本文選題：角膜損傷 + 眼表重建�。� 參考：《哈爾濱工業(yè)大學(xué)》2017年博士論文

【摘要】：眼表疾病是眼科的常見病、多發(fā)病,且復(fù)發(fā)率高,是眼科主要致盲疾病之一。對于各種眼表疾病所致角膜上皮缺損的治療,目前以眼表面重建術(shù)效果最好,常采用的方法是自體或異體角膜移植術(shù)。但自體角膜緣取材有限,并有造成健眼角膜緣干細(xì)胞缺乏的風(fēng)險。異體角膜要求新鮮,且存在來源不足、排斥反應(yīng)等問題。由于羊膜有其特有的結(jié)構(gòu)特點和促進(jìn)組織修復(fù)的生物學(xué)特性,研究者們開始轉(zhuǎn)向以羊膜為原材料治療眼表疾病的研究中。在對羊膜移植的研究基礎(chǔ)上,羊膜的研磨提取液—羊膜勻漿液保留了羊膜中的有效生物成分,在治療眼表疾病方面取得了一定的進(jìn)展,是一種較新的嘗試。隨著生物技術(shù)和其他相關(guān)技術(shù)的發(fā)展,以羊膜上皮細(xì)胞作為“種子細(xì)胞”構(gòu)建組織工程角膜上皮,在眼表疾病治療中亦顯示出良好的發(fā)展前景。本文以羊膜為原材料,從動物實驗、臨床研究、細(xì)胞培養(yǎng)三個層面,研究羊膜移植片、羊膜勻漿液、羊膜上皮細(xì)胞對角膜上皮缺損的治療作用及作用機制,為解決眼表重建材料缺乏及嚴(yán)重眼表疾病的治療提供新的方法和新的理論依據(jù)。首先建立兔角膜上皮損傷的動物模型,行以羊膜移植術(shù),以觀察羊膜對動物角膜傷口愈合的作用。結(jié)果發(fā)現(xiàn),羊膜移植組與對照組比較,角膜上皮愈合加快,顯微形態(tài)學(xué)觀察顯示,實驗組細(xì)胞排列水平、細(xì)胞形態(tài)、炎癥細(xì)胞數(shù)等方面較對照組均明顯改善。其次,在動物實驗的基礎(chǔ)上,對不同病因所致的眼表上皮缺損患者24人(25眼),行以羊膜移植術(shù),該臨床研究發(fā)現(xiàn):25只眼中21只眼眼表面得以重建,視力有不同程度的提高。再生的角膜上皮透明,結(jié)膜組織穩(wěn)定、無感染。平均觀察28.3周,所有21只眼無復(fù)發(fā),另外3只眼因并發(fā)癥致手術(shù)失敗。再次,以上皮細(xì)胞缺損的兔角膜作為研究對象,觀察不同濃度羊膜勻漿液對角膜上皮細(xì)胞增殖的作用。結(jié)果發(fā)現(xiàn),羊膜勻漿液對兔角膜上皮細(xì)胞增殖和修復(fù)具有明顯的促進(jìn)作用,且這種促進(jìn)作用在一定范圍內(nèi)隨濃度增加而增強,即角膜上皮細(xì)胞的增殖率對羊膜勻漿液具有濃度依從性。羊膜勻漿組與羊膜移植組比較作用效果相同,但可避免手術(shù)并發(fā)癥。最后,進(jìn)行羊膜及角膜上皮細(xì)胞培養(yǎng)以及二者共同培養(yǎng)的細(xì)胞學(xué)研究。結(jié)果顯示:羊膜上皮細(xì)胞在原代培養(yǎng)2 h后開始貼壁生長。細(xì)胞呈多角形、不規(guī)則形,排列呈鋪路石樣。體外可連續(xù)傳4~5代,CK3、CK18、CK19呈陽性表達(dá)。流式細(xì)胞儀檢測CK3、CK18、CK19表達(dá)率分別為40.84±3%,47.51±2%和32.41±3%。角膜上皮細(xì)胞在培養(yǎng)的第3天時開始爬出,呈多角形,排列均勻。經(jīng)誘導(dǎo)培養(yǎng)后的羊膜上皮細(xì)胞呈多角形、扁平狀,細(xì)胞體積增大。流式細(xì)胞學(xué)檢測CK3表達(dá)率為76.40±0.3%,與誘導(dǎo)前相比有顯著差異(P0.05),具有統(tǒng)計學(xué)意義。綜上所述,本文針對羊膜進(jìn)行了動物實驗、臨床研究、細(xì)胞培養(yǎng)三個層面的研究工作,實驗和臨床研究均證明了羊膜對角膜上皮缺損的修復(fù)具有促進(jìn)作用,為解決眼表重建材料缺乏及嚴(yán)重眼表疾病的治療提供了新的途徑和方法。
[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.

【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R779.6

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