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.     

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.     

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 (CECs) 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 vitro. CM was collected from CSCs, BEPCs, and BMSCs. 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 Ⅷ (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.     

Density and morphology of corneal endothelial cell after phacoemulsification using ringer lactate versus balanced salt solution as irrigating solutions

AIM: To compare the difference in corneal endothelial cell density and morphology after phacoemulsification using ringer lactate (RL) and balanced salt solution (BSS) irrigating solutions. METHODS: The prospective randomized controlled trial study was conducted between February 2017 and April 2017 in Dr. YAP Eye Hospital, Yogyakarta, Indonesia. There were a total of 52 subjects (52 eyes) who were senile cataract patients further grouped into two, 26 patients undergoing the phacoemulsification procedure using RL irrigating solution and the other 26 patients with BSS irrigating solution, both conducted by one operator. On the 1, 7, and 28d post operative, an evaluation was done to measure the density and corneal endothelial cell morphology, as well as the variable of inflammation in the two groups. RESULTS: Fifty-two eyes had undergone phacoemulsification with posterior intraocular lens implantation. Both groups were evaluated for the endothelial cell reduction and corneal endothelial cell morphology change, along with postoperative inflammation. On the 28d postoperative, endothelial cell reduction in the BSS group (173.96 cell/mm2, 8.12%) was lower than the RL group (253.20 cell/mm2, 10.25%), percentage of corneal endothelial cell variation coefficient increase in the BSS group (2.92%, 8.36%) was lower compared to the RL group (3.42%, 9.96%), decrease of hexagonal cells of corneal endothelium cells presentation percentage in the BSS group (4.30%, 8.17%) was lower compared to the RL group (4.84%, 8.97%), and the percentage increase of central corneal thickness in the BSS group (4.69 μm, 0.89%) was almost equal to the RL group (4.53 μm, 0.90%). All of the results regarding difference in density and corneal cell endothelium morphology between the two groups did not reveal any statistically significant difference (P>0.05). Inflammatory variable in the two groups were even. CONCLUSION: BSS and RL were equal in their capability of maintaining endothelial cell loss and endothelial cell morphologic change in senile cataract patients after phacoemulsification.     

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.     

Mechanism of Corneal Endothelial Cells Lesion during Phacoemulsification and Aspiration

Purpose: To evaluate the proportions of corneal endothelial lesion caused by differentfactors during phacoemulsification and aspiration.Methods: Fourteen cats (twenty eight eyes) were divided into four groups. The processedfactors were ultrasonic power, lens extraction by phacoemulsification or not, and lensextraction using different levels of ultrasonic power. The density of central cornealendothelial cells was measured before and after operation.Results: There is no statistic difference between pre-operation density and post-operationdensity for releasing ultrasonic power only without lens extraction group. But for the lensextraction group, there is difference in density of central corneal endothelial cells andthe higher level of ultrasonic power, the more the central corneal endothelial cells densitydecreased through operation.Conclusion: The primary factor that causes corneal endothelial lesion duringphacoemulsification and aspiration procedure is debris of lens nucleus, and the otherfactors cause t     

EXPERIMENTAL STUDY ON THE CORNEAL ENDOTHELIUM OF TRAUMATIC CATARACT

The cell morphology of corneal endothelium in 84 mice with experimental traumatic cataract was investigated with stained corneal buttons. In the experimental group, the boundaries between adjacent corneal endothelial cells were significantly distorted, some cell boundaries manifested degenerative changes that led to coalescence of the cells. The mean density and mean area of endothelial cells of the controls showed significant difference from those of the experimental group during the 12 weeks of observation. The density of endothelial cells decreased from 3312±337/mm~2 of the control group to 2944±418/mm~2 in the group of partially opaque lenses and 2713±472/mm~2 in the group of totally opaque lenses. Meanwhile, the area of endothelial cells and the coefficient of variation also significantly changed correspondingly, and the degree of damage in the corneal endothelium correlated with the degree of the lens opacity.     

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.     

Efficacy of poly (lactic-co-glycolic acid) and tetrandrine-loaded nanomicrospheres in the treatment of dry eye北大核心

Objective To discover a nano-drug with anti-inflammatory, sustained-release, and biocompatible properties aimed at blocking the dry eye formation pathway. Methods Nano-microspheres (Tet-ATS@PLGA) composed of tetrandrine (Tet) and poly (lactic-co-glycolic acid) (PLGA) were prepared using the thin-film hydration method, and their stability at room temperature (25 ℃), encapsulation efficiency, and drug loading were tested. Normal rabbit eyes without intervention were recruited in the normal group, and dry eye models were randomly divided into the control group (without any intervention), ATS group (intervened by artificial tears), Tet-ATS group (intervened by artificial tears and Tet), and Tet-ATS@PLGA group (intervened by Tet-ATS@PLGA). Flow cytometry was performed to detect the apoptosis of inflammatory corneal epithelial cells in each group after 24 hours of intervention. The staining of corneal epithelial cells, tear film break-up time (BUT), and surface tear secretion (detected by the Schirmer test strip) were recorded after 14 days of intervention. The thickness of corneal epithelial cells as well as the shape and number of bulbar conjunctival goblet cells were monitored by hematoxylineosin staining. Corneal proteins were extracted for the enzyme-linked immunosorbent assay to measure the expression levels of vascular endothelial growth factor (VEGF), interleukin-1β (IL-1β), prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α). The independent samples t-test was carried out for comparison among groups. Results The encapsulation efficiency and drug loading of Tet-ATS@PLGA nano-drug were 77.43% and 30.26%, respectively. The drug was stable at room temperature and easy to release when the ocular surface temperature stood at 33 ℃. Compared with other groups, BUT and the amount of tear secretion in the Tet-ATS@PLGA group were the largest, the thickness of corneal epithelial cells was close to the normal value, bulbar conjunctival goblet cells recovered the most, the apoptosis of inflammatory corneal epithelial cells after 24 hours of intervention was the most significant, and the expression levels of VEGF, IL-1β, TNF-α, and PGE2 were the lowest (all P<0.05). Conclusion Tet-ATS@PLGA nano-drug can effectively act on inflammatory corneal epithelial cells in rabbits, promote apoptosis of inflammatory cells, and block the inflammatory response of dry eyes by inhibiting the expression of VEGF, IL-1β, TNF-α, and PGE2, thus improving tear secretion on the ocular surface. © The Author(s) 2023.     

Effect of nintedanib thermo-sensitive hydrogel on neovascularization in alkali burn rat model

AIM: To investigate the effects of nintedanib thermo-sensitive hydrogel (NTH) on neovascularization and related markers in corneal alkali burns of Wistar rats. METHODS: NTH was prepared by grinding, and its phase-transition temperature was determined. Thirty specific-pathogen-free Wistar rats served as a model of corneal alkali burn in the right eye were randomly divided into 3 groups (n=10, each): model group treated with 0.9% saline once a day, NTH group with 0.2% nintedanib b.i.d, and dexamethasone group with dexamethasone ointment once a day. The left eye of rats served as the controls. The corneal transparency was observed under a slit-lamp microscope, and the area of neovascularization was calculated. On day 7, the rats were sacrificed, and the cornea was removed and embedded with paraffin, then stained with hematoxylin-eosin, and the expression of vascular endothelial growth factor receptor 2 (VEGFR-2) and CD31 in the corneal tissues of each group was detected by immunofluorescence. RESULTS: The phase-transition temperature of nintedanib obtained by grinding was 37℃ after adding artificial tears. The results of the alkali burn model indicated that the growth rate of neovascularization in the NTH group was slower than that in the model group, and the neovascularization area was significantly smaller than that in the model group (P<0.05). Moreover, CD31 and VEGFR-2 expression levels in the NTH group were significantly lower than those in the model group. CONCLUSION: NTH becomes colloidal at body temperature, which is beneficial for releasing the drug slowly and can significantly inhibit the neovascularization of corneal induced by alkali burn in rats.     

压力仿生培养对兔角膜内皮细胞调控的研究

目的 探讨体外压力仿生培养系统下不同梯度压力对角膜内皮细胞形态和功能的调控作用。设计 实验性研究。研究对象 兔角膜内皮细胞。 方法 将体外培养的第一代兔角膜内皮细胞分为五组：A组为无压力常规培养（空白对照）,B组为正常压力仿生培养（15 mmHg）,C组为压力波动组,将压力设为15mmHg、25 mmHg、20 mmHg、10 mmHg,D组30 mmHg压力培养,E组50 mmHg压力培养。细胞均培养24h,免疫法鉴定原代角膜内皮细胞,HE染色和电镜观察细胞形态的改变,流式细胞术检测细胞活性。主要指标 角膜内皮细胞的形态、存活率。 结果 获取的所有细胞证实为角膜内皮细胞表型,无角膜上皮细胞及基质细胞污染。五组细胞分别培养24h后,经HE染色和电镜检测发现正常压力微环境培养的角膜内皮细胞排列紧密,六边形细胞居多,细胞表面微绒毛丰富,细胞核染色质丰富,而高压力培养的角膜内皮细胞活性差,细胞间隙加大。经流式细胞术分析显示,正常压力组、30 mmHg组、压力波动组、50 mmHg组的角膜内皮细胞培养24 h后细胞存活率分别为（98.16±0.45）%、（78.83±1.65）%、（70.2±3.54）%、（41.33±0.25）%（P=0.016）。高压力培养组中随着压力的升高和持续时间延长,细胞活性显著下降。结论 正常压力微环境培养对角膜内皮细胞形态和功能具有正向调节作用,而高压力对角膜内皮细胞具有损伤性,并随时间延长而加重。（眼科,2017,26： 56-60）     

波动的压力对兔角膜内皮细胞的影响

目的　观察波动的压力对角膜内皮细胞的影响。方法　将体外培养的第一代兔角膜内皮细胞分为两组:Ａ组为压力波动组（压力设置为:１５ｍｍＨｇ～２５ｍｍＨｇ～２０ｍｍＨｇ～１０ｍｍＨｇ,每个压力持续６ｈ;１ｋＰａ＝７．５ｍｍＨｇ）;Ｂ组为３０ｍｍＨｇ压力组;Ｃ组为无压力组。三组细胞分别培养２４ｈ。免疫细胞化学染色法鉴定原代角膜内皮细胞形态;台盼蓝-茜素红联合染色检测细胞活性,ＨＥ染色观察细胞形态;Ｗｅｓｔｅｒｎ-ｂｌｏｔｔｉｎｇ检测细胞中Ｂｃｌ-２和Ｐ５３蛋白的表达水平。结果　获取的所有细胞证实为角膜内皮细胞表型,无角膜上皮细胞及基质细胞污染。三组细胞分别培养２４ｈ后,经台盼蓝-茜素红染色和ＨＥ染色证实:两个压力培养组的细胞活性较无压力培养组明显下降,其中压力波动组的细胞活性低于３０ｍｍＨｇ压力组。同时无压力组、３０ｍｍＨｇ压力组和压力波动组细胞中Ｐ５３蛋白的相对表达量分别为０．１５０±０．００５、０．２５３±０．０１４、０．６７０±０．０１９,差异有统计学意义（Ｐ＜０．０５）。结论　证实了高压力及非生理性波动的压力对角膜内皮细胞均具有损伤作用。     

角膜内皮细胞高压力损伤机制的实验研究

目的探讨青光眼急性高眼压对角膜内皮细胞的损伤机制。设计实验性研究。研究对象体外培养的角膜内皮细胞。方法采用后弹力层撕除联合酶消化法获取角膜内皮细胞,免疫组化法鉴定细胞。实验分两组：A组：急性压力增高组,压力为6．67kPa;B组：压力仿生培养,压力为2．0kPa。倒置显微镜定期观察细胞形态及生长规律;HE染色观察细胞形态结构变化;台盼兰一茜素红染色观察高压对细胞的损伤作用;流式细胞术分析细胞活性;免疫荧光检测细胞胞浆中细胞色素C（CytC）的表达。主要指标角膜内皮细胞形态结构、凋亡率及胞浆中CytC的表达。结果获取的细胞经免疫法证实为角膜内皮细胞表型。两组细胞分别培养24hr后,流式细胞术分析显示,高压力组的早、晚期细胞凋亡率分别为（16．40±0．95）％和（41．37±1．29）％;而正常压力组早、晚期细胞凋亡率分别为（1．07±0．40）％和（0．70±0．00）％,差异有统计学意义（P=0．000）。免疫荧光检测到高压力组角膜内皮细胞胞浆CytC呈阳性表达。结论高压力对角膜内皮细胞损伤呈时间敏感性,细胞的凋亡启动是其角膜内皮细胞损伤的机制之一。f眼科,2011,20：155-159）     

高压力培养下角膜内皮细胞凋亡的启动机制

目的 探讨高压力培养下角膜内皮细胞凋亡的启动机制.方法 原代兔角膜内皮细胞经免疫组织化学鉴定后,在50mmHg(1 kPa =7.5 mmHg)压力条件下,培养lh、2h、24 h,另设正常压力(15 mmHg)作为正常压力组,培养24 h.第一代兔角膜内皮细胞融合达70％～80％后,细胞在加压前lh分别使用浓度均为10-6 mol·L-l抗Caspase-8和抗Caspase-9预处理,然后置于压力装置中,压力设定为50 mmHg,培养24h后检测蛋白Bcl-2和P53表达,并且以无抑制剂处理的50 mmHg压力培养的细胞为对照组.各组培养的细胞均经Western blot检测Bcl-2和P53的表达.免疫荧光染色检测兔角膜内皮细胞胞浆细胞色素C的含量.结果 50 mmHg压力组角膜内皮细胞,加压lh、2h、24 h P53的表达量分别为0.651±0.007、0.805±0.006、0.839±0.011,较正常压力组(0.033±0.004)升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组随着加压时间的延长,角膜内皮细胞中P53蛋白表达量逐渐增加,各时间两两比较,差异均有统计学意义(均为P＜0.01).50 mmHg压力组中加压lh、2h、24 h Bcl-2的表达量分别为0.590±0.009、0.724±0.005、0.314±0.016,较正常压力组(0.081±0.013)反应性升高,差异均有统计学意义(均为P＜0.01);50 mmHg压力组各时间两两比较差异均有统计学意义(均为P＜0.01).实验发现正常压力组培养24h后,细胞核呈蓝色,胞浆中无细胞色素C释放;50 mmHg压力组培养lh可见部分细胞胞浆呈红色,提示细胞色素C释放进入到胞浆内,随着加压时间延长荧光强度增加,加压24 h见胞浆内出现广泛红色强荧光.抗Caspase-9和抗Caspase-8预处理组的角膜内皮细胞P53表达量分别为0.535±0.007、0.703±0.010,较对照组(0.727±0.021)下调,差异均有统计学意义(均为P＜0.0I);抗Caspase-9和抗Caspase-8预处理组中Bcl-2的表达量呈抑制性下降,分别为0.312±0.003、0.442±0.011,较对照组(0.501±0.011)下降,差异均有统计学意义(均为P＜0.01).抗Caspase-9预处理组的角膜内皮细胞P53和Bcl-2表达量较抗Caspase-8预处理组下降,差异亦均有统计学意义(均为P＜0.01),表明抗Caspase-9对高压下角膜内皮细胞凋亡的抑制作用更显著.结论 Caspase-9抑制剂可有效阻断高压力对角膜内皮细胞的促凋亡作用,高压力对角膜内皮细胞的损伤主要是触发了线粒体细胞色素C的释放,激活了Caspase-9参与的内源性酶联反应性凋亡途径.     

碱性成纤维细胞生长因子和表皮生长因子对角膜中期保存液保存人角膜内皮细胞的影响

目的探讨碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)对中期保存角膜内皮细胞活性的影响。方法角膜中期保存液保存人角膜环,对照组为角膜中期保存液,实验组分为A、B、C、D组,分别为角膜中期保存液中加入bFGF(5ng/ml)、bFGF(20ng/ml)、EGF及bFGF+EGF,在保存角膜第3、7、14天后分别取保存角膜环复温观察,锥虫蓝茜素红联合染色检测角膜内皮活细胞率,电镜检测细胞超微结构的改变。结果保存角膜环第14天角膜内皮活细胞率,对照组为(10.35±1.32)%,A组为(62.18±1.56)%,B组为(92.57±0.90)%,C组为(71.01±2.67)%,D组(82.59±1.45)%,与对照组比较,各保存时间各实验组活细胞率均高,差异有统计学意义(P<0.01)。保存第14天,对照组保存的角膜环明显水肿、混浊不透明、内皮细胞形态结构不完整,超微结构显示角膜内皮细胞Y型连接断裂;而实验B、D组,保存的角膜环轻度水肿、后弹力层皱褶少、角膜透明度高、内皮细胞形态结构完整,超微结构显示保存角膜内皮细胞连接紧密,Y型连接无明显断裂,细胞表面可见较丰富的微绒毛,胞体大,核突起明显。结论bFGF和EGF在角膜中期保存液中均有促进细胞增殖、保持角膜细胞活性的作用,以bFGF作用更明显。     

Study in cytotoxicity of gentamycin to corneal epithelium and endothelium in tissue culture

A study in cytotoxicity of gentamycin to tissue-cultured bovine corneal endothelial cells and rabbit corneal epithelial cells is reported. When the cultured cells reached confluence, they were exposed to tissue culture media containing gentamycin for 6 hours. We found that 0.5% gentamycin caused significant damage to corneal epithelial cells--diffuse plasmolysis, with scattered cell necrosis and 5% loss. While corneal endothelial cells were exposed to 1.6 mg/ml gentamycin, extensive cell loss (approximately 15%) was observed. The damaged cells recovered their normal morphology after 24 hours. When the concentration of gentamycin increased twice, serious damage to cells occurred. The area of cell loss reached 40%, and the recovery of cellular morphology was much slower. This study demonstrates that gentamycin potential cytotoxicity to corneal epithelium and endothelium, suggesting that gentamycin should be rationally used in the treatment of ocular diseases.     

羊膜载体培养标记兔角膜内皮细胞移植的研究

目的探讨以羊膜基底膜为载体培养兔角膜内皮细胞移植的可能性,为培养内皮细胞移植治疗角膜内皮失代偿疾病提供依据与方法。方法体外培养扩增兔角膜内皮细胞,采用亲脂性碳青染料(CM-DiI)对细胞进行标记,种植于去除上皮细胞的羊膜基底膜上,体外培养形成单层角膜内皮层,并进行形态学、组织学、超微结构及细胞标记情况的观察;将羊膜为载体培养的内皮层对切除后板层的兔角膜进行移植,同时以无培养内皮细胞的羊膜移植和单纯角膜后板层切除为对照,术后观察角膜透明度与厚度,对其进行组织学与细胞标记情况的检测。结果 5～7d 角膜内皮细胞即在羊膜基底膜上融合成单层,细胞为扁平多角形,排列紧密,密度可达(3202.84±347.77)个/mm~2,荧光显微镜下可见内皮细胞被 CM-DiI 标记后显现红色荧光;培养内皮层移植后角膜维持相对的透明与薄度,而无内皮细胞羊膜移植和单纯后板层切除两组对照角膜严重水肿、混浊,厚度明显超过实验组角膜;培养内皮移植后角膜形成新的内皮层,通过标记的细胞发现移植后细胞仍为培养移植的内皮细胞而非周边细胞的移行。结论羊膜基底膜是角膜内皮细胞生长和移植的良好载体,体外培养角膜内皮细胞移植有望代替供体角膜移植,具有广阔的应用前景。(中华眼科杂志,2006,42:925-929)     

改构aFGF在角膜内皮细胞中的分泌表达及意义

目的观察改构型人酸性成纤维细胞生长因子(m-haFGF)真核表达载体(pSecTag/m-haFGF)在培养的兔角膜内皮细胞中的分泌表达,探讨其表达产物对由生理盐水损伤的角膜内皮细胞的保护作用。方法采用脂质体转染的方法,将psecTag/m-haFGF转染培养的兔角膜内皮细胞,用免疫印迹法检测m-haFGF的表达情况,同时用生理盐水造成细胞损伤,用MTT法检测细胞存活状况。结果角膜内皮细胞在转染m-haFGF基因后能够表达目标蛋白,未转染组为阴性;MTT显示,转染后损伤的细胞存活状况明显好于未转染组(P<0.05)。结论转染pSecTag/m-haFGF能够在培养的兔角膜内皮细胞中分泌表达,其基因表达产物对生理盐水造成的角膜内皮细胞损伤具有保护作用。     

外伤性青光眼引流阀植入术与小梁切除术的疗效对比

目的:比较Ahmed引流阀植入术与小梁切除术治疗外伤性青光眼的效果。方法:前瞻性研究。选取2014年3月至2019年3月杭州市余杭第一人民医院外伤性青光眼120例(124眼),随机分为两组,A组60例(62眼),进行小梁切除术;B组60例(62眼),进行Ahmed引流阀植入术。术后随访12个月,比较两组手术疗效。结果:...     

The role of apoptosis in the early corneal wound healing after excimer laser keratectomy in the rat

Background: The potential role of apoptosis in corneal wound healing after excimer laser keratectomy was investigated in a rat model. Methods: Lewis rats underwent laser keratectomy using a 193-nm excimer laser. The central corneas were ablated in three depths: group A, epithelium; group B, superficial stroma; group C, deep stroma. Eyes were collected at 1, 12, 24, and 36 h and 1 week. Cellular markers associated with apoptosis – Fas, Fas ligand (FasL), Bcl-2, and Bax – were examined by immunohistochemistry. Keratocyte depletion and endothelial changes were evaluated histologically. In situ end labeling of double-stranded DNA breaks was used to demonstrate apoptosis in corneal sections. Results: Keratocyte depletion was observed in 6 (50%) of 12 rats (total from groups A, B, and C) at 12 h, 11 (73%) of 15 at 24 h, 3 (20%) of 15 at 36 h, and 2 (15%) of 13 at 1 week after laser surgery. Corneal endothelial edema was observed in the ablation zone. Expression of Fas, FasL, Bcl-2, and Bax in corneal cells showed dynamics similar to that of keratocyte depletion and endothelial changes. There was less expression of apoptotic molecules in newly generated epithelial cells and more in endothelial cells of the stromal ablation groups. Conclusions: Excimer laser keratectomy triggered apoptosis of corneal keratocytes and endothelial cells. More endothelial edema was observed in the stromal ablation than in the epithelial ablation group. The expression of apoptotic molecules coincided with the period of keratocyte depletion and regeneration and of endothelial recovery, suggesting that apoptosis is a dynamic part of corneal wound healing and remodeling after excimer laser keratectomy. Received: 9 November 1999 Revised: 18 May 2000 Accepted: 10 April 2000