人角膜内皮细胞株的建立与组织工程人角膜内皮的体外重建

为了体外重建出可用于角膜移植的组织工程人角膜内皮(TE-HCE),本文从业已建立的非转染人角膜内皮(HCE)细胞系中筛选出单克隆细胞株(mcHCE),并以其为种子细胞对TE-HCE的体外重建进行了研究。经有限稀释法从非转染HCE细胞系筛选出了mcHCE细胞株,形态结构、染色体分析以及细胞连接蛋白和膜运输蛋白的检测结果显示,mcHCE2401单克隆细胞株细胞具有正常而稳定的形态、结构和二倍染色体核型,并能表达细胞连接蛋白和膜运输蛋白,具有TE-HCE种子细胞的理想特征。以mcHCE2401细胞为种子细胞、以去上皮层修饰羊膜(mdAM)为载体支架体外重建出了TE-HCE,形态结构鉴定结果显示,多角形mcHCE2401种子细胞在mdAM 上形成了连续、完整的细胞单层,在细胞之间以及细胞与mdAM之间均形成了广泛的细胞连接,单层细胞密度高达3602.22±45.22个／mm²(相当于0～3岁孩童HCE的细胞密度),所重建单层角膜内皮的形态结构与在体HCE高度近似。可见,本文成功建立了形态结构、核型以及功能蛋白表达正常的单克隆细胞株,并利用mcHCE2401细胞和mdAM在体外成功重建出了形态结构与与在体HCE高度近似的最“年轻”的TE-HCE,有望作为捐献角膜内皮的等效替代物用于角膜内皮异常疾病的临床治疗。     

角膜移植及其排斥反应的内皮改变——角膜内皮显微镜观察

本文报告用角膜内皮显微镜观察兔穿透性角膜移植术前、术后和角膜移植排斥反应时的角膜内皮改变结果。发现在角膜移植术前,正常角膜的中央区和周边区的内皮细胞密度、细胞面积基本相同,没有显著差异。在角膜移植术后,术眼的植片和受主角膜的内皮细胞均出现密度减低、细胞面积增大,与术前比较有显著差异。在排斥反应期间,植片内皮细胞出现细胞肿胀、边界不清、甚至模糊不能窥见等异常改变,而受主角膜内皮细胞则表现正常。本文认为兔角膜内皮细胞的形态、密度等与人角膜内皮细胞基本相同。穿透性角膜移植术对角膜内皮有较大损伤,植片内皮细胞在排斥反应中遭受严重损害,提出在角膜移植术时,要注意保护角膜内皮;发现排斥反应时,要及时治疗,防止角膜内皮的过多损伤。     

壳聚糖植入兔角膜的生物学反应

目的：探讨壳聚糖植入兔角膜的生物相容性，以评价其作为人组织工程角膜材料的可行性。方法：将壳聚糖材料制成的膜植入新西兰兔角膜的板层内，进行裂隙灯观察并进行组织病理学检查。结果：术后1周有结膜睫状充血，角膜水肿；2周后充血水肿逐渐消失。兔眼的情况稳定，术后2周角膜半透明；术后8周新生血管消退，材料与角膜的生物相容性好，材料部分吸收溶解，角膜透明。电镜超微结构检查可见兔角膜标本中材料内有角膜基质细胞长入，细胞胞浆较多，细胞器丰富，有大量的新生胶原。结论：壳聚糖与角膜组织具有良好的生物相容性，且能自然溶解吸收，可用作组织工程角膜的载体材料。     

兔和牛角膜上皮、基质及内皮细胞体外培养和增殖的研究

目的：研究兔和牛角膜上皮、基质及内皮细胞等3种细胞体外培养的生物学特性，探索和改进这3种细胞体外培养的方法，为组织工程角膜奠定基础。方法：采用消化法分离培养兔和牛角膜上皮、内皮和基质细胞，观察细胞贴壁生长情况，同时对生长良好的原代细胞进行消化传代，进一步观察传代细胞的形态和生长情况。结果：角膜上皮、内皮和基质细胞均在培养48～72h贴壁生长，培养6～9d能形成良好单层，细胞形态典型，进行多次传代后细胞仍维持原有形态和功能，获得的细胞能进行长期培养。结论：为兔和牛角膜上皮、内皮和基质细胞体外培养，提供了简单高效经济的方法。     

人角膜细胞在体外培养的研究进展

人角膜是光通过的窗口，在维持眼压和对光路调节方面也起着重要的作用，由角膜缺损或病变引起的眼疾严重时会导致患者失明。目前治疗这类疾病的唯一办法就是角膜移植，但是及其匮乏的角膜供体使得多数患者失去重新获得光明的机会。近些年来兴起的组织工程人角膜体外重建技术给数以千万计的患者带来了福音。来自自体的人角膜细胞是上述组织工程所需的最理想的种子细胞，本文就人角膜细胞在体外的培养和扩增的进展和存在的问题进行综述。     

体外培养人角膜内皮细胞的UVB损伤及抗坏血酸的抗氧化保护作用研究

以非转染人角膜内皮(HCE)细胞系为体外实验模型系统研究了UVB氧化损伤、Asc抗氧化保护及其分子机理。体外培养的HCE细胞经UVB和/或Asc处理后,利用MTT和光镜对细胞的活力和形态进行了检测,利用8-羟基脱氧鸟苷免疫荧光染色对DNA的氧化损伤进行了检测,并利用二氢乙啶染色对胞内活性氧(ROS)的水平进行了检测。结果显示,100~800 mj/cm²的UVB辐射能剂量和时间依赖性地损伤HCE细胞的活力;200 mj/cm² UVB(自然太阳光中的平均辐射剂量)能引起HCE细胞发生皱缩,并显著增加细胞的DNA氧化损伤程度及胞内ROS水平;而1 mmol/L Asc不仅能显著增强HCE细胞的活力、促进细胞分裂,而且还能显著降低200 mj/cm² UVB所引起的DNA氧化损伤及胞内ROS水平。综上所述,UVB通过诱导ROS的产生进而引起DNA氧化损伤,对HCE细胞具有显著的氧化损伤作用;而Asc能够通过降低UVB诱导的ROS水平进而保护DNA免受氧化损伤,对HCE细胞的UVB损伤具有一定的抗氧化保护作用。本文研究结果对于利用Asc等抗氧化保护剂保护HCE细胞免受UVB氧化损伤具有一定的理论指导价值。     

保存人羊膜用于角膜缘缺陷兔眼表面重建

探讨羊膜移植术在角膜缘缺陷兔眼表面的作用,建立兔全角膜缘缺陷的动物模型,移植眼早期应用保存的人羊膜移植于受损区域,对照眼常规保守治疗,观察其角膜新生管及上皮层愈合情况。愈合后1个月,取角膜,做普通病理及扫描电镜检查。结果显示对照眼角膜上皮层愈合较慢,新生血管粗大丰富,愈合后上皮层不稳定,含有杯状细胞。羊膜移植的眼表面上皮层愈合快,新生血管细小,愈合后的上皮层光滑,稳定, 杯状细胞主要分布于周边角膜。表明羊膜移植能促进上皮细胞移行、增殖、获管稳定的眼表层。羊膜移植后,上皮仍为结膜型上皮。     

兔角膜真空冷冻干燥工艺及冻干角膜活性检测

利用真空冷冻干燥法对家兔角膜进行冻干贮藏·给出了真空冷冻干燥角膜工艺,主要是对角膜进行四步法冷平衡,两步法速率降温,采用变幅值、变周期循环压力法实现干燥室内压力的调节·对按该工艺冻干的兔角膜进行物理法检测,均符合检测标准;进行台盼蓝、茜素红联合染色法检测,内皮细胞呈六角形镶嵌排列;进行透射电镜、扫描电镜检测,细胞微观结构完好·检测结果表明冻干角膜具有活性,按照此工艺能够冻干出适合贮藏、再植的活性角膜·对真空冷冻干燥法贮藏的兔角膜进行了移植实验,手术成功,术后植片透明·实验结果表明冻干法保存的角膜可以用于角膜移植·     

FK506在同种异体兔角膜缘移植中的应用

目的：研究FK506对同种异体兔角膜缘移植术后免疫排斥反应的抑制效应。方法：建立48只新西兰白兔角膜缘于干细胞缺陷症动物模型，随机分成FK506治疗组、环孢霉素A治疗组和非治疗组，同种异体角膜缘移植术后分别给予质量分类为0．5％FK506滴眼液、1％环孢霉素A滴眼液和生理盐水点眼，用药4周。术后持续观察10周，以角膜缘植片水种、混浊程度、排斥反应发生时间、角膜新生血管和假性胬肉作为眼表评估指标。结果：观察期内，FK506组上皮排斥反应发生率、移植排斥指数均低于环孢霉素A组，差异有显著性（P＜0．05）；而两组成新生血管和假性胬肉指数方面，无显著性差异（P＞0．05）。结论：同种异体角膜缘移植术后早期应用FK506眼液点眼可以抑制排斥反应发生。     

角膜移植的免疫学研究——实验性角膜移植排斥反应的病理组织学观察

角膜移植排斥反应现已成为角膜移植手术失败的主要原因,研究角膜移植免疫反应具有重要意义。本文报告了用同一供体皮肤移植引起兔同种穿透性角膜移植排斥反应的动物模型,描述了角膜移植术后和皮肤移植术后的经过,排斥反应的临床表现,组织学标本,包括光镜、扫描电镜和透射电镜标本的制作方法,重点报告了排斥反应实验组的角膜和对照组透明角膜的组织学观察结果。发现对照组的透明角膜,除了在供、受体组织交界处形成疤痕外,植片和受主角膜表现与正常角膜相同。而在发生排斥反应的角膜,则有大量新生血管侵入,植片有大量的浸润细胞,这些细胞主要是淋巴细胞,还有巨噬细胞、浆细胞、多形核白细胞、成纤维细胞等。植片组织遭到不同程度的损害,受主角膜除了有新生血管生长和有少量浸润细胞附着以外,没有受损。本文讨论了有关动物模型、排斥反应的临床特征、病理组织学与免疫机理及功能的联系,新生血管在排斥反应中起的作用等问题。我们认为本组实验动物模型的临床表现和组织学改变均符合典型的特异性免疫反应。这种反应以细胞免疫为主,亦有体液免疫参与。血管在免疫排斥反应的传入、传出弧中均超重要作用。     

组织工程人角膜内皮的研究进展

从种子细胞的来源和载体支架的制备两个方面对组织工程人角膜内皮的研究进展及其前景进行综述。     

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

Recently, regenerative medicine has been focused on as next-generation definitive therapies for several diseases or injuries for which there are currently no effective treatments. These therapies have been rapidly developed through the effective fusion be- tween different fields such as stem cell biology and biomaterials. So far, we have proposed ＂cell sheet engineering＂ through our core technology that simply applies alterations of the temperature which allows regulation of the attachment or detachment of living cells on the culture surfaces grafted with the temperature-responsive polymer ＂poly（N-isoproplyacrylamide）＂. This tech- nology enables us to construct bioengineered sheet-like tissues, termed ＂cell sheets＂, without the need of using biodegradable scaffolds and protease treatments that are traditionally used. Therefore, our carrier-free cell sheets not only are independent of the issues observed in conventional methods, but also showed further advantages in the reconstruction of the corneal epithelium or endothelium （e.g. improvement of optical transparency and cell-cell interactions to host stroma in reconstructed tissues）. Moreo- ver, our approach does not have issues such as immune rejection or limited number of donors, due to the use of cell sheets derived from autologous limbal （in patients with unilateral disease） or oral mucosal epithelial cells （in patients with bilateral disorders）. Indeed, we have successfully performed the clinical application for the reconstruction of ocular surfaces through the transplanta- tion of our carrier-free corneal epithelial cell sheets, as evidenced by improved visual acuity as well as long-term maintenance of postoperative health conditions on ocular surfaces in all patients. We have also proposed a novel approach for the reconstruction of the corneal endothelium using corneal endothelial cell sheets by demonstrating its successful application. Thus, our cell sheet engineering technique provides a breakthrough in the field of regenerative medicine applied to the cornea.     

Oligopotent stem cells are distributed throughout the mammalian ocular surface

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants, and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals. Identification of slow cycling cells (label-retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.     

Measurement of corneal astigmatism

Cornea is the most important optical surface in the eye dioptric system. Three kinds of methods in measuring corneal astigmatism were used. Three hundred and sixty eyes suffered ametropia were selected on a random basis. Computer_assisted corneal topographic system was compared with keratometer and cycloplegic retinoscopy in measuring corneal curvature. There was no statistical difference in axes of astigmatism among the three groups. It is thought that ΔSim \%K\% could show the corneal regular astigmatism. As for diopters of astitigmatism, no difference was found between the groups of corneal topography and keratometer, but there was a significant difference between the group of cycloplegic retinoscopy and the other two groups. It was demonstrated that keratometer could function similarly as the corneal topography. However, it measured only four points from a small region of the cornea and was limited in measuring slight changes in corneal curvature. Some discussion was also made about the graphic patterns and ΔSim \%K\% in corneal topography.     

A study of chitosan blend film as a carrier of corneal endothelial cells

In order to evaluate the cytocompatibility and biocompatibility of a new kind of chitosan blend film as a carrier of corneal endothelial cell, rabbit corneal endothelial cells cultured in vitro were breeded onto the film. After a cell monolayer formed, the scanning electron micrography was performed. After inplanted into anterior chamber, slit lamp observation, thickness metering, specular microscopy and HE staining were performed at random time after operation to evaluate the biocompatibility. Inflammation in anterior, thickness of cornea, cell density, hexagonality and cell size of the surgical cornea were taken as the indexes of biocompatibility. The cultured cells exhibited a confluent monolayer 10 days after incubation, which proved the satisfactory cytocompatibility of this film. Biocompatibility assay results suggested the implantation feasibility of the film as a carrier of corneal endothelial cells.     

小鼠表皮生长因子对鸡胚角膜上皮组织的增殖效应

鸡胚角膜上皮组织离体培养试验表明，小鼠表皮生长因子对１０日龄鸡胚角膜上皮组织有显著的增殖效应。培养２４ｈ的实验组角膜上皮厚度较正常胚胎角膜上皮厚度增殖９．４３倍，提示鸡胚角膜上皮组织离体培养可作为评价表皮生长因子制剂活性的生物检定法。     

表皮生长因子对猕猴胚胎角膜上皮组织增殖效应的研究

离体培养试验表明，小鼠颌下腺表此生长因子对离体培养猕猴胚胎眼角膜上皮组织具有显著促进细胞增殖和分化的效应。培养４ｄ的实验组角膜上皮的厚度，上皮细胞增殖层烽以及上皮组织中基底层细胞核与表层细胞核两者的比率均较正常猴胚角膜增长３倍。与此同时培养４ｄ的实验组角膜上皮厚度和上皮细胞的层数，都达到成年猴正常角膜上皮的结构水平。