丝裂霉素对体外培养兔角膜内皮细胞的毒性影响

目的探讨丝裂霉素C(mitomycinC,MMC)对角膜内皮细胞的毒性作用。方法体外培养家兔角膜内皮细胞,接种于96孔板,加入不同浓度的MMC,分别为1×10-6g·L-1、1×10-5g·L-1、1×10-4g·L-1、1×10-3g·L-1、1×10-2g·L-1、1×10-1g·L-1,动态观察细胞的形态变化和增殖情况,加药22h后MTT法检测吸光度值,应用t检验比较各组间的差异。结果10-2g·L-1、10-1g·L-1浓度组8h后细胞部分死亡,20h细胞几乎全部死亡,其余各组变化不明显。统计学结果显示:10-6g·L-1、10-5g·L-1组和对照组比较无显著性差异(P>0.05),10-4g·L-1组有显著性差异(0.01相似文献   

Thrombin enhances release of tissue plasminogen activator from bovine corneal endothelial cells

The effect of thrombin on release of plasminogen activators (PAs) was studied using cultivated endothelial cells of the bovine cornea. Species of PAs released into the conditioned medium were determined by fibrin autography and immunological analyses. Chromogenic peptide (S-2251) microassay was used for a quantitative estimation of the PA activity in conditioned medium and enzyme-linked immunosorbent assay (ELISA) for tissue plasminogen activator concentration. Fibrin autography revealed that cultured bovine corneal endothelial cells released into the conditioned medium tissue plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). Addition of increasing concentrations (0.1 to 10.0 U/ml) of thrombin to the confluent cultures led to a dose-dependent increase in the rate of release of t-PA, while there was no significant increase in the release of u-PA. About a 2-fold increase in the t-PA concentration occurred when 10.0 U/ml thrombin was to the confluent cultures for 24 hr. Thrombin induced an increase in the release of t-PA, in a time-dependent manner. The addition of cycloheximide or actinomycin D to the thrombin-treated cultures resulted in a reduction of t-PA levels in the media. These findings indicate that the enhancing effect of thrombin is due to an increase in t-PA production, via protein synthesis. Thrombin inactivated with diisopropylfluorophosphate (DFP) did not induce an increase in t-PA levels. A 100-fold excess of DFP-treated thrombin did not inhibit the thrombin-induced increase. These findings indicate that binding ability and the effect of t-PA release depend on the enzymatically active site of thrombin.     

Toxic effects of mitomycin-C on cultured corneal keratocytes and endothelial cells.

Improper use of mitomycin-C in ocular medication may result in damage to corneal cells. In this study, the toxic effects of mitomycin-C on cultured porcine keratocytes and endothelial cells were estimated by MTT, 3H-thymidine uptake and cellular counting assay methods. It was found that mitomycin-C caused a dose-dependent toxic effect to keratocytes and endothelial cells. Both cells were treated with mitomycin-C at the concentration ranging from 100, 10, 1, 0.1 to 0.01 microg/ml for 3 min, 5 min or 100 min. The 50% inhibitory dose (ID50) of mitomycin-C to keratocytes and endothelial cells as measured by MTT assay was 0.40, 0.18, 0.16 mg/ml and 0.27, 0.15, 0.14 mg/ml, respectively, after 3, 5 and 100 minutes drug treatment. The ID50 for keratocytes and endothelial cells as measured by 3H-thymidine uptake immediately, 1 day and 7 days after 100 minutes mitomycin-C treatment was 0.3, 0.0002, 143.2 microg/ml and 45.1, 101.1, 450.2 microg/ml, respectively. The ID50 for keratocytes and endothelial cells as measured by cellular counting 1 day and 7 days after mitomycin-C treatment was 232.5, 109.7 microg/ml and 239.9, 367.5 microg/ml, respectively. It is concluded that mitomycin-C is more toxic to cellular proliferation in cultured corneal keratocytes than in endothelial cells.     

Synthesis of plasminogen activator by bovine corneal endothelial cells

Plasminogen activator activity in purified cultured bovine corneal endothelial cells grown in tissue culture was demonstrated by a solid-phase assay based on the hydrolysis of [¹²⁵I]fibrin. Plasminogen activation appears to take place on the surface of the corneal cells and not as a result of secretion of the activator. Only a small fraction of plasminogen activator, however, is associated with the membrane; 90% is located in the cytosol. As a result, after cell death or lysis, a large amount of plasminogen activator is released into the media. Plasminogen activator can be detected within 2 hr of adding plasminogen-containing serum to proliferating cells; fibrinolysis is maximal within 12 hr. The extent of fibrinolysis is dependent on the plasminogen concentration in the serum. Considerably more plasminogen activator is associated with endothelial cells derived from the cornea than with cells derived from bovine aorta.The clotting factor thrombin markedly inhibits corneal endothelial cell-mediated fibrinolysis. Since thrombin has no effect on the partially purified activator, inhibition must result from a direct effect on these cells.The ability to study the regulation of the synthesis and distribution of plasminogen activator by cultured corneal endothelial cells should aid in determining the normal physiology of fibrinolysis in the anterior chamber. In addition, this technique can be used to study fibrinolysis in other ocular tissues.     

Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor

Maintenance of patency of the trabecular meshwork, the major outflow channel of the anterior chamber of the eye, is necessary to prevent an excessive rise in intraocular pressure. Obstruction of flow due to clot formation results in severe glaucoma and damage to the optic nerve. We have found that human trabecular meshwork cells which have been passaged in tissue culture synthesize large amounts of tissue plasminogen activator (t-PA), based on functional, immunologic and molecular weight analysis. Trabecular cells express substantially more t-PA activity than vascular endothelium which produces t-PA for clot dissolution in the systemic circulation. Vascular cells produce excess t-PA inhibitor while trabecular cells make comparatively little. Trabecular meshwork cells are the first normal cell type reported in which the balance between t-PA and inhibitor is weighted towards the activator, indicating that fibrinolysis may be more important than clotting in the anterior chamber of the eye.     

Release of plasminogen activator by cultured corneal epithelial cells during differentiation and wound closure

The release of plasminogen activator (PA) by corneal epithelium was studied utilizing pure culture of rabbit corneal epithelial cells and a sensitive photometric assay for the enzyme. The activity of PA measured in conditioned medium collected from the cultured cells was plasminogen-dependent, acid-stable, and free of interference by endogenous PA inhibitors. When serum was included in the culture medium, it was necessary to acid-treat the conditioned medium before PA assay in order to inactivate plasma-derived inhibitors. During a month of culture in serum-free medium, the cells released a low basal level of PA in the first week of growth and confluency. During the second week the cells progressively increased PA secretion at the onset of cell differentiation, reaching a peak (18-fold increase) in the third week when focal multilayering of cells occurred. Thereafter, the cells declined release, concomitant with globular aggregation of cells. A delay in the elevated release of PA was observed when the confluent phase was extended to 3 weeks, after which a substantial rise in PA level occurred simultaneously with the onset of multilayering. An in vitro model of corneal wound closure was established on confluent epithelial cells (grown in serum-containing culture medium) by mechanically removing 35-40% of cells in a central circular area. Complete wound closure was effected within 2-4 days by cell migration. The release of PA during the first day of wound closure was four times that of unwounded culture. The results indicated that PA secretion by corneal epithelium was associated with cell differentiation and cytomobility, processes that occur during stratification or wound closure. This conclusion is discussed with respect to a previously hypothesized role of PA in corneal injury and ulceration.     

Enzymatic vitreolysis with recombinant microplasminogen and tissue plasminogen activator

PURPOSE: To generate microplasmin (microPlm) using recombinant microplasminogen (microPlg) and recombinant tissue plasminogen activator (rt-PA) before intravitreous injection and to investigate the efficacy of microPlm in inducing posterior vitreous detachment (PVD). METHODS: Forty-eight female or male New Zealand white rabbits were randomized into three groups. Recombinant human microPlg was incubated with rt-PA with a 200:1 molar ratio at 37 degrees C for 40 min. The right eyes of groups 1, 2, and 3, were injected with 0.5, 1.0, and 1.5 U microPlm in 0.1 ml respectively, and 0.1 ml balanced salt solution (BSS) was injected into the left eye as controls. Scanning electron microscopy (SEM), gross specimen examination, B-ultrasonography and optical coherence tomography (OCT) were performed to detect vitreoretinal interface. RESULTS: Over eighty percent of recombinant human microPlg could be activated to active microPlm by rt-PA after 40 min incubation. Complete PVD was found at vitreous posterior pole of microPlm-treated eyes without morphological change of retina. Complete PVD of 25, 75, and 87.5% rabbit eyes was induced by 0.5, 1.0 and 1.5 U recombinant microPlm respectively on day 1. The remnants of vitreous cortex at the posterior pole were dependent on the concentration of microPlm. Among the four approaches for detecting PVD, SEM, gross specimen examination, and B-ultrasonography were more effective methods than OCT. CONCLUSION: Intravitreous injection of 1.5 U microPlm can effectively induce complete PVD in rabbit eyes on day 1 without morphological change of retina.     

压力对体外培养的牛角膜内皮细胞的影响

目的观察压力对体外培养的牛角膜内皮细胞形态结构及细胞活力的影响。方法对体外培养的牛眼角膜内皮细胞施以2.67kPa、5.33kPa及8.00kPa的压力各6、12、24、36、48h,以不加压组作为对照组。用倒置相差显微镜及电镜观察细胞形态结构,以台盼蓝染色观察比较48h时各组细胞活力的差别,用单因素方差分析比较不同压力下阳性细胞率。结果当作用于细胞的压力为2.67kPa(1kPa=7.5mmHg),作用时间为6、12、24、36、48h时,与对照组比较,细胞形态结构均无明显变化;48h时台盼蓝计数与对照组比较差异无统计学意义(P=0.776)。当压力为5.33kPa,作用时间为24h时,细胞形态结构出现轻度的损伤,在36、48h时加重;48h时台盼蓝计数与对照组比较,差异有统计学意义(P=0.00)。当压力为8.00kPa时,6h时即出现明显细胞形态结构损害,随着压力和作用时间的进一步增加,细胞的损伤程度也进一步加重;48h时台盼蓝计数与对照组比较差异有统计学意义(P=0.000)。结论牛角膜内皮细胞在加压48h时只能承受2.67kPa的压力,当压力为5.33kPa及8.00kPa,施压48h时,将造成细胞...     

Toxic effects of ophthalmic preservatives on cultured rabbit corneal epithelium

We investigated the effects of the ophthalmic preservatives thimerosal and sorbic acid on the proliferation and survival of rabbit corneal epithelial cells in tissue culture. Normally, explants of corneal epithelium grow vigorously during the first 7 days in culture. With 0.004% thimerosal present in the culture medium, the normal proliferation of corneal cells is suppressed completely. When 0.1% sorbic acid is present, proliferation is delayed and the lifespan of the corneal cells is reduced. After a 1-h exposure to concentrations of thimerosal of 0.0005% or greater, virtually all corneal cells present in established cultures are killed. These results suggest that use of ophthalmic preparations containing these chemicals may affect the metabolic and proliferative capacity of the corneal epithelium adversely.     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

器官培养法保存角膜对角膜内皮细胞活性的影响

成功的角膜保存方法应能很好地维持角膜内皮细胞的活性.测定角膜内皮细胞的活性是器官培养保存角膜中最为重要的环节之一.本文总结了常用的角膜内皮细胞观察方法以及现阶段对其凋亡的研究,分析了保存液中主要成分、不同脱水剂以及保存时间对角膜内皮细胞活性的影响.     

Effect of donor age on morphologic variation of cultured human corneal endothelial cells

PURPOSE: To examine the effect of donor age on the morphologic variation of cultured human corneal endothelial cells (HCEC). METHODS: HCEC were obtained from the remaining corneoscleral rims of seven human corneas used for penetrating keratoplasty. The donor age ranged from 2 to 75 years. Primary cultures were established from explants of the endothelial cell layer, including the Descemet's membrane, and were propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. At the fourth passage, frequency distribution of cell area in the confluent monolayer was calculated and the effect of donor age on cell area was analyzed. RESULTS: The percentage of HCEC with cell area over 2000 microm2 significantly increased with donor age (r = 0.935, p = 0.0007). CONCLUSION: Cultured HCEC established from older donor tissue display greater heterogeneity. The use of HCEC from younger donors may be preferable to maximize the benefits of HCEC transplantation.     

Effect of bradykinin on cultured bovine corneal endothelial cells

PURPOSE: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). METHODS: The cytosolic free calcium concentration (Ca2+]i) was measured with the InCa(TM) Imaging System after the treatment of bradykinin (10(-11) to 10(-7) M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk1 and Bk2) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. RESULTS: In BCEC, [Ca2+]i in the resting state was 87 +/- 9 nM. Bradykinin induced an increment of [Ca2+]i in a concentration-dependent manner and its 50% effective concentration was approximately 5 x 10(-11) M. A [Ca2+]i increment at 10(-8) M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 x 10(-6) M) attenuated the bradykinin-induced [Ca2+]i increment. The pretreatment of HOE-140 (Bk2 antagonist) almost attenuated the bradykinin (10(-8) M)-induced [Ca2+]i increase, but des-Arg9-[Leu(8)]-bradykinin (Bk1 antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10(-8) M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk2 antagonist. CONCLUSIONS: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk2 type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.     

Cryopreservation and lentiviral-mediated genetic modification of human primary cultured corneal endothelial cells

PURPOSE: To determine the viability and potential usefulness of cryopreserved human primary cultured corneal endothelial cells by characterizing their morphology, gene expression, and ability for genetic modification by the lentiviral vector equine infectious anemia virus (EIAV). METHODS: Primary cultured endothelial cells were dissociated from human corneas and grown in organ culture medium. Corneal endothelial cell origin was confirmed by morphology and immunostaining with polyclonal anti-collagen VIII antibodies. Cells of different passages were cryopreserved in medium containing dimethyl sulfoxide and were assessed after thawing for morphology, proliferative capacity, gene expression, and ability to form cell-cell junctions. EIAV encoding enhanced green fluorescent protein (eGFP) was used to transduce cryopreserved human corneal endothelial cells. Transduced cells were then sorted by fluorescence-activated cell sorting (FACS) and imaged with fluorescence microscopy. RESULTS: Cryopreserved, primary, cultured human corneal endothelial cells are viable and retain their ability to proliferate, produce collagen VIII, and express ZO-1, a tight-junction protein. EIAV-based gene transfer of eGFP is highly efficient and nontoxic to cryopreserved human primary cultured corneal endothelial cells. These genetically modified cells can be selected to nearly pure populations with FACS sorting. CONCLUSIONS: Human primary cultured corneal endothelial cells retain their phenotypic properties after cryopreservation. The ability to store, genetically modify, and sort these cells through FACS to pure populations has the potential to greatly expand their future therapeutic application to treat corneal endothelial disorders.