RGDS肽对晶状体上皮细胞与细胞小基质蛋白粘附作用的影响

目的 探讨纤维连接蛋白、Ⅰ型胶原蛋白对晶状体上皮细胞粘附的影响,研究Arg-Gly-Asp-Ser(RGDS)肽抑制晶状体上皮细胞与基质蛋白粘附的作用。方法 应用MTT法观测纤维连接蛋白、Ⅰ型胶原蛋白对体外培养的牛晶状体上皮细胞的粘附作用,其次测定RGDS肽对牛晶状体上皮细胞与细胞外基质蛋白粘附和细胞增殖的影响。结果 牛晶状体上皮细胞 能够粘附到包被有不同浓度的纤维连接蛋白、Ⅰ型胶原蛋白培养板表面上,呈现浓度效应特点;RGDS肽能够抑制牛晶状体上皮细胞在基质蛋白上的粘附。结论 纤维连接蛋白、Ⅰ型胶原蛋白能够介导晶状体上皮细胞粘附到后囊上,在后囊混浊发生过程中起到重要的促进作用;在细胞水平上证实了RGDS肽通过竞争细胞外基质蛋白与晶状体上皮细胞表面的整合素体结合,阻止细胞粘附和增殖,具有潜在的防治后囊混浊的作用。     

小檗胺抑制兔Tenon囊和滤过道内成纤维细胞增殖的实验研究

目的:研究钙调素拮抗剂小檗胺(berbamine,BER)对兔Tenon囊成纤维细胞和兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。方法:体外培养兔Tenon囊成纤维细胞,经不同浓度BER处理不同时间后,细胞计数Kit8(CCK8)法检测BER对兔Tenon囊成纤维细胞增殖的抑制作用,流式细胞仪检测其凋亡率及细胞周期的变化。HE染色检测BER对兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。结果:兔Tenon囊成纤维细胞经不同浓度BER处理不同时间后细胞增殖受到抑制,并呈时间剂量依赖性。流式细胞仪检测发现BER处理后兔Tenon囊成纤维细胞呈现G1期阻滞,细胞凋亡率明显增加,20mg/LBER处理9h,细胞凋亡率从0.64%增加到31.86%。HE染色显示BER显著抑制兔小梁切除术后滤过道内成纤维细胞的增殖。结论:小檗胺在体内外均能抑制成纤维细胞的增殖,其可能是通过诱导细胞凋亡方式抑制兔Tenon囊和滤过道内成纤维细胞的增殖。     

氯化锂对人眼Tenon囊成纤维细胞增殖的影响

目的：观察氯化锂（Lithium chloride）对人眼Tenon囊成纤维细胞（human Tenons capsule fibroblasts, HTFs）体外生长的抑制效应,并探讨其作用机制。 方法：磺基罗丹明（SRB）法和BrdU法检测细胞生长抑制率;采用流式细胞技术（FCM）检测细胞凋亡和细胞周期的变化;Hoechst 33258染色后荧光显微镜观察细胞形态学的改变。结果：在40-160mmol/L范围内,氯化锂能够抑制HTFs的增殖,且呈剂量与时间依赖性。氯化锂使HTFs阻滞于G2/M期,且能诱导细胞凋亡,高浓度组尤为明显。结论：氯化锂能抑制HTFs增殖,机制为诱导细胞凋亡,并将细胞阻滞于G2/M期。     

曲尼司特对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖和移行的影响

目的 探讨曲尼司特(tranilast)对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖及移行的影响。方法 取青光眼患者滤过术中剪下的Tenon囊组织,在体外进行成纤维细胞原代及传代培养。分别采用MTT法、细胞计数法、免疫组化加图像分析法及划线法,研究不同浓度的曲尼司特对体外培养的成纤维细胞增殖及移行的影响及其与蛋白激酶C(PKC)表达的关系。结果 当浓度在12．5～100．0mg／L之间变化时,曲尼司特能明显抑制成纤维细胞的增殖,强度呈剂量依赖性;50．0mg／L和100．0mg／L的曲尼司特能明显抑制成纤维细胞的移行,并下调细胞内PKC的表达。结论 曲尼司特能抑制成纤维细胞的增殖及移行,这种作用可能与下调细胞内PKC的表达有关。     

RGD肽对体外培养的人晶状体上皮细胞粘附与增殖的影响

目的:研究RGD肽对体外培养的人晶状体上皮细胞粘附与增殖的影响,为RGD肽防治晶状体后囊混浊提供理论依据。方法:将在体外分离培养的人晶状体上皮细胞中分别加入系列浓度的RGD肽(50,125,250,500,1000mg/L)作为实验组,不含RGD肽的培养基作为对照组。以2×107个/L密度接种到预孵化含有纤维连接蛋白和I型胶原蛋白的96孔培养板中,于1h后应用MTT法检测RGD肽对细胞粘附的影响。细胞接种于培养板,加入系列浓度RGD肽(125,250,500,1000,2000mg/L)后的24,48,72h检测对细胞增殖的影响。结果:RGD肽对人晶状体上皮细胞粘附的抑制呈明显的剂量依赖性,随着其浓度增大,细胞粘附数就越低,500mg/L时,抑制作用最强(P<0.05);RGD肽对人晶状体上皮细胞增殖的抑制呈明显的时间剂量依赖性,在48h,1000mg/LRGD条件下,对细胞的抑制作用最强。结论:RGD肽可抑制晶状体上皮细胞的粘附与增殖,具有潜在的防治后囊混浊的作用。     

汉防己甲素对体外人眼Tenon囊成纤维细胞的抑制效应

目的:研究汉防己甲素(tetrandrine,Tet)对人眼Tenon囊成纤维细胞(Tenon’s capsule fibroblasts,TCFs)的抑制效应。方法:用组织块和混合消化液培养法体外培养人眼Tenon囊成纤维细胞,并对培养的传代细胞进行形态学的鉴定。采用MTT法检测不同浓度的汉防己甲素(10-4,10-5,10-6,10-7mol/L)及0.2g/L丝裂霉素(MMC)对人眼Tenon囊成纤维细胞的抑制作用。结果:人眼Tenon成纤维细胞体外生长良好,经光镜和免疫组化观察证明该细胞为成纤维细胞。MTT法证实,随着Tet药物浓度的增大,TCFs增生活性降低,对应的TCFs抑制率升高。结论:Tet对人眼Tenon囊成纤维细胞的增殖具有明显的抑制作用。     

氯化锂对人眼Tenon 囊成纤维细胞增殖的影响

目的：观察氯化锂（Lithium chloride）对人眼Tenon囊成纤维细胞（human Tenons capsule fibroblasts, HTFs）体外生长的抑制效应,并探讨其作用机制。 方法：磺基罗丹明（SRB）法和BrdU法检测细胞生长抑制率;采用流式细胞技术（FCM）检测细胞凋亡和细胞周期的变化;Hoechst 33258染色后荧光显微镜观察细胞形态学的改变。 结果：在40~160mmol/L范围内,氯化锂能够抑制HTFs的增殖,且呈剂量与时间依赖性。氯化锂使HTFs阻滞于G₂/M期,且能诱导细胞凋亡,高浓度组尤为明显。 结论：氯化锂能抑制HTFs增殖,机制为诱导细胞凋亡,并将细胞阻滞于G₂/M期。     

Bevacizumab对人Tenon囊成纤维细胞迁移抑制作用的研究

目的：观察bevacizumab对人Tenon囊成纤维细胞迁移能力的影响,探讨青光眼术后滤过泡瘢痕化的对策。方法：采用从陕西省人民医院中心实验室细胞库中取出冻存的人Tenon囊成纤维细胞,采用细胞复苏法,遵循无菌原则,进行常规培养。创伤划痕试验：待细胞融合度达到80%时在单层细胞表面划出一条无细胞的刮除带,空白对照组加入不含血清的DMEM培养液,bevacizumab处理组加入 bevacizumab 浓度为1 mg/mL 不含血清的DMEM培养液,分时段(0,24,48,72h)观察并测量划痕宽度。结果：人Tenon囊成纤维传代细胞显微镜下观察呈长梭形,细胞核位于细胞的中部,核较大,胞浆丰富,生长时呈漩涡状排列走形,增殖能力强,符合成纤维细胞的一般形态。冻存和复苏后细胞的形态结构和生物学特点维持不变。细胞划痕试验显示：0h时,两组细胞划痕初始宽度相等;24 h 时两组细胞迁移距离基本一致;48 h 时bevacizumab处理组细胞迁移距离明显小于空白对照组;72 h时空白对照组划痕基本愈合, bevacizumab处理组细胞迁移距离较48 h无明显变化,且细胞死亡数目较多。结论：利用细胞复苏法可成功培养形态结构和生物学特性稳定的传代人Tenon囊成纤维细胞,为实验研究奠定细胞学基础。成纤维细胞本身具有较强的迁移能力,外源性bevacizumab作用可以明显抑制细胞的迁移,作用时间过长时,会造成细胞的过多死亡。抗新生血管药物对成纤维细胞的迁徙具有一定的抑制作用,未来很有可能成为眼科临床抗击青光眼术后滤过泡瘢痕化的重要手段。     

维拉帕米对人眼Tenon''s囊成纤维细胞增殖的影响

为了探讨维拉帕米在防治眼部增殖性疾病中的价值，本文研究了维拉帕米对体外培养的人眼Ｔｅｎｏｎ’ｓ囊成纤维细胞增殖的影响及与ＣＡＭＰ的关系。结果发现：维拉帕米明显抑制人眼Ｔｅｎｏｎ‘ｓ囊成纤维细胞的增殖，药物浓度和细胞数量呈负相关，其５０％抑制率浓度（ＩＤ５０）为２８ｍｇ／ｌ；维拉帕米对人眼Ｔｅｎｏｌ’ｓ囊成纤维细胞的抑制作用不通过改变ＣＡＭＰ来实现。提示：维拉帕米可能是一种有价值的防治眼部增殖性疾病     

人工合成白细胞介素-1受体阻断剂CK-17对兔结膜下成纤维细胞的影响

目的研究非甾体类抗炎药物——人工合成白细胞介素-1受体阻断剂CK-17对体外培养的兔Tenon囊成纤维细胞(rabbit Tenon’s capsule fibroblast，RTCF)增生及细胞分泌功能的抑制作用。对CK-17在青光眼滤过术后抗瘢痕增生方面应用的可行性进行探讨。设计体外、对照干预实验研究。研究对象体外培养的兔RTCF。方法用细胞计数的方法检测RTCF增生的情况；用胶原检测试剂盒检测RTCF培养基中分泌的总胶原的含量。主要指标成纤维细胞记数，RTCF培养基中总胶原含量。结果(1)CK-17对RTCF的抑制呈浓度和时间依赖性；(2)CK-17对RTCF胶原分泌的抑制呈浓度和时间依赖性；(3)CK-17在实验浓度下对RTCF无细胞毒性。结论CK-17能够抑制体外培养的兔成纤维细胞增生及细胞总胶原的分泌。     

RNA干扰技术抑制体外培养的人眼球筋膜囊成纤维细胞增殖的初步研究

目的 探讨RNA干扰技术对体外培养的人眼球筋膜囊成纤维细胞增殖的抑制作用。方法 采用化学合成的靶向IKK-β的siRNA,转染体外培养的人眼球筋膜囊成纤维细胞,同时以对任何基因均无作用的siRNA作为阴性对照。以逆转录聚合酶链反应（RT—PCR）技术检测IKK-βmRNA表达水平,免疫印迹法检测IKK-β蛋白表达水平。将siRNA的转染浓度分别设为5、10、25、50、100、200nmol／L,进行转染,于转染后48h采用四甲基偶氮唑盐法检测其转染后对成纤维细胞的抑制作用。结果 经转染了靶向IKK-β的siRNA,其成纤维细胞IKK-β的mRNA和蛋白表达水平均受到抑制。各个转染浓度（5、10、25、50、100、200nmol／L）均可抑制成纤维细胞的增殖（P〈0．05）,抑制率分别为10．72％、23．35％、30．84％、51．25％、50．06％、49．63％,50nmol／L时已达最大抑制率。结论 靶向IKK-β的siRNA转染可以降低IKK-β的表达水平,同时对体外培养的人眼球筋膜囊成纤维细胞的增殖具有抑制作用。抗IKK-β的RNA干扰技术可能是抑制抗青光眼术后滤过道瘢痕化的新手段。     

拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞增殖的影响

目的研究拉坦前列素滴眼液对体外培养的人眼球筋膜囊成纤维细胞的增殖有无促进作用。方法体外培养人眼球筋膜囊成纤维细胞。按1∶10比例逐级稀释拉坦前列素滴眼液(0.005%),使药物浓度依次为5.00、0.50、0.05μg/ml直至5×10-8μg/ml。采用噻唑蓝比色法检测在不同浓度药物作用下的细胞增殖情况,并在光学显微镜下观察细胞的形态变化。结果较高浓度组(5.00μg/ml)拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞存在明显的杀伤作用,细胞几乎全部死亡,吸光度(A)值与对照组相比明显下降(P<0.01);低浓度组(<0.05μg/ml)A值较对照组无明显变化(P>0.05)。比较拉坦前列素滴眼液在不同作用时间下的剂量效应曲线,24、48及72h差异均无统计学意义(P>0.05)。结论拉坦前列素滴眼液不能促进体外培养的人眼球筋膜囊成纤维细胞的增殖,在较高浓度时具有细胞毒性,在较低浓度时则对细胞增殖无明显影响。     

Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts

PURPOSE: To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. METHODS: Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. RESULTS: Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. CONCLUSIONS: Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.     

Effect of growth factors on the activation of human Tenon's capsule fibroblasts

PURPOSE: To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1beta, TGF-beta1 and TGF-beta2 on the proliferation and myofibroblast transformation of cultured human Tenon's capsule fibroblasts and to characterize expression of PDGF- and TGF-beta-receptors in these cells. METHODS: To determine cell proliferation, cell number of 2nd passage cultured human Tenon's capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate alpha-smooth-muscle actin (alpha-SMA) expression. Expression of PDGF- and TGF-beta-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. RESULTS: A significant increase in proliferation (p < or = 0.05) was detected after exogenous stimulation with PDGF-AA (10 ng/ml and 100 ng/ml), PDGF-AB (10 ng/ml and 100 ng/ml), PDGF-BB (10 ng/ml and 100 ng/ml), bFGF (100 ng/ml), IL-1beta (1 ng/ml and 10 ng/ml), TGF-beta1 (0.5 ng/ml) and TGF-beta2 (0.5 ng/ml). Both TGF-beta1 and TGF-beta2 stimulated expression of alpha-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-beta1) and 500 ng/ml (TGF-beta2). Protein and mRNA of PDGF-receptor type alpha and type beta and TGF-beta-receptors type I, II and III are expressed in cultured human Tenon's capsule fibroblasts. CONCLUSIONS: The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon's capsule fibroblasts after glaucoma filtering surgery while TGF-beta-isoforms are essential for the transformation of Tenon's capsule fibroblasts into myofibroblasts.     

晶状体和玻璃体提取物及地塞米松影响Tenon囊成纤维细胞增殖和胶原合成的实验研究

目的　探讨晶状体和玻璃体提取物对猪眼筋膜囊(Tenon囊)成纤维细胞增殖和胶原合成的影响及其在糖皮质激素作用下的变化。方法　体外培养猪眼Tenon囊成纤维细胞,应用噻唑蓝(MTT)比色法和逆转录聚合酶链反应(RT -PCR)法观察在晶状体和玻璃体提取物及地塞米松单独或联合作用下,Tenon囊成纤维细胞增殖和胶原合成的变化。结果　与对照细胞吸光度(A值)(0 .305±0.013)比较, 10mg/L晶状体提取物( 0. 411±0. 000 )和10mg/L玻璃体提取物( 0. 349±0 .027)作用2d即具有促进细胞生长作用,差异有统计学意义(P=0. 00);且随着晶状体提取物、玻璃体提取物浓度的增高,A值相应增加,差异均有统计学意义(P=0. 00)。Ⅰ型胶原的相对含量在晶状体提取物作用细胞内为12 290±231,在玻璃体提取物作用细胞内为10 .853±231,与对照细胞(9389±178)比较,差异均有统计学意义(P<0. 01)。地塞米松对125mg/L晶状体提取物和125mg/L玻璃体提取物作用的Tenon囊成纤维细胞增殖分别具有一定抑制作用,差异有统计学意义(P=0. 00)。地塞米松和提取物联合作用细胞内Ⅰ型胶原的相对含量与提取物单独作用细胞比较,差异均无统计学意义(P>0 .05)。结论　晶状体和玻璃体提取物均可明显刺激Tenon囊成纤维细胞增殖,促进Tenon囊成纤维细胞内胶原合成,     

体外转染γ-干扰素基因抑制人眼球筋膜囊成纤维细胞的研究

目的探讨阳离子脂质体介导的γ-干扰素(IFN-γ)基因对体外培养的人眼球筋膜囊成纤维细胞增殖的抑制作用。方法采用脂质体介导的方法,以质粒pcDNA3 IFN-γ基因转染体外培养的人眼球筋膜囊成纤维细胞,同时以空载体重组质粒pcDNA3作为对照,G418筛选抗性克隆并扩增。以RT-PCR、免疫组化及流式细胞仪间接免疫荧光法检测IFN-γ基因的表达,应用流式细胞仪进行细胞周期分析,采用四甲基偶氮唑盐法检测IFN-γ基因对成纤维细胞的作用。结果转染pcDNA3 IFN-γ基因的成纤维细胞在转录水平和蛋白水平表达IFN-γ基因,转染的IFN-γ基因可明显抑制成纤维细胞的增殖。结论转染的IFN-γ基因对体外培养的人眼球筋膜囊成纤维细胞的增殖具有抑制作用。     

人Tenon囊成纤维细胞的体外培养及增殖能力研究

目的:离体培养人Tenon囊成纤维细胞(HTF)并观察细胞增殖能力。方法:取斜视患者手术切除Tenon囊组织进行成纤维细胞原代培养,培养的细胞通过免疫荧光染色法进行鉴定。CCK-8法描述细胞生长曲线,测定细胞活力。流式细胞术分析细胞周期,计算增殖指数。结果:通过组织块法成功培养出HTF。不同代次原代培养的HTF之间增殖能力无明显统计学差异(P>0.05)。结论:HTF在体外易于培养,经过数次传代,增殖能力稳定,是进行Tenon囊抗纤维化研究的良好靶细胞。     

Role of chymase on growth of cultured canine Tenon's capsule fibroblasts and scarring in a canine conjunctival flap model

Chymase is a chymotrypsin-like serine protease contained in the secretory granules of mast cells. Recently, we reported that chymase activity and the number of chymase-positive mast cells in conjunctival tissues were significantly increased during the wound healing process in a hamster model of glaucoma surgery. However, it has been unclear the role of chymase on conjunctival scarring. In the present study, we evaluated the effect of dog chymase on cell proliferation of fibroblasts established from canine Tenon's capsule and the effect of a chymase inhibitor on scarring in a canine conjunctival flap model. After a fibroblast cell culture was established from canine Tenon's capsules, the fibroblasts were incubated in the presence of dog chymase (5-20 ng ml(-1)). Cell proliferation was evaluated by bromodeoxyuridine incorporation. In a canine conjunctival flap model, a sponge treated with a chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), or placebo was placed in between the conjunctiva and sclera and the conjunctival incision was closed. One week after the surgery, adhesion degree was assessed, and chymase activities in the conjunctival lesion and in the areas of the conjunctiva and sclera were measured. In cultured canine Tenon's capsule fibroblasts, dog chymase significantly increased cell proliferation, and this chymase-dependent proliferation was completely suppressed by the chymase inhibitor. In the canine surgical model, chymase activity in placebo-treated eyes was significantly increased compared to control eyes, while it was significantly decreased by treatment with the chymase inhibitor. Scores for adhesion degree in the chymase inhibitor-treated eyes were significantly decreased in comparison with those in placebo-treated eyes. The conjunctival area in the chymase inhibitor-treated eyes was also suppressed to 52.6% compared with that in placebo-treated treated eyes. In conclusion, chymase stimulates proliferation of fibroblasts derived from canine Tenon's capsule and chymase may play an important role in scarring after glaucoma surgery.