异种角膜基质异位植入后免疫病理学研究

目的 观察异种角膜基质异位植入后的免疫病理变化，评价其免疫相容性。方法将一定大小的新鲜的和甘油脱水的猪角膜基质植入到小鼠皮下组织内，同时以异种皮肤、假手术为阳性和阴性对照；术后1个月取材进行组织病理学观察，并通过免疫荧光细胞化学法检测植入材料内T淋巴细胞总数和亚群的变化。结果新鲜的和甘油脱水的猪角膜基质皮下植入后1个月，可见少量的淋巴细胞、巨噬细胞、CD3＋细胞、CD4＋细胞、CD8＋细胞浸润．且CD4＋细胞多于CD8＋细胞，但未达到阳性对照水平（P〈0．05）。结论异种角膜基质异位植入后早期局部未引发免疫排斥反应，免疫相容性好。     

细胞间粘附分子在角膜疾病中的表达

目的：探讨细胞间粘附分子（Intercellular Adhesion Molecule-1,ICAM-1）在感染性角膜疾病和角膜移植免疫排斥中的表达及作用。方法：应用抗细胞间粘附分子ICAM-1的单克隆抗体，对42例角膜移植所取下的病变角膜进行免疫组织化学研究。角膜组织连续冰冻切片，进行SABC免疫组化及HE染色，研究细胞间粘附分子的表达与炎性细胞分布以及炎症程度的关系。结果：各种感染性角膜疾病、角膜变性以及角膜移植排斥过程中，ICAM-1在角膜上皮，尤其是基底细胞、角膜细胞、内皮细胞和血管化的基质层新生血管内皮细胞中均有表达，在炎性细胞包括单核细胞、巨噬细胞及淋巴细胞浸润部位表达增强；ICAM-1表达强度与炎症程度相平行。结论：ICAM-1在感染性、变性角膜病及角膜移植排斥中均有表达，在炎性细胞浸润部位表达增强；I-CAM-1对白细胞募集于炎症部位、炎症的发生、发展具有重要的中介作用。     

蛋白激酶Cα在人角膜基质细胞中的表达

目的 观察蛋白激酶Ca（PKCα）在原位及体外培养的人角膜基质细胞中的表达。方法标本来源于北京大学第一医院眼库。人角膜基质细胞体外原代及传代培养，应用免疫组织化学法检测PKCα在培养的人角膜基质细胞中的表达；制作人正常角膜组织冰冻切片，观察PKCα在角膜基质细胞中的表达。结果免疫组织化学法检测显示：正常人原位角膜基质细胞对PKCα无明显表达，而体外培养的人角膜基质细胞有明显阳性表达。结论特殊的角膜创伤模型——体外培养人角膜基质细胞中PKCα有明显阳性表达，提示PKC在角膜基质细胞增生过程中扮演了重要角色，抑制PKC的活性有可能成为治疗角膜瘢痕愈合的一个新途径。     

异种脱细胞角膜基质材料的生物相容性研究

目的：研究脱细胞猪角膜基质材料在角膜组织中的生物相容性,为组织工程角膜的构建寻找一种良好的支架材料。方法：将用去垢剂联合酶的方法脱细胞处理的猪角膜基质材料植入兔角膜基质囊袋中,进行裂隙灯观察并照相。不同时间取材HE染色,进行光镜检查。结果：脱细胞猪角膜基质材料植入兔角膜基质囊袋,观察4mo,生物相容性良好,材料逐渐降解吸收。结论：脱细胞猪角膜基质材料植入兔角膜中无炎症、无免疫排斥反应,生物相容性好,可作为组织工程角膜的支架材料。     

穿透性角膜移植术后慢性失功移植片的免疫学研究

目的检测穿透性角膜移植术后慢性失功移植片的免疫与非免疫相关细胞因子表达的变化,探讨免疫与非免疫因素在角膜植片慢性失功中的作用机制。设计临床实验研究。研究对象分为两组,角膜植片慢性失功（CCAD）组：穿透性角膜移植（PK）术后因CCAD导致移植失败的8例患者的角膜植片;正常对照组：由山东跟库提供的正常角膜供体3只。方法免疫组化法检测两组角膜片免疫相关细胞因子表达,结合患者的临床资料进行综合分析。主要指标角膜中CD4^＋、CD8^＋、F4／80、TGF-β、bFGF、α-SMA的表达。结果免疫组化检测显示正常对照组：角膜各层组织 、α-SMA、bFGF表达阴性,F4／80角膜基质偶见表达,TGF-β在角膜上皮层有表达。CCAD组：所有角膜植片全层均术见CD4^＋及CD8^＋T淋巴细胞浸润;5例失功植片基质层F4／80阳性表达,其余3例角膜植片全层表达阴性;所有角膜植片上皮层可见TGF-β阳性表达,基质层内可见TGF-β表达。所有失功角膜片基质层bFGF表达呈弱阳性;所有患者角膜基质内α-SMA表达阳性,后弹力层附近可见较强表达。结论穿透性角膜移植术后慢性失功移植片免疫组化检查未发现支持临床急性免疫排斥反应的证据,抗原递呈细胞及非免疫特异相关细胞因子的异常表达提示免疫与非免疫因素参与了CCAD的发生和发展。     

角膜缘干细胞对外周血淋巴细胞活化和增殖作用的实验研究

目的 观察角膜缘干细胞对外周血淋巴细胞活化和增殖的抑制活性，并分析其机制。方法 通过植片技术分离兔角膜上皮细胞以及角膜缘干细胞。存两种细胞单层上，观察淋巴细胞埘丝裂原刀豆蛋白A(ConA)的增殖反应。另外，将两种细胞培养上清直接转移到由ConA刺激的淋巴细胞增殖系统以观察两种细胞释放。某些因子的抑制效应。结果 单层角膜缘干细胞对ConA刺激的外周血淋巴细胞增殖反应可以施加有效的抑制效应。角膜缘干细胞以及角膜上皮细胞的培养上清对ConA刺激的外周血淋巴细胞增殖系统，仅在72h分离上清显示有抑制效应。结论 角膜缘干细胞通过细胞交互作用和(或)释放某些抑制性因子对淋巴细胞的活化和增殖发挥潜在的抑制作用。     

生物工程角膜移植术后排斥反应的临床及免疫病理观察:附5例报告

目的：对生物工程角膜移植术后发生免疫排斥反应患者进行激光共焦显微镜观察和免疫病理研究,初步探究生物工程角膜移植排斥反应的发生机制。

方法：回顾性病例研究。纳入2018-09/2020-12我院收治的感染性角膜炎行生物工程角膜移植术后发生免疫排斥患者5例5眼。裂隙灯检查角膜透明度、角膜新生血管; 激光共焦显微镜对角膜进行动态观察,记录免疫排斥反应后角膜上皮下朗格罕氏细胞和基质炎症细胞密度,并以对侧眼作为对照进行统计分析。行同种异体角膜移植时取下生物工程角膜,免疫组织化学染色后对CD4⁺细胞和CD8⁺细胞及炎症细胞的浸润和分布进行观察。

结果：激光共焦显微镜显示,术眼角膜上皮下可见大量活化郎格罕氏细胞,其活化比例与对侧眼比较有差异(χ²=38.29,P<0.001)。基质层大量炎症细胞,与对侧眼比较具有差异(t=32.5,P<0.05)。对生物工程角膜组织进行免疫组织化学染色和HE染色,角膜基质层均可见CD4⁺细胞和CD8⁺细胞,仅1眼角膜基质层可见大量嗜酸性粒细胞。

结论：生物工程角膜移植术后免疫排斥反应经免疫病理证实以T细胞介导的细胞免疫在发病机制中具有重要作用,对异种抗原的超敏反应可能参与其中。     

人类角膜缘干细胞对外周血淋巴细胞作用的实验研究

目的 观察体外培养的人类角膜缘干细胞对同种异体外周血淋巴细胞的作用。方法 通过植片技术分离人角膜上皮细胞以及角膜缘干细胞。在两种细胞单层上，观察同种异体外周血淋巴细胞对丝裂原刀豆蛋白A（ConA）的增殖反应。另外，将两种细胞培养上清直接转移到由ConA刺激的淋巴细胞增殖系统，以观察两种细胞释放某些因子的抑制效应。结果 单层角膜缘干细胞对ConA刺激的外周血淋巴细胞增殖反应可以施加有效的抑制效应。角膜缘干细胞培养上清对ConA刺激的外周血淋巴细胞增殖系统，在72h分离上清显示有明显抑制效应。结论 角膜缘干细胞通过细胞交互作用和（或）释放某些抑制性因子对淋巴细胞的活化和增殖发挥潜在的抑制作用。     

人体角膜移植内皮排斥反应的共焦显微镜研究

目的：应用共焦显微镜探讨活体角膜内皮排斥反应的早期形态学特征和变化。方法：19例(19只眼)角膜移植术后发生内皮排斥反应的人群根据角膜内皮排斥反应轻重的不同分为轻、中、重三组。应用共焦显微镜(Confoscan2．0)对角膜植片进行检查，并分析其相关因素。结果：活体角膜内皮排斥反应特有的共焦显微镜改变主要包括：①早期发现在KP及其附近的内皮细胞面有少量的免疫细胞附着，紧靠KP处的角膜基质细胞出现轻度水肿，细胞排列较混乱，胞浆中出现少量空泡状暗区。②中期发现在内皮细胞表面可见较多的免疫细胞聚集并被完全激活，基质细胞明显水肿，细胞核不清楚，基质中有免疫细胞进入；可见由高反光的免疫细胞和不明成份的组织碎片混合组成内皮排斥线。③在晚期排斥的患者中可见大量“激活”的免疫细胞在角膜内皮细胞面聚集，内皮细胞几乎完全破坏；基质细胞明显水肿，部分被“激活”且伴有大量免疫细胞聚集。结论：①内皮排斥反应的早期能够发现角膜内皮面有免疫细胞沉积；②角膜内皮面的免疫细胞沉积与角膜内皮排斥反应的轻重成正相关；③角膜内皮排斥反应实际上是一个混合排斥反应的过程；④共焦显微镜对角膜内皮排斥反应具有实时、无创、敏感、准确以及可莺蒽、可比较、可活体观察等临床价值。     

异种角膜基质材料的制备和体外细胞种植的实验研究

目的研究异种角膜基质生物支架材料的制备方法和体外种植兔角膜基质细胞后细胞在材料上的生长、增殖情况。方法将York猪全层角膜用去垢剂联合0．25％胰蛋白酶、DNA-RNA酶祛除猪角膜基质细胞并冻干制备成支架材料；将兔角膜组织块用胶原酶消化后，用含10％血清的DMEM培养液体外培养，并做波形丝蛋白，角蛋白免疫组织化学染色检测；将培养的兔角膜基质细胞的2～3代接种在材料上，培养5d后，做HE染色、扫描电镜观察。结果猪角膜基质片经脱细胞处理后，细胞成分祛除干净并保留了角膜组织的三维网格状结构，网状间隙明显增大，利于细胞生长；体外培养的兔角膜基质细胞波形丝蛋白染色为阳性、角蛋白染色为阴性；HE染色、扫描电镜结果显示兔角膜基质细胞在支架材料上生长、增殖良好。结论异源性角膜经祛脱细胞处理而获得的生物支架材料利于异种细胞的黏附和增殖，可进一步用于组织工程角膜的研究。     

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea

PURPOSE: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea. METHODS: Murine Genome_U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts. RESULTS: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing. CONCLUSIONS: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

氯化锂通过TGFBI促进角膜基质成纤维细胞增殖和自噬

目的：研究TGFBI和微管相关蛋白轻链3(LC3)在角膜营养不良患者中的表达,及氯化锂(LiCl)通过TGFBI对角膜基质成纤维细胞增殖能力的影响。

方法：用免疫组化和Western-blot方法检测角膜营养不良及正常角膜组织中TGFBI和LC3的表达。实验构建了TGFBI过表达载体并转染角膜基质成纤维细胞,分别以5、10、20、40mmol/L LiCl作用于突变型TGFBI转染的角膜基质成纤维细胞,检测不同时间(0、1、6、12h)后,TGFBI与LC3蛋白表达变化,并用CCK-8法检测细胞增殖活性。

结果：TGFBI和LC3在角膜营养不良患者角膜组织中显著高表达。TGFBI过表达抑制角膜基质成纤维细胞增殖活性(P<0.05)。LiCl抑制突变型TGFBI转染的角膜基质成纤维细胞中TGFBI和LC3蛋白表达,并增强其细胞增殖活性(P<0.05)。

结论：LiCl可以促进角膜基质成纤维细胞增殖活性和自噬,其作用机制与下调TGFBI和LC3的表达有关。     

Involvement of S100A4 in stromal fibroblasts of the regenerating cornea

PURPOSE. S100A4 is a member of the S100 family of calcium-binding proteins. Members of the S100 family have been implicated in a variety of cellular events, including growth, signaling, differentiation, and motility. It has been suggested that S100A4 modulates cell shape and motility by interacting with components of the cytoskeleton. In the present study, the distribution patterns of S100A4 were investigated in normal and regenerating mouse corneas. METHODS. Rabbit cDNA libraries were prepared from cultures of corneal fibroblasts. S100A4 was identified as the most abundant message present. Expression of S100A4 in the cornea was determined using Northern blot analysis, in situ hybridization, and immunohistochemistry. Distribution patterns of S100A4 in primary corneal fibroblast cultures treated with either FGF-2/heparin or TGFbeta1 were analyzed by immunofluorescence. RESULTS. S100A4 mRNA was rarely detected in keratocytes or epithelial cells of the normal rabbit cornea. Likewise, S100A4 antigen was not found in normal mouse corneas. However, after removal of the corneal epithelium, fibroblasts are activated and had readily detectable S100A4 expression 6 days after wounding. In the in vitro equivalent of activated keratocytes, cultured rabbit corneal fibroblasts, S100A4 was restricted to the cytoplasm. In contrast, in cultures treated with TGFbeta1, which induces a myofibroblast phenotype, more than 90% of the cells showed a nuclear localization of S100A4. CONCLUSIONS. The findings show that S100A4 is expressed in the keratocyte phenotypes that appear in stromal tissue of corneas recovering from damage, the fibroblasts, and myofibroblasts. Its expression and distinct subcellular redistribution patterns suggest that S100A4 may be involved in the interconversions that occur between keratocytes, fibroblasts, and myofibroblasts during corneal wound healing.     

Maspin increases extracellular plasminogen activator activity associated with corneal fibroblasts and myofibroblasts

Maspin, an inhibitor of cell migration and a stimulator of adhesion of cells to the ECM, is synthesized and released by corneal keratocytes into the extracellular matrix. When the cornea is wounded, the quiescent stromal keratocytes underlying the wound undergo apoptosis and cells adjacent to this apoptotic area convert to fibroblasts or myofibroblasts. This study explores the effect of extracellular maspin on the plasminogen–plasminogen activator system of corneal stromal cells following wounding. Treatment of corneal fibroblasts and myofibroblasts with r-maspin increased extracellular but not cell-associated tissue-type plasminogen activator (tPA), urinary-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1). Despite the extracellular increase in PAI-1, the net effect of maspin treatment was an increase in plasminogen activation. At physiological levels, maspin did not alter uPA or tPA mRNA levels, in these cells. The increase in pro and active uPA was due to decreased clearance in the presence of maspin for myofibroblasts but not for fibroblasts. The clearance of pro and active tPA was normal in fibroblasts indicating different mechanisms for the increase of these homologous enzymes in the two cell types. Increased generation of plasmin by maspin treated corneal stromal fibroblasts and myofibroblasts led to conversion of plasminogen to active plasmin degradation products and angiostatin-like molecules. This study suggests that extracellular maspin increased pro and active uPA and tPA released by corneal fibroblasts and myofibroblasts on the short time scale of 1–4 h, but by 24 h there was no increase over the levels produced without maspin. This augmentation of plasminogen activator activity increases plasmin activation and angiostatin generation. It further indicates that the effect of maspin on uPA and tPA levels is cell type dependent.     

大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞

目的:探讨骨髓间充质干细胞 (mesenchymal stem cell,MSC) 分化为角膜上皮细胞的可塑性及其重建角膜上皮的可能性.方法:用密度梯度离心法结合贴壁培养法分离纯化大鼠骨髓MSC,经体外与角膜基质细胞共培养诱导分化,免疫荧光法检测角膜上皮细胞特异标志物K12的表达.结果:体外培养的大鼠骨髓MSC表现出很强的增殖潜能,原代培养的骨髓MSC CD29免疫荧光染色阳性,CD34和CD45为阴性,符合骨髓MSC的特征.MSC与角膜基质细胞共培养1wk后大部分细胞分化为间质细胞,少部分细胞形态上相对偏小,免疫荧光检测这部分细胞表达角膜上皮细胞特异性标志角蛋白K12.结论:体外培养的MSC在角膜基质细胞的诱导下可横向分化为角膜上皮细胞.     

Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.

PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.     

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.