碱性成纤维细胞生长因子受体基因在牛眼晶体上皮细胞内的表达

目的探讨碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）对晶体上皮细胞促增殖作用的机理。方法从培养的第二代牛眼晶体上皮细胞提取ＲＮＡ，经逆转录反应合成ｃＤＮＡ。借助从人体胎盘纤维细胞ｂＦＧＦ受体序列合成的寡核苷酸引物，聚合酶链反应体外扩增ｃＤＮＡ。扩增的ｃＤＮＡ片段克隆后，Ｓａｎｇｅｒ双脱氧链终止法测定其序列。结果探测出牛眼晶体上皮细胞ｂＦＧＦ受体的ｍＲＮＡ，测序后发现其氨基酸序列片段中仅３个氨基酸与人体不同。结论晶体上皮细胞存在ｂＦＧＦ受体，当ｂＦＧＦ与其受体结合后，对促进晶体上皮细胞的增殖以及白内障术后后囊混浊具有重要作用。     

肿瘤坏死因子α和表皮生长因子对晶状体上皮细胞增殖的作用

目的研究肿瘤坏死因子α（ＴＮＦα）和表皮生长因子（ＥＧＦ）对人、牛晶状体上皮细胞增殖的影响。方法采用ＭＴＴ比色法检测ＴＮＦα和ＥＧＦ对晶状体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ，ＬＥＣ）增殖的作用。结果０．１Ｕ／ｍｌ的ＴＮＦα即可明显促进人、牛晶状体上皮细胞的增殖；ＥＧＦ为１、１０ｎｇ／ｍｌ时可明显促进牛晶状体上皮细胞增殖。结论细胞因子ＴＮＦα、ＥＧＦ通过促进ＬＥＣ的增殖参与后囊混浊的形成     

碱性成纤维细胞生长因子对人晶状体上皮细胞促增殖作用研究

研究了碱性成纤维细胞生长因子对人晶状体上皮细胞的促增殖作用。方法胎儿晶状体上皮细胞原代与传代培养。第２代培养细胞中添加ｂＦＧＦ，浓度１０＾－３－１０＾３ｎｇ／ｍｌ，用甲基噻唑基四唑测定法观察ｂＦＧＦ对ＨＬＥＣｓ的影响。结论ｂＦＧＦ在后囊混浊发生上起了生要作用。     

碱性成纤维细胞生长因子和胰岛素样生长因子-I及其协同作用对牛晶体上皮细胞增殖的影响

目的　研究碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）、胰岛素样生长因子Ｉ（ｉｎｓｕｌｉｎｌｉｋｅ ｇｒｏｗｔｈ ｆａｃｔｏｒＩ，ＩＧＦⅠ） 及二者的共同作用对体外牛晶体上皮细胞（ｂｏｖｉｎｅ ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｌ，ＢＬＥＣ）增殖的影响。方法　ＢＬＥＣ原代培养，取第４ 代细胞种入２４ 孔板，加入不同浓度的ｂＦＧＦ、ＩＧＦⅠ、ｂＦＧＦ＋ ２０ μｇ／ＬＩＧＦⅠ，２０ ｈ 后加入３ＨＴＤＲ，１６ ｈ 后作液闪计数。结果　一定浓度的ｂＦＧＦ（１～１００ μｇ／Ｌ） 、ＩＧＦⅠ（２０～１００ μｇ／Ｌ） 在体外有促进ＢＬＥＣ 增殖的作用（ Ｐ＜ ０－０１ ） ，２０ μｇ／Ｌ的ＩＧＦⅠ可增加ｂＦＧＦ的促ＢＬＥＣ增殖作用。结论　生长因子及它们之间的协同作用在促进晶体上皮细胞异常增殖的过程中起重要作用。     

表皮生长因子、碱性成纤维细胞生长因子对人视网膜色素上皮细胞DNA合成的影响

目的探索表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）、碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ，ｂＦＧＦ）对培养的人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞促进ＤＮＡ合成的最佳刺激浓度，并对两种因子的协同作用进行探讨。方法培养的人ＲＰＥ细胞第６代用于本实验。应用氚标胸腺嘧啶核苷（３Ｈ－ｔｈｙｍｉｄｉｎｅ，３Ｈ－ＴｄＲ）掺入试验及放射自显影检测ＥＧＦ、ｂＦＧＦ对ＲＰＥ细胞的促ＤＮＡ合成作用。结果ＥＧＦ、ｂＦＧＦ均可引起剂量依赖的促有丝分裂作用。在含２％血清的培养液中，ＥＧＦ、ｂＦＧＦ作用最佳浓度为１ｎｇ／ｍｌ，明显低于无血清培养液中ＥＧＦ、ｂＦＧＦ作用的最佳浓度（１０ｎｇ／ｍｌ）。联合应用１０ｎｇ／ｍｌＥＧＦ、１０ｎｇ／ｍｌｂＦＧＦ约提高ＲＰＥ细胞合成ＤＮＡ能力２．９６倍。结论ＥＧＦ、ｂＦＧＦ对培养的人ＲＰＥ细胞具有促进ＤＮＡ合成作用，且两者可产生协同效应。     

碱性成纤维细胞生长因子受体mRNA基因在人晶体上皮细胞内的检测研究

为研究碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）对晶体上皮细胞促增殖作用的机理,采用原位杂交,用合成的寡核苷酸探针来检测人晶体上皮细胞内的ｂＦＧＦ高亲和力受体—成纤维细胞生长因子受体１（ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒｒｅ－ｃｅｐｔｏｒ１,ＦＧＦＲ１）的ｍＲＮＡ基因,并用图像分析进行相对定量。结果：表明了人晶体上皮细胞表达ＦＧＦＲ１的ｍＲＮＡ基因。结论：人晶体上皮细胞存在ＦＧＦＲ１,当ｂＦＧＦ与其高亲和力受体结合后,对促进晶体上皮细胞的增殖以及白内障术后后囊混浊的形成具有重要作用。     

碱性成纤维细胞生长因子在人晶体上皮细胞的蛋白检测研究

研究成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）对人晶体上皮细胞（ｈｕｍａｎｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｓ,ＨＬＥＣｓ）的促增殖作用。方法：采用免疫组织化学检查（ＡＢＣ法）来检测人晶体上皮细胞上ｂＦＧＦ蛋白水平,并用图像分析进行相对定量。结果：定性及定量地证明了人晶体上皮细胞有ｂＦＧＦ蛋白存在。结论：阐明了人晶体上皮细胞本身存在的ｂＦＧＦ以蛋白的形式参与了术后后囊混浊的形成。     

IL-1和TNF-α对晶体上皮细胞增殖及胞浆内游离Ca^2＋浓度的影响

目的研究白细胞介素１（ｉｎｔｅｒｌｅｕｋｉｎ１,ＩＬ－１）、肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα,ＴＮＦ－α）对体外培养的小牛晶体上皮细胞（ｂｏｖｉｎｅｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ,ＢＬＥＣ）增殖和胞浆内游离Ｃａ２＋浓度的影响,探讨它们在后囊混浊形成中的作用及其影响细胞增殖的机理。方法采用ＭＴＴ比色法测定ＩＬ－１及ＴＮＦ－α对细胞增殖的影响,用荧光指示剂Ｆｕｒａ－２测定它们对胞浆内游离Ｃａ２＋的影响。结果ＩＬ－１１０２～１０５ｎｇ／ｍｌ、ＴＮＦ－α１０２～１０４Ｕ／ｍｌ可显著促进细胞增殖,并增加胞浆内游离Ｃａ２＋浓度。结论推测ＩＬ－１、ＴＮＦ－α参与了白内障术后的后囊混浊形成;它们影响细胞增殖的作用机理之一是通过胞浆内游离Ｃａ２＋。     

碱性成纤维细胞生长因子和胰岛素样生长因子—Ⅰ及其协同作用对？…

目的 研究碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）、胰岛素样生长因子－１（ｉｎｓｕｌｉｎ－ｌｉｋｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ－１,ＩＧＦ－１）及二者的共同作用对体外牛晶体上皮细胞（ｂｏｖｉｎｅ ｌｅｎｓｅｐｉｔｈｅｌｉａｌ ｃｅｌｌ,ＢＬＥＣ）增殖的影响。方法 ＢＬＥＣ原代培养,取第４例细胞种入２４孔板,加入不同浓度的ｂＦＧＦ、ＩＧＦ－１,     

碱性成纤维细胞生长因子对牛晶状体上皮细胞增殖的影响及其在牛晶状体上皮细胞中的蛋白表达

目的　研究碱性成纤维细胞生长因子（ｂＦＧＦ） 在体外对牛晶状体上皮细胞（ＢＬＥＣ） 增殖的影响及确定ＢＬＥＣ 是否表达ｂＦＧＦ 蛋白。 方法　ＢＬＥＣ 原代培养。用不同浓度的ｂＦＧＦ 及３Ｈ胸腺嘧啶（３ＨＴＤＲ） 处理ＢＬＥＣ３６ ｈ 后，液闪测定ｂＦＧＦ 对ＢＬＥＣ３ＨＴＤＲ 掺入率的影响。Ｗｅｓｔｅｒｎ 印迹（ Ｗｅｓｔｅｒｎ ｂｌｏｔ） 法确定ＢＬＥＣ 是否表达ｂＦＧＦ 蛋白。 结果　ｂＦＧＦ 在体外有促进ＢＬＥＣ 增殖作用，Ｗｅｓｔｅｒｎ ｂｌｏｔ 法表明ＢＬＥＣ 可表达低分子量（１８ ０００）ｂＦＧＦ 蛋白。 结论　ｂＦＧＦ 可能通过晶状体上皮细胞的自分泌，促进白内障术后残留晶状体上皮细胞的增殖，导致后发性白内障     

增殖细胞核抗原反义寡核苷酸对牛晶状体上皮细胞增殖的影响

目的　探讨增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)反义寡核苷酸对牛晶状体上皮细胞增殖的影响。方法　取体外培养的第 2代牛晶状体上皮细胞用于实验。A～F组分别加入PBS、30 μmol/LPCNA反义寡核苷酸、30 μmol/LPCNA正义寡核苷酸、10 μg/L碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)、10 μg/LbFGF及 30 μmol/L反义寡核苷酸、10 μg/LbFGF及30 μmol/L正义寡核苷酸 ;常规培养 2 4h后 ,使用流式细胞仪检测细胞周期的变化和PCNA蛋白的含量。提取细胞mRNA ,采用地高辛标记PCNA探针和Northen酶联吸附免疫杂交法 ,在酶标仪上测定吸光度 (A值 )以表达PCNAmRNA含量。结果　牛晶状体上皮细胞S期的细胞数量占细胞总数的百分比A组为 ( 15 7± 1 8) % ,B组为 ( 7 9± 0 4) % ,两组比较差异有非常显著意义 (P <0 0 1) ;PCNAmRNA含量A组A值为 0 2 0 6± 0 0 0 4,B组A值为 0 173± 0 0 14,两组比较差异有显著意义 (P<0 0 5 ) ;PCNA蛋白表达率A组为 ( 5 5 2 7± 2 6 4) % ,B组为 ( 12 32± 1 82 ) % ,两组比较差异有非常显著意义 (P <0 0 1)。牛晶状体上皮细胞S期的细胞数量占细胞总数的百分比D组为 ( 2 3 4± 2 8) % ,E组为( 19 9± 2 3) % ,两组比较差异有显著意     

Effect of heparin on proliferation of cultivated bovine lens epithelial cells]

The treatments proposed to date for the prevention of secondary cataract have shown limited efficacy or have not been satisfactory due to ocular toxicity. Since it has been demonstrated that heparin can inhibit the proliferative activity of smooth muscle cells and fibroblasts in vitro and in vivo, we examined the effect of heparin at concentrations ranging from 20 to 200 micrograms/ml on the proliferation of cultured bovine lens epithelial cells (BLEC) under various culture conditions: (1) serum-free medium (SFM); (2) SFM + aqueous humor 1:1; (3) SFM +1 and 10% fetal calf serum; (4) SFM +1% retinal extract; (5) SFM +50 micrograms/ml endothelial cell growth factor; (6) SFM +10 ng/ml epidermal growth factor; (7) SFM +10 ng/ml basic fibroblast growth factor. Heparin caused no cytotoxic effects in any of the experiments. With medium 2 and 3, heparin caused dose-dependent inhibition of cell proliferation at concentrations ranging from 10 to 50 micrograms/ml. Cells cultivated in medium 4-7 with the addition of 50 micrograms/ml heparin revealed increased proliferative activity when compared with the corresponding controls. The antiproliferative activity on BLEC in medium containing aqueous humor suggests that heparin is a valuable tool for the prevention of secondary cataract in vivo.     

Einfluss von Octreotid in Kombination mit Wachstumsfaktoren auf die Proliferation von RPE-Zellen in vitro

BACKGROUND: The long-acting somatostatin analogue octreotide is used as a therapeutic option for patients with diabetic retinopathy and age-related macular degeneration. Growth factors, such as EGF, bFGF, VEGF, and PDGF, have been implicated in the pathogenesis of these diseases. The aim of this study was to investigate the effect of octreotide on the growth-factor-induced proliferation of bovine retinal pigment epithelial (RPE) cells in vitro. METHODS: RPE cells were assayed for proliferation as measured by (3H)-thymidine uptake (ccpm). Bovine RPE cells at passage 3-8 were seeded in a 96-well plate and incubated for a 24-h period with a minimum medium followed by a 24-h incubation with the different growth factors at a concentration of 10 ng/ml with and without 5 x 10(-5) M octreotide. In a different assay the cells were incubated with octreotide at 5 x 10(-10)-5 x 10(-4) M. Afterwards the cells were pulsed with 5 microCi/ml (3H)-thymidine for 12 h, and the incorporated radioactivity was measured on glass fiber filters in a beta-counter (mean +/- SEM ccpm). The Wilcoxon Signed Rank test and the student's t-test were used for statistical analyses. RESULTS: There was a biphasic effect of octreotide on RPE cell proliferation. Exposure of RPE cells to PDGF (20,225 +/- 3304 ccpm) and bFGF (3441 +/- 539 ccpm) resulted in a significantly higher proliferation compared to control medium (1543 +/- 352 ccpm) (p < 0.05). No difference was found for EGF (2385 +/- 383 ccpm). For VEGF (776 +/- 83 ccpm) a significant reduction in RPE cell proliferation was found (P < 0.05). There was a significant reduction in proliferation of RPE cells with PDGF, bFGF, and EGF in combination with octreotide versus growth factors alone (octreotide in combination with PDGF 308 +/- 82 ccpm, with bFGF 229 +/- 88 ccpm, with EGF 2362 +/- 91 ccpm) (p < 0.005). VEGF in combination with octreotide resulted in a significant inhibition of RPE cell proliferation compared to VEGF alone (p < 0.0005). CONCLUSION: The data suggest that there is an inhibitory effect of octreotide on RPE cell proliferation of bovine RPE cells and on the increased proliferation of bovine RPE cells induced by PDGF and bFGF. An enhanced inhibitory effect is found for the combination of octreotide and VEGF.     

Effect of growth factors on bovine retinal pigment epithelial cell migration and proliferation

BACKGROUND: To examine the effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor beta 2 (TGF(beta2)) on bovine retinal pigment epithelial (RPE) cell migration and proliferation. MATERIALS AND METHODS: Cultured bovine RPE cells were treated with 10 ng/ml PDGF, bFGF, aFGF, IGF-1, EGF, or TGF(beta2). RPE cell migration studies were performed in multiwell plates confluently covered with RPE cells. After inhibition of proliferation and denudation of half of each well, cells were incubated with various growth factors. Migration was measured as the number of cells that had entered the denuded area after 20 h. RPE cell proliferation was determined by [(3)H]thymidine incorporation after growth factor stimulation for 24 h. RESULTS: RPE cell migration was significantly enhanced after incubation with PDGF (stimulation of 213% compared to the negative control, p = 0.002), bFGF (206%, p = 0.003), aFGF (175%, p = 0.003), IGF-1 (150%, p = 0.003), and EGF (144%, p = 0.003). RPE cell proliferation was stimulated by bFGF (322% compared to the negative control, p < 0.005), PDGF (119%, p < 0.005), aFGF (121%, p < 0.005), and EGF (94%, p < 0.005). IGF-1 showed no significant effect on RPE cell proliferation; TGF(beta2) displayed no effect on RPE cell migration nor on proliferation. CONCLUSIONS: The peptide growth factors PDGF, bFGF, aFGF, IGF-1, and EGF play an important role in initiating RPE cell migration. Basic FGF, PDGF, aFGF, and EGF stimulate RPE cell DNA synthesis.     

柔红霉素预防后囊膜混浊的研究

目的研究体外细胞培养中柔红霉素对各类晶体上皮细胞增殖的抑制作用及有效浓度,为后囊膜混浊的药物预防提供新线索。方法传代培养的牛、兔、人晶体上皮细胞经０．５、２．５、５．０、７．５、１０．０μｇ／ｍｌ柔红霉素３７℃孵育１０分钟后,观察细胞生长情况;用药后４８小时,采用Ｇｉｅｍｓａ染色－比色法检测细胞吸光值,并分别求得柔红霉素对三种晶体上皮细胞的半数抑制浓度（ＬＤ５０）。结果柔红霉素对体外培养牛、兔、人晶体上皮细胞的增殖均有显著抑制作用,呈浓度依赖性改变。对于人晶体上皮细胞,０．５μｇ／ｍｌ柔红霉素已有显著抑制效应,７．５μｇ／ｍｌ基本发挥最大作用。牛、兔、人晶体上皮细胞的ＬＤ５０值分别为０．４９、４．３０和４．０６μｇ／ｍｌ。结论柔红霉素低浓度短时间作用能有效抑制体外培养晶体上皮细胞的增殖,通过进一步在体研究,可能成为预防后囊膜混浊的理想药物。