MTT法测定低分子量肝素对兔结膜成纤维细胞增殖的抑制作用

观察低分子量肝素（ｌｏｗｍｏｅｃｕｌａｒｗｅｉｇｈｔｈｅｐａｒｉｎ,ＬＭＷＨ）对兔结膜成纤维细胞增殖的抑制作用,探讨其与地塞米松联合应用的价值,并与肝素的抑制作用相比较。方法应用ＭＴＴ法测定不同浓度的ＬＭＷＨ对结膜成纤维细胞增殖的抑制作用,同时比较单独应用ＬＭＷＨ、地塞米松、同一抗凝单位的肝素及ＬＭＷＨ联合地塞米松对成纤维细胞增殖的抑制作用。结果ＬＭＷＨ对兔结膜民纤维细胞增殖的抑制作用与浓度成正相     

阿霉素联合地塞米松抑制成纤维细胞增生的实验研究

研究阿霉素、地塞米松对成纤维细胞生长的抑制作用及有效浓度。为药物防治ＰＶＲ提供新线索。方法应用噻唑兰比色法检测二者联合应用对成纤维细胞的作用。结论阿霉素、地塞米松能有效抑制成纤维细胞生长，二者合用可能有实用价值。     

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）     

阿霉素联合地塞米松防治增殖性玻璃体视网膜病变的研究

研究体外细胞培养中阿霉素、地塞米松对兔成纤维细胞生长的抑制作用及有效浓度。为增殖性玻璃体视网膜病变（ＰＶＲ）的药物预防提供新线索。 方法将传代培养的细胞，在加药培养后４８ｈ，应用噻唑兰比色法（ＭＴＴ法）对阿霉素、地塞米松抑制成纤维细胞的作用进行检测，分别求得２种药物５０％抑制率浓度（ＩＤ５０）及 ２种药物半数抑制率浓度（ＩＤ５０）联合应用对细胞的增殖抑制率（％）。 结果阿霉素对细胞生长有明显的抑制作用，并呈浓度依赖性改变，其５０％抑制率浓度（ＩＤ５０）为０．５７ｍｇ·Ｌ－１；地塞米松对细胞增殖作用呈双重性，低浓度刺激细胞增生，高浓度则抑制细胞生长，其５０％抑制率浓度为１５４ｍｇ·Ｌ－１。上述２种药物５０％抑制率浓度联合应用，其细胞增殖抑制率为６６．３％，与２种药物半数抑制率浓度单用时细胞增殖抑制率相比，差异非常显著（Ｐ＜０．０１）。 结论阿霉素、地塞米松在一定浓度下能有效抑制体外成纤维细胞的生长，并提示二者合用可能是一种有价值的防治 ＰＶＲ的用药方式。     

汉防己甲素体外对正常结膜与翼状胬肉成纤维细胞抑制作用的比较

目的:观察汉防己甲素(tetrandrine,Tet)对正常结膜和翼状胬肉成纤维细胞抑制作用的异同。方法:在相同的培养条件下,传第3代的正常结膜与翼状胬肉成纤维细胞皆加入不同浓度的Tet,分别于加药后1和3d行MTT检测细胞的存活率。结果:用药后1d,4×10-5和2×10-5mol/LTet对正常结膜与翼状胬肉成纤维细胞存活率无显著性差异(P>0.05),其余各浓度的Tet对翼状胬肉成纤维细胞的抑制作用大于正常结膜成纤维细胞,正常结膜成纤维细胞的存活率大于翼状胬肉成纤维细胞(P<0.01)。用药后3d,4×10-5mol/LTet组翼状胬肉成纤维细胞的存活率大于正常结膜成纤维细胞(P<0.01);其余各浓度的Tet对两种成纤维细胞存活率无显著性差异(P>0.05)。结论:用药后3d,2×10-5mol/L及半数抑制量(10-5mol/L)以下浓度的Tet对翼状胬肉成纤维细胞起抑制作用,而对正常结膜成纤维细胞的影响较小。     

复方中药制剂抑制成纤维化增生作用的实验研究

目的观察复方中药制剂对结膜成纤维细胞增生的抑制作用,为临床防治增生性玻璃体视网膜病变提供新的治疗方法.方法以体外培养的兔结膜成纤维细胞作为研究对象,对照组用地塞米松、5-氟尿嘧啶、氢化可的松处理,实验组用丹苓(颗粒)冲剂及其君药(丹参、石决明)处理.48 h后,用MTT法观察成纤维细胞增生情况及其细胞形态变化特点.结果丹苓(颗粒)冲剂和丹参在一定浓度范围内可抑制成纤维细胞增生,且成剂量依赖关系;石决明对成纤维细胞增生未见明显抑制作用.5-氟尿嘧啶、氢化可的松在一定浓度范围内可明显抑制成纤维细胞增生;地塞米松在低浓度时轻度刺激成纤维细胞增生,但在高浓度时抑制成纤维细胞的增生.丹苓(颗粒)冲剂和丹参作用48 h后,部分成纤维细胞未能贴壁且收缩成圆形.结论丹苓(颗粒)冲剂可有效抑制成纤维细胞增生,有望成为防治增生性玻璃体视网膜病变的新药物.     

长期联合应用噻吗洛尔对结膜组织炎性反应的影响

目的:通过观察长期应用抗青光眼滴眼剂作用后结膜炎症反应的发生和炎性标记物ICAM-1在眼部的表达,探讨长期应用抗青光眼滴眼剂对结膜反应的影响。方法:从我科收住的原发性开角型青光眼患者90例,分为三组:应用5g/L噻吗洛尔与2g/L阿法根(联合用药组)30例;应用5g/L噻吗洛尔(单一用药组)30例;未用药组30例;单纯白内障患者作为空白对照组30例。按抗青光眼药物治疗时间及白内障患病时间分为三个时相(0～6mo,7～12mo,13～18mo)组,各个时相组10例,进行结膜上皮组织形态学观察及检测结膜上皮组织中ICAM-1炎性标记物的表达。结果:单一用药组在用药13～18mo时其球结膜组织学改变明显,表现为上皮形态不规则,表面不平或有上皮分离或增生,球结膜下纤维结缔组织排列不规则,小血管增多,巨噬细胞、淋巴细胞、成纤维细胞数增多。联合用药组在用药13～18mo时球结膜有明显的组织学改变,上皮有不同程度的增生或鳞状化生,上皮厚薄不均,表面不光滑,有上皮脱落,球结膜下组织中小血管增多,巨噬细胞、淋巴细胞、成纤维细胞数目明显增多、杯状细胞减少。单一用药组与联合用药组在13～18mo结膜组上皮组织中含ICAM-1抗原的阳性细胞数与其他组差别有统计学意义(P<0.01)。结论:长期联合应用抗青光眼药物可致结膜组织的炎性反应,结膜上皮组织中的炎性标记物ICAM-1表达明显增加。     

几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响

目的　研究几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响。方法　采用消化法培养结膜上皮细胞 ,组织块法培养结膜成纤维细胞。通过MTT比色法和细胞计数法测定几丁聚糖对两种细胞增殖的影响。结果　几丁聚糖在浓度 >0 0 3 %时能促进结膜上皮细胞的增殖 ,浓度为 0 12 %时促进作用最明显。结膜成纤维细胞在几丁聚糖浓度 >0 0 3 %时其增殖即受到抑制 ,抑制作用呈浓度依赖性。结论　几丁聚糖有望用于预防睑球粘连     

肝素抑制后发性白内障形成的细胞学研究

后发性白内障主要是白内障囊外摘除术后后囊膜上成纤维细胞的迁徙和增殖所致。我们用细胞培养的方法，通过细胞生长周期测定，细胞ＤＮＡ含量测定，细胞增长数及密度观察等，研究肝素对成纤维细胞生长的抑制作用，发现用肝素后成纤维细胞生长缓慢，肝素抑制成纤维细胞生长的抑制点在细胞周期的Ｇ１期和Ｓ期之间以及Ｍ期。     

正常球结膜与翼状胬肉成纤维细胞生长增殖状况的对比观察

目的对比观察相同的培养条件下翼状胬肉与正常结膜成纤维细胞的生长增殖状况,对两种成纤维细胞的生长增殖特性作出评价。方法用10%胎生牛血清的RPMI1640培养液,在相同的培养条件下分别培养正常球结膜与初发翼状胬肉组织成纤维细胞;倒置相差显微镜下观察培养的两种成纤维细胞的特点及生长增殖状况。对传第3代的两种成纤维细胞,分别于接种后2、5、8d行噻唑蓝[3-（4,5-dimethyl -2thiazol）-2,5-diphenyl-2H-tetrazolium bromid,MTT]比色试验,检测细胞增殖活力（细胞数）。结果翼状胬肉成纤维细胞较正常结膜成纤维细胞生长快,增殖旺盛;MTT检测结果显示：5d翼状胬肉成纤维细胞的细胞总数多于正常结膜成纤维细胞（P〈0.05）,8d更明显（P〈0.01）。结论体外翼状胬肉成纤维细胞的生长、增殖特性明显强于正常结膜成纤维细胞。提示治疗翼状胬肉药物的筛选和疗效评价的实验研究应直接应用翼状胬肉成纤维细胞为研究对象,才能作出较为符合客观实际的评价。     

Tryptase increases proliferative activity of human conjunctival fibroblasts through protease-activated receptor-2

PURPOSE: Tryptase that is released by mast cell degranulation has recently been thought to play a key role in wound healing in allergic bronchitis. Conjunctival fibroblasts secrete mediators and extracellular matrices that could exacerbate inflammation and papillary formation in allergic conjunctivitis. This study was conducted to investigate the effect of tryptase on the proliferation of conjunctival fibroblasts and studied whether this effect was mediated by protease-activated receptor (PAR)-2. METHODS: Conjunctival fibroblasts were cultured with or without tryptase (0.1 ng/mL to 1.0 microg/mL), and the proliferation rate was assessed after 48 hours. The effects of tryptase inhibitors (leupeptin, benzamidine) and a PAR-2 agonist (SLIGKV) were examined. The existence of PAR-2 mRNA and protein in conjunctival fibroblasts was examined by RT-PCR and Western blot analysis, respectively. The existence of PAR-2 in cultured conjunctival fibroblasts and conjunctival papillae from patients with vernal keratoconjunctivitis, as well as conjunctival tissue from normal subjects was examined by immunohistochemistry. RESULTS: Conjunctival fibroblast proliferation was upregulated by tryptase in a dose-dependent manner (P < 0.001). Leupeptin and benzamidine inhibited tryptase-induced fibroblast proliferation (P < 0.05), and SLIGKV mimicked tryptase's effect. PAR-2 mRNA and protein were detected in cultured conjunctival fibroblasts using RT-PCR and Western blot analysis. PAR-2 immunoreactivity in both the cultured conjunctival fibroblasts and in stromal cells in excised conjunctival tissues was observed. CONCLUSIONS: Tryptase increased conjunctival fibroblast proliferation and this response appeared to be mediated by PAR-2. Mast cells are the most likely source of tryptase in the conjunctiva and may play an important role in chronic exacerbations with conjunctival papillary formation in allergic conjunctivitis.     

Inhibition of human scleral fibroblast proliferation with heparin

Proliferation of fibroblasts is a serious problem in ocular trauma and surgical wound healing. Depending on the location of the injury, the growth of fibroblasts can lead to different problems. In glaucoma filtering surgery, fibroblast proliferation may contribute to scar tissue formation and premature wound closure. Fibroblastic growth in proliferative vitreoretinopathy may lead to the formation of preretinal membranes, which can contract, causing retinal detachment. In an effort to find a more effective method of inhibiting ocular fibroblast proliferation, we have investigated the effect of heparin, a sulfated polysaccharide, on the proliferation of fibroblasts obtained from the sclera of donor eyes. Heparin inhibits the incorporation of 3H-thymidine in a dose-dependent manner in the presence of fetal bovine serum (FBS). This inhibition is partially reversed by endothelial cell growth factor (ECGF). The heparin antagonist protamine sulfate causes a reversal of heparin inhibition and, in some instances, a significant increase in 3H-thymidine incorporation compared to serum controls. Heparin was equally effective in inhibiting cell proliferation in control and heparin-protamine sulfate-pretreated medium. These results were apparently unrelated to a direct toxic effect on cells, as a Trypan Blue exclusion assay showed no significant difference in viability when heparin treated cells were compared to control cells. Direct cell counts showed that heparin was effective in inhibiting cell proliferation over a long time period, but only if it was reinstilled every 2 days. Heparin treatment shows promise as a method for controlling fibroblast proliferation in the eye.     

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.     

Inhibition of vascular endothelial cell morphogenesis in cultures by limbal epithelial cells.

PURPOSE: To study the in vitro angiogenic activity of human conjunctival and limbal epithelial cells and conjunctival, limbal, and corneal fibroblasts in a three-cell-type coculture model. METHODS: Human umbilical vein endothelial cells (EC) were cocultured with epithelial cells, fibroblasts, or epithelial cells and fibroblasts to test their effect on EC morphogenesis. Neutralizing antibodies to some known angiogenic factors were added to the culture to see whether the EC morphogenesis may be blocked by a particular antibody. RESULTS: Conjunctival and limbal epithelial cells exhibited very little or no stimulatory effect on EC tube formation when examined in an EC- epithelial cell coculture system. In contrast, conjunctival, limbal, and corneal fibroblasts all promoted EC morphogenesis when examined under the same culture conditions. Fibroblast-induced EC morphogenesis was inhibited by addition of anti-vascular endothelial growth factor (VEGF) and/or anti-basic fibroblast growth factor (bFGF) antibodies to the culture medium. In the three-cell-type coculture system consisting of ECs, fibroblasts, and epithelial cells, limbal epithelial cells (but not conjunctival epithelial cells) exhibited a strong inhibitory effect on fibroblast-induced EC tube formation. CONCLUSIONS: The proangiogenic activity of ocular surface fibroblasts is probably mediated through a paracrine mechanism by VEGF and bFGF. Limbal epithelial cells, but not conjunctival epithelial cells, inhibit fibroblast-stimulated angiogenesis.     

The implications of the upregulation of ICAM-1/VCAM-1 expression of corneal fibroblasts on the pathogenesis of allergic keratopathy

OBJECTIVE: The present study investigated the expression of ICAM-1 and VCAM-1 on fibroblasts with interleukin (IL)-4 and/or tumor necrosis factor (TNF)-alpha stimulation and assessed the effect of eosinophil adhesion on fibroblast viability. METHODS: Primary cultured human corneal fibroblasts were incubated with IL-4, TNF-alpha, or their combination for 24 hours. Expression of ICAM-1 and VCAM-1 was examined by real-time quantitative PCR and flow cytometric analysis. Purified eosinophils were cocultured with activated fibroblasts, and the number of eosinophils adhered to fibroblasts and the number of damaged fibroblasts were counted using microscopy. In a separate trial, conjunctival and corneal impression cytology was performed on patients with atopic keratoconjunctivitis and corneal ulcers (eight eyes) to assess the status of the ocular surface epithelium and the presence of inflammatory cell infiltrates. RESULTS: Real-time quantitative PCR and flow cytometric analysis revealed that both mRNA and protein of VCAM-1 and ICAM-1 were upregulated by IL-4 and TNF-alpha. IL-5-primed eosinophils adhered to the corneal fibroblasts treated with IL-4 and TNF-alpha, and the fibroblasts were damaged by eosinophil adherence. Anti-ICAM-1 antibody and anti-VCAM-1 antibody inhibited the eosinophil adherence to fibroblasts and the fibroblast damage. Impression cytology revealed extensive infiltration of neutrophil and eosinophils among isolated ocular surface epithelial cells with advanced squamous metaplasia. CONCLUSIONS: Corneal fibroblasts expressed ICAM-1 and VCAM-1 when activated with IL-4 and TNF-alpha. Eosinophils can adhere to the activated fibroblasts and can induce subsequent fibroblast damage through these adhesion molecules. Eosinophil adhesion to fibroblasts may possibly contribute to the pathogenesis of severe persistent allergic corneal ulcers.     

Inhibition of cell proliferation by mitomycin C incorporated into P(HEMA) hydrogels

OBJECTIVES: The technique of mitomycin C (MMC) drug delivery and its application in glaucoma surgery are not standardized with resultant inconsistencies in the results. Also, one time application of MMC does not seem to have the same efficacy after glaucoma drainage device surgeries compared with trabeculectomies. This preliminary study examined the efficacy of a slow release form of MMC for its ability to inhibit cell proliferation in vitro. METHODS: MMC was incorporated into 1% P(HEMA) hydrogels using a redox polymerization method. For some experiments, unreacted low molecular weight components were removed from the hydrogels before the MMC was incorporated. Sterile disks (8 mm) of each polymer sample were affixed to 60 mm tissue culture dishes, and the dishes were inoculated with COS-1 cells or early passage human conjunctival fibroblasts. After 7 days in culture, the number of cells in each dish was determined. Cell morphology was assessed in replicate cultures after fixation and staining. RESULTS: Hydrogels with unreacted low molecular weight components slowed cell proliferation and induced morphologic changes. Early passage human conjunctival fibroblasts were more sensitive than COS-1 cells both to intrinsic contaminants in the hydrogels and to incorporated MMC. Once contaminants had been removed, MMC-loaded hydrogels inhibited conjunctival fibroblast proliferation in a dose-dependent fashion, with an IC50 of approximately 0.15 mg/g polymer. CONCLUSIONS: This study demonstrates that a slow release form of MMC can inhibit cell proliferation in vitro. Future experiments will focus upon the efficacy of this polymer-bound form during in vivo wound healing.