NCT02160327Unknown不适用
The Role of Cytokines and Mast Cell in the Pathogenesis of Ocular Surface Inflammation Diseases Including Superior Limbic Keratoconjunctivitis, Conjunctivochalasis, and Dry Eye
适应症
试验速览
- 阶段
- 不适用
- 入组人数
- 105
- 试验地点
- 1
- 主要终点
- Tear cytokines change measured by ELISA
研究概览
简要总结
The specific aims of the the investigators studies are as follows:
- To collect the tear samples from patients with different ocular surface disorders, including SLK, conjunctivochalasis, and keratoconjunctivitis sicca (KCS).
- To evaluate the differential expression of tear cytokines and pH values between different ocular surface disorders.
- To collect the surgical conjunctival specimens from the patients with SLK and conjunctivochalasis.
- To evaluate the factors inducing mast cell migration and how mast cell is activated in SLK via surgical specimens and cultivated fibroblast.
详细描述
- Detection of various tear cytokine levels in patients with superior limbic keratoconjunctivitis, conjunctivochalasis, and keratoconjunctivitis sicca
- Basal tear samples will be collected atraumatically from the inferolateral tear meniscus by using glass capillary tubes. Care is taken to avoid touching the corneal and conjunctival surface. Approximately 30 μl of tear samples is obtained, and then the samples are centrifuged for 10 minutes at 1500 rpm (225g). Tear sample are placed in microtubes (Eppendorf) and stored at -70°C. IL-1β, IL-17, TNF-α, and IFN-γ levels will be measured by enzyme-linked immunosorbent assay kits.
- Immunohistochemistry stain method for conjunctival specimens - The conjunctiva will be collected from SLK and conjunctivochalasis patients who received superior and inferior bulbar conjunctival resection as needed. The redundant conjunctiva noted after peritomy during cataract or retinal surgery will be excised to serve as a normal control. The conjunctival specimens of all the SLK, conjunctivochalasis, and control groups are sent to the pathology department for routine paraffin embedding and hematoxylin and eosin staining. The surgical specimens are placed into 4% paraformaldehyde at 4 C for one to two days and then stored in 1% sodium phosphate buffer at 4 C. They then are embedded in paraffin. To enhance tissue adhesion, 5-micrometer thick sections are mounted on glass slides pre- treated with Vectabond (Vector Laboratories, Burlingame, California, USA). The paraffin sections are deparaffinized, rehydrated and washed with phosphate-buffered saline (PBS; pH 7.4). Antigen retrieval is performed by immerse the sections in 0.1% trypsin in water bath for 15 mins. The endogenous peroxidase activity is quenched by placing the slides in 3% hydrogen peroxide. The slides are then blocked with serum followed by incubation with primary antibodies. Primary antibodies (TSL and SCF) are incubated overnight at 4°C. Thereafter, incubation with biotin conjugated secondary antibody is performed at room temperature for 60 min. Further, incubation with avidin-biotin horseradish peroxidase complex is then performed at room temperature as well for 30 min using Vectastain elite® ABC kit (Vector labs, CA USA). The location where the enzyme is bound will be visualized by the addition of the substrate 3,3'-diaminobenzidine (DAKO, CA, USA), a chromogen which produces a brown insoluble precipitate. The sections are then counterstained with haematoxylin, which is followed by dehydration and mounting with Vecta Mount Mounting medium was used to evaluate the slides.
研究设计
- 研究类型
- Observational
- 时间视角
- Prospective
入排标准
- 年龄范围
- 20 Years 至 85 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 是
入选标准
- 未提供
排除标准
- 未提供
结局指标
主要结局
Tear cytokines change measured by ELISA
时间窗: Tear will be collected before treatment, one month and six months follow-up visits
次要结局
未报告次要终点
研究者
研究点 (1)
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