非转染人角膜上皮细胞系的建立、鉴定及其克隆化研究

发布时间：2018-11-10 08:06

【摘要】：角膜盲是常见的致盲眼病,在我国有超过400万患者亟待治疗。其中许多患者是由于人角膜上皮(Human Corneal Epithelium, HCEP)发生不可逆病变而致盲。尽管可以通过捐献角膜移植来治愈,但限于捐献角膜数量的严重不足,致使绝大多数患者无法重见光明。组织工程人角膜上皮(Tissue-engineered Human Corneal Epithelium, TE-HCEP)作为HCEP的等效替代物,是解决供体角膜不足、绝大多数HCEP异常患者无法复明问题的有效途径。TE-HCEP体外重建的关键要素包括结构功能正常的HCEP种子细胞的获得和具有理想生物相容性的载体支架的制备。其中如何获得大量的符合用于TE-HCEP体外重建要求的种子细胞成为研究的热点之一。连续性人角膜上皮细胞系能够提供大量人角膜上皮细胞(Human Corneal Epithelial Cells, HCEPC),被认为是种子细胞的理想来源,目前已经建立的HCEPC细胞系均为癌基因转染的永生化细胞系,因其潜在致瘤性,无法应用于临床移植。用非转染方法建立HCEPC细胞系能为TE-HCEP体外重建提供足量的正常细胞,但仍未见HCEPC未经转染而建立细胞系的报道。为了解决TE-HCEP及全层角膜体外重建所需大量HCEP种子细胞的来源问题,本文利用非转染方法建立HCEPC细胞系,进而从此细胞系通过克隆化培养筛选出形态结构和功能蛋白表达正常的单克隆细胞株,旨在为TE-HCEP及全层角膜的体外重建创造条件。为了建立非转染HCEPC细胞系,本文根据人角膜组织的结构特点,用0.25%胰蛋白酶液对撕下的人角膜上皮层进行酶解后,再将上皮面朝下直接贴于0.01%明胶处理的24孔板中,12h后补加含bFGF、EGF、硫酸软骨素、羧甲基壳多糖和Ⅳ型胶原的DMEM/F12完全培养液(20%胎牛血清),置于37℃、5%CO2培养箱中培养,启动HCEPC的原代培养。在上述培养条件下,原代启动3d后即有细胞迁出,22d即可长成细胞单层,细胞为典型的上皮样细胞形态,且生长分裂旺盛,能稳定传代,冻存后再复苏的细胞依然可以维持良好的生长状态和增殖能力。经过连续的继代培养(已超过60代),细胞生长状况依然良好,完成了非转染HCEPC细胞系的建立。对连续传代HCEPC进行细胞属性鉴定是确定所建立细胞系确为HCEPC细胞系的关键。本文对已建立的非转染HCEPC细胞系第90代细胞进行了形态学观察、生长特性检测、染色体分析、细胞免疫荧光检测和致瘤性实验等鉴定。对细胞的形态学鉴定,包括光镜和扫描电镜观察,结果显示该细胞系细胞具备典型上皮样细胞形态,细胞表面富含微绒毛和伪足。生长特性分析结果显示,该细胞系细胞群体倍增时间为40.56h,细胞分裂以及代谢旺盛。染色体组型分析结果显示,该细胞系细胞特征性染色体数目为2n=46,并且具备人染色体核型特征,其中出现了染色体数目的非整倍性。对HCEP特异性标志蛋白免疫荧光检测结果显示,该细胞系细胞具有细胞角蛋白K3、K12和K19的阳性表达,结合染色体组型分析结果,可以初步确定本文所建立的非转染细胞系确为HCEPC细胞系。致瘤性检测结果显示,该细胞系细胞无任何致瘤性。由此可见,本文建立的细胞系是非转染、无致瘤性的HCEPC细胞系。为了筛选出染色体数目正常的HCEPC以用HCEP种子细胞,本文利用已建立的非转染HCEPC细胞系进行了克隆化研究。采用有限稀释法,以小鼠腹腔巨噬细胞为饲养层细胞对HCEPC细胞系细胞进行克隆化扩增,挑选出单克隆细胞株进行染色体组型分析,筛选一株具有2n=46正常核型的单克隆细胞株,命名为HCEP-5A1单克隆细胞株。该单克隆细胞株细胞经冻存然后复苏后其生长状态保持良好。通过免疫荧光检测该单克隆细胞株细胞的HCEP标志蛋白、连接蛋白和功能蛋白的表达情况,结果显示其具有K3和K12的阳性表达,证明其HCEPC属性；具有间隙连接蛋白-43和整联蛋白β1的阳性表达,说明其具备形成细胞通讯连接和锚定连接的潜能,可形成完整的人角膜上皮层；具有钙泵PMCA1/4蛋白和14-3-3s蛋白的阳性表达,说明其具备发挥正常HCEPC的跨膜运输和形成复层结构的功能。由此可见筛选出的HCEP-5A1单克隆细胞株符合用作HCEP种子细胞的条件,可以用于TE-HCEP的体外重建。综上所述,本文成功建立了非转染无致瘤性的HCEPC细胞系,并且筛选出了染色体组型正常的HCEP种子细胞,解决了HCEP种子细胞来源问题,为TE-HCEP体外重建,以及组织工程人全角膜的体外重建奠定了基础。
[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.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.18

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