基质蛋白Fibulin-5在眼眶lgG4相关性疾病中的表达

发布时间：2018-03-11 05:28

本文选题：Fibulin-5　切入点：IgG4-ROD　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨基质蛋白Fibulin-5与眼眶IgG4相关性疾病纤维化、炎症、硬化的关系。方法选取2014年3月至2016年6月在解放军第474医院收治的患者因眼眶IgG4相关性疾病(IgG4-related disease、IgG4-RD)(眼眶泪腺肿物摘除术中采集)的泪腺组织标本20例20眼(IgG4-ROD组),收集同期的20例20眼正常的泪腺组织标本(眼眶开放性粉碎性骨折清创术中采集)作为对照组。所有患者术前均未接受免疫和化学治疗,且在年龄方面具有可比性(P0.05)。在获得医院伦理委员会同意并取得患者的知情同意后,在术中所有组织均在行眼眶泪腺肿物摘除中留取。把泪腺组织标本分为大小均为约0.3cm×0.3cm×0.3cm三块,生理盐水充分冲洗干净。取一块组织放入4%多聚甲醛固定24h,然后取出放入自动组织脱水机,随后用石蜡包埋,制成蜡块备用,另外两块放入锡箔纸包裹和装有Trizol EP管中,放入-80℃冰箱保存备用。1对经-80℃冰箱冻存的标本采用实时荧光定量PCR检测Fibulin-5及mRAN表达和采用Western blot法检测Fibulin-5及蛋白表达,数据采用SPSS19.0统计软件进行统计学分析,数据资料经Shapiro-Wilk检验呈正态分布,结果进行t检验,均数±标准差(X±s)表示,组间均数经Levenne检验,以P0.05定为有显著性差异。2对经甲醛固定的标本采用免疫组织化学两步法检测Fibulin-5及蛋白的表达,其表达情况根据细胞染色面积和染色强度进行综合评分,所得结果采用Fisher确切概率法,相关性检验采用Sperman相关性分析法,以P0.05定为有显著性差异。结果1.实时荧光定量PCR检测:眼眶IgG4相关性疾病与正常泪腺的患者相比,眼眶IgG4相关性疾病泪腺组织中Fibulin-5mRNA较正常泪腺明显增高。IgG4-ROD组中Fibulin-5mRNA的表达水平为3.69±1.72,而对照组中1.69±0.45,依据P0.05的检验水准,两组比较差异有统计学意义(P0.05)。2 Western印迹法检测:Fibulin-5蛋白与Fibulin-5mRNA表达变化趋势较为一致。眼眶IgG4相关性疾病与正常泪腺的患者相比,眼眶IgG4相关性疾病泪腺组织中Fibulin-5蛋白较正常泪腺明显增高。IgG4-ROD组中Fibulin-5蛋白的表达为0.55±0.12,而对照组中Fibulin-5蛋白表达为0.19±0.11,按照P0.05的检验水准,两组比较差异有统计学意义(P0.01)。3免疫组织化学染色检测:Fibulin-5蛋白主要分布于细胞胞浆,阳性染色为黄色或棕黄色呈束状弥漫分布。免疫组化结果显示,IgG4-ROD组中泪腺细胞紊乱、部分破坏,染色为黄色、棕黄色的纤维化细胞排列紊乱,对照组中泪腺腺管规则,细胞规则排列,核染蓝色。IgG4-ROD组中Fibulin-5蛋白阳性率80%(16/20),对照组中Fibulin-5蛋白阳性率22.5%(9/40),按照P0.05的检验水准,两组比较差异有统计学意义,IgG4-ROD组Fibulin-5蛋白的阳性表达显著高于对照组。结论1在Fibulin-5mRNA和蛋白水平的检测结果中均显示,眼眶IgG4相关性疾病与正常泪腺的患者相比,眼眶IgG4相关性疾病泪腺组织中Fibulin-5mRNA和蛋白表达较正常泪腺显著升高。
[Abstract]:Objective to study the expression of matrix protein Fibulin-5 and IgG4 orbital diseases associated with fibrosis, inflammation, atherosclerosis. Methods from March 2014 to June 2016 in the 474th Hospital of PLA for patients with orbital IgG4 related disease (IgG4-related disease (IgG4-RD) of lacrimal gland tumor excision by collecting) lacrimal gland tissue specimens of 20 cases of 20 eyes (IgG4-ROD group). Collected in the same period in 20 cases 20 eyes of normal lacrimal gland tissue specimens (eyes open comminuted fracture debridement in the acquisition) as the control group. All patients were not receiving preoperative immune and chemical treatment, and is comparable in terms of age (P0.05). The hospital ethics committee and informed consent were obtained agree, in all the tissues in the lacrimal gland tumor in the lacrimal gland tissue specimens. The specimens were divided into size were about 0.3cm * 0.3cm * 0.3cm three, saline flush Clean. Take a piece of tissue were fixed in 4% paraformaldehyde 24h, then take out into the automatic tissue dehydration machine, followed by paraffin, paraffin spare, another two pieces into the package with Trizol foil and EP tube, placed in the refrigerator on the standby.1 -80 C by -80 C refrigerator frozen samples real time fluorescence quantitative PCR detection of Fibulin-5 and the expression of mRAN and blot using Western method to detect the expression of Fibulin-5 and protein data were statistically analyzed using SPSS19.0 statistical software, data by Shapiro-Wilk test of normal distribution, the results of t test, mean standard deviation (X + s) said that the two groups were tested by Levenne, to P0.05 is a significant difference of.2 expression by immunohistochemical detection of Fibulin-5 protein in two steps and formalin fixed specimens, the expression of cells according to the staining area and intensity in comprehensive evaluation, The results obtained using the Fisher exact test, correlation analysis using Sperman correlation analysis method, using P0.05 as a significant difference. The results of 1. real time fluorescence quantitative PCR detection: compared with normal orbital IgG4 related diseases of lacrimal gland in patients with orbital IgG4 related diseases of lacrimal gland tissue Fibulin-5mRNA than normal lacrimal gland significantly increased the expression level of Fibulin-5mRNA in the.IgG4-ROD group is 3.69 + 1.72, 1.69 + 0.45 in the control group, according to the P0.05 standard test, there was significant difference between two groups (P0.05): Fibulin-5 Western blot detection of.2 protein and Fibulin-5mRNA expression of the trend is more consistent. Compared with the orbital IgG4 disease patients and normal lacrimal gland, lacrimal gland tissue expression of IgG4 related diseases Fibulin-5 protein Fibulin-5 protein was significantly higher than normal lacrimal gland in.IgG4-ROD group was 0.55 + 0.12, while the control group in Fibulin-5 Protein expression was 0.19 + 0.11, according to the P0.05 standard test, there was significant difference between two groups (P0.01).3 immunohistochemical staining: Fibulin-5 protein mainly distributed in the cytoplasm, the positive staining for yellow or brown bundles of diffuse distribution. Immunohistochemical results show that the lacrimal gland cell disorder, IgG4-ROD group in the part of the damage, was yellow, brown and yellow fibrosis cells arranged in disorder, the control group in the lacrimal gland tube rules, cells arranged regularly, nuclear staining positive rate of Fibulin-5 protein in 80%.IgG4-ROD Blue Group (16/20). The positive rate of the control group in Fibulin-5 protein 22.5% (9/40), according to the P0.05 standard test, the comparison between the two groups there was significant difference in the positive expression of IgG4-ROD protein in Fibulin-5 group was significantly higher than the control group. Conclusion: 1 at the level of Fibulin-5mRNA and protein detection results showed that the orbital IgG4 related diseases and normal lacrimal gland The expression of Fibulin-5mRNA and protein in the lacrimal gland tissue of the orbital IgG4 related disease was significantly higher than that of the normal lacrimal gland.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.5

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