羊膜对角膜损伤的治疗作用及其机理研究

发布时间：2018-05-05 01:23

本文选题：角膜损伤 + 眼表重建　；参考：《哈尔滨工业大学》2017年博士论文

【摘要】：眼表疾病是眼科的常见病、多发病,且复发率高,是眼科主要致盲疾病之一。对于各种眼表疾病所致角膜上皮缺损的治疗,目前以眼表面重建术效果最好,常采用的方法是自体或异体角膜移植术。但自体角膜缘取材有限,并有造成健眼角膜缘干细胞缺乏的风险。异体角膜要求新鲜,且存在来源不足、排斥反应等问题。由于羊膜有其特有的结构特点和促进组织修复的生物学特性,研究者们开始转向以羊膜为原材料治疗眼表疾病的研究中。在对羊膜移植的研究基础上,羊膜的研磨提取液—羊膜匀浆液保留了羊膜中的有效生物成分,在治疗眼表疾病方面取得了一定的进展,是一种较新的尝试。随着生物技术和其他相关技术的发展,以羊膜上皮细胞作为“种子细胞”构建组织工程角膜上皮,在眼表疾病治疗中亦显示出良好的发展前景。本文以羊膜为原材料,从动物实验、临床研究、细胞培养三个层面,研究羊膜移植片、羊膜匀浆液、羊膜上皮细胞对角膜上皮缺损的治疗作用及作用机制,为解决眼表重建材料缺乏及严重眼表疾病的治疗提供新的方法和新的理论依据。首先建立兔角膜上皮损伤的动物模型,行以羊膜移植术,以观察羊膜对动物角膜伤口愈合的作用。结果发现,羊膜移植组与对照组比较,角膜上皮愈合加快,显微形态学观察显示,实验组细胞排列水平、细胞形态、炎症细胞数等方面较对照组均明显改善。其次,在动物实验的基础上,对不同病因所致的眼表上皮缺损患者24人(25眼),行以羊膜移植术,该临床研究发现:25只眼中21只眼眼表面得以重建,视力有不同程度的提高。再生的角膜上皮透明,结膜组织稳定、无感染。平均观察28.3周,所有21只眼无复发,另外3只眼因并发症致手术失败。再次,以上皮细胞缺损的兔角膜作为研究对象,观察不同浓度羊膜匀浆液对角膜上皮细胞增殖的作用。结果发现,羊膜匀浆液对兔角膜上皮细胞增殖和修复具有明显的促进作用,且这种促进作用在一定范围内随浓度增加而增强,即角膜上皮细胞的增殖率对羊膜匀浆液具有浓度依从性。羊膜匀浆组与羊膜移植组比较作用效果相同,但可避免手术并发症。最后,进行羊膜及角膜上皮细胞培养以及二者共同培养的细胞学研究。结果显示:羊膜上皮细胞在原代培养2 h后开始贴壁生长。细胞呈多角形、不规则形,排列呈铺路石样。体外可连续传4~5代,CK3、CK18、CK19呈阳性表达。流式细胞仪检测CK3、CK18、CK19表达率分别为40.84±3%,47.51±2%和32.41±3%。角膜上皮细胞在培养的第3天时开始爬出,呈多角形,排列均匀。经诱导培养后的羊膜上皮细胞呈多角形、扁平状,细胞体积增大。流式细胞学检测CK3表达率为76.40±0.3%,与诱导前相比有显著差异(P0.05),具有统计学意义。综上所述,本文针对羊膜进行了动物实验、临床研究、细胞培养三个层面的研究工作,实验和临床研究均证明了羊膜对角膜上皮缺损的修复具有促进作用,为解决眼表重建材料缺乏及严重眼表疾病的治疗提供了新的途径和方法。
[Abstract]:Ocular surface disease is one of the common diseases in the ophthalmology, which is frequently occurring and has a high recurrence rate. It is one of the main blindness diseases in the ophthalmology. For the treatment of corneal epithelial defects caused by ocular surface diseases, eye surface reconstruction is the best method. Autologous or allograft corneal transplantation is often used. The risk of limbal stem cell deficiency. Allograft cornea requires fresh, lack of source, rejection and other problems. The amniotic membrane has its unique structural characteristics and biological characteristics that promote tissue repair. Researchers began to turn to amniotic membrane for the treatment of ocular surface diseases. On the basis of amniotic membrane transplantation, amniotic membrane With the development of biotechnology and other related technologies, amniotic epithelial cells are constructed as "seed cells" to construct tissue engineered corneal epithelium with the development of biotechnology and other related technologies. With amniotic membrane as the raw material, this paper studies the therapeutic effect and mechanism of amniotic membrane transplantation, amniotic homogenate fluid and amniotic epithelial cells on corneal epithelial defect from three levels of animal experiment, clinical study and cell culture. It provides a new way to solve the deficiency of ocular surface reconstruction materials and the treatment of serious ocular surface diseases. Methods and new theoretical basis. First, an animal model of corneal epithelial injury in rabbits was established, and amniotic membrane transplantation was performed to observe the effect of amniotic membrane on the healing of corneal wound in animals. The results showed that the amniotic membrane transplantation group was compared with the control group, the corneal epithelial healing accelerated. The microscopic morphological observation showed that the cell arrangement level, cell morphology, and inflammation in the experimental group were in the experimental group. Secondly, on the basis of animal experiments, 24 patients (25 eyes) with ocular epithelia defect caused by different causes were treated with amniotic membrane transplantation. The clinical study found that the surface of 21 eyes in 25 eyes was reconstructed and the visual acuity was improved. The regenerated corneal epithelium was transparent and conjunctival tissue. The average observation was 28.3 weeks, all 21 eyes had no recurrence, and the other 3 eyes failed to operate due to complications. Again, the rabbit cornea with epithelial cell defect was used as the research object, and the effect of amniotic homogenate on the proliferation of corneal epithelial cells was observed. It has an obvious promoting effect, and the promotion effect is enhanced with the increase of concentration in a certain range, that is, the proliferation rate of corneal epithelial cells is dependent on the concentration of amniotic homogenate. The amniotic homogenate group has the same effect as amniotic membrane transplantation group, but it can avoid complications. Finally, the amniotic membrane and corneal epithelial cells are cultured. The results showed that the epithelial cells of the amniotic epithelial cells began to adhere to the wall after the primary culture of 2 h. The cells were polygonal, irregular, and arranged in the form of pave stone. In vitro, the cells could pass through 4~5 generation continuously, CK3, CK18, and CK19 were positive. The expression rate of CK3, CK18 and CK19 were 40.84 + 3%, 47.51 + 2% and 32. respectively. 41 + 3%. corneal epithelial cells began to climb out at third days of culture and were polygonal and evenly arranged. The amniotic epithelial cells after induction were polygonal, flat, and cell volume increased. The rate of expression of CK3 was 76.40 + 0.3%, compared with before induction (P0.05). The amniotic membrane has been studied in animal experiments, clinical studies, and cell culture at three levels. Both experimental and clinical studies have proved that amniotic membrane has a promoting effect on the repair of corneal epithelial defect. It provides a new way to solve the lack of ocular surface reconstruction materials and the treatment of serious ocular surface diseases.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R779.6

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