角膜上皮结构。提示：以羊膜为载体培养角膜缘上皮细胞可体外重建组织工程角膜上皮组织，自体角膜缘干细胞羊膜移植术可修复角膜缘干细胞缺损。
五．制作兔眼角膜缘干细胞完全缺损3个月的模型，将实验动物随机分为两组即自体移植组和异体移植组。自体移植组取对侧眼角膜缘组织，异体移植组取健康的兔眼角膜缘组织，均以去除上皮细胞的羊膜基底膜为载体，培养12天后行角膜缘干细胞羊膜移植术。术后观察3个月，以角膜上皮染色、角膜浑浊和新生血管三项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果显示：体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增殖，体外培养12天可形成复层。自体移植组和部分异体移植组术后角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS(+)，而移植后转为(-)；组织病理学显示：移植前角膜上皮大部分缺损，移植后呈现角膜上皮结构。部分异体移植组术后出现了免疫排斥反应。提示：自体角膜缘干细胞羊膜移植术可重建眼表；免疫排斥反应仍是异体角膜缘干细胞羊膜移植术失败的主要原因。
六．我们选择两例眼烧伤后重度睑球粘连患者，观察以去除上皮细胞的人羊膜基底膜为载体，，自体血清培养异体角膜缘上皮细胞后移植治疗眼烧伤后重度睑球粘连，术后观察结膜囊情况、眼球活动、角膜植片免疫排斥反应情况和转归情况。探讨以人自体血清、人羊膜为基底膜培养角膜缘上皮细胞后移植治疗眼烧伤后重度睑球粘连的眼表重建效果。结果显示：术后患者自觉症状轻，视力有所提高，角膜缘干细胞移植片紧密贴附，角膜上皮得到修复，浅层新生血管减少，睑球粘连得到松解。提示：自体血清培养异体角膜缘上皮细胞后移植是治疗眼烧伤后重度睑球粘连的有效方法。
本研究通过体外培养角膜缘上皮细胞生理特性，进一步证实了角膜干细胞存在于角膜缘的观点，验证了角膜缘干细胞对维持角膜的透明性和正常生理功能有重要意义的说法。通过观察并比较在完整羊膜与去上皮细胞层羊膜为载体培养兔角膜缘上皮细胞的生物学特性，表明去上皮细胞层羊膜可作为一个良好的载体用于移植。并以去上皮细胞羊膜作为载体，经培养后用于移植于全角膜缘干细胞缺损模型，可达到重建眼表。通过以去上皮细胞羊膜作为载体，人自体血清培养异体角膜缘上皮细胞后移植是治疗眼烧伤后重度睑球粘连的有效方法，避免了鼠源性3T3细胞饲养层和胎牛血清传播异种动物的风险，并降低了术后的免疫排斥反应，为眼表重建术提供另一研究新方向。
Corneal disease including keratitis, traumatic cornea, thermal and chemicalburning, genetous abnormity, recurrent pterygium and tumor could cause injury ofocular surface tissue, descent of vision, blindness even. The corneal blind accountedfor a considerable proportion in the blind, there was little effective treatment on thecorneal blindness caused by the disease of ocular surface and the absence of corneallimbal stem cell, so the work proceeded at a glacial pace. The reconstructionoperation of ocular surface using mucous, membrane or conjunctiva had little clinicaleffect, because of different phenotype of the mucous membrane conjunctiva and theepithelium of cornea, the reconstruction of ocular surface using the mucousmembrane or conjunctiva remained elusive result, and resulted in neovasculization.The Allogeneic corneal transplantation was limited by the donor eyes, and thedevitalization of the frozen corneal limbal stem cell could not cause the effectivereconstruction of the normal ocular surface.
It was realized that the corneal limbal stem cell was import to the ocular surface,and the deficiency or dysfunction of the corneal llimbal stem cell would causeepithelial healing obstacle and abnormal restoration, so the transplantation corneallimbal stem cell became hot. The human embryo stem cell had been successfullycultivated in vitro by American scientist, which reported on《science》magazine in1998. According to this research result, the stem cells can be transplanted to curesome diseases possibly. After one year, the American researcher discovered that the stem cell of mice myofiber tissue could be transversely differentiated into blood cell,which had been confirmed by other researcher. After that it was found so many stemcells had this kind of ability. All of this, the stem cell could be used to treat somekinds of disease. Tissue technology was born with the research of stem cell. It wasexcellent opportunity for oculist to take advantage of the tissue-engineered toreconstruct the ocular surface. The basic method of the tissue-engineered was to usea little limbal stem cell cultured in vitro. The germ of the corneal limbal stem cellwas planted in a good carrier which had good histocompatibility. This kind ofbiological tissue was transplanted in the damaged ocular surface. The advantage ofthis technology had: first, little material; second, little damage on the health eye;third, resolving the donor source and no immune rejection. The transplant of thecorneal stem cell could reconstruct the ocular surface because the HLA-DR antigenbecame more and more little in culture. So it resolved the stem cell source thatcorneal limbal stem cell was cultured in vitro, the allogeneic cultured corneal stemcell had been concentrated by most ophthalmologist. The transplantation of theallogeneic cultured stem cell might be the most suitable method to reconstruct thedamaged ocular surface.
At present the ideal cultural system of corneal limbal stem cell was beingexplored, and it was the first-line factor that corneal limbai tissue Was acquired andcultured exactly. The research went along due to the lack of masculine sign ofcorneal limbal stem cell, and much important content hadn't been found out, forexample, the restricting factors of cell proloferation, cell differentiation and celldivision. A major breakthrough in the research would bring great social andeconomic benefits, and bring happiness to tens of thousands of corneal blinders.
In summary, to attempt the corneal limbai stem cell was cultured in vitro, andthe tissue corneal limbal was be constructed successfully. To transplant the tissuecorneal limbal to animal models, ocular surface would be reconstructed, the smoothness and transparency of corneal surface was be restored, the normalanatomical structure and function of ocular surface was be restored, and thefoundations of theory and experimentation was be laid for further tissue ocularsurface reconstruction.
The main points of the results from this study are summarized below:
Ⅰ. To confirm the exact location of corneal precurcor epithelial cells or cornealstem cells (progenitor cells). Corneal of rabbits was assigned to 8 orientation zoneaccording to the basic reference of cornea and conjunctica limbus. Cornealspecimens were collected at 12, 6, 3 and 9 o'clock separately for the in vitro tissueculture. The morphology and area of the theca-form of corneal precursor epithelialcells in different limbus site were observed and measured daily for statisticalanalysis. It shew that: 1. Statistically significant differences were found in varioustissues at cells growth, theca-froming growth, theca-froming speed and theca-fromingshape. Close to the corneal conjunctiva limbus, the cells growth was the fastest, themembrane's growth was the earliest and best. 2. None of fibroblasts were seen insidethe corneal conjunctiva limbus. However, the fibroblasts were found during the culture.outside the corneal conjunctiva limbus and the numbers increased gradually outside. 3.No statistically significant difference was found in theca-forming shapeand rules ofcorneal precursor epithelium between horizontal level (3 and 9 o'clock) and verticallevel (12 and 6 o'clock). 4. The film with cultured in vitro was integrity,homogenate and clear. As the film was growing, it can resolve the fibroblasts oramniotic membrane epithelial cells. Tips: 1. Statistically significant differenceswere found in various tissues theca-froming physiological characteristic. Close to thecorneal conjunctiva limbus, the cells' growth was the fastest, the membrane's growthwas the earliest and best. 2. No a positive correlation is found between the richnessof palisade tissue and amount of corneal precursor epithelial cells 3: The film ofLimbal stem cells pesists the physiological characterigtic of the clear shape and the discrimination. 4. When the corneal precursor epithelial cells are cultured in vitro,they are better collcted closing to the corneal conjunctiva limbus.
Ⅱ. To cultivate the limbal epithelial cells at different places of rabbit limbus in vitroso as to explore the physiological characteristic of Corneal stem cells. It shew that:1. When the similar films from tissue blocks emerged, films were compatible andlinked up, namely, neither film inside cells penetrated into the other, nor cellsdeposited or formed to block the other cells from entering barrier structure, andultimately resulting in trace between film connections free. 2. When the films fromthe limbal cells and the fibroblasts from the conjunctiva cells linked up, they competedeach other. 3. There are some limbal stem cells in limbal epithelial cells, filmsformed by limbal cells have physiological characteristics of keeping transparency andexclusiveness. Tips: 1. The competition between limbal cells and conjunctiva cellsis the test base of corneal conjunctiva from lack of corneal epithelial stem cells. 2.There are some limbal stem cells in limbal epithelial cells, it is important to maintaincornea's transparency and normal physiology.
Ⅲ. To study and compare the physiological characteristic of intact and denudedamniotic membranes as a substrate for Corneal epithelial cells culture. Small biopsyspecimens of superficial cornea including epithelium were excised from the centraland. limbal, regions in rabbits. It shew that: 1. The theca-froming area andtheca-froming speed, of corneal epithelial cells with culture denuded amnioticmembrane was faster than intact amniotic membrane. The corneal epithelial cells'growing of denuded amniotic membrane was better. 2. During the process of filmgrowth, film forming cells would gradually dissolve amnion epithelial cells uponmeeting them, the film continued extending forward and maintained level, smooth andtransparent. Tips: 1. Different carriers, have some influences on the physiologicalcharacteristic of limbal stem cells. The corneal epithelial cells' growing of denudedamniotic membrane was better than intact amniotic membranes, it appears to be an excellent substrate for cultivation of Corneal epithelial cells, with a view totransplantation. 2. Corneal epithelial cells have physiological characteristics ofkeeping transparency and exclusiveness, it is important to maintain cornea'stransparency and normal physiology.
Ⅳ. To reconstruct corneal epithelium in vitro by tissue engineering technique andprovide effective method and materials for clinical transplantation. As model of totallimbal deficiency had been built in right eyes of 20 rabbits by surgical removal oflimbal rim for 3 months. As model of total limbal deficiency had been built in righteyes of 16 rabbits, They were randomly divided into 2 groups: experimental andcontrol group. Small biopsy specimens from the limbal region of experimental groupwere taken and cultured on denuded amniotic membrane. When it was culturedabout 12 days, experimental group received grafts of amniotic membrane containingautologous cultured epithelial cells, whereas control group received grafts ofdenuded amniotic membrane alone. To observe for 3 months after'transplantation,the restoration of corneal epithelium was evaluated by phthology, and the cellsphenotype of corneal epithelium was examined by impression cytology. It shew that:1. Limbal epithelial cells can be cultured and proliferated on the amniotic membrane.2. Corneas of experimental group gradually epithelialized and progressive decreaseof vascularity and stromal infiltration in the limbal and peripheral zone. Theexamination of impression cytology shew that corneal epithelium beforetransplantation: PAS (+) , corneal epithelium'after transplantation: PAS (-). Thehistopathology shew 'that corneal epithelium before transplantation was deficiencemostly, corneal epithelium after transplantation was restored. Tips: Tissue engineeredcorneal epithelium can be reconstructed in vitro by culturing limbal epithelial cellson denuded amniotic membrane, corneal transplantation of cultivated limbalcornealepithelium on denuded amniotic membrane can repair the limbal deficiency.
Ⅴ. To investigate the outcome of using human amniotic membrane as substrates for cultivating rabbit limbal corneal epithelial cells, and autologous or allogenoustransplanting them onto rabbits with total limbal deficiency. As model of total limbal.deficiency had been built in right eyes of 20 rabbits by surgical removal of limbalrim for 3 months. They were randomly divided into 2 groups of 10: Autologousgroup (5) and allogenous group (5). Small biopsy specimens from the limbal regionof 10 left eyes were taken and cultured on acellular amniotic membrane. When itwas cultured about 12 days, 5 of the right eyes then received grafts of amnioticmembrane containing Autologous cultured epithelial cells, 5 of the right eyes thenreceived grafts of amniotic membrane containing allogenous cultured epithelial cells.To observe for 3 months after transplantation, the restoration of corneal epitheliumwas evaluated by pathology, and the cells phenotype of corneal epithelium wasexamined by impression cytology. It shew that: Limbal epithelial cells can becultured and proliferated on the amniotic membrane, stratified layers of epitheliumwere formed after 12 days. Corneas of Autologous (5) or allogenous (3)transplanting gradually epithelialized and progressive decrease of vascularity andstromal infiltration in the limbal and peripheral zone. The examination of impressioncytology shew that corneal epithelium before transplantation: PAS (+), cornealepithelium after transplantation: PAS(-)The histopathology shew that cornealepithelium before transplantation was deficience mostly, corneal epithelium aftertransplantation was restored. However corneas of allogenous (2) transplanting hadhappened immunology rejection. Tips: Autologous transplantation of cultivatedlimbal corneal epithelium on amniotic membrane can reconstruct corneal epithelium.However, immunology rejection is main reason of limbal epithelial allogenoustransplantation.
Ⅵ. To observe the effects of human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation for severesymblepharon resulting from eye burns. Two patients with severe symblepharon visited our hospital, to observe the effects of human limbal stem cells cultured onhuman amniotic membrane with themselves serum using allograft transplantation forsevere symblepharon resulting from eye burns. Eye movement, diplopia,development of corneal grafts and recoverment were observed. It shew that: aftertransplantation, two patients felt better and visual acuity improving a little, graftincluding amniotic membrane and stem cells firmly attached on limbus, cornealepithelium become smoothly, superficial neovasculization reduced significantly andblepharostenosis vanished. Tips: Human limbal stem cells cultured on humanamniotic membrane with themselves serum using allograft transplantation, is anideal treatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.
To study the physiological characteristic of Corneal stem cells, it confirmed thatthe exact location of corneal stem cells located at limbus, it is important to maintaincornea's transparency and normal physiology. To study and compare thephysiological characteristic of intact and denuded amniotic membranes as a substratefor Corneal epithelial cells culture, it showed that denuded amniotic membraneappears to be an excellent substrate for cultivation of Corneal epithelial cells, with aview to transplantation. Corneal transplantation of cultivated limbal cornealepithelium on denuded amniotic membrane can repair the limbal deficiency, it can bereconstructed ocular surface. Human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation, is an idealtreatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.
随着对角膜缘干细胞在维持眼表中的重要作用的不断认识，角膜缘干细胞移植成为目前的研究热点。1998年12月美国科学家在《science》上报导，成功地在体外培养和扩增了人体胚胎干细胞，这一研究为利用干细胞治疗疾病提供了理论依据。一年之后，美国科学家发现小鼠肌肉组织的成体干细胞可以“横向分化”为血液细胞，这一发现陆续被世界各国科学家所证实。随着研究的深入，人们还发现人类的成体干细胞同样具有“横向分化”的功能，这也表明人类有望利用患者自身健康组织的干细胞，定向诱导分化成有功能的细胞用于治疗疾病。随着干细胞研究的深入，组织工程学也应运而生，组织工程技术的兴起为解决眼表重建的难题带来了希望。组织工程技术的基本方法是取少量自体健康角膜缘组织，体外培养扩增后种植于一种生物相容性良好的载体上，组成复合角膜上皮植片后移植到患侧眼表。其优点在于：取材量少、健眼损伤小、解决了供体来源问题、不存在免疫排斥反应。角膜缘干细胞经体外培养后其抗原性会有所降低，这就更有利于异体移植。因此，角膜缘干细胞培养后移植解决了干细胞来源不足的问题，对异体角膜缘干细胞的体外培养是目前研究的重点，是治疗眼表疾病最有前途的治疗方法。
目前角膜缘干细胞理想的培养体系还处于探索研究阶段，正确获取培养用角膜缘组织是体外成功培养角膜缘干细胞的首要因素，但由于角膜缘干细胞缺乏明确的阳性标志，对其的研究一直在进行中。还有角膜缘干细胞体外培养时，细胞增殖、分化等过程的调节受许多因素制约，其中许多环节人们还知之甚少。如能在这些方面有所突破，有望产生巨大的社会效益和经济效益，并为数以十万计的角膜盲患者带来福音。
基于上述，本实验研究利用组织工程学、细胞生物学和干细胞与干细胞工程学的基本原理与方法，试图在体外成功培养角膜缘干细胞、构建组织工程化角膜上皮，并将组织工程化角膜上皮移植于动物模型，以达到重建眼表、恢复角膜表面的光滑和透明，恢复眼表面较为正常的解剖结构和功能，为进一步组织工程化眼表重建奠定理论和实验基础。
在本研究中应用体外组织培养方法，对兔角膜上皮前体细胞或角膜缘干细胞(祖细胞)存在的确切部位和生理特性、在完整羊膜和去上皮层羊膜为载体培养的生物学特性和以去上皮层羊膜为载体培养后自体及异体移植治疗全角膜缘干细胞缺损以及临床应用等情况进行了实验，主要方法和结果概括如下：
一．应用体外组织培养方法，以兔角结膜分界线(Cornea-Conjuctiva Line CCL)为基准，分成8个定位区带，并分别以12点、3点、6点、9点为中心，取材进行体外培养。逐日观察细胞成膜状态，测量成膜面积并进行统计学分析。结果显示：1．角膜缘不同区域的上皮组织在细胞生长时间、成膜时间、成膜速率以及成膜类型方面具有明显差异(P＜0.01)，角膜缘附近的角膜上皮细胞生长最快、出膜最早、膜状态最好。2．CCL内各区组培养后均无成纤维细胞出现；而CCL外各区组培养后可见成纤维细胞，距CCL线越远成纤维细胞数量越多。3．水平方位与垂直方位即12点位、6点位、3点位和9点位的角膜上皮前体细胞成膜形态与成膜规律的比较，发现二者不存在明显差异(p＞0.05)。4．角膜缘附近的角膜上皮细胞体外培养形成的膜都是完整、均质、透明的，当膜在其增长过程中，可将扩展范围内的成纤维细胞化解。提示：1．角膜缘不同定位区组织细胞形成膜的特性不同，含有干细胞的角膜缘附近的角膜上皮细胞生长最快、出膜最早、膜状态最好。2．角膜缘部栅栏组织的丰富程度与角膜上皮前体细胞的数量及功能未必存在一致关系。3．角膜缘干细胞形成的膜有保持其透明性及排它性的生理特性。4．进行角膜缘干细胞体外培养，从CCL附近取材较为适宜。
二．对兔角巩膜缘不同定位区的角结膜上皮细胞进行体外组织培养，探讨了角膜缘干细胞的生理特性。结果显示：1．当角膜缘上皮细胞形成的膜片相互融合时，他们之间是兼容连接，最终形成的膜片之间无连接的痕迹。2．当角膜缘上皮细胞形成的膜片与结膜上皮细胞形成的成纤维细胞融合时出现相互竞争现象。3．角膜缘存在有角膜缘干细胞，角膜缘上皮细胞形成的膜有保持其透明性及排它性的生理表现。提示：1．角膜缘上皮细胞和结膜上皮细胞存在相互竞争机制，为角膜缘干细胞缺乏症引起的角膜结膜化提供了必要的实验基础。2．角膜缘存在有角膜缘干细胞，角膜缘干细胞对维持角膜的透明性和正常生理功能有重要意义。
三．采用组织块培养法，将角膜缘及角膜中心组织块分别种植于完整羊膜和去上皮层羊膜上进行培养，观察并比较在完整羊膜、去上皮层羊膜为载体培养兔角膜上皮细胞的生物学特性。结果显示：1．角膜上皮细胞在去上皮层羊膜上培养成膜速率与成膜面积方面均快于在完整羊膜上培养，去上皮层羊膜更利于角膜上皮细胞的生长。2．角膜上皮细胞在完整羊膜上培养，当膜在其增长过程中，需将扩展范围内的羊膜上皮细胞推开，膜才能继续增长。提示：1．不同性质羊膜为载体对角膜上皮细胞的生物学特性有一定的影响，去上皮层羊膜更利于角膜上皮细胞的生长，可作为一个良好的载体用于移植。2．角膜上皮细胞具有排它性的生理特性，对维持角膜的透明性和正常生理功能有重要意义。
四．制作兔眼(以右眼为实验眼)角膜缘干细胞完全缺损模型3个月，将实验动物随机分为两组即实验组和对照组。实验组取对侧眼角膜缘组织以去除上皮细胞的羊膜基底膜为载体制成组织工程角膜上皮，约培养后12天行角膜缘干细胞羊膜移植术；对照组行单纯羊膜移植术(仅将刮除上皮的羊膜)，术后观察3个月。以角膜上皮染色，角膜浑浊和新生血管三项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果显示：1．体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增殖。2．自体角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS(+)，而移植后角膜上皮细胞PAS(-)；组织病理学显示：移植前角膜上皮大部分缺损，移植后角膜上皮为角膜上皮结构。提示：以羊膜为载体培养角膜缘上皮细胞可体外重建组织工程角膜上皮组织，自体角膜缘干细胞羊膜移植术可修复角膜缘干细胞缺损。
五．制作兔眼角膜缘干细胞完全缺损3个月的模型，将实验动物随机分为两组即自体移植组和异体移植组。自体移植组取对侧眼角膜缘组织，异体移植组取健康的兔眼角膜缘组织，均以去除上皮细胞的羊膜基底膜为载体，培养12天后行角膜缘干细胞羊膜移植术。术后观察3个月，以角膜上皮染色、角膜浑浊和新生血管三项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果显示：体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增殖，体外培养12天可形成复层。自体移植组和部分异体移植组术后角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS(+)，而移植后转为(-)；组织病理学显示：移植前角膜上皮大部分缺损，移植后呈现角膜上皮结构。部分异体移植组术后出现了免疫排斥反应。提示：自体角膜缘干细胞羊膜移植术可重建眼表；免疫排斥反应仍是异体角膜缘干细胞羊膜移植术失败的主要原因。
六．我们选择两例眼烧伤后重度睑球粘连患者，观察以去除上皮细胞的人羊膜基底膜为载体，，自体血清培养异体角膜缘上皮细胞后移植治疗眼烧伤后重度睑球粘连，术后观察结膜囊情况、眼球活动、角膜植片免疫排斥反应情况和转归情况。探讨以人自体血清、人羊膜为基底膜培养角膜缘上皮细胞后移植治疗眼烧伤后重度睑球粘连的眼表重建效果。结果显示：术后患者自觉症状轻，视力有所提高，角膜缘干细胞移植片紧密贴附，角膜上皮得到修复，浅层新生血管减少，睑球粘连得到松解。提示：自体血清培养异体角膜缘上皮细胞后移植是治疗眼烧伤后重度睑球粘连的有效方法。
本研究通过体外培养角膜缘上皮细胞生理特性，进一步证实了角膜干细胞存在于角膜缘的观点，验证了角膜缘干细胞对维持角膜的透明性和正常生理功能有重要意义的说法。通过观察并比较在完整羊膜与去上皮细胞层羊膜为载体培养兔角膜缘上皮细胞的生物学特性，表明去上皮细胞层羊膜可作为一个良好的载体用于移植。并以去上皮细胞羊膜作为载体，经培养后用于移植于全角膜缘干细胞缺损模型，可达到重建眼表。通过以去上皮细胞羊膜作为载体，人自体血清培养异体角膜缘上皮细胞后移植是治疗眼烧伤后重度睑球粘连的有效方法，避免了鼠源性3T3细胞饲养层和胎牛血清传播异种动物的风险，并降低了术后的免疫排斥反应，为眼表重建术提供另一研究新方向。
Corneal disease including keratitis, traumatic cornea, thermal and chemicalburning, genetous abnormity, recurrent pterygium and tumor could cause injury ofocular surface tissue, descent of vision, blindness even. The corneal blind accountedfor a considerable proportion in the blind, there was little effective treatment on thecorneal blindness caused by the disease of ocular surface and the absence of corneallimbal stem cell, so the work proceeded at a glacial pace. The reconstructionoperation of ocular surface using mucous, membrane or conjunctiva had little clinicaleffect, because of different phenotype of the mucous membrane conjunctiva and theepithelium of cornea, the reconstruction of ocular surface using the mucousmembrane or conjunctiva remained elusive result, and resulted in neovasculization.The Allogeneic corneal transplantation was limited by the donor eyes, and thedevitalization of the frozen corneal limbal stem cell could not cause the effectivereconstruction of the normal ocular surface.
It was realized that the corneal limbal stem cell was import to the ocular surface,and the deficiency or dysfunction of the corneal llimbal stem cell would causeepithelial healing obstacle and abnormal restoration, so the transplantation corneallimbal stem cell became hot. The human embryo stem cell had been successfullycultivated in vitro by American scientist, which reported on《science》magazine in1998. According to this research result, the stem cells can be transplanted to curesome diseases possibly. After one year, the American researcher discovered that the stem cell of mice myofiber tissue could be transversely differentiated into blood cell,which had been confirmed by other researcher. After that it was found so many stemcells had this kind of ability. All of this, the stem cell could be used to treat somekinds of disease. Tissue technology was born with the research of stem cell. It wasexcellent opportunity for oculist to take advantage of the tissue-engineered toreconstruct the ocular surface. The basic method of the tissue-engineered was to usea little limbal stem cell cultured in vitro. The germ of the corneal limbal stem cellwas planted in a good carrier which had good histocompatibility. This kind ofbiological tissue was transplanted in the damaged ocular surface. The advantage ofthis technology had: first, little material; second, little damage on the health eye;third, resolving the donor source and no immune rejection. The transplant of thecorneal stem cell could reconstruct the ocular surface because the HLA-DR antigenbecame more and more little in culture. So it resolved the stem cell source thatcorneal limbal stem cell was cultured in vitro, the allogeneic cultured corneal stemcell had been concentrated by most ophthalmologist. The transplantation of theallogeneic cultured stem cell might be the most suitable method to reconstruct thedamaged ocular surface.
At present the ideal cultural system of corneal limbal stem cell was beingexplored, and it was the first-line factor that corneal limbai tissue Was acquired andcultured exactly. The research went along due to the lack of masculine sign ofcorneal limbal stem cell, and much important content hadn't been found out, forexample, the restricting factors of cell proloferation, cell differentiation and celldivision. A major breakthrough in the research would bring great social andeconomic benefits, and bring happiness to tens of thousands of corneal blinders.
In summary, to attempt the corneal limbai stem cell was cultured in vitro, andthe tissue corneal limbal was be constructed successfully. To transplant the tissuecorneal limbal to animal models, ocular surface would be reconstructed, the smoothness and transparency of corneal surface was be restored, the normalanatomical structure and function of ocular surface was be restored, and thefoundations of theory and experimentation was be laid for further tissue ocularsurface reconstruction.
The main points of the results from this study are summarized below:
Ⅰ. To confirm the exact location of corneal precurcor epithelial cells or cornealstem cells (progenitor cells). Corneal of rabbits was assigned to 8 orientation zoneaccording to the basic reference of cornea and conjunctica limbus. Cornealspecimens were collected at 12, 6, 3 and 9 o'clock separately for the in vitro tissueculture. The morphology and area of the theca-form of corneal precursor epithelialcells in different limbus site were observed and measured daily for statisticalanalysis. It shew that: 1. Statistically significant differences were found in varioustissues at cells growth, theca-froming growth, theca-froming speed and theca-fromingshape. Close to the corneal conjunctiva limbus, the cells growth was the fastest, themembrane's growth was the earliest and best. 2. None of fibroblasts were seen insidethe corneal conjunctiva limbus. However, the fibroblasts were found during the culture.outside the corneal conjunctiva limbus and the numbers increased gradually outside. 3.No statistically significant difference was found in theca-forming shapeand rules ofcorneal precursor epithelium between horizontal level (3 and 9 o'clock) and verticallevel (12 and 6 o'clock). 4. The film with cultured in vitro was integrity,homogenate and clear. As the film was growing, it can resolve the fibroblasts oramniotic membrane epithelial cells. Tips: 1. Statistically significant differenceswere found in various tissues theca-froming physiological characteristic. Close to thecorneal conjunctiva limbus, the cells' growth was the fastest, the membrane's growthwas the earliest and best. 2. No a positive correlation is found between the richnessof palisade tissue and amount of corneal precursor epithelial cells 3: The film ofLimbal stem cells pesists the physiological characterigtic of the clear shape and the discrimination. 4. When the corneal precursor epithelial cells are cultured in vitro,they are better collcted closing to the corneal conjunctiva limbus.
Ⅱ. To cultivate the limbal epithelial cells at different places of rabbit limbus in vitroso as to explore the physiological characteristic of Corneal stem cells. It shew that:1. When the similar films from tissue blocks emerged, films were compatible andlinked up, namely, neither film inside cells penetrated into the other, nor cellsdeposited or formed to block the other cells from entering barrier structure, andultimately resulting in trace between film connections free. 2. When the films fromthe limbal cells and the fibroblasts from the conjunctiva cells linked up, they competedeach other. 3. There are some limbal stem cells in limbal epithelial cells, filmsformed by limbal cells have physiological characteristics of keeping transparency andexclusiveness. Tips: 1. The competition between limbal cells and conjunctiva cellsis the test base of corneal conjunctiva from lack of corneal epithelial stem cells. 2.There are some limbal stem cells in limbal epithelial cells, it is important to maintaincornea's transparency and normal physiology.
Ⅲ. To study and compare the physiological characteristic of intact and denudedamniotic membranes as a substrate for Corneal epithelial cells culture. Small biopsyspecimens of superficial cornea including epithelium were excised from the centraland. limbal, regions in rabbits. It shew that: 1. The theca-froming area andtheca-froming speed, of corneal epithelial cells with culture denuded amnioticmembrane was faster than intact amniotic membrane. The corneal epithelial cells'growing of denuded amniotic membrane was better. 2. During the process of filmgrowth, film forming cells would gradually dissolve amnion epithelial cells uponmeeting them, the film continued extending forward and maintained level, smooth andtransparent. Tips: 1. Different carriers, have some influences on the physiologicalcharacteristic of limbal stem cells. The corneal epithelial cells' growing of denudedamniotic membrane was better than intact amniotic membranes, it appears to be an excellent substrate for cultivation of Corneal epithelial cells, with a view totransplantation. 2. Corneal epithelial cells have physiological characteristics ofkeeping transparency and exclusiveness, it is important to maintain cornea'stransparency and normal physiology.
Ⅳ. To reconstruct corneal epithelium in vitro by tissue engineering technique andprovide effective method and materials for clinical transplantation. As model of totallimbal deficiency had been built in right eyes of 20 rabbits by surgical removal oflimbal rim for 3 months. As model of total limbal deficiency had been built in righteyes of 16 rabbits, They were randomly divided into 2 groups: experimental andcontrol group. Small biopsy specimens from the limbal region of experimental groupwere taken and cultured on denuded amniotic membrane. When it was culturedabout 12 days, experimental group received grafts of amniotic membrane containingautologous cultured epithelial cells, whereas control group received grafts ofdenuded amniotic membrane alone. To observe for 3 months after'transplantation,the restoration of corneal epithelium was evaluated by phthology, and the cellsphenotype of corneal epithelium was examined by impression cytology. It shew that:1. Limbal epithelial cells can be cultured and proliferated on the amniotic membrane.2. Corneas of experimental group gradually epithelialized and progressive decreaseof vascularity and stromal infiltration in the limbal and peripheral zone. Theexamination of impression cytology shew that corneal epithelium beforetransplantation: PAS (+) , corneal epithelium'after transplantation: PAS (-). Thehistopathology shew 'that corneal epithelium before transplantation was deficiencemostly, corneal epithelium after transplantation was restored. Tips: Tissue engineeredcorneal epithelium can be reconstructed in vitro by culturing limbal epithelial cellson denuded amniotic membrane, corneal transplantation of cultivated limbalcornealepithelium on denuded amniotic membrane can repair the limbal deficiency.
Ⅴ. To investigate the outcome of using human amniotic membrane as substrates for cultivating rabbit limbal corneal epithelial cells, and autologous or allogenoustransplanting them onto rabbits with total limbal deficiency. As model of total limbal.deficiency had been built in right eyes of 20 rabbits by surgical removal of limbalrim for 3 months. They were randomly divided into 2 groups of 10: Autologousgroup (5) and allogenous group (5). Small biopsy specimens from the limbal regionof 10 left eyes were taken and cultured on acellular amniotic membrane. When itwas cultured about 12 days, 5 of the right eyes then received grafts of amnioticmembrane containing Autologous cultured epithelial cells, 5 of the right eyes thenreceived grafts of amniotic membrane containing allogenous cultured epithelial cells.To observe for 3 months after transplantation, the restoration of corneal epitheliumwas evaluated by pathology, and the cells phenotype of corneal epithelium wasexamined by impression cytology. It shew that: Limbal epithelial cells can becultured and proliferated on the amniotic membrane, stratified layers of epitheliumwere formed after 12 days. Corneas of Autologous (5) or allogenous (3)transplanting gradually epithelialized and progressive decrease of vascularity andstromal infiltration in the limbal and peripheral zone. The examination of impressioncytology shew that corneal epithelium before transplantation: PAS (+), cornealepithelium after transplantation: PAS(-)The histopathology shew that cornealepithelium before transplantation was deficience mostly, corneal epithelium aftertransplantation was restored. However corneas of allogenous (2) transplanting hadhappened immunology rejection. Tips: Autologous transplantation of cultivatedlimbal corneal epithelium on amniotic membrane can reconstruct corneal epithelium.However, immunology rejection is main reason of limbal epithelial allogenoustransplantation.
Ⅵ. To observe the effects of human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation for severesymblepharon resulting from eye burns. Two patients with severe symblepharon visited our hospital, to observe the effects of human limbal stem cells cultured onhuman amniotic membrane with themselves serum using allograft transplantation forsevere symblepharon resulting from eye burns. Eye movement, diplopia,development of corneal grafts and recoverment were observed. It shew that: aftertransplantation, two patients felt better and visual acuity improving a little, graftincluding amniotic membrane and stem cells firmly attached on limbus, cornealepithelium become smoothly, superficial neovasculization reduced significantly andblepharostenosis vanished. Tips: Human limbal stem cells cultured on humanamniotic membrane with themselves serum using allograft transplantation, is anideal treatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.
To study the physiological characteristic of Corneal stem cells, it confirmed thatthe exact location of corneal stem cells located at limbus, it is important to maintaincornea's transparency and normal physiology. To study and compare thephysiological characteristic of intact and denuded amniotic membranes as a substratefor Corneal epithelial cells culture, it showed that denuded amniotic membraneappears to be an excellent substrate for cultivation of Corneal epithelial cells, with aview to transplantation. Corneal transplantation of cultivated limbal cornealepithelium on denuded amniotic membrane can repair the limbal deficiency, it can bereconstructed ocular surface. Human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation, is an idealtreatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.
引文
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