人热敏瞬时受体通道1基因转染对兔角膜内皮细胞的影响

目的：探讨人热敏瞬时受体通道1基因转染对培养的兔角膜内皮细胞增殖能力的影响。

方法：研究组为人热敏瞬时受体通道1基因通过脂质体介导的方法转染到体外培养的兔角膜内皮细胞中,采用MTT方法观察对细胞增殖的影响,免疫组织化学染色法和计算机图像分析系统检测对细胞增殖细胞核抗原(proliferation cell nucleus antigen,PCNA)表达的影响。

结果：热敏瞬时受体通道1基因转染后内皮细胞增殖增加,实验组与对照组比较差异有统计学意义(t=3.01,P=0.013); 实验组细胞PCNA表达明显增加,与对照组比较差异有统计学意义(t=3.21,P=0.007)。

结论：人热敏瞬时受体通道1基因转染可以促进兔角膜内皮细胞增殖。     

转化生长因子β1对牛角膜内皮细胞DNA合成的影响

目的：研究外源性转化生长因子β1(transforming growth factor，TGF-β1)对牛角膜内皮细胞(bovine corneal emdothelial cells，BCECs)增殖过程的影响。方法：在体外培养的牛角膜内皮细胞经不同浓度(0．10、1．00、10．00、100．00μg／L)TGF-β1处理后，用流式细胞术测量分析细胞周期，用甲基噻唑基四唑(methyl thiazolyl tetrazolium，MTT)法检测TGF-β1对角膜内皮细胞增殖功能以及DNA合成的影响。结果：一定浓度(0．10、1．00、10．00μg／L)的TGF-β1对角膜内皮细胞有明显的促增殖作用，并呈剂量依赖性。但在较高浓度(100．00μg／L)时对内皮细胞表现出一定的抑制作用。结论：一定浓度外源性的TGF-β1与其受体结合后可以促进牛角膜内皮细胞DNA的合成，可以参与角膜内皮细胞的损伤修复再生过程。     

碱性成纤维细胞生长因子促进角膜碱烧伤内皮修复的实验研究

目的 观察碱性成纤维细胞生长因子对角膜碱烧伤内皮修复的作用.方法 120只兔角膜碱烧伤眼随机分成5组,分别以滴眼剂溶媒、3种不同剂量的bFGF滴眼液和素高捷疗滴眼治疗,锥兰联合茜素红内皮细胞染色后照相,经计算机图象系统分析,并进行光镜、电镜检查.结果 bFGF治疗组1周内损伤区角膜内皮几乎全部被移行的内皮细胞覆盖,各时间点内皮愈合率也明显高于溶媒组.电镜下bFGF治疗组再生的内皮细胞胞体较长,胞质内粗面内质网、核糖体明显增多.结论bFGF能促进角膜内皮细胞的增生和移行,加速角膜内皮的修复.     

神经生长因子对兔角膜内皮细胞增殖的影响

目的 探讨神经生长因子（NGF）对角膜内皮细胞增殖的影响。方法 在培养兔角膜内皮细胞的培养液中分别添加5U／ml,50U／ml和500u/ml的NGF,加药后第3,7天采用四甲基偶氮唑盐（MTT）法,在酶标仪上测定570nm波长处的吸光度值来观测细胞增殖情况。结果 与对照组比较,加药后第3,7天,3组的NGF对培养的兔角膜内皮细胞增殖均促进作用（P＜0．01）,且呈剂量依赖性。其中50U／ml组及500U／ml组作用强于5U／ml（P＜0．05）,而50U／ml组和500U／ml组比较作用无差异（P＞0．05）。结论 外源性NGF对培养的兔角膜内皮细胞增殖有明显的促进作用。     

层粘连蛋白促进兔角膜内皮细胞bcl-2表达的最适浓度测定

目的　探索层粘连蛋白促进兔角膜内皮细胞bcl 2表达的最适浓度。方法　给于体外培养的兔角膜内皮细胞以不同浓度的层粘连蛋白 ,采用酶联免疫技术找到最适作用浓度。结果　在一定的浓度范围内 (2 0～ 6 0mg·L-1)层粘连蛋白能够促进家兔角膜内皮细胞bcl 2的表达 ,并且成量效依赖关系 ,在 6 0mg·L-1时有最佳的促进作用。结论　层粘连蛋白可以促进兔角膜内皮细胞bcl 2基因表达的作用 ,最适作用浓度为 6 0mg·L-1。     

庆大霉素对组织培养角膜细胞毒性的影响

目的探讨临床眼部应用庆大霉素的安全剂量。方法采用体外培养的人角膜上皮细胞、内皮细胞、基质细胞分别在生长旺盛形成单层时加入不同浓度的庆大霉素，于不同时相在光镜和扫描电镜下观察细胞形态。结果浓度为１ｍｇ／ｍｌ不含保存剂的庆大霉素液对培养的３种细胞作用４８小时无损害；当药物浓度增加到２ｍｇ／ｍｌ和４ｍｇ／ｍｌ时，光镜下显示，不同的角膜细胞在不同时相均出现细胞病变，其中以内皮细胞损害最为严重，其次是上皮细胞，基质细胞损害最轻，损害程度与庆大霉素浓度的增加和作用时间的延长有关。结论临床眼部应用庆大霉素的安全剂量为１ｍｇ／ｍｌ。     

不同染色剂对兔眼角膜内皮细胞影响的研究

目的 观察并比较荧光素钠、吲哚靛青绿，台盼蓝，亚甲蓝，龙胆紫5种不同的前囊膜染色剂对兔眼角膜内皮细胞的影响。方法 新鲜离体兔眼角膜内皮滴入各实验试剂，0.25%台盼蓝，0.2%茜素红双重染色，光镜下观察角膜内皮细胞的活性，计数内皮细胞的死亡数，透射电镜检查，观察角膜内皮细胞超微结构的变化。结果 1%亚甲蓝组角膜内皮细胞失去正常的六边形结构，可见较多着色死亡细胞，细胞的死亡率与对照组相比，有统计学差异（P〈0.01）。电镜下，1%亚甲蓝组可观察到内皮细胞的胞膜不完整，胞浆内有空泡形成等变化。结论 1%亚甲蓝溶液对兔眼角膜内皮细胞的损伤作用。     

缓激肽对体外培养兔角膜内皮细胞增殖状况及对闭锁小带蛋白-1与相关性核酸结合蛋白表达的影响

目的　观察缓激肽（bradykinin，BK）对体外培养家兔角膜内皮细胞增殖及对紧密连接相关蛋白闭锁小带蛋白-1（zonula occludens-1，ZO-1）、闭锁小带蛋白-1相关性核酸结合蛋白（zonula occludens-1-associated nucleic-acid-binding protein，ZONAB）表达的影响，初步探讨BK促进角膜内皮细胞增殖的作用机制。方法　选择对数生长期的兔角膜内皮细胞，向培养基中分别加入0.01、0.10、1.00、10.00 μmol·L^-1 BK(BK组)，对照组不做加药处理，倒置相差显微镜下观察细胞的形态变化和增殖状况，MTT法检测各组细胞在24 h、48 h、72 h、96 h的吸光度(A)值，Western blot检测72 h各组细胞中紧密连接处ZO-1与核内ZONAB蛋白的表达。结果　加药 72 h，各组细胞融合成片并紧密连成单层，加药96 h后各组细胞生长受限，细胞间隙变大，脱落细胞增多。与对照组相比，除0.01 μmol·L^-1 BK组24 h外，其余浓度BK处理后A值升高、增殖活力增强，差异均有统计学意义 （均为P<0.001），并呈现出一定的浓度依赖性，其中1 μmol·L^-1 BK作用最强 （P<0.001）。Western blot结果显示BK可促进紧密连接处ZO-1与核内ZONAB蛋白的表达，且呈一定的浓度依赖性。结论　BK体外刺激可促进兔角膜内皮细胞的增殖，具有浓度和时间的依赖性，其作用机制可能与ZO-1与ZONAB介导的信号通路有关。     

表皮生长因子对人角膜内皮细胞损伤修复的影响

目的　观察人表皮生长因子 (epidermal growth factor,EGF)对人角膜内皮细胞损伤修复的影响 ,检测人角膜内皮细胞表皮生长因子受体 (epidermal growth factor recep-tor,EGFR)的表达。方法　胎儿角膜内皮细胞体外培养传一代融合后 ,定量损伤细胞 ,倒置显微镜下观察不同浓度 EGF对角膜内皮细胞损伤修复的影响 ,并于损伤前及损伤后 1、3、7、14 d用间接免疫荧光的方法检测角膜内皮细胞 EGFR的表达。结果　 EGF促进人角膜内皮细胞损伤愈合 ,并显示剂量依赖性 ,EGF在 10 μg· L- 1时促进作用达高峰 ,浓度再增高促进作用下降。人角膜内皮细胞损伤前及损伤后均在细胞膜上表达 EGFR,表现为细胞膜上绿色荧光 ,在未损伤区的细胞及缺损区修复的细胞均见表达。结论　 EGF促进人角膜内皮细胞损伤修复 ,人角膜内皮细胞表达 EGFR     

人工角膜植入术后EGF和bFGF局部应用的实验研究

目的 在兔眼局部应用上皮生长因子(EGF)及碱性成纤维细胞生长因子(bFGF)，观察对人工角膜植入术后植床角膜的影响。方法 于PMMA襻状支架人工角膜植入兔眼后，结膜下注射EGF或bFGF，术后1月取植床角膜制作石蜡切片，应用免疫组织化学技术ABC法，检测植床角膜组织增生细胞核抗原(PCNA)的表达情况及细胞凋亡情况；并检测c—myc基因的表达情况。结果 bFGF组植床角膜基质细胞可见PCNA阳性表达；EGF组植床角膜上皮细胞及基质细胞均可见PCNA阳性表达；对照组未见PCNA表达；各组植床角膜基质及上皮细胞均可见c—myc基因表达，角膜内皮细胞未见表达；各组均未见阳性凋亡细胞。结论 EGF和bFGF对人工角膜植入兔眼后植床角膜组织细胞的增生过程有促进作用，对减少人工角膜植入术后并发症，提高成功率有积极意义。     

以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究

目的探讨以异种角膜脱细胞基质为载体,体外构建生物角膜的可能性和方法。方法应用去垢剂1％TritonX-100冷冻干燥处理制备猪角膜脱细胞基质载体,在其上皮面和内皮面分别接种兔角膜上皮细胞和内皮细胞,体外培养2周。将复合物制成组织切片,在光镜下观察组织形态（HE染色）,采用免疫组织化学方法检测角膜上皮特异性细胞角蛋白3（CK3）,使用锥虫蓝联合茜素红染色观察内皮细胞,在扫描电镜下观察上皮面和内皮面的超微结构。结果体外培养生物角膜获得上皮、无细胞基质和内皮三层复合结构。4或5层复层上皮中以扁平细胞为主,胞质内特异性CK3表达阳性;内皮层为一连续的单层扁平细胞,细胞活性良好,锥虫蓝联合茜素红染色显示组织呈典型的蜂巢样镶嵌结构。扫描电镜下观察,在载体的上皮面细胞呈复层样生长,形态近似扁平与梭形之间;内皮面为多边形单层细胞,表面具有微绒毛结构。结论构建的生物角膜为上皮一脱细胞基质载体一内皮复合物,初具角膜的雏形结构。异种角膜脱细胞基质提供了良好的细胞生长界面,有望成为体外构建角膜的载体材料。     

Endothelin-induced changes of secondary messengers in cultured corneal endothelial cells.

The effect of endothelins on corneal endothelial cells is not well understood. We have investigated the effects of endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) on bovine corneal endothelial cellular proliferation and the secondary messenger changes in cells in the presence of ET-1. It was found that the 3H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. ET-1 remarkably affects the increase of corneal endothelial cells on 3H-thymidine, 3H-leucine, and 3H-uridine uptakes in a dose-dependent manner. The 50% effective concentrations (EC50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake, and 3H-leucine uptake were 10(-8.78) M, 10(-8.53) M and 10(-8.04) M, respectively. It was found that endothelin-1 increased intracellular calcium concentration by using the method of preloading with Fura-2-AM and assaying with spectrophotometry. The cellular IP1, IP2, and IP3 were also stimulated in the presence of ET-1. Moreover, ET-1 enhanced the basal cellular cAMP and cGMP concentrations in corneal endothelial cells in a dose-dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed protein distribution in corneal endothelial cells. These findings indicate that endothelin-1 increases in cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation, and fibronectin protein synthesis.     

Smad7 suppresses the inhibitory effect of TGF-beta2 on corneal endothelial cell proliferation and accelerates corneal endothelial wound closure in vitro

PURPOSE: The inhibitory activity of transforming growth factor-beta2 (TGF-beta2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-beta signaling, regulated the inhibitory activity of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation. METHODS: The effect of Smad7 on TGF-beta2- or aqueous humor-mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle-related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. RESULTS: Overexpression of Smad7 abolished the inhibitory effect of TGF-beta2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. CONCLUSION: Overexpression of Smad7 suppressed the inhibitory effect of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.     

Proteoglycans on normal and migrating human corneal endothelium

Proteoglycans are of fundamental importance to the normal functioning of the cornea. They consist of a core protein to which one or more glycosaminoglycan chains are attached. Cell surface proteoglycans are known to mediate many aspects of cell behaviour including cell adhesion, control of extracellular matrix deposition, cell proliferation, cell migration, leukocyte adhesion and modulation of growth factor activity. This paper describes the first investigation into the distribution and function of the three main classes of proteoglycans on human corneal endothelium. Immuno-gold labelling techniques were used at the light, scanning and transmission electron microscope level to localise heparan sulphate, chondroitin sulphate and keratan sulphate proteoglycans on human corneal endothelium. Human corneas were freeze-wounded and kept in organ culture for 3 days in order to study the distribution of proteoglycans on migrating corneal endothelium. An Optimas image analysis system was used to quantify the change in proteoglycan labelling during cell migration. Labelling for chondroitin sulphate and heparan sulphate was at very low levels on normal corneal endothelium while keratan sulphate labelling was at high levels. The wound healing experiments showed that migrating cells had increased labelling for heparan sulphate and chondroitin sulphate with greatly decreased labelling for keratan sulphate. Statistical analysis showed these changes were highly significant (P<0.001). Transmission electron microscopy revealed that chondroitin sulphate and keratan sulphate were present throughout Descemet's membrane while heparan sulphate was concentrated at the interface of Descemet's membrane and the migrating corneal endothelial cells. The pattern of occurrence of chondroitin sulphate, heparan sulphate and keratan sulphate on the human endothelium in normal and wounded cornea suggests that these proteoglycans are linked to the process of cell migration.     

Characterization of corneal endothelium cell cultured on microporous membrane filters.

In these experiments we characterize rabbit and bovine corneal endothelia cell cultured on microporous membrane filters (0.6cm2). Cell cultured bovine or rabbit corneal endothelial cells (subcultures 1-3) were seeded onto Millicell-HA filter inserts. Electrical resistance measured across the cultured monolayers increased steadily through 14 days of culture, reaching 34.2 +/- 0.8 ohm-cm2 (mean +/- SE) for rabbit cells and 33.1 +/- 1.1 ohm-cm2 for bovine cells. Alizarin red staining of the monolayers showed a polygonal morphology comparable to that observed in situ. Transmission electron microscopy showed well developed apical junctional complexes and flaps. Exposure of the monolayers to calcium-free medium resulted in the disruption of intercellular junctions, rounding-up of the cells and a decrease in electrical resistance (to near 0). Transmonolayer fluxes of inulin and dextran correlated well to the resistance measurements. Results of this study demonstrate that corneal endothelium, both bovine and rabbit, grown on filter inserts is comparable in morphology and ultrastructure to corneal endothelium in situ. The cells cultured in this system form functional apical junctional complexes that effect a barrier function comparable to that of the endothelium in situ.     

Electron microscopic study of corneal epithelial-endothelial interactions in organ culture

The interactions between rabbit corneal epithelium and endothelium were investigated in an organ culture model using scanning electron microscopy and correlative light microscopy. Following mechanical removal of corneal endothelium, corneal epithelium was observed to migrate across the cut surface of the corneal stroma and onto the denuded Descemet's membrane after 48 hours in organ culture. By 72 hours, a distinct line of contact between the migrated epithelium and endothelium was established. Following epithelial-endothelial contact, no cellular migration occurred. Corneal endothelial inhibition of epithelial migration may be a major factor in the prevention of surface epithelial invasion and proliferation following surgery and trauma.     

体外兔胚胎成纤维细胞为饲养层克隆兔角膜缘干细胞

目的研究兔胚胎成纤维细胞体外克隆兔角膜缘干细胞。方法兔胚胎成纤维细胞经丝裂霉素C(MMC)处理成饲细胞,用消化法培养兔角膜缘基底层细胞并接种于含有经MMC处理饲细胞的12孔培养板,进行原代培养。观察其光镜特征、电镜结构,用间接免疫细胞化学染色等对克隆的细胞进行综合鉴别。结果克隆的细胞呈典型的上皮细胞形态,电镜下可见微绒毛、桥粒、张力丝等典型上皮细胞结构,单克隆抗体AE1、PCNA染色后细胞大多数阳性,单克隆抗体AE5染色后细胞染色偶见阳性。结论体外兔胚胎成纤维细胞为饲养层成功地克隆出兔角膜缘干细胞。     

双氢青蒿素对体外培养兔角膜上皮细胞毒性作用的实验研究

目的 评价双氢青蒿素对体外培养的兔角膜上皮细胞的毒性作用。设计 实验研究。研究对象 体外培养兔角膜上皮细胞。方法 体外培养兔角膜上皮细胞,加入不同稀释浓度（1.6%、0.8%、0.4%、0.2%、0.1%、0.05%和0.025%）的双氢青蒿素共同孵育,同时以洗必泰和聚六亚甲基双胍（PHMB）做对照药物。在培养后2、6、24小时,光学显微镜下观察细胞形态,LIVE/DEAD染色观察药物作用后兔角膜上皮细胞的活性改变。主要指标 细胞形态改变。结果 培养后2、6小时,0.4%双氢青蒿素作用后的细胞出现圆化,细胞间隙加宽;0.0125%洗必泰作用后的细胞开始出现细胞间隙加宽,边界不清;0.003%PHMB作用后的细胞开始出现细胞边界不清,细胞内颗粒样改变。培养后24小时,0.1%的双氢青蒿素作用的角膜上皮细胞尚存在活性,0.025%的双氢青蒿素对角膜上皮细胞无明显影响。0.006%洗必泰和0.003%PHMB可导致角膜上皮细胞的死亡。结论 双氢青蒿素对体外培养的兔角膜上皮细胞的毒性较低,其对活体角膜细胞的作用尚待进一步研究。     

Induction of cellular toxicity in cultured porcine corneal keratocytes by endothelin-1.

The effect of endothelin-1 (ET-1) on corneal cells is not well understood. We investigated the biochemical changes of cultured porcine corneal keratocytes under exposure to ET-1. The results indicate that ET-1 has remarkable effects to inhibit corneal keratocytes on 3H-thymidine, 3H-leucine, 3H-uridine uptakes and cellular migration. It is in a dose-dependent manner at concentrations ranging from 10(-7) M to 10(-9) M. The 50% inhibitory dose (ID50) for ET-1, as measured by 3H-thymidine uptake, 3H-uridine uptake and 3H-leucine uptake, were 10(-7) M, 10(-0.52) M and 10(-11.8) M, respectively. The dead and living cells were estimated with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of ET-1, the cellular MTT values were also decreased. The ID50 for ET-1 with cell migration assay and MTT assay were measured at 10(-7.86) M and 10(-5.1) M. Endothelin-1 (10(-6) M) promptly changed cellular morphology and attenuated adhesion observed with laser scanning cytometer. Endothelin-1-induced characteristic apoptosis cells were observed using a TUNEL assay that detected fragmented DNA of apoptosis. Western blot assay revealed that endothelin-1 induced proteolysis and decreased in fibronectin protein. These findings indicate that endothelin-1 may lead keratocytes to death resulting from induction of apoptosis and functional loss.