SMILE来源的人角膜基质透镜联合纤维蛋白胶构建组织工程角膜基质的实验研究

发布时间：2017-12-31 19:00

本文关键词：SMILE来源的人角膜基质透镜联合纤维蛋白胶构建组织工程角膜基质的实验研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:角膜是眼屈光系统的重要组成部分,同时也是重要的机体防御屏障。由外伤性、感染性或免疫性角膜病所致的角膜穿孔为眼科急症,如不及时给予正确有效的治疗,很可能进一步发展导致严重的并发症,最终造成视力下降或丧失,严重者还可能面临眼球摘除的后果。而角膜移植是目前治疗角膜穿孔的主要手段。然而,目前我国角膜供体严重匮乏,远远不能满足需要。此外,角膜溃疡穿孔时往往伴有强烈而持续的炎症反应,容易引起角膜移植术后免疫排斥反应导致手术失败,这部分病人往往需要多次的角膜移植手术和更多的供体角膜。因此,供体角膜来源匮乏和术后免疫排斥是目前亟需解决的问题。近年来,组织工程角膜研究的兴起和发展为角膜盲患者带来新的希望,其中,脱细胞猪角膜基质因具有较好的透光率、生物力学性能及生物相容性,在动物实验和临床治疗方面取得了较好的效果。然而,脱细胞猪角膜基质是异种移植物,脱细胞处理后保留的细胞外基质具有潜在的抗原性;同时还具有传播潜在的动物相关性疾病的危险,因此其安全性尚需要长期的随访研究。此外,宗教信仰等因素也限制了脱细胞猪角膜基质的临床应用。飞秒激光小切口角膜基质透镜取出术(small incision lenticule extraction,SMILE)不仅可以矫正近视及散光,而且取出的角膜基质透镜这一手术"副产品"还可以再利用。我们的研究团队自2012年开始首次利用多层SMILE来源的人角膜基质透镜移植的方法治疗角膜穿孔,获得较为满意的治疗效果。随后,国内外许多学者也成功将这种方法用于角膜基质缺损或穿孔的治疗。然而,由于单片角膜基质透镜的中央厚度约为60-150μm,边缘厚度为10-30 μm,需要将多层角膜基质透镜叠加后才能达到足够的厚度用于移植。手术操作的难度较大,同时缝合的过程中透镜片可以滑动,易引起植片的扭曲变形及边缘成角不一致,造成术后散光和植片混浊,而且单纯叠加的角膜基质透镜的生物力学强度还有待进一步加强。此外,虽然基质细胞参与角膜组织损伤的修复,但也可能与术后的免疫排斥反应有关,而这些细胞成分可以通过合适的脱细胞方法去除。纤维蛋白胶广泛用于眼科手术及组织工程角膜的构建,然而,能否将纤维蛋白胶用于SMILE来源的人角膜基质透镜的层间粘合,将两者构建成具有一定厚度和生物力学强度的组织工程角膜基质用于角膜基质损伤的修复有待进一步研究,尤其是其移植术后的组织学和超微结构的观察尚无报道。因此,本研究旨在探讨以SMILE来源的含有活性角膜基质细胞的人角膜基质透镜联合纤维蛋白胶体外构建组织工程前板层角膜的可行性,同时,我们将制备脱细胞的人角膜基质透镜,然后联合纤维蛋白胶构建组织工程角膜基质,探讨其移植后的生物学特性和功能。方法:在无菌条件下去除角膜缘环组织中残余的巩膜、虹膜、结膜,并撕去内皮层后,分别用2mg/mLdispaseⅡ和0.25%胰酶-0.02%EDTA消化,获取人角膜缘上皮细胞(human limbal epithelial cells,hLECs)。将纤维蛋白胶用于SMILE来源的含有活性角膜基质细胞的人角膜基质透镜(直径6.6mm,中央厚度大于100μm)的层间粘合,将两者重建成角膜基质。将重建的角膜基质置于Transwell小室内,加入hLECs培养基培养24 h,然后将hLECs细胞悬液滴加在角膜基质表面,孵育2 h后,进行2周的浸没培养和2周的气液界面培养。最后取材进行HE染色观察构建物表面细胞生长情况及组织结构;免疫组织荧光检测角膜上皮细胞特异性标志物CK12和角膜缘干细胞标志物ABCG2和p63α;扫描电镜(scanning electron microscopy,SEM)及透射电镜(transmission electron microscopy,TEM)检测构建物的超微结构。采用高浓度NaCl溶液联合核酸酶的方法制备脱细胞人角膜基质透镜(直径6.6 mm,中央厚度大于100μm),通过HE染色、DAPⅠ染色及TEM检测细胞脱除情况并通过DNA定量分析检测脱细胞效率。将纤维蛋白胶用于脱细胞人角膜基质透镜的层间粘合,将两者重建成组织工程角膜基质,用于新西兰大白兔的板层角膜移植。制作直径为4.0mm,深度约250 μm的板层植床,用环钻钻取组织工程角膜基质中央直径为4.25 mm的基质片作为植片,采用10-0尼龙线间断缝合,将植片固定于植床。术毕球结膜下注射庆大霉素加地塞米松;术后典必殊滴眼液每天4次滴术眼,维持2周。分别于术后1、7、15、30、60、90天评价并记录角结膜反应;于术后7、15、30、60、90天行荧光素钠染色及眼前节照相记录;于术后15、60、90天行眼前节光学相干断层扫描(anterior segment-optical coherence tomography,AS-OCT)检查;于术后90天行角膜地形图检查;于术后15、60、90天取材经4%多聚甲醛及3%戊二醛固定,分别进行组织学检查及TEM检测。结果:经过2周的浸没培养和2周的气液界面培养,HE染色显示接种在重建的角膜基质表面的hLECs可形成排列紧密的复层上皮结构,与正常中央部角膜上皮相似;免疫组织荧光显示其表层细胞高表达角膜上皮细胞标记物CK12,而基底部细胞高表达角膜缘干细胞标记物ABCG-2及p63α,说明重建的角膜基质能为角膜缘干细胞特性的维持提供适宜的环境支持;基质内可见较稀少的角膜基质细胞分布;纤维蛋白胶作为重建的角膜基质的一部分,与角膜基质透镜紧密黏附,未被降解。SEM显示接种在重建的角膜基质表面的hLECs呈铺路石样形态,黏附生长良好,细胞间紧密连接,细胞表面布满微绒毛;TEM显示hLECs之间形成桥粒结构,并在角膜基质表面形成了半桥粒和基底膜结构,与正常人角膜上皮组织相似。经过高浓度NaCl溶液联合核酸酶脱细胞处理后,人角膜基质透镜呈半透明样,但经过100%无菌甘油脱水后,能够完全恢复透明。HE染色及DAPI染色显示脱细胞处理后人角膜基质透镜内无细胞成分残留。TEM显示脱细胞人角膜基质透镜结构完整,胶原间隙并未受到严重的破坏,可见细胞脱离后残留的空隙。DNA定量分析显示去除了人角膜基质透镜中94.2%的DNA成分,脱细胞效果显著。术后3个月观察期间内,所有动物均存活,术眼未出现感染和出血等并发症;未见植片脱离、植片和植床新生血管、植片融解、角膜钙化和免疫排斥等现象。植片完全上皮化的时间为16±2天,至术后3月,植片基本恢复透明。AS-OCT结果显示术后15天,植片和植床均处于水肿状态;至术后2月和3月植片仍呈较高信号,但无明显水肿。术后3月,角膜地形图检查显示术眼未出现圆锥角膜及其他角膜形变表现。HE染色显示术后2月,植片与受体角膜已经融合良好,两者间无明显界限;植片内有角膜基质细胞分布,未见中性粒细胞和淋巴细胞浸润现象;胶原排列整齐、规则;植片表面有4-5层角膜上皮细胞分布,具有正常角膜上皮特征。TEM显示术后2月,角膜上皮与组织工程角膜基质之间开始出现新合成的基底膜结构和半桥粒,术后3月,胶原纤维排列整齐,间距一致,激活的角膜基质细胞恢复静止状态,与正常角膜相似。结论:SMILE来源的人角膜基质透镜联合纤维蛋白胶构建的组织工程前板层角膜具有与正常角膜组织类似的表型和结构,重建的角膜基质能够支持角膜上皮细胞黏附、增殖和形成复层结构;使用高浓度NaCl溶液联合核酸酶的脱细胞方法有效的去除了基质内的细胞成分,没有造成胶原纤维的明显破坏;脱细胞人角膜基质透镜联合纤维蛋白胶构建的组织工程角膜基质能够用于动物板层角膜移植以恢复角膜的完整性,具有良好的生物学性能和功能特征,可以为角膜基质损伤或穿孔的治疗提供新途径。
[Abstract]:Background: the corneal refractive system is an important part of the body is also an important defensive barrier. By traumatic, infectious or autoimmune disease caused by corneal perforation of cornea for ophthalmic emergencies, if not timely correct and effective treatment, further development is likely to cause serious complications, resulting in decline or loss of vision. Severe cases may also face eyeball consequences. Corneal transplantation is the main means in the treatment of corneal perforation. However, at present China's shortage of donor cornea, can not meet the needs. In addition, corneal perforation often accompanied by a strong and persistent inflammation, can cause immune rejection after corneal transplantation surgery due to failure in this part, patients often require multiple corneal transplant surgery and more donor cornea. Therefore, the lack of sources of donor cornea and postoperative immune rejection is present To solve the problem. In recent years, the rise and development of corneal tissue engineering research has brought new hope for patients with corneal blindness, the acellular porcine corneal matrix because of its good transmittance, biomechanical properties and biological compatibility, it has a good effect in animal experiments and clinical treatment. However, acellular the corneal stroma is xenograft, extracellular matrix after acellular treatment retention has potential risk of antigenicity; also has the potential for the spread of animal disease correlation, so its safety still needs long-term follow-up studies. In addition, religious beliefs and other factors also limit the clinical application of acellular porcine corneal stroma by femtosecond laser. Corneal small incision lenticule extraction (small incision lenticule extraction, SMILE) not only can correct myopia and astigmatism and corneal stromal lens removed the surgery side " The product can also be utilized again. Our research team began treatment method of human corneal stromal corneal lens implanted multilayer SMILE source for the first time by perforation since 2012, obtain satisfactory treatment effect. Subsequently, many domestic and foreign scholars have also successfully applied to the corneal defect or perforation treatment. However, due to the central thickness of single chip corneal lens is about 60-150 m, the edge thickness is 10-30 m, will need to multilayer corneal stromal lens after superposition can achieve sufficient thickness for transplantation. The operation is difficult, and the lens suture process can slide, distortion and edge of the graft angle caused by inconsistent. Postoperative astigmatism and graft failure, and biomechanical strength of the simple superposition of corneal lens remains to be further strengthened. In addition, although the matrix cells in corneal tissue damage Wound repair, but also may be associated with immune rejection after operation, and these cellular components through appropriate removal methods. Acellular fibrin glue is widely used in construction, eye surgery and corneal tissue engineering. However, whether fibrin glue for SMILE derived from human corneal stromal lens adhesion between layers will be the construction of tissue engineering corneal stroma with a certain thickness and biomechanical strength for further study to repair damage to the corneal stroma, especially after transplantation to observe the histology and ultrastructure has not been reported. Therefore, this study aims to investigate the source of SMILE to contain active corneal stromal cells of human corneal stromal lens combined with fibrin construction of colloidal feasibility, corneal tissue engineering at the same time, we will prepare acellular corneal stroma lens, then combined with fiber protein Glue the construction of tissue engineering corneal stroma, explore its biological characteristics and function after transplantation. Methods: under aseptic condition. In addition to residual tissue limbal ring in the iris, sclera, conjunctiva, and tear to the endothelial layer, respectively with 2mg/mLdispase II and -0.02%EDTA 0.25% trypsin digestion, obtaining human limbal epithelial cells (human limbal epithelial cells, hLECs). The fibrin glue for the origin of SMILE containing active corneal stromal cells of human corneal stromal lens (6.6mm diameter, central thickness greater than 100 m) of the adhesive layer, the corneal matrix. The reconstruction of corneal stroma in Transwell chamber, adding hLECs medium for 24 h then, the hLECs cell suspension was dripped on the corneal surface after 2 h incubation of submerged culture 2 weeks and 2 weeks of training. The gas-liquid interface finally were observed by HE staining to construct cell surface growth. Situation and organization structure; immunohistochemistry detection of corneal epithelial cell specific markers CK12 and limbal stem cell markers ABCG2 and p63 alpha; scanning electron microscope (scanning electron, microscopy, SEM) and transmission electron microscopy (transmission electron microscopy, TEM) ultrastructural detection constructs. By using the method of high concentration NaCl solution combined with nuclease the preparation of acellular corneal stroma lens (6.6 mm in diameter, the central thickness is greater than 100 m), by HE staining, DAP staining and TEM 1 detection of cell removal and detection of acellular efficiency by DNA quantitative analysis. The fibrin glue for acellular human corneal stromal lens adhesion between layers of the two reconstruction of tissue engineering corneal stroma, for New Zealand rabbits lamellar keratoplasty. Making the diameter of 4.0mm, in depth about 250 m planting bed, with a trephine drill for tissue engineering cornea matrix central diameter As the matrix tablets 4.25 mm as grafts by 10-0 nylon suture, the graft fixed to the graft bed. Subconjunctival injection of gentamicin plus dexamethasone; postoperative TobraDex eye drops 4 times a day, for 2 weeks respectively after 1,7,15,30,60,90 days to assess and record the angle conjunctival reaction; after 7,15,30,60,90 days of sodium fluorescein staining and anterior segment photography records; in coherencetomography 15,60,90 days after the operation of anterior segment optical (anterior Segment-Optical coherence tomography, AS-OCT); after 90 days of corneal topography examination; 15,60,90 days after the operation taken by 4% paraformaldehyde and 3% glutaraldehyde. Respectively for histological examination and TEM detection. Results: after 2 weeks of incubation and immersion of gas-liquid interface 2 weeks of training, HE staining showed that the inoculation can form a stratified epithelium arranged closely in corneal surface reconstruction hLECs The structure, similar to normal central corneal epithelium; immunohistochemistry showed that the expression of corneal epithelial cell marker CK12 the surface cells, and basal cells with high expression of limbal stem cell markers ABCG-2 and p63 alpha, for limbal stem cell character support can provide a suitable environment to maintain corneal stroma reconstruction; the matrix can be seen in the distribution of rare corneal stromal cell; fibrin glue as a part of the corneal stroma reconstruction, close adhesion and corneal lens, not by degradation of.SEM vaccination showed cobblestone morphology in corneal surface reconstruction of hLECs, good adhesion and growth, tight junctions between cells, cell surface covered with microvilli; TEM showed desmosome structure formed between hLECs, and the formation of hemidesmosome and basal membrane structure in the corneal surface, similar to normal human corneal epithelial tissue. After high concentration NaCl solution Combined with nuclease after acellular treatment, human corneal stromal lens was translucent, but after 100% sterile glycerol dehydration, can completely restore the staining of acellular corneal stroma after lens no cell components of residual.TEM showed acellular corneal stroma lens structure complete transparent.HE and DAPI staining, collagen gap has not been seriously damaged void.DNA, quantitative analysis of the residual cells from the DNA show that the removal of components of 94.2% human corneal stromal lens, acellular effect significantly. 3 month postoperative observation period, all animal were survived without infection and postoperative complications; no graft from the graft and the graft bed of newborn vascular graft melting, corneal calcification and immune rejection phenomenon. The graft complete epithelization time was 16 + 2 days, until after March, the grafts recovered transparent.AS-OCT results showed that after 15 days, The graft and the graft bed are in a state of edema; after surgery in February and March the grafts were still high signal, but no obvious edema. After March, corneal topography examination showed that the eye does not appear keratoconus and other corneal deformation showed.HE staining showed that after February, the graft and recipient cornea has good fusion. No significant difference between the two; graft in distribution of corneal stromal cells, granulocytes and lymphocytes infiltration was neutral; collagen arranged rules; graft surface 4-5 layer of corneal epithelial cells with normal distribution, corneal epithelial features.TEM display after February, between corneal epithelium and tissue engineering corneal stroma appeared new synthesis the basement membrane structure and hemidesmosomes, after March, collagen fibers arranged neatly, the spacing between the corneal stromal cells, activate the recovery of static state, similar to the normal cornea. Conclusion: SMILE derived from human corneal stromal lens Combined with fibrin glue to construct tissue engineering anterior lamellar structure with similar phenotype and normal cornea, corneal stroma reconstruction can support corneal epithelial cell adhesion, proliferation and formation of double layer structure; acellular method using high concentration NaCl solution combined with nuclease can remove the cellular components in the matrix, no cause obvious damage of collagen fibers; integrity of tissue engineering corneal stromal cells and human corneal stromal lens combined with fibrin glue can be used to construct animal corneal transplantation to restore the cornea, has good biological properties and functional characteristics, can provide a new way for the treatment of injury or perforation of the corneal stroma.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08

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