Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

Local anesthetic lidocaine induces apoptosis in human corneal stromal cells in vitro

AIM:To demonstrate the apoptosis-inducing effect of iidocalne on human corneal stromal(HCS)cells fn vitm,and provide experimental basis for safety anesthetic usage In clinic of ophthalmology.METHODS:In vitro cultured HCS cells were treated with lidocaine at different doses and times,and their morphology was monitored successively with inverted phase contrast microscopy.The membrane permeability of them was detected by acridine orange/ethidium bromide(AO/EB)double staining.The DNA fragmentation of them was examined by agarose gel electrophoresis,and their ultrastructure was observed by transmission electron microscopy(TEM),respectively.RESULTS:Exposure to lidocaine at doses from0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation,cellular shrinkage,and turning round,and elevated membrane permeability of these cells in AO/EB staining.The change of morphology and membrane permeability was doseand time-dependent,while lidocaine at dose below0.15625g/L could not induce these changes.Furthermore,lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation,structural disorganization,chromatin condensation,and apoptotic body appearance of the cells.CONCLUSION:Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose-and time-dependent manner by inducing apoptosis of these cells.The established experimental model and findingsbased on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.

Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

AIM：To investigate the impact of adipose-derived mesenchymal stem cells（ADSCs） on cell viability and extracellular matrix（ECM） synthesis of corneal stromal cells（CSCs）. METHODS：ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate（FITC）/proliferation indices（PI） assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase（MMP）, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS：ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis（early and late） between the negative control group（6.34% and 2.06%） and the ADCSs-treated group（4.69% and 1.59%）. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type（I, II, III, IV, and V） in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.CONCLUSION：ADSCs play a promotive role in CSCs＇ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Effects of corneal stromal cell-and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM： To explore the effects of conditioned media on the proliferation of corneal endothelial cells （CECa） and to compare the efficiency of different conditioned media （CM）. METHODS： Rat CECs, corneal stromal cells （CSCs）, bone marrow -derived endothelial progenitor cells （BEPCs）, and bone marrow-derived mesenchymal stem cells （BMSCs） were isolated and cultured in vitra CM was collected from CSCs, BEPCs, and BMSCSo CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed.~ RESULTS： After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone- like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na＋/K＋-ATP, aquaporin 1 （AQP1）, and zonula occludens 1 （ZO-1）. Based on quantitative polymerase chain reaction （qPCR） analysis, Na ＋/K ＋-ATP expression in CSC-CM was notably upregulated by 1.3-fold （＋0.036） （P〈0.05, n=3）. The expression levels of ZO-1, neuron specific enolase （NSE）, Vimentin, paired homebox 6 （PAX6）, and procollagen type VII （COL8A1） were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation.~ CONCLUSION： CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. KEYWORDS： conditioned medium; corneal endothelial cell; corneal stromal cell; bone marrow-derived endothelial progenitor cell; proliferation

Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms

AIM： To examine the cytotoxic effect of pilocarpine, an anti-glaucoma drug, on human corneal stromal（HCS）cells and its underlying cytotoxic mechanisms using an in vitro model of non-transfected HCS cells.· METHODS： After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L,their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability,DNA fragmentation and ultrastructure were examined by acridine orange（AO）/ethidium bromide（EB） double-staining. DNA electrophoresis and transmission electron microscopy（TEM）, cell cycle, phosphatidylserine（PS）orientation and mitochondrial transmembrane potential（MTP） were assayed by flow cytometry（FCM）. And the activation of caspases was checked by ELISA.· RESULTS： Morphology observations and viability assay showed that pilocarpine at concentrations above0.625 g/L induced dose- and time-dependent morphological abnormality and viability decline of HCS cells. AO/EB double-staining, DNA electrophoresis and TEM noted that pilocarpine at concentrations above 0.625 g/L induced dose- and/or time-dependent membrane permeability elevation, DNA fragmentation, and apoptotic body formation of the cells. Moreover, FCM and ELISA assays revealed that 2.5 g/L pilocarpine also induced S phase arrest, PS externalization, MTP disruption, and caspase-8,-9 and-3 activation of the cells.· CONCLUSION： Pilocarpine at concentrations above0.625 g/L（1/32 of its clinical therapeutic dosage） has a dose- and time-dependent cytotoxicity to HCS cells by inducing apoptosis in these cells, which is most probably regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

Profile of biological characterizations and clinical application of corneal stem/progenitor cells

Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role.Corneal stem/progenitor cells include mainly corneal epithelial stem cells,corneal endothelial cell progenitors and corneal stromal stem cells.Since the discovery of corneal epithelial stem cells(also known as limbal stem cells)in 1971,an increasing number of markers for corneal stem/progenitor cells have been proposed,but there is no consensus regarding the definitive markers for them.Therefore,the identification,isolation and cultivation of these cells remain challenging without a unified approach.In this review,we systematically introduce the profile of biological characterizations,such as anatomy,characteristics,isolation,cultivation and molecular markers,and clinical applications of the three categories of corneal stem/progenitor cells.

树鼩角膜基质永生化细胞系的构建及其在病毒感染性方面的研究

目的建立树鼩永生化角膜基质细胞(corneal stromal cells,CSCs)系,并探讨其在病毒感染中的应用。方法利用组织块贴壁培养法分离培养树鼩原代CSCs,将携带SV40T基因的慢病毒转染细胞后,再挑取单克隆进行传代培养,传至50代以上进行形态学观察并与40代细胞形态进行比较,波形蛋白(vimentin)和SV40T基因免疫荧光鉴定、核型鉴定、细胞增殖曲线测定。用单纯疱疹病毒1型(herpes simplex virus-1,HSV-1)(McKrae株)、寨卡病毒(Zika virus,ZIKV)(GZ01株)、登革病毒Ⅱ型以及甲型流感病毒H1N1(PR8株)在该细胞上进行病毒感染实验。结果传至50代以上的树鼩永生化CSCs呈梭形,细胞形态结构与40代相比仍较好。vimentin和SV40T基因免疫荧光表达阳性。增殖曲线结果显示:细胞生长旺盛,第4~5天时处于对数生长期。原代细胞核型的染色体数稳定为62条,而永生化细胞第21、56代突变为64条且保持稳定。病毒感染实验显示:树鼩永生化CSCs对HSV-1(McKrae株)、ZIKV(GZ01株)、登革病毒Ⅱ型以及H1N1(PR8株)病毒敏感,产生较高感染滴度,分别为1.32×105、5.62×106、2.69×107、7.76×104CCID50/mL。结论成功建立了树鼩永生化CSCs细胞系,提示该细胞系可用于单纯疱疹病毒、寨卡病毒、登革病毒和甲型流感病毒感染眼角膜疾病的作用机制及抗病毒药物的研究。

姜黄素调节PI3K/AKT/mTOR信号通路对LPS诱导的角膜基质细胞自噬的影响

目的:探讨姜黄素调节磷脂酰肌醇3-激酶(PI3K)/丝氨酸-苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对LPS诱导的角膜基质细胞自噬的影响。方法:小鼠角膜基质细胞分为对照组、LPS组(1μg/μl LPS)、L-Cur组(15μmol/L姜黄素)、M-Cur组(30μmol/L姜黄素)、H-Cur组(50μmol/L姜黄素)、H-Cur+740Y-P组(50μmol/L姜黄素+10μmol/L PI3K激活剂740Y-P);MTT检测姜黄素对角膜基质细胞毒性和细胞活力的影响;ELISA检测角膜基质细胞炎症因子水平;免疫荧光染色检测小鼠角膜基质细胞Beclin1、LC3水平;Western blot检测角膜基质细胞自噬相关蛋白及通路相关蛋白表达。结果:0~90μmol/L姜黄素对角膜基质细胞无明显毒性。与对照组相比,LPS组A570nm、Beclin1、LC3Ⅱ/Ⅰ水平显著降低(P<0.05),IL-6、IL-8水平、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平显著升高(P<0.05);与LPS组相比,L-Cur组、M-Cur组、H-Cur组A570 nm、Beclin1、LC3Ⅱ/Ⅰ水平依次显著升高(P<0.05),IL-6、IL-8水平、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平依次显著降低(P<0.05);H-Cur+740Y-P消除了姜黄素对角膜基质细胞的有利作用。结论:姜黄素可能通过抑制PI3K/AKT/mTOR信号通路促进LPS诱导的角膜基质细胞自噬。

多西环素对TGF-β1诱导的人角膜基质细胞纤维化的抑制作用

目的:探讨多西环素(Doxy)对转化生长因子-β1(TGF-β1)诱导的人角膜基质细胞纤维化的抑制作用。方法:收集飞秒激光微小切口基质透镜取出术(SMILE术)后角膜基质透镜提取人角膜基质细胞,利用TGF-β1诱导人角膜基质细胞建立体外角膜纤维化模型,设置对照组、TGF-β1组、DOXY组。采用CCK-8法检测各组细胞增殖能力、划痕实验检测增殖能力;酶联免疫吸附实验(ELISA)测定胶原合成关键因子羟脯氨酸(HYP)含量;实时荧光定量PCR(RT-qPCR)检测纤维化关键因子Thy-1、Vimentin mRNA表达情况;蛋白免疫印迹(western blotting)检测Thy-1、Vimentin蛋白表达水平。结果:与对照组相比,TGF-β1组能促进人角膜基质细胞细胞增殖、迁移,增加HYP含量、Thy-1和Vimentin的表达(均P<0.05)。与TGF-β1组比较,DOXY组细胞增殖、细胞迁移和胶原合成能力明显降低,Thy-1和Vimentin的表达减少(均P<0.05)。结论:Doxy可抑制TGF-β1诱导的人角膜基质细胞细胞增殖、迁移和胶原合成,下调Thy-1、Vinmentin表达水平,从而抑制角膜纤维化。

应用活体共聚焦显微镜观察糖尿病患者的角膜细胞

活体角膜共聚焦显微镜是一种无创、快速且全层面的技术,可以实时、动态地观察角膜组织的所有层面。共聚焦显微镜可以通过直接可视化检查角膜不同层面的形态和细胞密度。随着糖尿病患病率的不断上升,眼部并发症已经变得越发常见,并引起眼科临床工作者和科研工作者越来越多的兴趣和逐渐深入的研究。文章旨在通过采用活体角膜共聚焦显微镜观察糖尿病患者角膜组织各层的研究进展进行综述。

Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis

Background： Corneal stromal cells （CSCs） are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells （ADSCs） have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear. Methods： ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis （NTA） were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/ propidium iodide assays were used to assess the effect of ADSC-derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects ofADSC-derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases （MMPs） and collagens. Results： ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles （40-200 nm）, and Western blotting confirmed positive expression of CD9, CDSI, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins （all P〈 0.01 ）. ADSC-derived exosomes （50μg/ml and 100μg/ml） significantly promoted proliferation and inhibited apoptosis （mainly early apoptosis） of CSCs versus non-exosome-treated CSCs （all P 〈 0.05）. Interestingly, MMPs were downregulated and extracellular matrix （ECM）-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs （MMPI： t = 80.103, P 〈 0.01; MMP2： t = 114.778, P 〈 0.01; MMP3： t = 56.208, P 〈 0.01; and MMP9： t = 60.617, P〈 0.01; collagen I： t = -82.742, P〈 0.01; collagen II： t = -72.818, P〈 0.01; collagen III： t = -104.452, P〈 0.01; collagen IV： t = - 133.426, P 〈 0.01, and collagen V： t - -294.019, P 〈 0.01 ; and fibronectin： t = -92.491, P 〈 0.01, respectively）. Conclusion： The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

The anti-scarring effect of corneal stromal stem cell therapy is mediated by transforming growth factorβ3

Background:Corneal stromal stem cells(CSSC)reduce corneal inflammation,prevent fibrotic scarring,and regenerate transparent stromal tissue in injured corneas.These effects rely on factors produced by CSSC to block the fibrotic gene expression.This study investigated the mechanism of the scar-free regeneration effect.Methods:Primary human CSSC(hCSSC)from donor corneal rims were cultivated to passage 3 and co-cultured with mouse macrophage RAW264.7 cells induced to M1 pro-inflammatory phenotype by treatment with interferonγand lipopolysaccharides,or to M2 anti-inflammatory phenotype by interleukin-4,in a Transwell system.The timecourse expression of human transforming growth factorβ3(hTGFβ3)and hTGFβ1 were examined by immunofluorescence and qPCR.TGFβ3 knockdown for>70%in hCSSC[hCSSC-TGFβ3(si)]was achieved by small interfering RNA transfection.Naïve CSSC and hCSSC-TGFβ3(si)were transplanted in a fibrin gel to mouse corneas,respectively,after wounding by stromal ablation.Corneal clarity and the expression of mouse inflammatory and fibrosis genes were examined.Results:hTGFβ3 was upregulated by hCSSC when co-cultured with RAW cells under M1 condition.Transplantation of hCSSC to wounded mouse corneas showed significant upregulation of hTGFβ3 at days 1 and 3 post-injury,along with the reduced expression of mouse inflammatory genes(CD80,C-X-C motif chemokine ligand 5,lipocalin 2,plasminogen activator urokinase receptor,pro-platelet basic protein,and secreted phosphoprotein 1).By day 14,hCSSC treatment significantly reduced the expression of fibrotic and scar tissue genes(fibronectin,hyaluronan synthase 2,Secreted protein acidic and cysteine rich,tenascin C,collagen 3a1 andα-smooth muscle actin),and the injured corneas remained clear.However,hCSSC-TGFβ3(si)lost these anti-inflammatory and anti-scarring functions,and the wounded corneas showed intense scarring.Conclusion:This study has demonstrated that the corneal regenerative effect of hCSSC is mediated by TGFβ3,inducing a scar-free tissue response.

蒙花苷对CXCL12诱导人角膜上皮细胞表达VEGF的影响

目的:探讨蒙花苷对基质细胞衍生因子(CXCL12)诱导人角膜上皮细胞(HCEC)表达血管生成因子(VEGF)的抑制作用。方法:培养HCEC,ELISA检测不同滴度CXCL12慢性病毒基因转染HCEC上清液中VEGF含量,并建立细胞模型。ELISA检测不同浓度的蒙花苷干预后细胞上清液VEGF含量,Western Blot检测细胞中的VEGFA蛋白的表达,实时荧光定量PCR(Q-PCR)检测细胞中的VEGFA mRNA水平的表达。结果:在CXCL12慢性病毒基因滴度为1.775 TU/mL时,HCEC分泌的VEGF的含量明显高于正常细胞组及阴性对照组(P<0.05),具有显著性差异。选用1.775 TU/mL滴度的CXCL12慢性病毒基因对HCEC进行造模。与模型组及阴性对照组比较,不同浓度的蒙花苷均能减少HCEC分泌VEGF,减少VEGFA蛋白含量和VEGFA mRNA表达量(P<0.05)。不同浓度的蒙花苷作用比较差异有统计学意义(P<0.05),随蒙花苷浓度增加,呈先增强后减弱的趋势,且当浓度为66.7μmol/L时对VEGFA蛋白及mRNA表达抑制力最为显著。结论:蒙花苷能够抑制CXCL12诱导HCEC分泌VEGF,减少VEGFA蛋白及VEGFA mRNA的表达,这为中药密蒙花“消目中赤脉”作用提供实验佐证。

角膜基质细胞诱导分化的脂肪间充质干细胞羊膜片移植治疗兔角膜碱烧伤的疗效及其机制

背景角膜化学烧伤是致盲眼病之一，先前的各种疗法在临床上的应用均受到一定的限制，而脂肪间充质干细胞（ADSCs）移植疗法受到密切关注。目的观察诱导分化的ADSCs羊膜片移植治疗兔角膜碱烧伤的疗效及其机制。方法无菌条件下剪取兔角膜，用悬浮基质片法分离并培养兔角膜基质细胞（CSCs）。取兔腹股沟脂肪组织，以酶消化法（质量分数0．25％胰蛋白酶）分离及培养兔ADSCs，并用流式细胞术鉴定ADSCs表面抗原的表达。将CSCs与ADSCs共培养，采用免疫荧光技术和逆转录PCR（RT-PCR）鉴定CSCs诱导后ADSCs向角膜基质样细胞分化的表达，分别将诱导或未诱导的ADSCs种植于羊膜上制备成细胞羊膜片。选择60只新西兰白兔，右眼用浸有质量分数1％NaOH的滤纸贴敷于角膜中央50S制备兔角膜碱烧伤模型，按随机数字表法将模型眼随机分为诱导ADSCs＋羊膜移植组、未诱导ADSCs＋羊膜移植组、单纯羊膜移植组及模型组。分别于术后1周、2周和1个月在裂隙灯显微镜下观察各组兔模型眼角膜的混浊程度和新生血管形成情况，并对角膜炎症进行评分，采用ELISA法检测术后1个月角膜组织匀浆中CD45、叮干扰素（IFN-γ）、白细胞介素．10（IL-10）蛋白及房水中血管内皮生长因子（VEGF）蛋白的质量浓度。结果从脂肪组织分离培养的ADSCs表面抗原CDl05、CD29、CD44的阳性表达率分别为90．23％、88．56％、98．88％；分离培养的CSCs波形蛋白呈阳性表达；CSCs与ADSCs共培养后多数细胞双标记染色阳性。苏木精一伊红染色显示，ADSCs种植于羊膜后生长良好。术后1个月，模型组角膜混浊呈瓷白色，新生血管面积最大，而诱导ADSCs＋羊膜移植组角膜透明，新生血管最少。术后1个月，诱导ADSCs＋羊膜移植组、未诱导ADSCs＋羊膜移植组、单纯羊膜移植组和模型组兔角膜混浊程度评分分别为1．65±0．18、2．05±0．17、2．68±0．25和2．904-0．18，新生血管面积分别为（10．59±1．78）、（22．58±1．63）、（37．98±1．90）和（45．37±1．65）mm^2，术后1周、2周及1个月4个组间角膜混浊程度评分和角膜新生血管（CNV）面积的差异均有统计学意义（F=280．826、330．172、465．707，均P=0．000；F=60．020、670．811、1510．231，均P=0．000），其中诱导ADSCs＋羊膜移植组角膜炎症评分睨显低于其他3个组，CNV面积小于其他3个组，差异均有统计学意义（均P〈0．01）。术后1个月，4个组间角膜组织匀浆中CD45、IL-10、IFN-γ质量浓度的差异均有统计学意义（F=916．545、1739．358、462．134，均P=0．000），其中与未诱导ADSCs＋羊膜移植组、单纯羊膜移植组和模型组比较，诱导ADSCs＋羊膜移植组CD45、IFN-γ质量浓度明显降低，而IL-10质量浓度明显升高，差异均有统计学意义（均P〈0．01）。4个组兔房水中VEGF质量浓度的总体比较差异有统计学意义（F=129．126，P=0．000），其中诱导ADSCs＋羊膜移植组兔房水中VEGF质量浓度明显低于未诱导ADSCs＋羊膜移植组、单纯羊膜移植组和模型组，差异均有统计学意义（均P〈0．01）。结论CSCs诱导的ADSCs羊膜片移植法治疗兔角膜碱烧伤有较好的疗效，其作用机制可能是ADSCs在CECs诱导下分化为具有角膜上皮细胞特征的细胞，同时羊膜可减轻免疫炎症反应，抑制新生血管形成。

不同脱乙酰度对壳聚糖膜与角膜基质细胞相容性的影响

以分子量为30万,脱乙酰度分别为63.3%、73.7%、83%和97%的壳聚糖制备不同的壳聚糖膜,在不同脱乙酰度的壳聚糖膜上培养兔角膜基质细胞,通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性,研究壳聚糖分子脱乙酰度对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖脱乙酰度越高,壳聚糖膜对细胞的损伤越小,越有利于细胞在膜上的生长和贴附,反之,低脱乙酰度的壳聚糖膜与角膜细胞的相容性较差。

角膜基质细胞Nrf2-ARE信号通路活化缺陷在圆锥角膜发病中的作用

背景 氧化应激在圆锥角膜发病过程中具有重要的作用,核因子E2相关因子2-抗氧化反应元件（Nrf2-ARE）信号通路是介导细胞氧化应激反应的关键通路,但其在圆锥角膜发病中的作用及其机制鲜见报道. 目的 研究正常角膜与圆锥角膜基质细胞中Nrf2-ARE信号通路活化的区别及Nrf2-ARE通路对角膜基质降解酶表达水平的影响,探讨圆锥角膜发病的具体机制.方法 于2012年11月至2013年6月在青岛眼科医院收集圆锥角膜患者术中的角膜组织样本和正常供体角膜样本,采用中性蛋白酶和胶原蛋白酶联合消化法分离角膜基质细胞并用含质量分数10％胎牛血清的DMEM/F12培养基培养细胞,待细胞80％融合后在培养基中加入200 μmol/L H2O2处理1h以模拟氧化应激微环境.采用DCFH-DA荧光底物孵育法检测细胞内活性氧簇（ROS）含量,分别采用Western blot和实时定量PCR法检测细胞核内Nrf2mRNA及其蛋白、Nrf2-ARE信号通路下游抗氧化蛋白、尿激酶型纤溶酶原激活物（uPA）、uPA受体（uPAR） mRNA及其蛋白的相对表达水平,采用明胶酶谱法检测细胞中基质金属蛋白酶2（MMP-2）活性.结果 正常培养条件下,圆锥角膜基质细胞中ROS荧光强于正常角膜基质细胞,细胞核内Nrf2蛋白表达水平均明显高于正常角膜基质细胞,差异有统计学意义（t=18.155,P＜0.01）,但在H2O2处理条件下,圆锥角膜基质细胞中ROS荧光强度明显强于正常角膜基质细胞,且圆锥角膜基质细胞核内Nrf2表达水平明显低于正常培养条件下的圆锥角膜基质细胞,差异有统计学意义（t=62.123,P＜0.01）.正常培养条件下,圆锥角膜基质细胞间还原型烟酰胺腺嘌呤二核苷酸磷酸氧化还原酶1（NQO-1）、血红素氧合酶1（HO-1）、超氧化物歧化酶2（SOD2） mRNA及其蛋白的相对表达量明显低于正常角膜基质细胞,差异均有统计学意义（均P＜0.01）;但在H2O2培养条件下2种细胞间未见明显变化（NQO-1：t=2.209,P=0.092;HO-1：t=0.293,P=0.784;SOD2：t=0.749,P=0.495）;圆锥角膜基质细胞uPA、uPAR表达量和MMP-2活性均明显高于正常角膜基质细胞,差异均有统计学意义（t=19.164、15.458、4.818,均P＜0.01）. 结论 圆锥角膜基质细胞在Nrf2-ARE信号通路活化方面存在缺陷,且这种缺陷与其表达基质降解酶的水平密切相关,说明该通路异常可能是圆锥角膜发病的机制之一.

新型可降解支架材料植入角膜的生物学反应研究

目的 探讨可降解交联剂交联乙烯基吡咯烷酮对兔角膜组织的生物学反应的影响。方法 将乙烯基吡咯烷酮植入兔角膜囊袋内 3个月 ,观察生物材料在角膜内生物学反应。结果 可降解交联剂交联乙烯基吡咯烷酮合成材料植入兔角膜囊袋 3个月 ,生物相容性良好 ,材料逐渐降解 ,材料内有胶原和角膜基质细胞长入。结论 新型可降解交联剂交联乙烯基吡咯烷酮合成材料植入后未见兔角膜有明显的炎症反应 ,材料的组织相容性好 ,可作为组织工程角膜的支架材料。

细菌纤维素构建组织工程角膜基质的方法及其评价

目的:探讨以细菌纤维素(BC)为支架构建组织工程角膜基质的可行性。方法:体外分离培养兔和人角膜基质细胞,种植到BC膜中,构建完成后进行兔和人角膜基质细胞-BC复合膜的生物检测,并将兔角膜细胞生物复合物进行同种异体移植。术后1、4和8周时分别活体行前节OCT、角膜共焦显微镜检查,离体后组织学及免疫组织化学检查。结果:人和兔角膜基质细胞长入BC的网架结构,细胞生长状态良好。移植术后1周兔眼无炎症反应及新生血管,4周后植片边缘出现新生血管,表面无水肿及溃疡形成。术后8周角膜共焦显微镜显示:移植片周围基质细胞增生活跃,邻近的角膜内皮细胞数量和形态正常。前节OCT显示:移植后复合膜逐渐降解,4周时复合膜边界模糊,8周时密度接近正常角膜组织。离体组织学检查显示:BC复合膜与正常角膜基质相似,有炎细胞浸润和血管形成,角膜组织无坏死和溶解。结论:BC无细胞毒性,具有良好的组织相容性和可降解性,可作为构建组织工程角膜基质的生物支架。

吡非尼酮对大鼠角膜基质细胞增殖的影响

目的:探讨吡非尼酮(Pirfenidone,PFD)对体外培养大鼠角膜基质细胞增殖的抑制效果及其对转化生长因子-β1(transforming growth factor-β1,TGF-β1)表达的影响。方法:分离培养大鼠角膜基质细胞,根据PFD用药的不同浓度分为对照组(0mg/m L)、实验组Ⅰ(0.15mg/m L)、实验组Ⅱ(0.3mg/m L)、实验组Ⅲ(1mg/m L),加药48h后应用CCK-8法检测其对角膜基质细胞增殖能力的影响,免疫细胞化学和Western-blot分别检测ki-67和TGF-β1表达的变化。结果:CCK-8结果显示,相比对照组,各实验组对角膜基质细胞的增殖均有明显的抑制作用,且随浓度的增大其抑制作用明显增强(均P<0.05);免疫细胞化学显示PFD能明显降低ki-67阳性指数(P<0.05);Western-blot结果显示,PFD能降低TGF-β1的表达,且呈剂量依赖关系(P<0.05)。结论:PFD对大鼠角膜基质细胞的增殖具有明显抑制作用,抑制机制与下调TGF-β1表达密切相关,在减轻角膜创伤愈合方面具有潜在的运用前景。

吡非尼酮抑制转化生长因子β1诱导的大鼠角膜基质细胞纤维化

目的观察吡非尼酮(PFD)对转化生长因子β1(TGF-β1)诱导的大鼠角膜基质细胞向成纤维细胞转化过程的影响,探讨PFD抗角膜基质纤维化的作用。方法分离培养大鼠角膜基质细胞,免疫细胞化学法检测波形蛋白(Vimentin)进行细胞鉴定。实验分为对照组(DMEM+10%FBS)、TGF-β1组(2 ng/mL TGF-β1+DMEM+10%FBS)和PFD组(1 mg/mL PFD+2 ng/mL TGF-β1+DMEM+10%FBS)。CCK-8法检测细胞增殖能力,Western blot检测细胞CollagenⅠ、CollagenⅢ、Keratocan和CD90蛋白表达。结果大鼠角膜基质细胞呈Vimentin胞浆阳性。与对照组及TGF-β1组比较,PFD组细胞的增殖OD值显著减低(P<0.05)。Western blot显示PFD组的细胞CollagenⅠ、Keratocan蛋白表达增强而CollagenⅢ和CD90蛋白表达减弱(P<0.05)。PFD组的CollagenⅢ/CollagenⅠ比值在3组中最低(P<0.05)。结论 PFD抗角膜基质细胞纤维化是通过抑制TGF-β分子途径,从而影响胶原合成和Keratocan、CD90蛋白表达实现的。