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 co-cultured 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.     

Human β-NGF gene transferred to cat corneal endothelial cells

AIM: To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS: The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco’s modified Eagle''s medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS: The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the percentage of G1cells in CEC-AAV-NGF group was 76.8% while that in normal CEC control group and CEC-AAV control group was 46.6% and 49.8%. CONCLUSION: Recombinant AAV-β-NGF promotes proliferation in cat CECs by expressing bioactive β-NGF protein in high efficiency and suggests that its modulation can be used to treat vision loss secondary to corneal endothelial dysfunction.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

人角膜内皮细胞压力调节培养的初步研究

Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.     

Correlation of the history of stroke and the retinal artery occlusion: a nested case-control study

AIM: To investigate the promotion effect and explore its potential mechanism of leucine-rich α-2-glycoprotein-1 (LRG1) on corneal angiogenesis and lymphangiogenesis. METHODS: Corneal neovascularization and lymphatics were induced by establishing alkali burn mouse model. Immunofluorescence staining was performed to detect the location of LRG1 in cornea tissues and to verify the source of LRG1-positive cells. Corneal whole-mount staining for CD31 (a panendothelial cell marker) and lymphatic endothelial hyluronan receptor-1 (LYVE-1; lymphatic marker) was performed to detect the growth of blood and lymphatic vessels after local application of exogenous LRG1 protein or LRG1 siRNA. In addition, expressions of the proangiogenic vascular endothelial growth factor (VEGF) related proteins were detected using Western blot analysis. RESULTS: LRG1 was dramatically increased in alkali burned corneal stroma in both the limbal and central areas. LRG1-positive cells in the corneal stroma were mainly derived from Vimentin-positive cells. Local application of exogenous LRG1 protein not only aggravated angiogenesis but also lymphangiogenesis significantly (P<0.01). LRG1 group upregulated the levels of VEGF and the VEGF receptor (VEGFR) family when compared with the phosphate-buffered saline (PBS) control group. We also found that LRG1-specific siRNA could suppress corneal angiogenesis and lymphangiogenesis when compared with the scramble siRNA-treated group (P<0.01). CONCLUSION: LRG1 can facilitate corneal angiogenesis and lymphangiogenesis through heightening the stromal expression of VEGF-A, B, C, D and VEGFR-1, 2, 3; LRG1-specific siRNA can suppress corneal angiogenesis and lymphangiogenesis in corneal alkali burn mice.     

Protection of tight junction between RPE cells with tissue factor targeting peptide

AIM: To investigate the effect of tissue factor targeting peptide (TF-TP) on retinal pigment epithelium (RPE) cells tight junctions. METHODS: Cell counting kit-8 (CCK-8) was used to measure the proliferation of ARPE-19 cells. Expression of tight junction, ZO-1 in ARPE-19 cells was measured by Western blot and immunofluorescent staining. Western blot was also used to detect the expression of tissue factor (TF). CEC Transmigration Assay was used to measure the migration of ARPE-19 cells. The transport of fluorescent markers [fluorescein isothiocyanate dextrans of 4, 10, 20 (FD4, FD10, FD20)] and the transepithelial electrical resistance (TEER) were used to measure in ARPE-19 cell. RESULTS: CCK-8 assay showed that 5 μmol/L TF-TP can inhibit ARPE-19 cells abnormally proliferation stimulated by lipopolysaccharide (LPS; P<0.05). LPS increased the transport of fluorescent markers (FD4, FD10, FD20) and decreased TEER levels in ARPE-19 cells, respectively, which were prevented by 5 μmol/L TF-TP pretreatment (P<0.05). Furthermore, LPS significantly up-regulated the expression of TF and downregulated the expression of ZO-1 (P<0.05) in ARPE-19 cell which was inhibited by the TF-TP (P<0.05). In addition, TF-TP inhibited the abnormal migration induced by LPS in ARPE-19 cell (P<0.05). CONCLUSION: Our findings suggest that TF-TP suppressed proliferation and migration of ARPE-19 cells induced by LPS, and maintained the RPE tight junctions through inhibition of TF expression and increased expression of ZO-1.     

Down-regulation of histone deacetylase 7 reduces biological activities of retinal microvascular endothelial cells under high glucose condition and related mechanism

AIM: To investigate the expression and effect of histone deacetylase 7 (HDAC7) in human retinal microvascular endothelial cells (HRMECs) under high glucose condition and related mechanism, and the expression of HDAC7 in the retinal tissue in diabetic rats. METHODS: The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot. LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities. Cell count kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), flow cytometry, scratch test, Transwell test and tube formation assay were used to examine the ability of cell proliferation, migration, and angiogenesis. Finally, a preliminary exploration of its mechanism was performed by Western blot. RESULTS: The expression of HDAC7 was both up-regulated in retinal tissues of diabetic rats and high glucose-treated HRMECs. Down-regulation of HDAC7 expression significantly reduced the ability of proliferation, migration, and tube formation, and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs. CONCLUSION: High glucose can up-regulate the expression of HDAC7 in HRMECs. Down-regulation of HDAC7 can inhibit HRMECs activities. HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.     

小梁切除手术前后角膜内皮细胞观察

目的:观察小梁切除术对角膜内皮细胞有无影响.方法:采用非接触型角膜内皮显微镜,对40例56眼行小梁切除术的患者,做术前术后角膜内皮细胞密度和细胞形态学的检测.结果:行青光眼小梁切除术的患者40例56眼,除了4眼有2度浅前房的患者外,其余52眼术前角膜内皮细胞密度均值为2 580.90±323.20个/mm2,术后1 wk均值为2 558.28±341.83/mm2,细胞形态学方面,最大细胞面积、最小细胞面积、平均细胞面积、细胞面积标准差、细胞面积变异系数、六角形细胞百分数术前术后无显著性差异(P＞0.05).结论:在通常情况下,小梁切除术不会对角膜内皮细胞产生不良影响.     

角膜葡萄膜炎患者角膜内皮细胞相关性分析

目的:探讨角膜葡萄膜炎患者角膜内皮细胞在治疗前后相关参数的变异和临床意义。方法:对我院眼科2012-10/2013-12收治的角膜葡萄膜炎患者52例52眼,在治疗前后分别应用非接触型角膜内皮细胞仪测量角膜内皮细胞的相关参数,并进行统计学分析。结果:与正常组比较,患病组内皮细胞水肿明显,变异大;治疗时间越短,内皮细胞恢复越好,细胞丢失越少;反之,愈合时间越长,正常六边形细胞比率越小,变异系数较大。治疗前后各参数比较,差异有统计学意义(P<0.05)。结论:患者病程直接影响角膜内皮细胞功能的恢复。     

前葡萄膜炎患者角膜内皮细胞的计算机图像分析

目的探讨前葡萄膜炎患者角膜内皮细胞相关参数的变化及其临床意义。方法选择我院收治的前葡萄膜炎患者115例(115眼),分为首次发病组(45例)、首次复发组(35例)、多次复发组(35例),并将首次发病组按病程分为2～4周组、4～8周组、8～12周组,另设正常对照组35例(35眼)。应用非接触型角膜内皮细胞仪测量角膜内皮细胞的相关参数,并对结果进行统计学分析。结果首次发病组六边形细胞百分比与细胞面积变异系数分别为(53.8±5.4)%、(33.8±5.4)%,与正常对照组(55.7±5.7)%、(31.6±2.9)%比较,差异均有统计学意义(均为P<0.05);首次复发组最大细胞面积、细胞面积变异系数、六边形细胞百分比分别为(769.8±103.0)μm2、(34.3±6.1)%、(53.2±6.9)%,与正常对照组比较,差异均有统计学意义(均为P<0.05);多次复发组各参数与正常对照组比较,差异均有统计学意义(均为P<0.05)。8～12周组细胞面积变异系数与2～4周组、4～8周组比较,差异均有统计学意义(均为P<0.05);8～12周组六边形细胞百分比与2～4周组比较,差异有统计学意义(P<0.05);各组间其余参量比较,差异均无统计学意义(均为P>0.05)。结论前葡萄膜炎对角膜内皮有明显影响,角膜内皮细胞损害与前葡萄膜炎发病频数呈正相关,与病程及疾病严重程度有直接关系。     

无内皮移植的后弹力层剥离手术促进中央角膜内皮再生的研究进展

角膜内皮功能损害传统上被认为是不可逆的,角膜内皮移植几乎是目前唯一的治疗方式。然而,近来出现的仅剥离Descemet膜而无内皮移植的手术(DWEK)可使Fuchs角膜内皮营养不良患者中央角膜内皮细胞再生,而局部Rho相关激酶抑制剂可以促进其疗效。     

选择性角膜内皮移植术

长期以来，治疗角膜内皮病变标准的方法为穿透性角膜移植术(PK)。虽然总体上说，PK手术成功率高，但由于术后视力恢复时间长，且可出现多种并发症，如高度散光／不规则散光、植片排斥反应以及与缝线和切口愈合相关的并发症等，因此并非一种理想的手术方式。过去十几年内，出现了多种选择性角膜内皮移植术，如经角膜瓣途径的内皮移植术、经角巩膜袋途径的内皮移植术及带活性内皮细胞的Descemet’s膜移植术等。与PK手术相比，这些新的手术方法理论：有着多方面的优势，初步的临床结果亦令人鼓舞，有可能成为替代传统PK手术治疗角膜内皮病变的更有效的方法。     

脂质体介导兔角膜内皮细胞基因转染的观察

目的观察阳离子脂质体Lipofectamine2000能否介导目的基因转移至兔角膜内皮细胞内及脂质体潜在的毒副作用。方法RT-PCR检测特异性胶原Ⅷ进行细胞鉴定,通过体外细胞转染实验优化阳离子脂质体与质粒DNA的浓度和比例,选择Lipo-fectamine^TM 2000/pEGFP-N1比值3∶1,脂质体体积分别为0μL、6μL、9μL、12μL转染兔角膜内皮细胞,荧光显微镜观察目的基因的表达,透射电镜观察细胞超微结构。结果RT-PCR扩增得到单一产物,与预期的扩增序列大小完全一致,角膜内皮细胞内可见脂质体介导的绿色荧光蛋白表达,Lipofectamine^TM 2000浓度能显著影响其对兔角膜内皮细胞的转染效率,存在最佳浓度,在35mm培养皿中,3μg DNA与9μL脂质体转染效率最高,透射电镜显示12μL脂质体可导致细胞器出现肿胀、崩解现象。结论阳离子脂质体可有效介导目的基因转移至角膜内皮细胞内,在35mm培养皿中,12μL脂质体对角膜内皮细胞有一定的毒副作用。     

高血压患者角膜内皮细胞非接触角膜内皮显微镜观察

目的:应用内皮显微镜对原发性高血压患者进行角膜内皮细胞形态学定量分析。方法:2005/2006年我院拟行白内障手术患者164例184眼。其中原发性高血压患者56例70眼;非高血压对照组108例114眼。所有对象均排除青光眼、糖尿病、高度近视、眼外伤及眼部手术史,并按年龄分为50～59岁,60～69岁及≥70岁3层。应用SPSS10.0统计分析软件对各组角膜内皮细胞的平均面积、平均密度、变异系数及六角型细胞比例进行成组设计t检验。结果:在50～59岁组高血压患者角膜内皮平均细胞面积、细胞密度较对照组无显著性差异,但细胞变异系数增大(t=3.26,P<0.05),六角形细胞比例下降(t=3.86,P<0.05);在60～69岁组中细胞密度较对照组无显著性差异,但平均细胞面积增大(t=2.14,P<0.05),细胞变异系数增大(t=4.08,P<0.05),六角形细胞比例下降(t=5.19,P<0.05);在≥70岁年龄组中细胞密度、平均细胞面积、变异系数较对照组无显著性差异,六角形细胞比例下降(t=3.23,P<0.05)。结论:高血压患者角膜与正常对照组患者相比,正六角形细胞比例下降、内皮细胞变异系数增大。     

角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

角膜内皮细胞培养的研究进展

角膜内皮功能失代偿引起的失明可通过角膜移植来治疗.人们通过体外培养角膜内皮细胞并使其增殖,进行角膜内皮细胞移植和角膜组织工程学研究.目前常用的分离角膜内皮细胞的方法是从供体上撕除后弹力层和角膜内皮层,利用酶消化作用使角膜内皮细胞分离.常用的培养基为基础培养基加各种生长因子.这些生长因子在一定程度上促进细胞增殖,但培养的角膜内皮细胞长期传代后仍难以维持正常的细胞形态和功能.基因转染技术建立了永生化角膜内皮细胞系,有关细胞周期、信号通路和离子通道的研究成果也促进了角膜内皮细胞体外培养的研究进展.本文综述了角膜内皮细胞体外培养各个环节的最近进展.     

低密度角膜内皮细胞白内障的超声乳化术效果分析

目的评价超声乳化治疗低密度角膜内皮细胞白内障的安全性及临床效果。方法低密度角膜内皮细胞（〈1500／mm^2）的白内障22例（26眼）,其中伴青光眼者8例（10眼）,伴眼前段炎症者5例（5眼）,玻璃体切除术后4例（4眼）。进行超声乳化吸出联合人工晶状体植人手术,观察术后视力及角膜水肿的情况。结果术后视力均有不同程度的提高。最佳矫正视力0．1～0．3者8眼,〉0．3者18眼。术后第1天角膜水肿≤I级者11眼,Ⅱ级者10眼,Ⅲ级者5眼。无角膜失代偿者。结论随着人工晶状体的改善和手术技巧的提高,低密度角膜内皮细胞的白内障可以行超声乳化吸出及人工晶状体植入术。