【摘要】：角膜盲是常見的致盲眼病,在我國(guó)有超過400萬(wàn)患者亟待治療。其中許多患者是由于人角膜上皮(Human Corneal Epithelium, HCEP)發(fā)生不可逆病變而致盲。盡管可以通過捐獻(xiàn)角膜移植來治愈,但限于捐獻(xiàn)角膜數(shù)量的嚴(yán)重不足,致使絕大多數(shù)患者無(wú)法重見光明。組織工程人角膜上皮(Tissue-engineered Human Corneal Epithelium, TE-HCEP)作為HCEP的等效替代物,是解決供體角膜不足、絕大多數(shù)HCEP異�；颊邿o(wú)法復(fù)明問題的有效途徑。TE-HCEP體外重建的關(guān)鍵要素包括結(jié)構(gòu)功能正常的HCEP種子細(xì)胞的獲得和具有理想生物相容性的載體支架的制備。其中如何獲得大量的符合用于TE-HCEP體外重建要求的種子細(xì)胞成為研究的熱點(diǎn)之一。連續(xù)性人角膜上皮細(xì)胞系能夠提供大量人角膜上皮細(xì)胞(Human Corneal Epithelial Cells, HCEPC),被認(rèn)為是種子細(xì)胞的理想來源,目前已經(jīng)建立的HCEPC細(xì)胞系均為癌基因轉(zhuǎn)染的永生化細(xì)胞系,因其潛在致瘤性,無(wú)法應(yīng)用于臨床移植。用非轉(zhuǎn)染方法建立HCEPC細(xì)胞系能為TE-HCEP體外重建提供足量的正常細(xì)胞,但仍未見HCEPC未經(jīng)轉(zhuǎn)染而建立細(xì)胞系的報(bào)道。為了解決TE-HCEP及全層角膜體外重建所需大量HCEP種子細(xì)胞的來源問題,本文利用非轉(zhuǎn)染方法建立HCEPC細(xì)胞系,進(jìn)而從此細(xì)胞系通過克隆化培養(yǎng)篩選出形態(tài)結(jié)構(gòu)和功能蛋白表達(dá)正常的單克隆細(xì)胞株,旨在為TE-HCEP及全層角膜的體外重建創(chuàng)造條件。 為了建立非轉(zhuǎn)染HCEPC細(xì)胞系,本文根據(jù)人角膜組織的結(jié)構(gòu)特點(diǎn),用0.25%胰蛋白酶液對(duì)撕下的人角膜上皮層進(jìn)行酶解后,再將上皮面朝下直接貼于0.01%明膠處理的24孔板中,12h后補(bǔ)加含bFGF、EGF、硫酸軟骨素、羧甲基殼多糖和Ⅳ型膠原的DMEM/F12完全培養(yǎng)液(20%胎牛血清),置于37℃、5%CO2培養(yǎng)箱中培養(yǎng),啟動(dòng)HCEPC的原代培養(yǎng)。在上述培養(yǎng)條件下,原代啟動(dòng)3d后即有細(xì)胞遷出,22d即可長(zhǎng)成細(xì)胞單層,細(xì)胞為典型的上皮樣細(xì)胞形態(tài),且生長(zhǎng)分裂旺盛,能穩(wěn)定傳代,凍存后再?gòu)?fù)蘇的細(xì)胞依然可以維持良好的生長(zhǎng)狀態(tài)和增殖能力。經(jīng)過連續(xù)的繼代培養(yǎng)(已超過60代),細(xì)胞生長(zhǎng)狀況依然良好,完成了非轉(zhuǎn)染HCEPC細(xì)胞系的建立。 對(duì)連續(xù)傳代HCEPC進(jìn)行細(xì)胞屬性鑒定是確定所建立細(xì)胞系確為HCEPC細(xì)胞系的關(guān)鍵。本文對(duì)已建立的非轉(zhuǎn)染HCEPC細(xì)胞系第90代細(xì)胞進(jìn)行了形態(tài)學(xué)觀察、生長(zhǎng)特性檢測(cè)、染色體分析、細(xì)胞免疫熒光檢測(cè)和致瘤性實(shí)驗(yàn)等鑒定。對(duì)細(xì)胞的形態(tài)學(xué)鑒定,包括光鏡和掃描電鏡觀察,結(jié)果顯示該細(xì)胞系細(xì)胞具備典型上皮樣細(xì)胞形態(tài),細(xì)胞表面富含微絨毛和偽足。生長(zhǎng)特性分析結(jié)果顯示,該細(xì)胞系細(xì)胞群體倍增時(shí)間為40.56h,細(xì)胞分裂以及代謝旺盛。染色體組型分析結(jié)果顯示,該細(xì)胞系細(xì)胞特征性染色體數(shù)目為2n=46,并且具備人染色體核型特征,其中出現(xiàn)了染色體數(shù)目的非整倍性。對(duì)HCEP特異性標(biāo)志蛋白免疫熒光檢測(cè)結(jié)果顯示,該細(xì)胞系細(xì)胞具有細(xì)胞角蛋白K3、K12和K19的陽(yáng)性表達(dá),結(jié)合染色體組型分析結(jié)果,可以初步確定本文所建立的非轉(zhuǎn)染細(xì)胞系確為HCEPC細(xì)胞系。致瘤性檢測(cè)結(jié)果顯示,該細(xì)胞系細(xì)胞無(wú)任何致瘤性。由此可見,本文建立的細(xì)胞系是非轉(zhuǎn)染、無(wú)致瘤性的HCEPC細(xì)胞系。 為了篩選出染色體數(shù)目正常的HCEPC以用HCEP種子細(xì)胞,本文利用已建立的非轉(zhuǎn)染HCEPC細(xì)胞系進(jìn)行了克隆化研究。采用有限稀釋法,以小鼠腹腔巨噬細(xì)胞為飼養(yǎng)層細(xì)胞對(duì)HCEPC細(xì)胞系細(xì)胞進(jìn)行克隆化擴(kuò)增,挑選出單克隆細(xì)胞株進(jìn)行染色體組型分析,篩選一株具有2n=46正常核型的單克隆細(xì)胞株,命名為HCEP-5A1單克隆細(xì)胞株。該單克隆細(xì)胞株細(xì)胞經(jīng)凍存然后復(fù)蘇后其生長(zhǎng)狀態(tài)保持良好。通過免疫熒光檢測(cè)該單克隆細(xì)胞株細(xì)胞的HCEP標(biāo)志蛋白、連接蛋白和功能蛋白的表達(dá)情況,結(jié)果顯示其具有K3和K12的陽(yáng)性表達(dá),證明其HCEPC屬性；具有間隙連接蛋白-43和整聯(lián)蛋白β1的陽(yáng)性表達(dá),說明其具備形成細(xì)胞通訊連接和錨定連接的潛能,可形成完整的人角膜上皮層；具有鈣泵PMCA1/4蛋白和14-3-3s蛋白的陽(yáng)性表達(dá),說明其具備發(fā)揮正常HCEPC的跨膜運(yùn)輸和形成復(fù)層結(jié)構(gòu)的功能。由此可見篩選出的HCEP-5A1單克隆細(xì)胞株符合用作HCEP種子細(xì)胞的條件,可以用于TE-HCEP的體外重建。 綜上所述,本文成功建立了非轉(zhuǎn)染無(wú)致瘤性的HCEPC細(xì)胞系,并且篩選出了染色體組型正常的HCEP種子細(xì)胞,解決了HCEP種子細(xì)胞來源問題,為TE-HCEP體外重建,以及組織工程人全角膜的體外重建奠定了基礎(chǔ)。
[Abstract]:Corneal blindness is a common cause of blindness, and more than 4 million patients are in need of treatment in China. Many of these patients were blind due to irreversible changes in human corneal epithelium (HCEP). Although it can be cured by the donation of a corneal graft, it is limited to a serious shortage of the amount of the donor cornea, which makes the vast majority of the patients unable to see the light. Tissue engineering human corneal epithelium (TE-HCEP), as an equivalent substitute for HCEP, is an effective way to solve the problem of insufficient donor cornea and most of the HCEP abnormal patients. The key elements of TE-HCEP in vitro reconstruction include the preparation of the HCEP seed cells with normal structural function and the preparation of the carrier stent with the desired biocompatibility. How to obtain a large number of seed cells that meet the requirements for in vitro reconstruction of TE-HCEP is one of the hot spots in the study. Consecutive human corneal epithelial cells can provide a large number of human corneal epithelial cells (HCEPC), considered to be the ideal source of seed cells, and the HCEPC cell line that has been established is an immortalized cell line transfected with an oncogene due to its potential tumorigenicity, Can't be applied to clinical transplant. The establishment of HCEPC cell line by non-transfection method can provide a sufficient number of normal cells for the in vitro reconstruction of TE-HCEP, but no report of the cell line is established without the transfection of the HCEPC. In order to solve the source problem of the large number of HCEP seed cells required for the in vitro reconstruction of TE-HCEP and the whole-layer cornea, the HCEPC cell line is established by the non-transfection method, in order to create the conditions for the in vitro reconstruction of the TE-HCEP and the whole-layer cornea. In order to establish a non-transfected HCEPC cell line, according to the structural characteristics of human corneal tissue, after enzymatic hydrolysis of the torn human corneal epithelial layer with 0.25% trypsin solution, the upper leather surface was directly applied to the 24-hole plate treated with 0. 01% gelatin, and then added with bFGF, EGF and sulfuric acid after 12h. DMEM/ F12 complete culture solution (20% fetal bovine serum) of osteopsin, permethyl shell polysaccharide and type IV collagen, cultured in a 37 & deg; C, 5% CO2 incubator, and the primary culture of HCEPC was initiated in that culture condition, after the primary start of 3d, i. e., the cell is removed, and the cell monolayer is grown, and the cell is a typical epithelial-like cell, and the cell is a typical epithelial-like cell, and the growth and division are vigorous, and the cells can be stably passaged, and the cells that are recovered after the freeze-storage can still maintain good growth state and proliferation. The cell growth was still good after successive successive generations (over 60 generations), and the non-transfection of the HCEPC cell line was completed. Establishment. The cell property identification of the continuous passage HCEPC is to determine that the established cell line is a HCEPC fine The key of the cell line is that the 90th generation cells of the non-transfected HCEPC cell line, which have been established, were observed by morphological observation, growth characteristic detection, chromosome analysis, cell immunofluorescence and tumorigenicity. The morphological identification of the cell, including the observation of the light and the scanning electron microscope, showed that the cell line of the cell line had a typical epithelial-like cell morphology, and the cell surface was rich in micro-organisms. The results of the analysis of the growth characteristics of the cell line showed that the population doubling time of the cell line was 40. 56h, and the cell division was The number of the characteristic chromosomes of the cell line is 2n = 46, and the chromosome number of the cell line of the cell line is 2n = 46, and the number of the chromosomes has been found. The results of the immunofluorescence test of the HCEP-specific marker protein showed that the cell line cells had positive expression of the cytokeratin K3, K12 and K19, and combined with the results of the karyotype analysis, it was possible to preliminarily determine that the non-transfected cell line established in this paper is the HCE. PC cell line. The results of the tumorigenicity test showed that the cell line did not The cell line established in this article is a non-transfected, non-tumorigenic HCE. PC cell line. In order to screen the normal HCEPC for the number of chromosomes to use HCEP seed cells, this paper uses the established non-transfected HCEPC cell line The cloning of HCEPC cell line was carried out by using a limited dilution method. The cell of HCEPC cell line was cloned and amplified by the mouse peritoneal macrophages, and the monoclonal cell line was selected for chromosome group analysis. A monoclonal cell line with 2n = 46 normal karyotype was selected, named HCEP-5. A1, the cell of the monoclonal cell line is frozen and then recovered, The growth state is good. The expression of the HCEP marker protein, the connexin and the functional protein of the monoclonal cell line cell is detected by immunofluorescence, and the result shows that it has the positive expression of K3 and K12 to prove the HCEPC property, and has the gap connection protein-43 and the whole-linked egg. The positive expression of the white antigen 1 indicates that it has the potential to form a cell communication connection and an anchor connection, can form a complete human corneal epithelial layer, and has a positive expression of a calcium pump PMCA1/ 4 protein and a 14-3-3s protein, indicating that it is provided with a cross-membrane transport and a shape of a normal HCEPC. thus, the screened HCEP-5A1 monoclonal cell line meets the condition that the HCEP-5A1 monoclonal cell line is used as the HCEP seed cell, and can be used for TE-H In conclusion, the non-transfectively non-tumorigenic HCEPC cell line was successfully established, and the normal HCEP seed cells of the chromosome group were selected, the origin of the HCEP seed cell was solved, the in vitro reconstruction of TE-HCEP and the full-angle of the tissue engineering were solved.
【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.18

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