【作者】 杨洪收；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2010， 硕士

【摘要】 角膜是由角膜上皮层、前弹力层、基质层、后弹力层和内皮层组成,而角膜上皮层在防止角膜水分散失和外界病原体入侵中具有不可替代的作用。但由于角膜上皮层直接与外界接触,故易受到损伤和感染而引起结构和功能异常,使角膜上皮发生不可逆病变,严重者则引起角膜上皮盲。在我国约400余万、全世界约5500余万角膜盲患者中,许多患者为角膜上皮盲。目前,角膜上皮移植是角膜上皮盲治愈的唯一途径,但由于捐献的供体角膜数量的严重不足,致使绝大多数角膜上皮盲患者无法通过角膜上皮移植而重见光明。组织工程角膜上皮作为供体角膜上皮的替代物,是目前解决供体角膜材料不足的一种新的可行途径,也是众多角膜上皮盲患者早日重见光明的唯一希望。为了建立组织工程角膜上皮的体外重建条件,本文拟对业已建立的人角膜上皮细胞系进行鉴定,并以非转染、无致瘤性人角膜上皮细胞系细胞为种子细胞、以去上皮层羊膜为载体支架,采用气-液界面培养法对组织工程人角膜上皮的体外重建进行了研究。首先,本文利用光镜观察、生长特性分析、染色体分析、免疫荧光检测以及BALB/c裸鼠致瘤性检验等方法对业已建立的人角膜上皮细胞系进行了初步鉴定。光镜观察结果显示,体外培养的人角膜上皮细胞透明度高,形态饱满,呈现出稳定的上皮样细胞形态,3天左右即可传代1次；一生长特性分析结果显示,该细胞系细胞的群体倍增时间为45.42小时,表明该细胞系细胞的生长分裂旺盛；染色体计数与核型分析结果显示,该细胞系虽然出现了染色体数目的非整倍性(42～54条),但其特征性染色体数目仍为46条,约占细胞总数的56%,并具有典型的人染色体特征,包括6对中央着丝粒染色体、11对亚中着丝粒染色体、5对近端着丝粒染色体和1对X染色体；免疫荧光检测显示,具有角膜上皮细胞特异性的标志蛋白——角蛋白K3的阳性表达；该细胞系细胞致瘤性检验结果显示,该细胞系细胞皮下接种后没有引发肿瘤的形成,表明其没有任何致瘤性。由此可见,现有人角膜上皮细胞系细胞可以用作组织工程人角膜上皮体外重建的种子细胞。其次,本文进行了去上皮层羊膜的制备研究。对新鲜羊膜采用0.25%胰蛋白酶-0.02%EDTA(1:1)混合液消化30分钟,经细胞刮刀刮擦上皮面后用适量D-Hanks轻轻清洗,获得去上皮层羊膜,用作组织工程人角膜上皮体外重建的载体支架。最后,本文以非转染、无致瘤性人角膜上皮细胞系细胞为种子细胞、以去上皮层羊膜为载体支架,采用气-液界面培养法进行了组织工程人角膜上皮的体外重建研究。先将去上皮羊膜贴在插入式培养皿底部,37℃5%CO2培养箱中干贴2天,加入100μL0.01%Ⅳ型胶原,待胶原溶液自然干燥后,向每个插入式培养皿中接种100μL密度为3×106个/mL的种子细胞,24小时候转至气-液界面,含10%胎牛血清的DMEM培养液在5%C02培养箱中培养。光镜观察结果显示,接种后2小时后,角膜上皮细胞即粘附在羊膜上皮面,24小时形成单层,第9天即可形成大约5-8层细胞。组织工程人角膜上皮石蜡切片的HE染色结果显示,培养5天时即可形成由3层角膜上皮细胞构成的复层上皮、培养7天时即可形成由4-5层角膜上皮细胞构成的复层上皮、培养9天时即可形成由5-8层角膜上皮细胞构成的复层上皮,其透明度好、表面光滑平整,各层细胞之间连接紧密,且底层细胞与去上皮层羊膜连接紧密；对第9天体外重建组织工程人角膜上皮的免疫荧光检测结果显示,组织工程人角膜上皮的种子细胞具有角膜上皮细胞特异性的标志蛋白——角蛋白K3的阳性表达,同时还能表达整联蛋白β1；对第9天体外重建组织工程人角膜上皮的扫描电镜观察结果显示,组织工程人角膜上皮的种子细胞绝大多数仍保持有角膜上皮细胞的原有形态,与在体角膜上皮细胞一样其表面具有微绒毛结构,且表层细胞之间结合紧密,形成了连续致密的角膜上皮细胞层。上述结果表明,种子细胞在体外重建后仍保持有角膜上皮细胞的属性,能形成类似在体角膜上皮的由5-8层细胞构成的复层角膜上皮,并具有与载体支架形成锚定连接的能力。综上所述,本文以非转染、无致瘤性人角膜上皮细胞系细胞为种子细胞、以去上皮层羊膜为载体支架,采用气-液界面培养法成功体外重建出了在形态和属性上与在体人角膜上皮近似的组织工程人角膜上皮,为组织工程人角膜上皮的规模化体外重建及其临床应用,以及组织工程人全角膜的体外重建奠定了基础。更多还原

【Abstract】 Cornea is composed of epithelium, Bowman,stroma, descemet and endothelium and play irreplaceable role to prevent water loss and pathogen invasion. Since the epithelium of cornea contacts with external environment directly,it is prone to be hurted and infected resulted in structure and function abnormity which perhaps bring on ireversible lesions or even cause bind corneal epithelium.There are many paients caused by bind corneal epithelium in four million people in China and 55 million bind corneal patients. At present, corneal epithelial transplantation is the sole method to cure bind corneal epithelium, however most of the patients could not accecpt corneal epithelial transplantation for the serious shortage of donated corneas. Corneal epithelial tissue engineering, as succedaneum of corneal epithelial, is a new feasible method to solve the lack of corneal material and also the hope of bind corneal epithelium patients. In order to bulid corneal epithelial tissue engineering vitro rebuild conditions, current study plan to identify human corneal epithelial clones that has been built and study human corneal epithelial tissue vitro rebuild utilizing air-liquid interface culture method.First of all,current thesis coducted preliminary identification on established human corneal epithelial clone using microscope observation, growth characteristics and chromosome analysis, BALB/c tumorigenicity test. According to the results of optical microscope observation, vitro-cultured human corneal epithelial own higher transparency and plump conformation as stable epithelial-like cell morphology that could passage when cultured 3 days.Proliferation time of the cells is 45.42 h indicated keep strong ability to cleavage. Chromosome analysis showed that the cell lines have their Characteristic chromosome number in 46, although some cells are chromosomal aneuploidy.Karyotype analysis showed that the cells have a typical diploid karyotype(12m+22s+10t+XX).Cells subcutaneous injection of the cell line has not caused tumorigenesis implied no any tumorigenicity as tumorigenicity test results showed. Thus it can be seen current cells of human corneae epithelium cell line could be used as seed cells of vitro-rebuilt human corneal epithelial tissue engineering.Meanwhile, current article conducted preparing research eliminating amniotic epithelium.To ingest fresh amnion using 0.25% Trypsin-0.02% EDTA mixture, washing utilizing D-Hanks solution after cleaning pellicle to gain amniotic eptthelium to served as carrier bracket of vitro-rebuilt human corneal epithelial tissue engineering.At last, vitro-rebuilt human corneal epithelial tissue engineering is studied using air-liquid interface culture method, non-transfected, non-oncogenic cells of human corneal epithelial cell line as seed cells and cleaning pellicle amnion as carrier bracket. According to the results of optical microscope, corneal epithelial cells attached to the leather of amniotic membrane could form single layer for 24 h and could form 5-8 layers at the 9th day. HE staining results of human corneal epithelial tissue engineering slices showed it could form stratified epithelium consisted of 3 layers corneal epithelial cells after 5 days, stratified epithelium consisted of 4-5 layers corneal epithelial cells after 7 days, and stratified epithelium with preferable transparency and connectivity, consisted of 5-8 layers corneal epithelial cells after 9 days. According to the immunofluorescence test results, the seed cells possess Keratin K3 positive expression which is the marker proteins of corneal epithelial cell specificity, meanwhile expresses integrinβ1.Based on scanning electron microscopy results, the seed cells still keep original form of corneal epithelial cells, possess microvilli structure at surface and forms continuous dense layer of corneal epithelial cells.These results indicate that seed cells still could maintain the properties of corneal epithelial cells after vitro reconstrction,form 5-8 layers of stratified corneal epithelium similar to corneal epithelial cells and possess anchor connective ability. In conclusion, current article successfully rebuilt tissue engineering human corneal epithelial similar to forms and proporties of vivo human corneal epithelial using air-liquid interface culture method, non-transfected, non-oncogenic cells of human corneal epithelial cell line as seed cells and cleaning pellicle amnion as carrier bracket and supply reference for large scale vitro-reconstruction, clinical application and vitro-reconstruction of tissue engineering of human whole cornea.更多还原