腺病毒介导人内皮抑素感染视网膜色素上皮细胞的可行性研究

目的探讨以腺病毒为载体对体外培养的人视网膜色素上皮（human retinal pigment epithelial，hRPE）细胞进行人内皮抑素（human endostatin，hES）感染，获得高效表达hES转基因细胞的可行性。方法常规培养hRPE细胞，传代至第3～第5代后．应用携带有绿色荧光蛋白（green fluorescent protein．GFP）的hES重组腺病毒按感染复数30进行感染。培养24h后，在荧光显微镜下计数培养的hRPE细胞的GFP表达阳性率：用免疫组织化学法观察hES在已感染hRPE细胞中的表达情况；提取hRPE细胞总RNA，采用RT—PCR技术对产物行琼脂糖凝胶电泳：用Western blot法检测感染hRPE细胞培养上清液中hES的表达水平。结果hES重组腺病毒感染后，hRPE细胞形态正常，感染后24h，GFP即呈100％表达．弥漫整个胞质；免疫组织化学法观察可见，hRPE细胞浆内呈棕黄色的阳性反应．而在感染空载腺病毒的对照组中细胞胞浆无染色；RT—PCR反应可见，细胞裂解产物中有hES阳性条带：Western blot法检测结果表明，培养上清液中有hES蛋白的表达。结论hES重组腺病毒载体能有效感染体外培养的hRPE细胞．并稳定表达hES蛋白。     

分泌人内皮抑素的微囊化基因工程细胞系的构建

目的 建立能稳定分泌人内皮抑素(hES)的基因工程细胞系,观察内皮抑素(ES)蛋白和hES的表达.方法 以hES重组质粒pcDNA3.0(pcDNA3-Endo)为模板,通过聚合酶链反应(PCR)扩增获得hES基因片段,且在基因前加信号肽序列,将其定向插入真核表达载体绿色荧光蛋白(pEGFP-N1)质粒中,获得重组质粒pEGFP-N1-Es;利用阳离子脂质体介导将其转染到人胚胎肾细胞(HeK-293)细胞中,G418筛选后得到阳性克隆hES/293,采用蛋白免疫印迹(Western blot)法检测转染细胞上清中ES蛋白的表达;用海澡酸钠壳聚糖(ACA)微囊包裹hES/293细胞,分别收集培养3、7、21、35 d的ACA微囊化hES/293细胞的培养上清液,Western blot法检测包裹后培养液上清中hES的表达.结果 重组质粒pEGFP-N1-ES经限制性核对内切酶HindⅢ和限制性核酸内切酶BamH Ⅰ双酶切得到4700碱基对(bp)和600 bp 2条带;PCR扩增出600 bp条带;测序结果与NCBI上序列比对软件(BLAST)比对,同源性达到100%.pEGFP-N1-ES转染HeK-293细胞,经G418筛选后获得阳性克隆,选取筛选的10株单克隆细胞培养上清液进行Western blot分析,在相对分子质量为20×103处出现蛋白条带.在ACA微囊内hES/293细胞随着培养时间的延长,细胞团逐渐长大,充满整个囊内空间.培养3、7、21、35 d时,在相对分子质量为20×103处出现蛋白条带.结论 重组pEGFP-N1-ES真核表达载体构建正确,转染HeK-293细胞后可有效的表达hES蛋白,并能分泌到细胞外;微囊化hES/293细胞产生的ES蛋白可以自由扩散出微囊膜外,并呈持续性表达.     

整合素连接激酶对人视网膜色素上皮细胞增殖的影响

目的研究整合素连接激酶(integrin linked kinase,ILK)小分子干扰RNA(siRNA)对人视网膜色素上皮(human retinal pigment epithelium,hRPE)细胞增殖的影响。方法培养hRPE细胞,角蛋白鉴定。以ILK为靶基因设计合成特异性的siRNA干扰片段转染hRPE细胞。荧光显微镜下观察转染效率,选择转染效率最高的干扰片段进行细胞转染。RT-PCR检测siRNA对ILK基因表达的抑制作用,MTT法检测转染前后hRPE细胞增殖活性。结果 ILK-siRNA可以成功转染至hRPE细胞,转染24h后hRPE细胞中ILK mRNA的相对表达水平在正常对照组、单纯脂质体组、阴性对照组及转染组中分别为0.44±0.48、0.43±0.38、0.42±0.47、0.32±0.71,正常对照组、单纯脂质体组和阴性对照组中ILKmRNA表达差异无统计学意义(P>0.05),转染组ILKmRNA的表达较正常对照组明显降低,差异有显著统计学意义(F=21.78,P<0.01)。不同浓度ILK-siRNA转染组在不同时间点细胞增殖较正常培养组明显降低(P<0.01),50nmol·L-1浓度转染组对hRPE细胞的生长抑制最明显。结论 hRPE细胞表达ILK,siRNA抑制ILKmRNA的表达,在一定范围呈时间浓度依赖性抑制hRPE细胞增殖。     

应用转基因技术体外培养表达人碱性成纤维细胞生长因子的视网膜色素上皮细胞

目的 探讨应用转基因技术体外培养稳定表达人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的视网膜色素上皮(retinal pigment epithelium,RPE)细胞的可行性。方法 构建以绿色荧光蛋白(green fluorescent protein,GFP)作为标记基因的人bFGF真核表达载体pcFG。应用脂质体介导法转染人RPE细胞,以G418筛选出表达bFGF的RPE细胞,并进行细胞克隆,传代培养4周。于荧光显微镜下观察GFP的表达情况,用原位杂交和免疫组织化学染色法检测bFGF在RPE细胞的表达情况。结果 酶切结果证实含有GFP的真核表达载体pcFG构建正确。在荧光显微镜下可见RPE细胞表达绿色荧光蛋白。经原位杂交和免疫组织化学染色证实,转染pcFG后的RPE细胞内有大量bFGF—mRNA的转录蛋白表达。结论 应用转基因技术可体外培养稳定表达bFGF的RPE细胞。     

缺氧对培养的人视网膜色素上皮细胞中NOX4表达的影响

目的:观察缺氧对培养的人视网膜色素上皮细胞中NADPH氧化酶4(NOX4)表达的影响,探讨NADPH氧化酶与脉络膜新生血管(choroidal neovascularization,CNV)形成的关系.方法:人视网膜色素上皮细胞(human retinal pigment epithelium,hRPE)体外常规培养和传代,取3～6代细胞用于实验.将培养的人RPE细胞分为常氧对照组和缺氧处理组,常氧对照组用DMEM完全培养液培养细胞,缺氧处理组培养液中含有终质量浓度为200μmoL/LCoCl2,分别观察缺氧后4、6、8、l2、24h后终止.采用细胞免疫荧光染色技术及Western-blot方法鉴定人RPE细胞中NOX4的表达以及缺氧不同时间对NOX4表达的影响.结果:常氧对照组及缺氧处理组人RPE细胞生长良好,形态呈梭形.缺氧处理组细胞的形态及排列方式与空白对照组相比无明显改变.免疫荧光染色显示,常氧对照组RPE细胞内有少量的NOX4表达,细胞质呈绿色荧光,细胞核呈蓝色荧光;缺氧处理后NOX4的表达量增加,一直持续到缺氧后期;Western-bolt显示缺氧条件下人RPE细胞中NOX4的表达明显增加,与缺氧时间呈正比,与常氧对照组相比,差异具有统计学意义(P＜0.05):结论:NADPH氧化酶在人RPE细胞中有表达,缺氧条件下NOX4的表达显著提高,且与时间呈正比.提示NADPH氧化酶可能在脉络膜新生血管形成中发挥重要调节作用.     

整合素连接激酶对人视网膜色素上皮细胞增殖和迁移的影响

目的:探讨RNA干扰抑制整合素连接激酶（integrin-linked kinase, ILK）对人视网膜色素上皮（human retinal pigment epithelium,hRPE）细胞增殖和迁移的影响。方法:体外培养hRPE细胞,设计并合成特异性人ILK的RNA干扰片段,阳离子脂质体转染入人视网膜色素上皮细胞,利用RT-PCR及Western blot半定量检测siRNA对ILK基因及蛋白表达的抑制作用,MTT方法检测转染前后siRNA对细胞增殖的抑制作用。损伤愈合实验和Transwell实验观察人视网膜色素上皮细胞迁移能力的改变。结果:培养的hRPE细胞存在ILK的基因转录表达,siRNA显著抑制hRPE细胞ILK的mRNA和蛋白表达（P<0.01）。MTT、损伤愈合实验和Transwell实验提示转染ILK-siRNA后人视网膜色素上皮细胞的增殖、迁移能力有明显下降,与正常对照组相比有统计学差异（P<0.05）。结论:特异性ILK-siRNA能有效抑制ILK在hRPE的mRNA和蛋白的表达并显著降低hRPE的增殖和迁移能力。     

培养人视网膜色素上皮细胞在PVR患者视网膜下液作用下c-fos和c-jun的表达

目的 观察培养的人视网膜色素上皮细胞(human retinal pigment epithelium，hRPE)在不同程度增生性玻璃体视网膜病变(proliferative vitreoretinopathy，PVR)患者视网膜下液(subretinal fluid，SRF)作用下细胞增生与c—fos和c—jun的表达。方法 不同程度PVR患者SRF作用于培养的hRPE细胞后利用细胞计数和MTT比色实验观察SRF对培养hRPE细胞的作用，同时采用SP法在不同时间点作兔抗人c—fos多抗和兔抗人c—jun多抗的免疫组织化学染色。结果 PVR患者SRF作用组与对照组相比可明显促进培养hRPE细胞的增殖，同时实验组hRPE细胞c—fos和c—jun均表达增高，视网膜下液作用lh后，Fos蛋白抗体染色阳性，细胞核呈明显棕黄色染色；3h表达达高峰，持续约12h；视网膜下液作用lh后，Jun蛋白抗体染色阳性，细胞核呈明显棕黄色染色；6h表达达高峰，持续约48h，对照组hRPE细胞(未经SRF作用仅加入等量PBS)Fos蛋白抗体和Jun蛋白抗体染色均为阴性。结论 PVR患者SRF中含有能诱导c—fos和c—jun等“立早基因”转录的细胞外生长或分化因子；c—fos和c—jun的表达是SRF促进RPE细胞的增殖过程中一个非常重要的早期分子事件。     

溶血磷脂酸对人类视网膜色素上皮细胞生物学功能的影响研究

目的:研究溶血磷脂酸(lysophosphatidic acid,LPA)对体外培养人类视网膜色素上皮(human retinal pigment epithelia-lium,hRPE)细胞增殖活力、分化特性及免疫活性等生物学功能的影响。方法:采用MTT法检测体外培养hRPE细胞增殖活力;角蛋白及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)免疫细胞化学染色方法分析体外培养hRPE细胞分化特性;流式细胞术检测hRPE细胞表面HLA-ABC、HLA-DR、ICAM-1的表达。结果:加药后第2d开始LPA组吸光度明显高于对照组(P<0.001);角蛋白18表达对照组较LPA组强(uc=-12.683,P<0.001),α-SMA表达对照组较LPA组弱(uc=10.402,P<0.001);hRPE细胞表面HLA-ABC、HLA-DR、ICAM-1的阳性细胞率和平均荧光强度组间差异无统计学意义(各指标均P>0.05)。结论:LPA明显促进hRPE的增殖活力,促进hRPE转分化,在PVR形成过程中起潜在的促进作用,但对hRPE免疫活性无影响。     

P21-GFP融合基因表达载体的构建与在视网膜色素上皮细胞的表达定位

目的构建绿色荧光蛋白GFP—p21融合基因表达载体,并观察其在人视网膜色素上皮细胞中的表达及定位,为研究细胞周期蛋白激酶抑制因子p21对视网膜色素上皮细胞细胞周期的影响提供实验基础。方法以含有p21的全长序列的质粒为模板PCR扩增p21 cDNA,应用基因重组技术与GFP基因融合,构建以绿色荧光蛋白GFP为报告基因的重组表达质粒pGFP—p21。利用脂质体转染体外培养的人视网膜色素上皮细胞,在活细胞状态下用荧光显微镜直接观察pGFP—p21融合蛋白在细胞中的分布和定位,用Western Blot方法分析其蛋白的表达。结果①酶切、DNA测序均证实插入片断的正确性。②荧光显微镜观察结果显示在空载体pEGFP—C1转染组中,细胞内绿色荧光呈弥散分布;重组质粒pGFP—p21转染组中,绿色荧光主要集中在细胞核内。③Western Blot证实了pGFP—P21融合基因的表达。结论成功的构建了pGFP—p21质粒。视网膜色素上皮细胞能高效表达pGFP—p21融合基因,且主要定位于细胞核内,与p21基因的表达方式相同。     

视网膜特异性表达人血管内皮生长因子 基因载体的构建

目的构建在视网膜组织特异性表达的人血管内皮生长因子（VEGF）₁₆₅基因。方法用聚合酶链反应(PCR)方法从BLAB/C鼠全基因组扩增能在视网膜组织特异性表达的rho启动子，经限制性内切酶纯化后克隆于质粒pcDNA^3.1+-VEGF₁₆₅中，建立重组质粒pcDNA^3.1+-rho-VEGF₁₆₅，通过限制性内切酶酶切分析及PCR鉴定筛选出正确重组质粒pcDNA^3.1+-rho-VEGF₁₆₅,由jetPEI介导转染人视网膜色素上皮细胞和人脐静脉内皮细胞，并通过免疫组织化学染色以及绘制细胞生长曲线检测在人视网膜色素上皮细胞和人脐静脉内皮细胞中VEGF蛋白的表达。结果在人视网膜色素上皮细胞中，重组质粒pcDNA^3.1+-rho-VEGF₁₆₅比质粒pcDNA^3.1+-rho-VEGF₁₆₅的VEGF蛋白表达强，在人脐静脉内皮细胞，两者的表达量无明显差别。结论pcDNA^3.1+-rho-VEGF₁₆₅载体的构建为进一步研究VEGF在视网膜新生血管形成中的致病机理提供基础材料，并为进一步建立视网膜特异性表达VEGF转基因鼠模型建立了基础。(中华眼底病杂志,2005,21:106-109)     

Circulating antibodies to human and bovine cornea in human keratoplasty

PURPOSE: To compare anti-human cornea and anti-bovine cornea serum reactivity in corneal transplantation and pathology. METHODS: We investigated the presence of IgG and IgM antibodies to bovine corneal extract (BCE) and human corneal extract (HCE) by ELISA in 48 healthy adults and in 30 corneal allograft recipients before and after grafting. Baseline levels of antibodies in healthy controls served to establish a positivity threshold. Anti-HLA antibodies were investigated in all patients. RESULTS: Preoperatively, five and one patient were positive respectively for IgG and IgM anti-BCE; one and ten patients were positive for IgG and IgM anti-HCE. The presence or absence of antibodies was not modified after grafting, and was not associated with rejection. Anti-HCE of the IgM class were more frequent in patients with keratoconus. Two patients had anti-HLA antibodies. CONCLUSIONS: Antibodies to non-HLA corneal antigens are detectable in corneal allograft recipients. Reactions against bovine and human substrates differ in frequency and class. This reactivity is not modified after grafting or rejection, but is increased in corneal disorders, including keratoconus.     

Ocular human bite

We report on a case of an ocular bite occurring during a rape attempt. The patient suffered a conjunctival laceration and a stab injury in the upper eyelid, the point of entry of a severe deep infection that caused limitations in extraocular motility. Antibiotic therapy resulted in a complete cure. Psychopathologic considerations about intentional eye injuries are discussed.     

Establishment of human corneal epithelial cells stably expressing human connexin43

Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium.     

Ultraviolet transmittance of human limbal epithelial cells cultured on human amniotic membranes

PURPOSE: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. METHODS: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 microW/cm2, and UV transmittance was measured with a UV light meter. RESULTS: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3-4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbal epithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. CONCLUSIONS: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3-4 layers are formed in order to prevent ocular damage by UV light after transplantation.     

In vivo confocal microscopy of human cornea covered with human amniotic membrane

Purpose  Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Methods  Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22–24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Results  Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. Conclusions  We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.