层黏连蛋白对人翼状胬肉成纤维细胞生长的抑制作用

目的:探讨层黏连蛋白对体外培养的人翼状胬肉成纤维细胞增殖的影响.方法:体外培养的第3代翼状胬肉成纤维细胞,接种于层黏连蛋白包被培养皿(Ln组)和空白培养皿(对照组)中,倒置显微镜观察细胞生长状态,MTT法检测细胞增殖状态,作出生长曲线;免疫荧光法检测α-SMA在细胞内的表达;RT-PCR法检测TGF-β1和cytokeratin mRNA的表达水平.结果:Ln组成纤维细胞数量少于对照组;3～7 d,Ln组A570明显低于对照组(0.20 vs 0.28,0.22 vs 0.30,0.28 vs0.34,0.31 vs0.36,0.33 vs 0.36,P＜0.05);α-SMA在对照组细胞内表达良好,在Ln组的表达受到抑制;与对照组相比,Ln组TGF-β1 mRNA的表达下降,cytokeratin mRNA表达增加.结论:层黏连蛋白能抑制翼状胬肉中成纤维细胞的增殖.     

兔眼虹膜色素上皮细胞的培养与鉴定

目的:体外培养和观察兔眼虹膜色素上皮细胞(IPEC),为临床和实验研究IPE细胞提供基础。方法:采用酶消化加酶辅助机械分离法分离IPE细胞,观察培养细胞的生长情况,并采用免疫荧光法对其进行鉴定。结果:原代培养的IPE细胞多数呈圆形或六边形,细胞内充满黑色颗粒。传代培养的IPE细胞呈梭形或纤维细胞样生长,细胞中的色素颗粒也逐渐减少。免疫荧光法对培养细胞进行cytokeratin染色显示IPE细胞成阳性反应,阳性率达100%。结论:采用酶消化加酶辅助机械分离法可成功地分离培养出IPE细胞。     

兔眼虹膜色素上皮细胞体外生物学特性研究

目的观察体外培养兔眼虹膜色素上皮(irispigmentepithelium,IPE)细胞的生物学特性,为IPE移植治疗老年性黄斑变性等疾病打下基础。方法应用扫描电镜、透射电镜对所培养IPE细胞的超微结构进行观察,并通过细胞的贴壁率、伸展率、倍增时间、总分裂次数的测定以及生长曲线的绘制对其在体外生长的生物学特性进行研究。结果体外培养的IPE细胞呈多角形,细胞内有色素颗粒和丰富的细胞器,细胞表面有长短粗细不等的微绒毛,生长成为具有尖基底极性的细胞单层。IPE细胞伸展率为81.5%±3.7%;原代IPE细胞的贴壁率为32.18%±3.18%;传代培养的IPE细胞的贴壁率为86.20%±2.84%;细胞倍增时间为2～7d,平均为(3.46±2.10)d;细胞总分裂次数为6～10代。结论体外培养IPE细胞与体内生长IPE细胞有着相似的生物学特性,通过对IPE细胞体外生物学特性的研究,为进一步探索其功能和自体移植提供了必要的实验基础。     

层黏连蛋白和纤维连接蛋白对传代人晶状体上皮细胞生长和Cx43表达的影响

目的:观察层黏连蛋白(Ln)和纤维连接蛋白(Fn)对传代培养的人晶状体上皮细胞(hLECs)形态学和连接蛋白43(Cx43)表达的影响。方法:以层黏连蛋白(Ln)和纤维连接蛋白(Fn)处理培养皿表面,以倒置显微镜观察第3代培养的hLECs生长的形态,MTT法记录其生长曲线,免疫荧光法检测Cx43表达,比较两组表达阳性率的变化。结果:在层黏连蛋白处理组,传代细胞以典型的六角形上皮细胞形态为主,而纤维连接蛋白处理组则表现为以梭形的成纤维细胞为主。MTT实验显示,传代后4～7d纤维连接蛋白处理组细胞数多于层黏连蛋白处理组(P<0.05)。免疫荧光反应中,两组Cx43表达的阳性率有明显差别(23/28vs16/28,P<0.05)。结论:层黏连蛋白可以提供适宜hLECs保持上皮细胞状态的体外培养微环境,而纤维连接蛋白可以促进hLECs的增殖和分化。     

酶-机械分离-酶消化法分离培养兔眼虹膜色素上皮细胞

目的　建立兔眼虹膜色素上皮细胞体外培养体系。方法　采用酶 机械分离 酶消化法分离兔眼IPE细胞 ,台盼蓝染色法测定细胞活力 ,在体外对其进行原代及传代培养扩增 ,倒置相差显微镜观察IPE细胞的生长情况。取第 2次传代的IPE细胞行AE1/AE3及S 10 0抗体细胞免疫组化染色对其进行鉴定。结果　酶 机械分离 酶消化法分离得到IPE细胞的活力为 70 %～ 85 % .原代细胞 16～ 4 8h贴壁 ,72h后开始生长 ,细胞大多呈多角形或方形 ,少数为梭形 ,12～ 16d生长融合为单层细胞。第 1次传代的IPE细胞 6～ 12h贴壁 ,5～ 7d生长融合为细胞单层。IPE细胞在体外培养可传 5～ 6代。免疫组化染色显示 ,几乎所有细胞胞浆呈现棕黄色阳性反应 ,对照组胞浆不染色。结论　酶 机械分离 酶消化法分离培养兔眼IPE细胞易于操作 ,联合应用细胞角蛋白、S 10 0蛋白单克隆抗体的免疫组化技术可用来鉴定IPE细胞。     

兔虹膜和视网膜色素上皮细胞的体外培养比较

目的 观察比较有色素兔虹膜色素上皮(IPE)及视网膜色素上皮(RPE)细胞的体外生长状况。方法 采用酶消化法及酶辅助机械分离法分别分离IPE和RPE细胞。观察培养的两种细胞的生长状况，并对其进行免疫组化鉴定。结果 原代IPE及RPE细胞大多呈圆形或六边形，细胞内充满了黑色素颗粒。随着传代的增加，呈梭形或纤维细胞样生长的细胞增多，细胞中的色素颗粒也逐渐减少。细胞角蛋白免疫组化染色显示IPE及RPE细胞绝大多数呈棕黄色阳性反应。Desmin兔疫组化染色显示IPE细胞中阳性细胞约占5％。结论 分离培养的IPE和RPE细胞纯度很高。IPE细胞中绝大多数为后层IPE细胞。IPE和RPE细胞的形态及体外生长特点类似。     

层黏连蛋白促进人晶状体上皮细胞蛋白激酶B的活性

目的:探讨层黏连蛋白(Ln)对传代培养的人晶状体上皮细胞(hLECs)的蛋白激酶B(PKB)活性的影响,以及Ln激活PKB的信号分子途径。方法:选取第5代hLECs,分为对照组和处理组,在处理组的培养液中加入层黏连蛋白,终浓度为5mg/L。分别在处理后0,10,20,40,60min取出细胞,[γ-32P]-ATP掺入法测定胞膜和胞质PKB比活性。以PI3K的特异性抑制剂Wortmannin(终浓度100nmol/L)预处理1h,再加入层黏连蛋白,0,10,20,40,60min测定胞膜和胞质PKB比活性。结果:hLECs胞膜和胞质PKB比活性在层黏连蛋白作用40min时达到最高值,分别为空白对照的2.72和2.00倍。层黏连蛋白处理的各组胞膜和胞质PKB比活性显著高于对照组(P<0.05)。经Wortmannin预处理后,再加入层黏连蛋白,胞膜和胞质PKB比活性与未经预处理的各时间组相比均显著下降(P<0.05)。结论:层黏连蛋白通过PI3K途径激活人晶状体上皮细胞的PKB活性,比活性随时间变化而发生改变。     

体外分离培养猪虹膜色素上皮细胞的生物学评价

目的 :培养猪的虹膜色素上皮 (IPE)细胞并进行IPE的生物学评价 ,为IPE移植治疗视网膜色素变性 (RP)打下基础。方法 :采用酶辅助的显微分离法对 5 0只猪眼进行IPE的原代和传代培养 ,免疫组化方法鉴定细胞 ,并对其生物学性状进行评价。结果 :从IPE生长曲线上看 ,细胞于接种后 2天开始进入细胞对数生长期 ,经过 3～ 4天的对数期生长后进入生长缓慢的平台期。细胞培养成功率为 85 0 %。原代细胞的贴壁率为 31 8%± 3 7% ,细胞的活力为 82 5 %± 5 1% ,传代细胞的贴壁率为 86 4 %± 4 3% ,细胞的活力为 91 2 %± 3 7%。伸展率为 83 6 %± 5 3% ,分裂时间为 2～ 7天 ,平均 3 5± 0 96天 ,总分裂次数为 6～ 10代。免疫组织化学显示 ,培养获得的IPE细胞的细胞角蛋白 (AE1/AE3)和S 10 0抗原染色均为阳性。结论 :对IPE细胞的生物学评价 ,不仅证实了IPE细胞高效纯化培养的实现 ,而且系列完整的数据为IPE移植治疗RP的相关实验提供了必要的基础保证     

体外AGEs诱导培养的视网膜Müller细胞增殖活性和分泌VEGF的改变

目的:探讨体外Mller细胞在糖基化终末产物(AGEs)作用下增殖活性的变化。方法:采用本实验室培养及鉴定的新生大鼠Mller细胞,设置正常生长组和添加AGEs培养组,AGEs浓度在250～1000mg/L范围内逐步递增。MTT法测定AGEs对体外纯化培养Mller细胞增殖的影响和分泌VEGF的改变。结果:高浓度AGEs对Mller细胞有明显的促增殖作用,并且促进Mller细胞分泌VEGF,并在一定的浓度范围内(250～1000mg/L)细胞增殖活性呈剂量依赖关系。结论:体外高浓度AGEs可促进Mller细胞异常增殖和VEGF表达增加,与临床上观察到增殖性糖尿病视网膜病变时胶质细胞反应性增生情况一致,因此可以在体外用高浓度AGEs模拟体内糖尿病环境,观察胶质细胞形态和功能的改变。     

体外培养的兔虹膜色素上皮细胞吞噬功能及细胞连接的观察

目的;观察体外培养和虹膜色素上皮（iris pigment epithelium IPE）细胞是否具有吞噬功能及屏障作用。方法：将鸡红细胞悬液加入体外培养的IPE细胞内,计算其吞噬数量,电镜下观察IPE细胞间连接。结果：体外培养状态下,IPE细胞对鸡红细胞的吞噬率为5．4％,视网膜色素上皮（retinal pigment epithelium RPE)细胞为8．8％（P＜0．01）;IPE同RPE一样具有紧密连接结构。结论：兔IPE在体外培养状态下,具有吞噬功能及屏障作用。     

Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2. 1×108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro. Eye Science, 2003; 19: 49 - 53.     

活体染料羧基荧光素乙酰乙酸对兔眼虹膜色素上皮细胞体外染色的研究

目的 探讨活体染料羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）标记兔眼虹膜色素上皮（IPE）细胞的可行性。 方法 使用酶消化加机械分离的方法分离培养兔眼IPE细胞，将传3～4代的IPE细胞用2.5、5、10、20、40 μmol/L浓度的CFSE分别作用1、5、10 min进行染色，通过观察染色后的细胞荧光强度和细胞贴壁率筛选出最理想的标记条件。采用流式细胞术追踪检测标记的IPE细胞的荧光强度变化。用免疫荧光抗体标记法检测此染色剂有无渗漏以及是否对周围细胞着染。 结果 浓度为20 μmol/L的CFSE 37℃下水浴作用1 min为IPE细胞染色、观测、研究的最适条件。流式细胞仪和荧光显微镜观察结果显示，CFSE对IPE细胞染色可持续4周以上，未发生染料渗漏或转染其它细胞，且此浓度的CFSE不影响细胞的性状。 结论 活体染料CFSE是一种染色率高，追踪时间长，应用简便安全可靠的标记兔IPE细胞的新方法。 (中华眼底病杂志, 2006, 22: 261-264)     

虹膜周边切除手术标本的色素上皮细胞体外培养与超微结构研究

目的：体外培养虹膜周边切除标本中的色素上皮细胞(iris pigment epithelium，IPE)，并进行生物学检测。方法：用酶-显微解剖-酶分离法培养IPE细胞并传代，免疫组化方法鉴定，透射电镜观察超微结构。结果：酶-显微解剖-酶分离法在小标本可成功获得较高纯度和密度的IPE细胞。其形态与免疫标志物表达与视网膜色素上皮(retinal pigment epithelium，RPE)细胞相似。结论：培养的IPE细胞为自体移植提供了较可靠的细胞来源。     

Isolation and cultivation of human iris pigment epithelium.

There have been very few attempts to isolate and culture human iris pigment epithelium (IPE). Earlier efforts that used whole iris explant methods did not achieve pure cultures of IPE. We have developed methods for separating the IPE from the iris stroma of post-mortem eyes that avoid contamination by other cell types. Three different isolation methods were studied: direct dissection, enzyme digestion, and enzyme-assisted microdissection. The latter method yielded the best results. After treatment with enzyme solution, the IPE was easily separated from the stroma under the stereomicroscope and subsequently cultured with supplemented F12 medium. With this method, approximately 2.3 x 10(5) cells were isolated from each iris with an average viability of 90.2%. IPE cells isolated from 19 of 24 eyes grew to confluence in primary culture. The IPE could be maintained in pure culture for many generations over several months with up to 20 population doublings. Cultured IPE demonstrated cytokeratin and S-100 protein by immunocytochemistry. Some of these cells also displayed desmin, indicating origin from the anterior IPE. Cultured IPE cells retained most of the characteristics of IPE in vivo, such as apical/basal polarization, microvilli, and many cell junctions. Gradual dilution of pigment occurred in the dividing IPE cells, suggesting an inability to produce melanin in vitro. A subpopulation of the IPE cells contained myofilaments by electron microscopy, also indicating a anterior IPE origin. This method provides a source for large numbers of human IPE cells and could be useful in studies of the biology of IPE and the role of IPE in pathogenesis of several eye diseases, most notably exfoliation syndrome and its associated glaucomas.     

杆状病毒介导LacZ基因转染体外培养的人虹膜色素上皮细胞

目的：研究重组杆状病毒(baculovinus)载体介导β—半乳糖苷酶基因(LacZ)对体外培养虹膜色素上皮(IPE)细胞的转染和表达情况。方法：将质粒pCMV—β中的CMV—LacZ表达盒插入杆状病毒表达栽体pFast BacI的Eco RI／Hind Ⅲ位点，构建重组质粒pBac—β，并利用Bac—to—Bac表达系统在Sf9细胞中产生出重组杆状病毒BV—β。将MOI为200的BV—β感染经酶消化加显微法分离培养的IPE细胞，24h后进行X-gal染色。在显微镜下观察IPE中LacZ表达阳性的情况，记录阳性细胞所占的百分比。结果：限制性酶切分析及测序结果表明，我们已成功构建表达质粒pBac—β及重组杆状病毒BV—β。X-gal染色证实杆状病毒介导LacZ在细胞中的表达呈蓝色，弥漫于整个胞浆。感染后24h时表达阳性率达85％。结论：重组杆状病毒可有效介导报告基因在体外培养的IPE细胞中表达，为利用杆状病毒构建可供移植的基因工程化虹膜色素上皮细胞提供了实验依据。     

腺相关病毒介导绿色荧光蛋白基因对体外培养IPE细胞的转染及毒性

目的 研究腺相关病毒(recombinant adeno-associated virus,rAAV)载体介导的绿色荧光蛋白基因(green fluorescent protein，GFP)对体外培养虹膜色素上皮细胞(iris pigment epithelial，IPE)的转染和病毒载体对IPE细胞的毒性，为rAAV携带目的基因治疗视网膜色素变性提供理论依据。方法酶辅助的显微分离方法体外培养IPE细胞并鉴定。按转染倍数(multiple of infection,MOI)为10^3,10^4,10^5,10^6转染已培养3d的传二代IPE细胞，流式细胞仪检测rAAV—GFP对IPE的转染效率。MTT法检测rAAV—GFP对IPE细胞的毒性。结果 rAAV-GFP对IPE的转染效率，MOI=10^3时为26．7％、MOI=10^4时为53．6％、MOI=10^5时为60．2％、MOI=10^6时是63．7％。AAV—GFP转染IPE细胞后，细胞生长正常。MTT法显示各转染组与未转染组的OD值无统计学差异。结论 rAAV—GFP对体外培养的IPE细胞转染效率可达60％以上，而且腺相关病毒对细胞生长无明显抑制，腺相关病毒作为视网膜色素变性基因治疗的载体是安全可行的。     

Effect of interleukin-1 blockers, CK112, and CK116 on rat experimental choroidal neovascularization in vivo and endothelial cell cultures in vitro.

PURPOSE: The aim of this study was to investigate the effect of interleukin-1 blockers, CK112 and CK116, on laser-induced experimental choroidal neovascularization (CNV) in rat models in vivo and endothelial cell proliferation in vitro. METHODS: Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch's membrane. CK112, CK116, and prednisolone were given once-daily through intraperitoneal (i.p.) injection after laser treatment for 4 weeks. The development of CNV was determined by fluorescein angiography performed on weeks 2 and 4. Human umbilical vein endothelial cells (HUVEC) were tested with proliferation assay with CK112, CK116, and prednisolone at different concentrations. RESULTS: The intensity of fluorescein leakage from the photocoagulated lesions decreased significantly, compared to the control group (treated with dimethyl sulfoxide [DMSO] only), following CK112, CK116, and prednisolone treatment. Four (4) weeks after administration, CK112, at 10 mg/kg and 30 mg/kg, inhibited CNV development to 75% and 77% of the control group, respectively (P < 0.01). Both CK116, 10 mg/kg, and prednisolone, 5 mg/kg, inhibited the CNV development to 85% of the control group (P < 0.05). All three compounds interfered with the endothelial cell proliferation significantly. The reduction of the endothelial cells was 50.5% (P < 0.01), 28.5% (P < 0.05), and 23.1% (P < 0.05), respectively, in 500 microg/mL, 300 microg/mL, and 100 microg/mL of the CK112-treated group. CK116 inhibited the cell proliferation significantly to 77.2% of the control group at 500 microg/mL (P < 0.05). CONCLUSIONS: CK112 and CK116 inhibited the development of CNV in the rat model and interfered with vascular endothelial cell proliferation in vitro. Our results suggest that CK112 and CK116 may be good candidates to inhibit ocular neovascularization related to age-related macular degeneration (ARMD).     

In vitro culture of iris cells

BACKGROUND: The biological behaviour of iris cells in vitro was not yet completely investigated. For a more detailed study of the scope of cultivation of iris cells in vitro we isolated human iris pigment epithelium (IPE) cells and iris fibroblasts. MATERIALS AND METHODS: For the purpose of this study iris cells were isolated from 19 donor eyes. A method was established for isolation and cultivation of IPE cells by means of fibronectin coating and the use of a special cell culture medium. Additionally, a method was developed for the selection of fibroblasts from iris stroma (IS) in vitro by means of fibronectin coating, passaging and proliferation in cell culture medium. RESULTS: The IPE and IS cells could be cultivated successfully. The IPE cells started to divide after a mean interval of 5.4 +/- 0.7 days in culture. The mitosis of IS cells was observed after 3.3 +/- 0.87 days in culture. Confluency of IPE cells was reached after 14.7 +/- 4.92 days and by IS cells after 8.1 +/- 1.45 days. Immunocytochemical staining using two antibodies for cytoceratin and one for human fibroblast showed that the IPE cell culture was pure and that the IS culture consisted of fibroblasts. Furthermore, electron microscopy of IPE and IS cultures confirmed the results of the immunocytochemical staining. CONCLUSIONS: The use of human IS and IPE cells in vitro has established a novel model for the research on iris cells. The model might possibly be applied in the research of metabolic structures and diseases of the iris.     

人胚胎和兔结膜成纤维细胞对抗肿瘤药物敏感性的比较

比较了体外培养条件下人胚胎和兔结膜成纤维细胞对5种抗肿瘤药的敏感性。结果表明长春新碱和阿霉素在0.001~10mg/L、5-FU在1~1000mg/L、顺铂在0.01~10mg/L的浓度范围内时，人胚胎结膜成纤维细胞对这几种药物的敏感性显著低于兔结膜成纤维细胞（P＜0.01）；两种细胞对VP-16的敏感性差别不明显（P＞0.05）.提示在进行眼内增殖性疾病防治药物的体外筛选时，用人结膜成纤维细胞较好；将动物实验结果用于临床时，要考虑种属之间的药物敏感性差异。 （中华眼底病杂志，1994，10：223-225）