基质金属蛋白酶及其组织抑制剂在结膜松弛症成纤维细胞中的表达

目的探讨基质金属蛋白酶1、3（MMP．1、MMP．3）及其组织抑制因子1、3（TIMP-1、TIMP．3）在结膜松弛症（CCh）成纤维细胞培养上清液中的表达,揭示四者表达水平变化与CCh的内在联系。方法体外原代培养CCh成纤维细胞,取第3—6代细胞,以2．5×10^5个／mL浓度接种于24孔板中培养,24h后提取细胞上清液,采用ELISA检测MMP-1、MMP-3、TIMP-1、TIMP-3的表达水平。不同组间比较采用单因素方差分析。结果MMP-1的表达：CCh组成纤维细胞的蛋白浓度明显高于正常对照组,差异有统计学意义（P〈0．05）。MMP-3的表达：CCh组成纤维细胞的蛋白浓度明显高于正常对照组,差异有统计学意义（P〈0．01）。TIMP．1的表达：CCh组成纤维细胞的蛋白浓度与正常对照组比较,差异无统计学意义（P〉0．05）。TIMP-3的表达：CCh组成纤维细胞的蛋白浓度明显低于正常对照组,差异有统计学意义（P〈0．05）。结论成纤维细胞MMP．1／TIMP-1及MMP-3／TIMP-3的表达失衡可能参与了CCh的发生、发展。     

多西环素对人翼状胬肉成纤维细胞血管生成拟态形成的影响

目的：探讨多西环素(DOX)对人翼状胬肉成纤维细胞(HPFs)血管生成拟态(VM)形成的影响及其可能的分子机制。

方法：采用波形蛋白(Vimentin)和角蛋白(CK)免疫细胞化学染色鉴定原代培养的HPFs。分别设置对照组、DOX低、中、高浓度(50、100、200mg/L)组,应用CCK-8法和划痕实验检测各组HPFs活力及迁移能力,利用细胞三维培养及过碘酸-雪夫(PAS)染色观察各组细胞VM生成情况,并通过Western blot法检测各组细胞基质金属蛋白酶(MMP)-9及血管内皮生长因子(VEGF)的表达情况。

结果：免疫细胞化学染色结果显示,细胞呈梭形且Vimentin阳性表达,CK阴性表达,符合成纤维细胞特性。与对照组相比,DOX组细胞活力、迁移能力、VM密度及MMP-9和VEGF蛋白的表达均显著降低(P<0.05),DOX各浓度组组间相比均有差异(P<0.05)。相关性分析提示HPFs形成的VM密度与MMP-9和VEGF蛋白表达水平呈显著正相关(r=0.949、0.960,均P<0.05)。

结论：DOX可通过减少MMP-9及VEGF的表达抑制HPFs的活力及迁移能力,减少VM的形成,提示MMP-9和VEGF可能是翼状胬肉形成VM的分子机制。     

Growth factors in cultured pterygium fibroblasts: immunohistochemical and ELISA analysis

· Background: In order to study growth factors in the pathogenesis and recurrence of pterygium, we grew pterygium tissues in culture and compared fibroblasts from primary and from recurrent pterygia with reference to the fibroangiogenic growth factors basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α). · Methods: We used indirect immunohistochemical procedures against human b-FGF, PDGF, TGF-β and TNF-α. As controls, we used cultured normal human conjunctival fibroblasts. A serum-free conditioned medium (CM) from confluent fibroblasts derived from primary and recurrent explants was assessed by enzyme-linked immunosorbent assay to determine the level of the above-mentioned growth factors. · Results: Immunoreactivity of b-FGF was stronger in recurrent than in primary pterygium fibroblasts. PDGF immunolabeling was stronger in primary than in recurrent pterygium fibroblasts. TGF-β and TNF-α immunolabeling was weak in both pterygia. All these growth factors were very sparse in normal conjunctival fibroblasts. Basic-FGF and TGF-β₁ were found in the CM from both primary and recurrent pterygium, while PDGF and TNF-α were not detectable. · Conclusion: The strong immunoreactivity and the release of b-FGF in cultured fibroblasts of recurrent pterygia suggest that fibroblasts may play an important role in the recurrence of pterygium. Received: 7 May 1997 Revised version received: 3 November 1997 Accepted: 8 December 1997     

阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制

目的 探讨不同浓度阿托品对大鼠巩膜成纤维细胞增殖和凋亡的影响及其机制。方法 取出生24 h的健康雄性SD大鼠眼巩膜组织(均为右眼)进行原代培养。取细胞进行分组：对照组(正常巩膜成纤维细胞),0.1 g·L^-1、1.0 g·L^-1及5.0 g·L^-1阿托品处理组(正常巩膜成纤维细胞分别加入三种不同浓度阿托品溶液),每组5只大鼠原代培养细胞。加药后24 h, CCK-8法检测各组巩膜成纤维细胞活力，流式细胞仪检测各组巩膜成纤维细胞凋亡率，Western blot法检测各组巩膜成纤维细胞光蛋白聚糖、基质金属蛋白酶(MMP)-2、MMP-14和MMP抑制剂(TIMP)-2蛋白表达情况。结果 CCK-8实验结果显示：与对照组相比，0.1 g·L^-1、1.0 g·L^-1及5.0 g·L^-1阿托品处理组细胞活力均增高，其中以1.0 g·L^-1阿托品处理组增高最显著，差异均有统计学意义(均为P<0.05)。流式细胞仪检测结果表明：阿托品处理组各...     

核转录因子抑制剂对脂多糖诱导角膜基质细胞分泌细胞因子的影响

目的探讨二硫代氨基甲酸吡咯烷（PDTC）对绿脓杆菌脂多糖（LPS）诱导的人角膜基质细胞核因子κB（NF—κB）活化与细胞因子释放的干预作用及其机制。方法为实验研究。原代培养人眼角膜基质细胞（HCFs）,选取生长良好的传代HCFs分为对照组、LPS刺激组及PDTC预处理组。LPS组给予单纯LPS刺激,PDTC预处理组则在LPS刺激前先加PDTC培养30min,再给予相同浓度的LPS。在LPS刺激1、2、4及8h时,分别收集各组细胞和上清液,Western blot检测HCFs NF—κB的活化表达;酶联免疫吸附实验（EHSA）检测HCFs培养上清液中自细胞介素6（IL-6）和IL-8的分泌情况;逆转录聚合酶链反应（RT—PCR）检测HCFs IL-6和IL-8 mRNA的表达。结果与对照组相比较,LPS刺激后,HCFs胞核NF—κB p65的表达明显升高,HCFs中IL-6与IL-8蛋白的分泌和mRNA的表达均显著升高。PDTC预处理30min后再给予LPS刺激,可部分抑制HCFs胞核中NF—κB p65的活化表达（t1h=9．3766,t2h=15．9011,t4h=12．5851,t8h=10．8346;均P〈0．01）;PDTC预处理可不同程度的下调LPS诱导的HCFs分泌IL-6、IL-8蛋白及IL-6、IL-8 mRNA的表达,二者均明显低于同时间点LPS组HCFs的表达,差异均有统计学意义（IL-6蛋白：t1h=7．9154,t2h=10．8630,t4h=8．2451,t8h=13．5063;IL-8蛋白：t1h=8．5663,t2h=20．5169,t4h=25．1580,t8h=34．8699;IL-6 mRNA：t1h=12．0235,t2h=13．2894,t4h=24．0799,t8h=27．2261;IL-8 mRNA：t1h=20．9424,t2h=24．1314,t4h=29．8580,t8h=47．9442;均P〈0．01）。结论绿脓杆菌LPS可引起HCFs NF—κB活化,促进细胞因子的表达,PDTC可部分抑制LPS对NF-κB的激活,下调相应细胞因子的表达。（中华眼科杂志,2008,44：6146）     

Expressions of matrix metalloproteinases 1 and 3 and their tissue inhibitors in the conjunctival tissue and fibroblasts cultured from conjunctivochalasis

AIM: To investigate the expression of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) and their tissue inhibitors of metalloproteinases 1 and 3 (TIMP-1 and TIMP-3) in the conjunctiva of eyes with conjunctivochalasis (CCh). METHODS: The conjunctival tissue was obtained from the CCh patients and controls, the MMPs/TIMPs expression concentration was determined by enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence staining. The expression levels of MMPs/TIMPs in the CCh fibro-blasts were determined by analyzing its concentration in the cellular supernatant that was abstracted from the in vitro cultured CCh fibroblasts. RESULTS: MMP-1 and MMP-3 levels determined by ELISA were both significantly higher in the CCh group than that in the control group (P=0.042, 0.022, respectively), so was the levels of TIMP-1 (P=0.010). No significant difference in the expression of TIMP-3 in conjunctiva was found between the two groups (P=0.298). The expression of MMP-1 and MMP-3 were both up-regulated significantly in the CCh group (P=0.040, 0.001, respectively) on immuno-fluorescence staining. MMP-1 and MMP-3 expression in the fibroblasts were both significantly higher in the CCh group than that in the control group (P=0.027, 0.001, respectively), while neither the TIMP-1 nor TIMP-3 expression was significantly different between the two groups (P=0.421, 0.237, respectively). CONCLUSION: The overexpression of MMP-1 and MMP-3 in conjunctival tissue and fibroblasts may play an important role in the pathogenesis and development of CCh.     

Effects of hepatocyte growth factor on MMP-2 expression in scleral fibroblasts from a guinea pig myopia model

AIM:To investigate the effects of hepatocyte growth factor (HGF) on MMP-2 expression in scleral fibroblasts from guinea pig with LIM.METHODS:Sixty 1-week-old guinea pigs were chosen for the study. The right eyes were treated with -10.0 D lenses as the LIM group; the left eyes remained untreated as the control group. The refraction and axial length were measured by streak retinoscopy and A-scan ultrasonography respectively prior to and 4 weeks after the experiment. Four weeks later, the guinea pigs were sacrificed and primary scleral fibroblasts were taken for tissue culture. The 3^rd-5th generation scleral fibroblasts were chosen for the experiments. The expression levels of HGF and MMP-2 protein in the scleral fibroblasts were analyzed by Western blotting. After HGF with different doses acted on the scleral fibroblasts of the control group, MMP-2 protein expression in the scleral fibroblasts was analyzed by Western blotting. HGF siRNA was transfected into the scleral fibroblasts of the LIM group and the protein expressions of HGF and MMP-2 were analyzed by Western blotting.RESULTS:The LIM group became myopic with a significant increase in axial length (7.97±0.29 mm vs 7.01±0.26 mm, P<0.05), and a significant decrease in refraction (-5.06±0.31 D vs 0.55±0.25 D, P<0.05) compared with the control group. The protein expression of HGF in the scleral fibroblasts of the LIM group was significantly higher compared with the control group ( 1.26±0.04 vs 0.32 ±0.04, P<0.05). The protein expression of MMP-2 in the scleral fibroblasts of the LIM group was significantly higher compared with the control group (0.89±0.06 vs 0.42±0.05, P<0.05). In the scleral fibroblasts of the control group, HGF(0, 0.1, 1, 10 ng/mL) upregulated MMP-2 protein expression in a dose-dependent manner (0.35±0.03, 0.44±0.02, 0.91±0.03, 1.33±0.04, all P<0.05). In the scleral fibroblasts of the LIM group transfected with HGF siRNA, MMP-2 protein expressions were significantly decreased compared with the negative control group (0.29±0.03 vs 0.81±0.05, P<0.05).CONCLUSION:HGF is a upstream mediator of MMP-2 in scleral fibroblasts from guinea pig.     

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures

PURPOSE: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. METHODS: Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. RESULTS: The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. CONCLUSIONS: PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.     

水蛭提取液通过VEGF/PI3K/AKT通路抑制WERI-RB-1细胞表达VEGF

目的：研究水蛭提取液对视网膜母细胞瘤细胞(WERI-RB-1细胞)表达血管内皮生长因子(VEGF)的影响及相关分子机制。

方法：将体外培养的WERI-RB-1细胞分为对照组、0.04U/mL水蛭提取液组、0.08U/mL水蛭提取液组,对照组采用完全培养基培养48h,水蛭提取液组分别采用含0.04、0.08U/mL含药培养基培养48h。采用ELISA法检测各组细胞培养基上清液中VEGF表达水平; 采用RT-PCR法检测各组细胞缺氧诱导因子-1a(HIF-1a)和基质金属蛋白酶-9(MMP-9)mRNA相对表达水平; 采用Western Blot法检测各组细胞HIF-1a、MMP-9、磷脂酰肌醇3-激酶(PI3K)、人磷酸化蛋白激酶(p-AKT)相对表达水平。

结果：与对照组比较,水蛭提取液组细胞培养基上清液中VEGF表达均降低(P<0.05),0.04、0.08U/mL水蛭提取液对VEGF表达抑制率分别为32.43%、38.92%。与对照组比较,水蛭提取液组细胞HIF-1a和MMP-9 mRNA相对表达水平均明显降低(P<0.05),0.04、0.08U/mL水蛭提取液对HIF-1a mRNA表达抑制率分别为27.64%、24.75%,对MMP-9 mRNA表达抑制率分别为43.97%、51.48%。与对照组比较,水蛭提取液组细胞HIF-1a、MMP-9、PI3K、p-AKT蛋白相对表达水平均明显降低(P<0.05),0.04、0.08U/mL水蛭提取液对HIF-1a蛋白表达抑制率分别为55.81%、43.85%,对MMP-9蛋白表达抑制率分别为39.49%、47.23%,对PI3K蛋白表达抑制率分别为33.27%、29.83%,对p-AKT蛋白表达抑制率分别为52.07%、30.21%。

结论：水蛭提取液可能通过VEGF/PI3K/AKT通路及HIF-1a、MMP-9因子抑制WERI-RB-1细胞VEGF的表达。     

银杏叶提取物对体外培养翼状胬肉成纤维细胞增殖的抑制作用

目的：研究不同浓度的银杏叶提取物（ginkgo biloba extract,GBE）对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblasts,HPFs)增殖和凋亡的影响。 方法：用噻唑蓝比色法(MTT)检测不同浓度(12.5,25.0,50.0,100.0mg/L) GBE在不同作用时间(24,48,72h)对体外培养的HPFs增殖的影响。用流式细胞仪检测HPFs的细胞周期及凋亡率。 结果：MTT结果显示,GBE浓度为12.5mg/L组作用24h后对HPFs的增殖无明显抑制作用（P>0.05）,其余各时间段各组均可见明显抑制HPFs的增殖,且各组间比较差异有统计学意义(P<0.05);流式细胞仪检测发现GBE作用48h后G₀/G₁期细胞比例增加,S期细胞比例下降,计算不同浓度GBE作用下凋亡率分别为4.37%,8.14%,11.09%,15.18%,组间比较差异有统计学意义(P<0.05)。 结论：GBE对HPFs的增殖有明显抑制作用,并与用药剂量和持续时间有关。     

Effect of Avastin on the migration and invasion of pterygium fibroblasts

Purpose:.To evaluate the effect of Avastin on human pterygium fibroblast migration and invasiveness.Methods:.VEGF secretion was compared between human pterygium fibroblasts and conjunctival fibroblasts by measuring VEGF-A by ELISA. The influence of Avastin on HPF migration and invasiveness was observed by wound scratch and Transwell migration assays..The expression of p-ERK1 / 2 and p-FAK was analyzed by western blotting.Results:(1)VEGF was secreted in higher amounts by human pterygium fibroblasts than by conjunctival fibroblasts.(2)Avastin treatment decreased HPF migration and invasion.(3)Avastin significantly decreased the expression of p-ERK1 / 2and p-FAK in human pterygium fibroblasts.Conclusion:.Avastin can inhibit migration and invasion of HPFs by decreasing the expression of p-ERK1 / 2 and p-FAK.