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     

人工晶体植入术后房水肿瘤坏死因子和白细胞介素1含量的研究

目的从兔眼人工晶体植入术后房水肿瘤坏死因子（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ，ＴＮＦ）和白细胞介素１（ｉｎｔｅｒｌｅｕｋｉｎ１，ＩＬ－１）的活性及动态变化，探讨它们对术后眼内炎症反应的影响。方法青紫蓝兔２７只，分为晶体囊外摘除及后房型人工晶体囊袋内植入术组；晶体囊外摘除术组；正常对照组。于术后１、３、７和１４天抽取房水，采用ＥＬＩＳＡ双层夹心法检测ＴＮＦ，采用ＭＴＴ比色法检测ＩＬ－１。结果房水ＴＮＦ含量和ＩＬ－１活性在人工晶体植入术组术后第１、３、７及１４天高于正常对照组，ＴＮＦ含量在术后３、７、１４天，ＩＬ－１活性在术后１、３天高于晶体囊外摘除术组（Ｐ＜０．０５）。人工晶体植入术后第７～１４天房水ＴＮＦ含量最高，术后第３～１４天ＩＬ－１活性最高。结论人工晶体植入术后房水ＴＮＦ和ＩＬ－１在术后早期眼内炎症反应中作为炎症介质，起着重要作用。     

肿瘤坏死因子对晶状体上皮细胞原癌基因表达的作用

目的研究肿瘤坏死因子α （ＴＮ－α）对体外培养的牛眼晶状体上皮细胞中原癌基因 ｆｏｓ，ｊｕｎ， ｍｙｂ， ｍｙｃ， ｒａｓ等蛋白产物表达的作用。方法细胞经 １０ Ｕ／ｍｌ ＴＮＦ－α作用不同时间后，采用卵白素－生物素－过氧化物酶复合物（ＡＢＣ）免疫酶标技术测定蛋白表达。结果经ＴＮＦ－α作用后，几种原癌基因表达的阳性细胞百分率均明显增高（Ｐ＜０．０１）。 ｆｏｓ，ｊｕｎ，ｍｙｂ，ｒａｓ在刺激１ｈ后即达到顶峰，ｍｙｃ在刺激２ｈ后达到顶峰，随后很快下降，恢复至正常水平。同时，细胞的阳性反应强度增加，细胞核内变化更为明显。结论ＴＮＦ－α通过激活细胞内原癌基因的表达而促进晶状体上皮细胞的增殖。     

兔眼内窥镜下睫状体光凝联合白内障超声乳化手术对房水成分的影响

目的　观察眼内窥镜下睫状体光凝术（ｅｎｄｏｓｃｏｐｉｃｃｙｃｌｏｐｈｏｔｏｃｏａｇｕｌａｔｉｏｎ,ＥＣＰ）联合白内障超声乳化及人工晶状体植入术（ｐｈａｃｏｅｍｕｌｓｉｆｉｃａｔｉｏｎａｎｄｉｎｔｒａｏｃｕｌａｒｌｅｎｓ,Ｐｈａｃｏ＋ＩＯＬ）后不同范围睫状体光凝对前房蛋白质浓度、ＴＮＦ-α含量、ＩＬ-１β含量改变的影响,并分析对前房炎症反应的影响。方法　将２４只（４８眼）成功建立慢性青光眼模型的灰兔随机分成Ａ组、Ｂ组、Ｃ组、Ｄ组,其中Ａ组、Ｂ组、Ｃ组分别给予１８０°、２７０°、３６０°三种不同范围睫状体光凝的ＥＣＰ＋Ｐｈａｃｏ＋ＩＯＬ手术,Ｄ组给予小梁切除（ｔｒａｂｅｃｕｌｅｃｔｏｍｙ,ＴＲＡＢ）＋Ｐｈａｃｏ＋ＩＯＬ手术,分别对术前、术后房水蛋白质浓度、ＴＮＦ-α、ＩＬ-１β含量检测;观察术后炎症反应;术中、术后并发症的发生情况。结果　Ａ组、Ｂ组、Ｃ组、Ｄ组术后１ｄ、７ｄ、１４ｄ房水中蛋白质浓度、ＴＮＦ-α含量、ＩＬ-１β含量升高,３０ｄ基本恢复至术前水平;术前及术后１ｄ、７ｄ、１４ｄ、３０ｄ房水中蛋白质浓度、ＴＮＦ-α含量、ＩＬ-１β含量:不同组间含量随时间变化趋势相同,差异均无统计学意义（Ｐ＝０．１５３、０５９３、０．２０３）;相同时间点不同组间含量差异均无统计学意义（均为Ｐ＞００５）。４组术后蛋白质浓度与前房炎症反应均呈正相关（均为Ｐ＜０．０５）。结论　ＥＣＰ＋Ｐｈａｃｏ＋ＩＯＬ与ＴＲＡＢ＋Ｐｈａｃｏ＋ＩＯＬ两种不同联合手术方式术后早期均引起蛋白质浓度、ＴＮＦ-α和ＩＬ-１β含量升高,３０ｄ基本恢复正常;而在同一时间点不同的手术方式对蛋白质浓度、ＴＮＦ-α和ＩＬ-１β含量影响不大。     

巨噬细胞诱发的实验性增殖性玻璃体视网膜病变中玻璃体内的肿瘤坏死因子-α

目的：检测增殖性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ，ＰＶＲ）发生过程中玻璃体内肿瘤坏死因子－α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα，ＴＮＦ－α）的含量变化。方法：用同种巨噬细胞诱发兔眼ＰＶＲ，在第７，１４，２１，２８天抽取玻璃体液，每组４只眼，用双抗体夹心法酶联免疫检测（ｅｎｚｙｍｅｌｉｎｋｅｄｉｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙ，ＥＬＩＳＡ）试剂盒检测其ＴＮＦ－α含量。结果：ＴＮＦ－α的含量１～２１天逐渐上升，２１天时达最高，为４３４ｐｇ／ｍｌ，２８天时下降至１２２ｐｇ／ｍｌ。结论：ＴＮＦ－α在此ＰＶＲ模型中玻璃体内的含量变化与ＰＶＲ形成的自然病程相吻合，提示可能在ＰＶＲ启动和调控中有重要作用。     

8—氯腺苷对生长因子诱导的视网膜色素上皮细胞增殖抑制作用的研究

目的探讨８氯腺苷（８ｃｈｌｏｒｏａｄｅｎｏｓｉｎｅ,８ＣＬＡ）对生长因子诱导的视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）细胞增殖的抑制作用。方法通过ＲＰＥ细胞培养,采用３ＨＴｄＲ掺入测定８ＣＬＡ对肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ,ＴＮＦα）、白细胞介素１β（ｉｎｔｅｒｌｅｕｋｉｎ,ＩＬ１β）和碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）诱导的ＲＰＥ细胞ＤＮＡ合成的抑制作用。结果三种因子单独使用均可使ＲＰＥ细胞３ＨＴｄＲ掺入每分钟计数（ｃｏｕｎｔｐｅｒｍｉｎｕｔｅ,ＣＰＭ）值明显增高（Ｐ＜０．０５）,８ＣＬＡ在其终浓度为２～１６μｍｏｌ／Ｌ时可使三种因子刺激的ＲＰＥ细胞３ＨＴｄＲＣＰＭ值明显降低（Ｐ＜０．０５）。在ＴＮＦα、ＩＬ１β、ｂＦＧＦ和８ＣＬＡ组,分别于１６、８、２μｍｏｌ／Ｌ时,ＣＰＭ值与基础值相近（Ｐ＞０．０５）。结论８ＣＬＡ可抑制生长因子诱导的ＲＰＥ细胞增殖,为抗增殖药物提供了新的途径     

肿瘤坏死因子-α对RF/6A细胞自噬促进细胞增殖、迁移和管腔形成的影响

目的　本研究观察肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ-ａｌｐｈａ,ＴＮＦ-α）对体外培养的恒河猴脉络膜／视网膜内皮细胞（ＲＦ／６Ａ）表达自噬蛋白Ｂｅｃｌｉｎ-１及细胞增殖、迁移和管腔形成的影响,探讨ＴＮＦ-α参与新生血管生成的可能机制。方法　将生长良好的ＲＦ／６Ａ细胞随机分为空白对照组、ＴＮＦ-α组和ＴＮＦ-α＋３-ＭＡ组。培养２４ｈ、４８ｈ后采用Ｗｅｓｔｅｒｎｂｌｏｔ检测细胞Ｂｅｃ-ｌｉｎ-１蛋白的表达,ＭＴＴ法检测细胞增殖,细胞划痕法检测细胞迁移,Ｍａｔｒｉｇｅｌ法检测管腔形成。结果　培养２４ｈ和４８ｈ,各组Ｂｅｃｌｉｎ-１／β-ａｃｔｉｎ比值分别是ＴＮＦ-α组（２４ｈ）０．６７３±０．０１７、ＴＮＦ-α＋３-ＭＡ组（２４ｈ）０．４９１±０．０１７、ＴＮＦ-α组（４８ｈ）０．７９２±０．００６、ＴＮＦ-α＋３-ＭＡ组（４８ｈ）０．５０４±０．００７、空白对照组０．２６８±０．０１７。ＴＮＦ-α组ＲＦ／６Ａ细胞Ｂｅｃｌｉｎ-１的表达量均明显高于空白对照组（Ｐ＜０．０５）,ＴＮＦ-α＋３-ＭＡ组Ｂｅｃｌｉｎ-１的表达量较ＴＮＦ-α组明显减少（Ｐ＜０．０５）。各组细胞的相对增殖率分别是ＴＮＦ-α组（２４ｈ）１．４１０±０．０１０、ＴＮＦ-α＋３-ＭＡ组（２４ｈ）１．２９０±０．００４、ＴＮＦ-α组（４８ｈ）１．３２０±０．０１１、ＴＮＦ-α＋３-ＭＡ组（４８ｈ）０．１８０±０．０１５、对照组（２４ｈ）１．０００±０．０２０、对照组（４８ｈ）１．０００±０．０１１;ＴＮＦ-α组细胞的相对增殖率均较空白对照组升高（Ｐ＜０．０５）,３-ＭＡ预处理后,ＴＮＦ-α促进ＲＦ／６Ａ细胞增殖的能力在两个时间点均受到抑制（均为Ｐ＜０．０５）。各组细胞的相对迁移距离分别是ＴＮＦ-α组（２４ｈ）（３４５±２８）μｍ、ＴＮＦ-α＋３-ＭＡ组（２４ｈ）（２５９±７７）μｍ、ＴＮＦ-α组（４８ｈ）（７６２±５５）μｍ、ＴＮＦ-α＋３-ＭＡ组（４８ｈ）（６５９±４８）μｍ、空白对照组（２４ｈ）（１９５±６３）μｍ、空白对照组（４８ｈ）（４１２±９４）μｍ;ＴＮＦ-α处理组ＲＦ／６Ａ细胞２４ｈ的迁移明显强于对照组（Ｐ＜０．０５）,加入３-ＭＡ预处理后,这种增强作用有所下降,但仍然明显高于对照组（Ｐ＜０．０５）;培养４８ｈ,更多细胞迁移入划痕区域,较２４ｈ明显增加;不同组之间的差异与２４ｈ时的结果类似,差异有统计学意义（Ｐ＜０．０５）;培养２４ｈ各组细胞管腔形成个数:ＴＮＦ-α组（１１．８０±０．８１）个、ＴＮＦ-α＋３-ＭＡ组（７．５０±０．７２）个、空白对照组（４．３０±１．１２）个,ＴＮＦ-α组及ＴＮＦ-α＋３-ＭＡ组管腔形成个数均高于对照组（均为Ｐ＜０．０５）,ＴＮＦ-α＋３-ＭＡ组较ＴＮＦ-α组明显减少（Ｐ＜０．０５）。结论　ＴＮＦ-α能够促进ＲＦ／６Ａ细胞的增殖、迁移和管腔样结构的形成,且ＴＮＦ-α促进内皮细胞自噬在血管新生中发挥重要的调控作用。     

视网膜前膜内相关炎性细胞因子的免疫组化研究

目的 研究增生性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅ ｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ,ＰＶＲ）的视网膜前膜（ｅｐｉｒｅｔｉｎａｌ ｍｅｍｂｒａｎｅｓ,ＥＲＭ）中炎性细胞因子白细胞介素－１β（ｉｎｔｅｒｌｅｕｋｉｎ－１β,ＩＬ－１β）、白细胞介素－６（ｉｎｔｅｒｌｅｕｋｉｎ－６,ＩＬ－６）、白细胞介素－８（ｉｎｔｅｒｌｅｕｋｉｎ－８,ＩＬ－８）和肿瘤坏死因子－α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ－α,ＴＮＦ－α）的表达及其作用。方法 用免疫组织化学方法对１９例复杂性视网膜脱离行玻璃体切割术时剥离的视网膜前膜进行观察。结果 ＩＬ－１β、ＩＬ－６、ＩＬ－８和ＴＮＦ－α分别在９、１２、１１和１５例视网膜前膜中表达,尤其以细胞外基质中明显,４种细胞因子在４例膜中同时表达,仅有１例膜中人表达ＩＬ－６。     

多次玻璃体取样对实验性PVR中炎性细胞因子的影响

为观察多次玻璃体取样造成的手术干扰对炎性ＰＶＲ模型早期炎源性细胞因子的含量变化的影响，及其与ＰＶＲ形成时相的关系，采集由巨噬细胞诱发的兔眼ＰＶＲ模型的玻璃体液和静脉血，用双抗体夹心法ＥＬＩＳＡ试剂盒，检测样本中肿瘤坏死因子－α（ＴＮＦ－α）、白细胞介素－８和６（ＩＬ－８和ＩＬ－６）的含量，并进行多元回归拟合曲线分析。结果：玻璃体内ＴＮＦ－α含量在巨噬细胞注入后７天上升，２１天达峰值５８０ｐｇ／ｍｌ，至７０天仍维持在２２２ｐｇ／ｍｌ高水平。ＩＬ－８７天起迅速上升，至７０天呈线性上升状态（４９４ｐｇ／ｍｌ）。ＩＬ－６在７～２８天呈一高水平状态８４～１８８ｐｇ／ｍｌ，２１天达高峰３７４ｐｇ／ｍｌ。结论：ＴＮＦ－α、ＩＬ－８和ＩＬ－６在本模型的炎症期和细胞增生期均呈明显的高水平，提示它们对眼内细胞增生和修复具有重要的启动和调控作用。也证实眼内局部较长时间、较高含量细胞因子可促进ＰＶＲ形成的观点，多次手术干扰或外源性刺激致血—眼屏障破坏在ＰＶＲ形成中的重要作用     

趋化素、肿瘤坏死因子-α与2型糖尿病视网膜病变的相关性

目的　探讨２型糖尿病视网膜病变（ｔｙｐｅ２ｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,Ｔ２ＤＲ）患者血清中趋化素（ｃｈｅｍｅｒｉｎ）、肿瘤坏死因子-α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ-α,ＴＮＦ-α）水平及其临床意义。方法　将１６０例研究对象分为增殖性糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａ-ｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＰＤＲ）组４０例,非增殖性糖尿病视网膜病变（ｎｏｎ-ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ,ＮＰＤＲ）组４０例,单纯糖尿病（ｄｉａｂｅｔｅｓｍｅｌｌｉｔｕｓ,ＤＭ）组患者４０例以及健康对照（ｎｏｒｍａｌｃｏｎｔｒｏｌｓ,ＮＣ）组４０例。观察４组研究对象的体检指标,并检测空腹胰岛素、血糖、血脂、糖化血红蛋白及血清中ｃｈｅｍｅｒｉｎ、ＴＮＦ-α等含量。计算稳态模型评估的胰岛素抵抗指数（ｉｎｓｕｌｉｎｒｅｓｉｓｔａｎｃｅｉｎ-ｄｅｘ,ＨＯＭＡ-ＩＲ）。结果　ＤＭ组［ｃｈｅｍｅｒｉｎ为（３．８３±０．４６）ｍｇ?Ｌ－１、ＴＮＦ-α为（３７．６９±５．０７）ｎｇ?Ｌ－１］、ＮＰＤＲ组［ｃｈｅｍｅｒｉｎ为（４．６８±０．７４）ｍｇ?Ｌ－１、ＴＮＦ-α为（４０．６９±５．９０）ｎｇ?Ｌ－１］、ＰＤＲ组［ｃｈｅｍｅｒｉｎ为（５．８６±１．２９）ｍｇ?Ｌ－１、ＴＮＦ-α为（４４．１７±６．６３）ｎｇ?Ｌ－１］血清中ｃｈｅｍｅｒｉｎ、ＴＮＦ-α水平均较ＮＣ组［ｃｈｅｍｅｒｉｎ为（２．０１±０．５４）ｍｇ?Ｌ－１、ＴＮＦ-α为（２２．６０±９．７８）ｎｇ?Ｌ－１］升高,且随着ＤＲ病情的进展逐渐升高,差异均有统计学意义（均为Ｐ＜０．０５）。相关分析显示血清中ｃｈｅｍｅｒｉｎ水平与收缩压、空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白胆固醇、糖化血红蛋白、胰岛素抵抗指数均呈正相关（ｒ＝０．３３１、０．３６１、０．２５１、０．３４８、０．３０６、０．５２３、０．６４４,均为Ｐ＜０．０５）;血清中ＴＮＦ-α水平与收缩压、舒张压、空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白胆固醇、糖化血红蛋白、胰岛素抵抗指数均呈正相关（ｒ＝０．２９９、０．１５９、０．６０５、０．２６２、０．４０７、０．２８２、０．６１９、０．８０９,均为Ｐ＜０．０５）;血清中ｃｈｅｍｅｒｉｎ与ＴＮＦ-α水平呈正相关（ｒ＝０．７３８,Ｐ＜０．０５）。结论　血清中ｃｈｅｍｅｒｉｎ和ＴＮＦ-α水平的升高是Ｔ２ＤＲ的危险因子,可能共同参与了Ｔ２ＤＲ的发生发展。     

晶体上皮细胞在体外的分化规律及血清和眼组织对其增殖的影响

目的从细胞培养水平探讨后囊混浊形成机理和术后血－房水屏障破坏、晶体皮质残留等对其的影响。方法用考马斯亮蓝（ＣｏｏｍａｓｓｉｅＢＢ）染色、倒置显微镜和电镜观察培养的牛晶体上皮细胞（ｂｏｖｉｎｅｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｓ，ＢＬＥＣ）的增殖和分化规律，用Ｇｉｅｍｓａ染色比色法观察胎牛血清、房水、晶体皮质等对ＢＬＥＣ增殖的影响。结果在体外培养条件下ＢＬＥＣ可在第１～７代内分化为晶体纤维细胞，表现为细胞体积增大，形状渐趋长条形和梭形，细胞骨架逐渐增多；胎牛血清呈浓度依赖性促进ＢＬＥＣ增殖（Ｐ＜０．０５）；高浓度房水（占培养液１／３）抑制ＢＬＥＣ增殖（Ｐ＜０．０１），晶体皮质、晶体核、玻璃体均促进ＢＬＥＣ增殖（Ｐ＜０．０１）。结论晶体上皮细胞的分化在后囊混浊形成中起重要作用；术后血－房水屏障破坏、晶体皮质残留和玻璃体脱出可通过刺激晶体上皮细胞增殖促进后囊混浊形成。     

地塞米松和柔红霉素抑制后囊混浊的体外模拟研究

为评价地塞米松和柔红霉素防治后囊混浊的价值，在体外模拟其在体内的可行性用药方式观察其对牛晶体上皮细胞增殖的抑制作用，结果地塞米松模拟在术后用药７２小时，在房水有效浓度内对晶体上皮细胞无明显抑制作用；提示其防治后囊混浊的作用主要是通过加速血－房水屏障的恢复，减少和控制术后炎症反应而间接实现。柔红霉素模拟加入皮质冲洗液中作用１０分钟，在眼部允许剂量内可呈浓度依赖性抑制晶体上皮细胞增殖，认为按这种方式用药可达到抑制后囊混浊作用。     

碱性成纤维细胞生长因子诱导牛晶状体上皮细胞增殖细胞核抗原的表达及对晶状体上皮细胞增殖作用的研究

目的 观察碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)诱导牛晶状体上皮细胞（bovine lens epithelial cell,BLEC)增殖细胞核抗原（proliferating cell nuclear antigen,PCNA)表达及对BLEC增殖的作用。方法 取体外培养的第2代BLEC,加入不同浓度的bFGF(0.01-100.00μg/L）共同培养24－96h后,用^3H-TdR掺入法测定BLEC DNA合成率,用流式细胞仪检测细胞周期和PCNA的表达。结果 不同浓度的bFGF有促进BLEC增殖作用,并呈剂量时间依赖性,当浓度为10.00μg/L的bFGF作用BLEC24－48h后,晶状体上皮细胞的^3H-TdR掺入值分别为11772．5和10988．2,较对照组的6550．5增加约一倍,两组比较差异有显著意义（P＜0．05）。bFGF作用BLEC可使PCNA表达率上升（77．40％）,并使BLEC周期发生变化,S期细胞和G2／M期细胞比例分别提高至23．41％和20．76％,与对照组比较,差异有显著意义（P＜0．05）。结论 bFGF通过上调BLEC的PCNA表达,改变细胞周期,致使BLEC进入增殖状态。     

人角膜上皮细胞Toll样受体介导的炎性细胞因子的表达

目的探讨人角膜上皮组织和细胞系(THCE)对Toll样受体(TLR)2和TLR4的表达及后者介导THCE对烟曲霉菌(AF)抗原炎性反应的影响。方法用免疫印迹(Western blot)和免疫细胞化学方法检测人角膜上皮组织和细胞系THCE的TLR2和TLR4蛋白质表达与分布;采用自制的AF菌丝体片段(5×106／ml)和培养上清提取物(牛血清白蛋白当量浓度为10μg／ml)抗原,刺激培养的THCE细胞,于刺激1、2、4及8 h收集培养上清,用酶联免疫吸附试验(ELISA)方法检测白细胞介素(IL)8和肿瘤坏死因子(TNF)α的水平。于刺激后30 min、1及2 h收集细胞,Western blot检测IκBα的表达变化以评价核转录因子κB的活化;采用抗体封闭实验分析封闭TLR2和TLR4对THCE细胞表达IL-8和TNF-α的影响。结果Western blot和免疫细胞化学结果显示人角膜上皮组织和THCE细胞均表达TLR2和TLR4蛋白质;AF菌丝体或培养上清抗原刺激THCE细胞后,培养上清液中IL-8和TNF-α于1 h后开始升高,至8 h分别达到(64．71±5．15)pg／ml和(32．46±3．28)pg／ml(菌丝体刺激组)及(94．94±11．92)pg／ml和(48．70±3．32)pg／ml(上清刺激组),分别为对照组THCE细胞培养上清中IL-8和TNF-α浓度的3．0倍和2．5倍及4．5倍和3．5倍(均P<0．01);同时Western blot检测AF菌丝体和AF上清刺激30 min后THCE细胞的IκBα表达(平均灰度值)分别由对照组的51．57±5．58和49．23±3．49下降为10．31±1．30和8．15±2．37(均P<0．01),2 h后恢复至对照组水平。在AF菌丝体刺激组,单纯封闭TLR2或TLR4部分抑制THCE细胞IL-8和TNF-α的分泌(均P<0．05),同时封闭两种受体则明显抑制了IL-8和TNF-α分泌(均P<0．01);AF上清抗原刺激组,单纯封闭TLR4和联合封闭两个受体均可明显抑制IL-8和TNF-α的分泌(均P<0．01),而单纯封闭TLR2未明显抑制二者的分泌(均P>0．05)。结论AF可能经过TLR-NF-κB信号转导通路诱导人角膜上皮细胞表达IL-B和TNF-α等炎性细胞因子。TLR2和TLR4可能介导人眼角膜上皮细胞对AF菌丝体的识别,而对AF上清抗原的识别则可能主要由TLR4介导。     

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.     

Multiplex cytokine detection versus ELISA for aqueous humor: IL-5, IL-10, and IFNgamma profiles in uveitis

PURPOSE: The purpose of this study was to determine levels of IL-2, -4, -5, -10, TNF-alpha, and IFN-gamma in aqueous humor (AH) from patients with active panuveitis, anterior uveitis (AU), and noninflammatory controls by using a flow cytometric mutiplex array (CBA) and to compare with results from ELISA. METHODS: Pooled normal AH was spiked with six cytokines at decreasing concentrations for evaluating the CBA. AH was also obtained from 10 controls (cataract patients) and 36 patients with active uveitis. Cell-free supernatants were added to a cocktail of capture beads and detector antibodies or to antibody-coated wells for CBA and ELISA determination, respectively. RESULTS: CBA demonstrated greater sensitivity for detecting IL-4, IL-10, and TNF-alpha than with ELISA. Increased IFN-gamma was detected in both AU and panuveitis groups compared with controls (P < 0.01). IL-10 was higher in the panuveitis group on steroids (P < 0.01). IL-5 was detected in the control (P < 0.01) and AU groups (P < 0.05) but was undetectable in the panuveitis group (n = 10). Correlations between IFN-gamma and IL-10 were found in all uveitis groups (P < 0.01) but not in controls, whereas TNF-alpha correlations with IL-4/IFN-gamma were obtained in controls but not in the uveitis groups (P < 0.01). CONCLUSIONS: It was possible to measure cytokines titrated into normal AH specimens by CBA, and a greater number of cytokines were detected with increased sensitivity than with ELISA. Elevated IFN-gamma in active uveitis and decreased IL-5 in posterior uveitis suggest Th1 polarity is more marked, with greater uveal tract involvement. The increased IL-10 in the steroid treated group suggests glucocorticoid-induced IL-10 upregulation.     

巨噬细胞诱发的实验性增生性玻璃体视网膜病变中的几种早期炎 …

观察在增生性玻璃体视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｖｉｔｒｅｏｒｅｔｉｎｏｐａｔｈｙ,ＰＶＲ）模型中几种早期炎性细胞因子的含量变化及其与ＰＶＲ形成时相的关系。方法采集巨噬细胞诱发的兔眼ＰＶＲ模型的玻璃体液用双抗体夹心法酶联免疫吸际测试（ｅｎｚｙｍｅｌｉｎｋｅｄｉｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙ,ＥＬＩＳＡ）试剂盒,检测样本中肿瘤坏死因子－α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ－α,ＴＮ     

Comparison of serum NO, TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels with grades of retinopathy in patients with diabetes mellitus

BACKGROUND: Vitreal interleukin (IL)-1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels have previously been determined in patients with proliferative diabetic retinopathy (PDR). However, at present there is no cohort study linking serum levels of NO and many inflammatory cytokines such as TNF-alpha, IL-1beta, soluble IL-2 receptor (sIL-2R), IL-6 and IL-8 to the grade of the microvascular complications. PURPOSE: To determine the relation between the stages of DR and the levels of serum NO, TNF-alpha, IL-1beta, sIL-2R, IL-6 and chemokine IL-8 in patients with diabetes compared with healthy controls. METHODS: Fifty-three consecutive patients with diabetes (25 men, 28 women) with or without DR and 15 non-diabetic healthy subjects (seven men, eight women) as controls were included in this prospective study. As an indicator for NO, serum total nitrite (NO2- + NO3-) levels (end-product of NO) were measured by the Griess reaction. Serum TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels were determined by a spectrophotometric technique using an Immulite chemiluminescent immunometric assay. The patients with diabetes were classified into three groups according to the stage of DR: no DR (NDR; n = 16), non-proliferative DR (NPDR; n = 18) and PDR (n = 19). The data were analysed using a Mann-Whitney U-test and the results were expressed as mean +/- SE (range). RESULTS: The levels of IL-1beta and IL-6 were below the detection limits of the assay (for each, <5.0 pg/ml) in all patients with diabetes and controls. Soluble IL-2R levels ranged from 260 to 958 U/ml, with the highest values observed in the patients with PDR. In 47 of the 53 samples (89%) tested for diabetic patients, IL-8 levels were above the detection limits of the assay (5.0 pg/ml). IL-8 levels ranged from <5.0 to 25.0 pg/ml, with the highest mean values observed in PDR patients. TNF-alpha was detectable in 46 of 53 patients with diabetes (87%), ranging from <4.0 to 26.4 pg/ml, with again the highest values obtained in the patients with PDR. Serum NO levels ranged from 80 to 188 micromol/l, with the highest values obtained in patients with PDR. Taken together, the mean serum NO, sIL-2R, IL-8 and TNF-alpha levels increased with the stage of DR and the highest levels were found in patients with PDR. The PDR patients had significantly (for each, P < 0.001) higher serum NO (166.8 +/- 3.2 micromol/l), sIL-2R (807.9 +/- 33.3 U/ml), IL-8 (17.9 +/- 0.4 pg/ml) and TNF-alpha (15.0 +/- 0.8 pg/ml) levels compared with NPDR patients (149.5 +/- 2.1, 659.4 +/- 23.4, 12.9 +/- 1.1, 11.5 +/- 0.6, respectively), NDR patients (115.9 +/- 5.8, 373.8 +/- 15.0, 8.3 +/- 1.0, 6.6 +/- 0.9, respectively) and controls (116.6 +/- 2.3, 392.4 +/- 16.6, 7.2 +/- 0.3, 7.3 +/- 0.5, respectively). Serum levels of these parameters for NPDR patients were also significantly (for each, P < 0.01) higher compared with those of NDR patients and controls. On the other hand, serum NO, sIL-2R, IL-8 and TNF-alpha levels of patients with NDR were comparable with those of controls (for each, P > 0.05). CONCLUSION: The results of the present study suggest that NO, sIL-2R, IL-8 and TNF-alpha may play important roles in the pathophysiology and progression of DR. We think that these potentially inflammatory cytokines and NO with their endothelial implications may act together during the course and progression of DR. These molecules may serve as therapeutic targets for the treatment and/or prevention of diabetes with its systemic and ocular microvascular complications.     

Calcium influx induced by activation of receptor tyrosine kinases in SV40-transfected human corneal endothelial cells

This study was undertaken to investigate electrophysiological properties of immortalized SV40-transfected human corneal endothelial cells (HCEC-SV40) combined with the analysis of intracellular Ca(2+) responses mediated by ligands for receptor tyrosine kinases (RTK). In addition, the effects of several tyrosine kinase inhibitors were tested on Ca(2+) inflow mediated by induction of capacitative calcium entry (CCE). Patch-clamp techniques and measurements of the intracellular free Ca(2+) ([Ca(2+)](i)) by fura-2 were performed using HCEC-SV40. Stimulation of fibroblast growth factor receptors (FGFR) (e.g. by basic-FGF) (10 ng ml(-1)) elicited activation of Ca(2+) permeable channels and a subsequent increase of cytosolic free Ca(2+) in HCEC-SV40. This effect could be disrupted by the L-type Ca(2+) channel blocker nifedipine (5 microM). In addition, nifedipine significantly reduced the magnitude of CCE. Inhibition of protein tyrosine kinases (PTKs) by genistein, lavendustin A, or tyrphostin 51 (all 5 microM) also led to a reduction of CCE in HCEC-SV40. This study demonstrates for the first time that L-type Ca(2+) channel activity in HCEC-SV40 is linked to the activity of FGF receptor tyrosine kinases. These data regarding Ca(2+) inflow through Ca(2+) channels could be useful for investigation of culture and vitality conditions of HCEC.