Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells

PURPOSE: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. METHODS: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. RESULTS: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exhibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. CONCLUSIONS: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.     

Comparative effects of estrogen on matrix metalloproteinases and cytokines in immortalized and primary human corneal epithelial cell cultures

PURPOSE: Recently, we found that 17beta-estradiol induces cytokine and MMP gene expression in SV40 immortalized human corneal epithelial cells (SV40 HCEs). The purpose of this study was to determine if 17beta-estradiol stimulates MMP secretion by cultures of SV40 HCEs and primary human corneal epithelial cells. Further, we determined if estrogen influenced cytokine and MMP gene expression in the primary cultures. METHODS: Gelatin zymography was used to identify MMP-2 and MMP-9 gelatinase activity in the culture medium from SV40 HCEs and primary human corneal epithelial cell cultures exposed to vehicle or 17beta-estradiol for 6 or 24 hour. In addition, 17beta-estradiol effects on cytokine and MMP gene expression in primary cultures were evaluated by real-time PCR methods. RESULTS: 17beta-estradiol had no significant effects on MMP activity in the culture media of either SV40 HCEs or primary corneal epithelial cell cultures. SV40 HCEs secreted predominantly MMP-2, whereas the primary cultures secreted predominantly MMP-9. In primary cultures, 17beta-estradiol caused a significant decrease of IL-6 and IL-8 gene expression, but had no effect on the levels of IL-1beta, MMP-2, and MMP-9 gene expression. CONCLUSIONS: Our findings suggest that 17beta-estradiol effects on gelatinase gene expression in SV40 HCEs are not translated into changes in secreted MMP activity. Estrogen influences on gene expression of cytokine and MMPs in primary cultures are different from those in SV40 HCEs.     

Synthesis of type II interleukin-1 receptors by human corneal epithelial cells but not by keratocytes

PURPOSE. The purpose of this study was to determine whether human corneal epithelial cells and keratocytes synthesize both the soluble and membrane forms of the type II IL-1 receptor (IL-1RII). METHODS. Primary cell cultures of human corneal epithelial cells and keratocytes were established from human corneas. RT-PCR was used to analyze cell cultures for expression of IL-1RII mRNA. The capacity of corneal cells to synthesize membrane-bound IL-1RII was determined by immunofluorescence microscopy, whereas ELISA was used to quantitate synthesis of soluble IL-1RII after IL-1alpha and TNF-alpha stimulation. RESULTS. Corneal epithelial cells expressed IL-1RII mRNA. The cells also stained positive for membrane-bound IL-1RII, and media harvested from epithelial cell cultures contained up to 50 pg/ml of soluble IL-1RII. Both IL-1alpha and TNF-alpha significantly enhanced the amounts of soluble IL-1RII released from epithelial cell surfaces. In contrast to epithelial cells, corneal keratocytes did not express IL-1RII mRNA. Membrane-bound IL-1RII was not detected on keratocytes, nor was soluble IL-1RII detected in culture media harvested from these cells. CONCLUSIONS. Human corneal epithelial cells but not corneal keratocytes synthesize both membrane and soluble forms of IL-1RII. Because both forms of IL-1RII can function as IL-1alpha antagonists, the results suggest that human corneal epithelial cells but not corneal keratocytes have evolved the capacity to dampen IL-1alpha responses through the production of IL-1RII.     

Growth and characterization of rabbit corneal cells in vitro

Primary organ cultures of rabbit corneal epithelium, stroma, and endothelium were established by microdissection. Secondary cultures of epithelial cells, keratocytes, and endothelial cells were established by serial passage. The doubling time for epithelial cells and keratocytes was 18 h, and endothelial cells doubled their number in 5 days. Ultrastructural studies demonstrated characteristic morphological, nuclear, and cytoplasmic features of corneal epithelial cells, keratocytes, and endothelial cells and confirmed the identity of the cell lines. The purity of the three distinct cell types was ascertained by indirect immunofluorescence techniques, using antibodies against keratin, which identified epithelial cells, and fibronectin, which identified keratocytes and endothelial cells. The indirect immunofluorescence technique represents a simple method to screen an aliquot of a cell suspension and determine the purity of corneal cells grown in vitro.     

Tropoelastin biosynthesis by corneal cells. Epithelial inhibition of keratocyte tropoelastin biosynthesis

The vertebrate cornea is an avascular tissue and does not contain elastic fibers. We tested the capacity of corneal epithelial cells and stromal keratocytes to synthesize tropoelastin. Explant cultures and cell cultures were obtained from these two cell types in standard culture conditions. Their elastin-synthetic activity was compared to skin explant cultures and to dermal fibroblast cell cultures. Both corneal cell types synthesized tropoelastin as shown by the incorporation of a radioactive precursor followed by immunoprecipitation of tropoelastin. When serial cultures of keratocytes were tested, tropoelastin biosynthesis strongly increased after the 3rd passage and was at the 9th passage more than the double of that of the first passage. When cocultures were studied with or without cell contact, epithelial cells partially inhibited tropoelastin biosynthesis by keratocytes. This inhibition was somewhat stronger (-36%, p < 0.005) with cell-to-cell contact than keeping separate epithelial cells and keratocytes bathing in the same medium (-18%, p < 0.005). When human skin fibroblasts were substituted for keratocytes with cell-to-cell contact, their tropoelastin biosynthesis was also inhibited by corneal epithelial cells (-42%, p < 0.005), to the same extent as for keratocytes. In Transwell culture, this inhibition was again somewhat lower (-36%, p < 0.005). Some diffusible factor produced by epithelial cells is apparently involved. The epithelial inhibition of tropoelastin biosynthesis by stromal keratocytes might represent one of the mechanisms keeping corneal stroma exempt of elastin fibers.     

Comparison of EphA receptor tyrosine kinases and ephrinA ligand expression to EphB-ephrinB in vascularized corneas

PURPOSE: Eph cell surface receptors and their ligands, ephrins, are involved in neuronal patterning and neovascularization. Our purpose is to compare and characterize the expression of ephrinA ligands and EphA receptors to ephrinB ligands and EphB receptors in excised mouse corneal tissue, in corneal epithelial and keratocyte cell lines, and during corneal angiogenesis. METHODS: Mouse corneal epithelial cells and keratocytes were immortalized using SV40T antigen viral infection of primary cultures. The immortalized epithelial cells and keratocytes were cloned and characterized using antibodies to keratin, vimentin, integrin alpha5beta1, and alpha-smooth muscle actin. Basic fibroblast growth factor pellets were implanted to induce corneal neovascularization. The eyes of wild-type, ephrinB2(tlacZ/+), and EphB4(tlacZ/+) heterozygous mice were harvested and sectioned 7 days after pellet implantation. Confocal immunohistochemistry was performed to compare the expression of the Eph/ephrinA family (EphA1-8, ephrinA1-5) and Eph/ephrinB family (EphB1-4, EphB6 ephrinB1-3). RESULTS: EphA1, EphA3, ephrinA1, ephrinA2, EphB1, EphB4, ephrinB1, and ephrinB2 were detected in wild-type mouse corneal epithelial cells and keratocytes. EphA2 was immunolocalized only in epithelial cells. Also, EphA3, ephrinA1, EphB1, EphB4, and ephrinB1 were immunolocalized to the corneal epithelium and stroma. In the vascularized corneas, ephrinB1 was immunolocalized mainly to the keratocytes around the vessels, and ephrinB2, EphB1, and EphB4 were colocalized mainly with CD31 to the vascular endothelial cells. CONCLUSIONS: The characterization of ephrin ligand and Eph receptor expression during cornea angiogensis in this study suggests that the Eph/ephrin family of receptor tyrosine kinases and their ligands may play a role in the regulation of corneal angiogenesis.     

IL-4 regulates chemokine production induced by TNF-α in keratocytes and corneal epithelial cells

BACKGROUND: Eosinophils are thought to play a major role in the pathogenesis of corneal lesions in ocular allergies. The regulation of chemokine production in corneal cells by the Th2 cytokine, interleukin 4 (IL-4), was examined in order to investigate its role in ocular allergies. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to tumour necrosis factor alpha (TNF-alpha) and/or IL-4. 24 hours after exposure, culture supernatants were removed and concentrations of IL-8 and RANTES were quantified by ELISA assay. RESULTS: Simultaneous addition of IL-4 inhibited TNF-alpha induced IL-8 production in both corneal epithelial cells and keratocytes. TNF-alpha and IL-4 synergistically stimulated the production of RANTES in keratocytes. CONCLUSION: Differential regulation of chemokine production from corneal cells by IL-4 may play a role in the selective recruitment of predominantly eosinophils to the ocular surface in ocular allergies.     

Bcl-2 expression in the human cornea.

The purpose of this study was to determine the localization of Bcl-2 protein in the human cornea. Anti-human Bcl-2 monoclonal antibodies (MAbs) against selective Bcl-2 peptide sequences were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas (n = 4). Specificity of each MAb was determined by Western blot analysis of pooled protein extracted from human corneal epithelium (n = 3). Expression of Bcl-2 protein in apoptotic surface epithelial cells was detected by co-labeling with TUNEL assay and anti-Bcl-2 antibody staining. Two MAbs specific for amino acids residues (aa) 41-54 within the loop domain of Bcl-2 protein stained nuclei of all corneal epithelial cell layers. MAb specific for aa 61-76, also within the loop domain, produced faint nuclei and nuclear envelope staining. Occasional corneal surface epithelial cells however, consistently lacked anti-Bcl-2 nuclear staining with these three MAbs; concomitant TUNEL assay revealed that all TUNEL positive-surface cells were Bcl-2 negative. In the stroma, keratocytes showed similar but weak anti-Bcl-2 staining. All corneal endothelial cells showed intense nuclear staining with MAbs, with no gradient or absence of staining. In summary, Bcl-2 protein can be localized to the nuclei and nuclear envelope of corneal epithelial cells, keratocytes and endothelial cells with the use of MAbs specific for the loop domain of Bcl-2. TUNEL-labeled surface epithelial cells did not stain with MAbs to Bcl-2, suggesting degradation or epitope masking perhaps by specific phosphorylation of the loop domain during apoptosis. Taken together, these findings suggest that Bcl-2 protein may play a critical role in modulating apoptotic cell desquamation in the human corneal epithelium.     

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA4 vector to construct recombinant eukaryotic expression plasmid pcDNA4-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA4-PDGF-B eukaryotic expression vector was transferred into cat corneal endothelial cells by EffecteneTM lipofectine. The transfection efficiency of EffecteneTM lipofectine in pcDNA4-B was detected with pcDNA4-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MTT) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. EffecteneTM lipofectine transfection technique could be effectively used to transfer pcDNA4-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.     

Nuclear ferritin in corneal epithelial cells: tissue-specific nuclear transport and protection from UV-damage

We have identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form that is indistinguishable from the cytoplasmic form of ferritin found in other cell types. Thus it most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and other tissues were UV irradiated, and damage to DNA was detected by an in situ 3'-end labeling assay. Consistent with the hypothesis, corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than the other cells types examined. However, when the expression of nuclear ferritin was inhibited the cells now became much more susceptible to UV-induced DNA damage. Since ferritin is normally cytoplasmic, corneal epithelial cells must have a mechanism that effects its nuclear localization. We have determined that this involves a nuclear transport molecule which binds to ferritin and carries it into the nucleus. This transporter, which we have termed ferritoid for its similarity to ferritin, has at least two domains. One domain is ferritin-like and is responsible for binding the ferritin; the other domain contains a nuclear localization signal that is responsible for effecting the nuclear transport. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. In addition, since ferritoid is structurally similar to ferritin, it may represent an example of a nuclear transporter that evolved from the molecule it transports (i.e., ferritin).     

人转化生长因子β诱导基因真核表达载体的构建及其对人角膜上皮细胞增生的影响

背景人转化生长因子-β诱导（TGFBI）基因是第一个被确定的角膜营养不良的致病基因,而TGFBI引起角膜营养不良的机制目前尚不清楚,研究TGFBI的功能对于揭示角膜营养不良的发病机制及了解角膜生理及病理功能都具有重要意义。目的构建人TGFBI基因的真核表达载体并将其转染于人角膜上皮细胞,探讨其对角膜上皮细胞增生及相关基因表达的影响。方法从角膜移植后剩余的供体角膜中提取人正常角膜组织总RNA,经逆转录聚合酶链反应（RT—PCR）合成TGFBI cDNA,将TGFBI胶回收产物与载体pCMV—N-HA分别进行双酶切,取连接产物10μl转化100μl大肠杆菌感受态DH5α克隆入真核表达载体pCMV—N—HA,以菌落PCR和EcoRV、XhoI双酶切法测序鉴定。设置重组质粒pCMV—N—HA—TGFBI转染组、空质粒pCMV—N—HA转染组、阴性对照组及pGFP—C2转染对照组,以测定重组质粒转染率。重组质粒pCMV-N—HA—TGFBI转染人角膜上皮细胞,激光共焦显微镜下观察转染pGFP—C2后增强型绿色荧光蛋白（EGFP）的表达,用细胞计数试剂盒8（CCK-8法）检测转染细胞的增生情况,SYBR荧光实时定量PCR和Western blot法检测TGFBI、基质金属蛋白酶（MMPs）、组织金属蛋白酶抑制剂（TIMP）蛋白及其mRNA在转染后角膜上皮细胞的表达。结果pCMV—N-HA—TGFBI阳性克隆质粒进行经EcoRV和XhoI双酶切鉴定测序结果显示,扩增的TGFBI cDNA以正确序列和方式插入载体,pGFP—C2载体转染人角膜上皮细胞后48h共焦显微镜下可见EGFP的表达,转染效率为70％。重组质粒pCMV—N—HA-TGFBI转染组TGFBI mRNA的表达明显高于空质粒pCMV—N—HA转染组和阴性对照组,TGFBI蛋白仅在pCMV—N-HA．TGFBI转染组表达。CCK-8法显示重组质粒pCMV—N-HA-TGFBI转染组、空质粒pCMV—N—HA转染组和阴性对照组间角膜上皮的吸光度（A450）值的差异无统计学意义（F=3．34,P〉0．05）。荧光实时定量PCR结果显示,重组质粒pCMV-N—HA-TGFBI转染组中MMP,mRNA、MMP,mRNA转录水平比空质粒pCMV—N—HA转染组和阴性对照组均升高（P〈0．05）,而TIMP,mRNA则明显下降（P〈0．05）;Western blot结果证实MMP1、MMP3、TIMP1蛋白表达规律相同（P〈0．05）。结论人TGFBI基因真核表达载体可被成功构建并在人角膜上皮细胞中呈过表达。TGFBI可能是通过MMP1、MMP3及TIMP1的表达变化来增加细胞外基质的降解,从而参与角膜的某些生理病理过程。     

氮酮促进脂质体介导质粒DNA转染兔角膜内皮细胞及毒性的实验研究

目的探讨氮酮(AZONE)促进脂质体介导质粒DNA转染兔角膜内皮细胞的作用及其对细胞活性的影响。方法采用细胞培养方法,利用绿色荧光蛋白基因做报告基因,配制不同转染液,脂质体联合pEGFP-N1、氮酮联合脂质体及pEGFP-N1、氮酮联合pEGFP-N1、单纯pEGFP-N1、单纯脂质体、单纯氮酮,分别处理兔角膜内皮细胞,荧光显微镜观察细胞中绿色荧光蛋白的表达。用RT-PCR方法检测EGFPmRNA表达情况。采用MTT法研究不同转染液对细胞活性的影响。结果单纯氮酮、单纯脂质体及对照组处理的细胞不表达绿色荧光蛋白。氮酮联合脂质体及pEGFP2N1对兔角膜内皮细胞的转染效率为42.6%,脂质体联合pEGFP2N1组的转染效率为22.4%,氮酮联合PEGFP-N1组的转染效率为7.2%,单组质粒组的转染效率为1%。氮酮加脂质体加PEGFP-N1组转染效率高于其他转染组(P<0.01)。各组转染液对角膜内皮细胞活性无影响。结论氮酮可促进脂质体介导质粒DNA转染兔角膜内皮细胞,在有效的转染浓度下对细胞无明显毒性,有望成为促进角膜内皮细胞基因转染的新型试剂。     

β1整合素过表达抑制角膜上皮细胞凋亡的实验研究(英文)

目的:探讨β1整合素功能性过表达对角膜上皮细胞凋亡的影响及机制,为角膜细胞移植提供理论依据。方法:构建β1整合素-绿色荧光蛋白(GFP)融合基因并转染兔角膜上皮细胞。观察融合基因在角膜上皮细胞的表达及对各细胞外基质蛋白的黏附能力。检测β1整合素功能性转染对角膜上皮细胞凋亡及丝裂素活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)磷酸化的影响。结果:β1整合素-GFP融合基因成功转染至兔角膜上皮细胞并过表达;β1整合素过表达能够明显增加角膜上皮细胞对各细胞外基质蛋白的黏附力并抑制角膜上皮细胞凋亡及促使MAP激酶磷酸化。结论:β1整合素过表达能有效抑制角膜上皮细胞凋亡,MAP激酶磷酸化可能在这一过程中起重要作用。     

转化生长因子β诱导基因在人角膜组织及细胞中的表达

背景 临床研究表明多数角膜营养不良的发病与转化生长因子β诱导(TGFBI)基因突变有关,但其发病的分子机制尚不完全清楚. 目的 研究TGFBI基因在人角膜组织及体外培养的角膜上皮细胞和基质细胞中的表达,为进一步研究角膜营养不良的发病机制奠定基础. 方法 对人角膜上皮细胞和基质细胞进行培养和传代,应用逆转录聚合酶链反应( RT-PCR)法检测TGFBI mRNA在人角膜组织及细胞中的表达,将供体角膜组织制成石蜡包埋切片,利用免疫组织化学法检测TGFBI蛋白在角膜组织、人角膜上皮细胞和角膜基质细胞中的表达,采用免疫荧光技术检测TGFBI蛋白在细胞爬片的表达. 结果 RT-PCR检测显示人角膜组织和基质细胞中在1274 bp处可见TGFBI mRNA的清晰条带,而角膜上皮细胞中亦有TGFBImRNA表达.免疫组织化学检测显示,TGFBI蛋白在人角膜组织中基质细胞的细胞质中呈阳性表达,但人角膜上皮细胞中未见TGFBI蛋白表达.免疫荧光检测技术显示,人角膜基质细胞胞质中TGFBI蛋白呈红色荧光,而角膜上皮细胞未见TGFBI蛋白表达. 结论 TGFBI主要在人角膜基质层表达,而在上皮层几乎不表达,有助于进一步研究TGFBI在角膜营养不良发病机制中的作用.     

Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo

PURPOSE: To determine whether subunits of VEGF receptor-1 coupled with an endoplasmic reticulum retention signal can block hypoxia-induced upregulation of VEGF secretion in corneal epithelial cells and block murine corneal angiogenesis induced by corneal injury. METHODS: Human corneal epithelial cells, transfected with plasmids encoding Flt23K or Flt24K (the VEGF-binding domains of the Flt-1 receptor coupled with the endoplasmic reticulum retention peptide KDEL), were subjected 2 days after transfection to 5% hypoxia for 24 hours. Supernatant was sampled at 24 hours and assayed for VEGF by ELISA. For in vivo models, mouse corneas underwent intrastromal injections of plasmids encoding Flt23K or Flt24K, and 2 days later, sustained injury induced by topical NaOH and mechanical scraping. Corneas were assessed 2 days later for VEGF ELISA and leukocyte counting or 1 week later for quantification of neovascularization. RESULTS: Hypoxia induced VEGF by human corneal epithelial cells was sequestered by both Flt23K and Flt24K; Flt-1 23K suppressed VEGF secretion as well. Intrastromal delivery of plasmid Flt23K suppressed VEGF by 40.4% (P = 0.009), leukocytes by 49.4% (P < 0.001), and neovascularization by 66.8% (P = 0.001). Flt24K suppressed VEGF expression by 30.8% (P = 0.042), leukocytes by 25.8% (P < 0.001), and neovascularization by 49.5% (P = 0.015). CONCLUSIONS: Flt-1 intraceptors, which are endoplasmic reticulum retention signal-coupled VEGF receptors, significantly suppress hypoxia-induced VEGF secretion by corneal epithelial cells in vitro. In vivo, delivery of naked plasmids expressing these intraceptors inhibits injury-induced upregulation of VEGF, leukocyte infiltration, and corneal neovascularization.