本文關(guān)鍵詞：SMILE來源的人角膜基質(zhì)透鏡聯(lián)合纖維蛋白膠構(gòu)建組織工程角膜基質(zhì)的實(shí)驗(yàn)研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小切口角膜基質(zhì)透鏡取出術(shù) 角膜基質(zhì)透鏡 纖維蛋白膠 脫細(xì)胞 組織工程角膜基質(zhì) 角膜穿孔

【摘要】：背景:角膜是眼屈光系統(tǒng)的重要組成部分,同時(shí)也是重要的機(jī)體防御屏障。由外傷性、感染性或免疫性角膜病所致的角膜穿孔為眼科急癥,如不及時(shí)給予正確有效的治療,很可能進(jìn)一步發(fā)展導(dǎo)致嚴(yán)重的并發(fā)癥,最終造成視力下降或喪失,嚴(yán)重者還可能面臨眼球摘除的后果。而角膜移植是目前治療角膜穿孔的主要手段。然而,目前我國角膜供體嚴(yán)重匱乏,遠(yuǎn)遠(yuǎn)不能滿足需要。此外,角膜潰瘍穿孔時(shí)往往伴有強(qiáng)烈而持續(xù)的炎癥反應(yīng),容易引起角膜移植術(shù)后免疫排斥反應(yīng)導(dǎo)致手術(shù)失敗,這部分病人往往需要多次的角膜移植手術(shù)和更多的供體角膜。因此,供體角膜來源匱乏和術(shù)后免疫排斥是目前亟需解決的問題。近年來,組織工程角膜研究的興起和發(fā)展為角膜盲患者帶來新的希望,其中,脫細(xì)胞豬角膜基質(zhì)因具有較好的透光率、生物力學(xué)性能及生物相容性,在動(dòng)物實(shí)驗(yàn)和臨床治療方面取得了較好的效果。然而,脫細(xì)胞豬角膜基質(zhì)是異種移植物,脫細(xì)胞處理后保留的細(xì)胞外基質(zhì)具有潛在的抗原性;同時(shí)還具有傳播潛在的動(dòng)物相關(guān)性疾病的危險(xiǎn),因此其安全性尚需要長期的隨訪研究。此外,宗教信仰等因素也限制了脫細(xì)胞豬角膜基質(zhì)的臨床應(yīng)用。飛秒激光小切口角膜基質(zhì)透鏡取出術(shù)(small incision lenticule extraction,SMILE)不僅可以矯正近視及散光,而且取出的角膜基質(zhì)透鏡這一手術(shù)"副產(chǎn)品"還可以再利用。我們的研究團(tuán)隊(duì)自2012年開始首次利用多層SMILE來源的人角膜基質(zhì)透鏡移植的方法治療角膜穿孔,獲得較為滿意的治療效果。隨后,國內(nèi)外許多學(xué)者也成功將這種方法用于角膜基質(zhì)缺損或穿孔的治療。然而,由于單片角膜基質(zhì)透鏡的中央厚度約為60-150μm,邊緣厚度為10-30 μm,需要將多層角膜基質(zhì)透鏡疊加后才能達(dá)到足夠的厚度用于移植。手術(shù)操作的難度較大,同時(shí)縫合的過程中透鏡片可以滑動(dòng),易引起植片的扭曲變形及邊緣成角不一致,造成術(shù)后散光和植片混濁,而且單純疊加的角膜基質(zhì)透鏡的生物力學(xué)強(qiáng)度還有待進(jìn)一步加強(qiáng)。此外,雖然基質(zhì)細(xì)胞參與角膜組織損傷的修復(fù),但也可能與術(shù)后的免疫排斥反應(yīng)有關(guān),而這些細(xì)胞成分可以通過合適的脫細(xì)胞方法去除。纖維蛋白膠廣泛用于眼科手術(shù)及組織工程角膜的構(gòu)建,然而,能否將纖維蛋白膠用于SMILE來源的人角膜基質(zhì)透鏡的層間粘合,將兩者構(gòu)建成具有一定厚度和生物力學(xué)強(qiáng)度的組織工程角膜基質(zhì)用于角膜基質(zhì)損傷的修復(fù)有待進(jìn)一步研究,尤其是其移植術(shù)后的組織學(xué)和超微結(jié)構(gòu)的觀察尚無報(bào)道。因此,本研究旨在探討以SMILE來源的含有活性角膜基質(zhì)細(xì)胞的人角膜基質(zhì)透鏡聯(lián)合纖維蛋白膠體外構(gòu)建組織工程前板層角膜的可行性,同時(shí),我們將制備脫細(xì)胞的人角膜基質(zhì)透鏡,然后聯(lián)合纖維蛋白膠構(gòu)建組織工程角膜基質(zhì),探討其移植后的生物學(xué)特性和功能。方法:在無菌條件下去除角膜緣環(huán)組織中殘余的鞏膜、虹膜、結(jié)膜,并撕去內(nèi)皮層后,分別用2mg/mLdispaseⅡ和0.25%胰酶-0.02%EDTA消化,獲取人角膜緣上皮細(xì)胞(human limbal epithelial cells,hLECs)。將纖維蛋白膠用于SMILE來源的含有活性角膜基質(zhì)細(xì)胞的人角膜基質(zhì)透鏡(直徑6.6mm,中央厚度大于100μm)的層間粘合,將兩者重建成角膜基質(zhì)。將重建的角膜基質(zhì)置于Transwell小室內(nèi),加入hLECs培養(yǎng)基培養(yǎng)24 h,然后將hLECs細(xì)胞懸液滴加在角膜基質(zhì)表面,孵育2 h后,進(jìn)行2周的浸沒培養(yǎng)和2周的氣液界面培養(yǎng)。最后取材進(jìn)行HE染色觀察構(gòu)建物表面細(xì)胞生長情況及組織結(jié)構(gòu);免疫組織熒光檢測角膜上皮細(xì)胞特異性標(biāo)志物CK12和角膜緣干細(xì)胞標(biāo)志物ABCG2和p63α;掃描電鏡(scanning electron microscopy,SEM)及透射電鏡(transmission electron microscopy,TEM)檢測構(gòu)建物的超微結(jié)構(gòu)。采用高濃度NaCl溶液聯(lián)合核酸酶的方法制備脫細(xì)胞人角膜基質(zhì)透鏡(直徑6.6 mm,中央厚度大于100μm),通過HE染色、DAPⅠ染色及TEM檢測細(xì)胞脫除情況并通過DNA定量分析檢測脫細(xì)胞效率。將纖維蛋白膠用于脫細(xì)胞人角膜基質(zhì)透鏡的層間粘合,將兩者重建成組織工程角膜基質(zhì),用于新西蘭大白兔的板層角膜移植。制作直徑為4.0mm,深度約250 μm的板層植床,用環(huán)鉆鉆取組織工程角膜基質(zhì)中央直徑為4.25 mm的基質(zhì)片作為植片,采用10-0尼龍線間斷縫合,將植片固定于植床。術(shù)畢球結(jié)膜下注射慶大霉素加地塞米松;術(shù)后典必殊滴眼液每天4次滴術(shù)眼,維持2周。分別于術(shù)后1、7、15、30、60、90天評價(jià)并記錄角結(jié)膜反應(yīng);于術(shù)后7、15、30、60、90天行熒光素鈉染色及眼前節(jié)照相記錄;于術(shù)后15、60、90天行眼前節(jié)光學(xué)相干斷層掃描(anterior segment-optical coherence tomography,AS-OCT)檢查;于術(shù)后90天行角膜地形圖檢查;于術(shù)后15、60、90天取材經(jīng)4%多聚甲醛及3%戊二醛固定,分別進(jìn)行組織學(xué)檢查及TEM檢測。結(jié)果:經(jīng)過2周的浸沒培養(yǎng)和2周的氣液界面培養(yǎng),HE染色顯示接種在重建的角膜基質(zhì)表面的hLECs可形成排列緊密的復(fù)層上皮結(jié)構(gòu),與正常中央部角膜上皮相似;免疫組織熒光顯示其表層細(xì)胞高表達(dá)角膜上皮細(xì)胞標(biāo)記物CK12,而基底部細(xì)胞高表達(dá)角膜緣干細(xì)胞標(biāo)記物ABCG-2及p63α,說明重建的角膜基質(zhì)能為角膜緣干細(xì)胞特性的維持提供適宜的環(huán)境支持;基質(zhì)內(nèi)可見較稀少的角膜基質(zhì)細(xì)胞分布;纖維蛋白膠作為重建的角膜基質(zhì)的一部分,與角膜基質(zhì)透鏡緊密黏附,未被降解。SEM顯示接種在重建的角膜基質(zhì)表面的hLECs呈鋪路石樣形態(tài),黏附生長良好,細(xì)胞間緊密連接,細(xì)胞表面布滿微絨毛;TEM顯示hLECs之間形成橋粒結(jié)構(gòu),并在角膜基質(zhì)表面形成了半橋粒和基底膜結(jié)構(gòu),與正常人角膜上皮組織相似。經(jīng)過高濃度NaCl溶液聯(lián)合核酸酶脫細(xì)胞處理后,人角膜基質(zhì)透鏡呈半透明樣,但經(jīng)過100%無菌甘油脫水后,能夠完全恢復(fù)透明。HE染色及DAPI染色顯示脫細(xì)胞處理后人角膜基質(zhì)透鏡內(nèi)無細(xì)胞成分殘留。TEM顯示脫細(xì)胞人角膜基質(zhì)透鏡結(jié)構(gòu)完整,膠原間隙并未受到嚴(yán)重的破壞,可見細(xì)胞脫離后殘留的空隙。DNA定量分析顯示去除了人角膜基質(zhì)透鏡中94.2%的DNA成分,脫細(xì)胞效果顯著。術(shù)后3個(gè)月觀察期間內(nèi),所有動(dòng)物均存活,術(shù)眼未出現(xiàn)感染和出血等并發(fā)癥;未見植片脫離、植片和植床新生血管、植片融解、角膜鈣化和免疫排斥等現(xiàn)象。植片完全上皮化的時(shí)間為16±2天,至術(shù)后3月,植片基本恢復(fù)透明。AS-OCT結(jié)果顯示術(shù)后15天,植片和植床均處于水腫狀態(tài);至術(shù)后2月和3月植片仍呈較高信號(hào),但無明顯水腫。術(shù)后3月,角膜地形圖檢查顯示術(shù)眼未出現(xiàn)圓錐角膜及其他角膜形變表現(xiàn)。HE染色顯示術(shù)后2月,植片與受體角膜已經(jīng)融合良好,兩者間無明顯界限;植片內(nèi)有角膜基質(zhì)細(xì)胞分布,未見中性粒細(xì)胞和淋巴細(xì)胞浸潤現(xiàn)象;膠原排列整齊、規(guī)則;植片表面有4-5層角膜上皮細(xì)胞分布,具有正常角膜上皮特征。TEM顯示術(shù)后2月,角膜上皮與組織工程角膜基質(zhì)之間開始出現(xiàn)新合成的基底膜結(jié)構(gòu)和半橋粒,術(shù)后3月,膠原纖維排列整齊,間距一致,激活的角膜基質(zhì)細(xì)胞恢復(fù)靜止?fàn)顟B(tài),與正常角膜相似。結(jié)論:SMILE來源的人角膜基質(zhì)透鏡聯(lián)合纖維蛋白膠構(gòu)建的組織工程前板層角膜具有與正常角膜組織類似的表型和結(jié)構(gòu),重建的角膜基質(zhì)能夠支持角膜上皮細(xì)胞黏附、增殖和形成復(fù)層結(jié)構(gòu);使用高濃度NaCl溶液聯(lián)合核酸酶的脫細(xì)胞方法有效的去除了基質(zhì)內(nèi)的細(xì)胞成分,沒有造成膠原纖維的明顯破壞;脫細(xì)胞人角膜基質(zhì)透鏡聯(lián)合纖維蛋白膠構(gòu)建的組織工程角膜基質(zhì)能夠用于動(dòng)物板層角膜移植以恢復(fù)角膜的完整性,具有良好的生物學(xué)性能和功能特征,可以為角膜基質(zhì)損傷或穿孔的治療提供新途徑。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.08

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