γ-干扰素上调桥本甲状腺炎Fas表达介导甲状腺破坏

发布时间：2018-04-15 08:06

本文选题：桥本甲状腺炎 + IFN-γ　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的1.探讨桥本甲状腺炎(Hashimoto thyroiditis,HT)甲状腺组织Th1细胞因子γ-干扰素(interferon-γ,IFN-γ)与凋亡蛋白Fas之间的关系。2.探讨Th1细胞因子IFN-γ对大鼠甲状腺细胞系(FRTL-5细胞)Fas表达的调控。方法1.桥本甲状腺炎甲状腺组织来源于甲状腺病理组织切片,收集甲状腺腺瘤旁正常甲状腺组织作为正常对照组;采用免疫组织化学方法检测HT和正常甲状腺组织中IFN-γ及Fas的表达和分布。2.对体外培养的FRTL-5细胞,采用两种干预方法:(1)使用终浓度为40 ng/ml的IFN-γ分别干预FRTL-5细胞6、12、24、48、72h为IFN-γ刺激组,未经IFN-γ作用的FRTL-5细胞为对照组,收集细胞用于后期检测;(2)终浓度分别为5、10、20、40、80 ng/ml的IFN-γ干预FRTL-5细胞为IFN-γ刺激组,未经IFN-γ作用的FRTL-5细胞为对照组,培养48h后收集细胞用于后期检测。3.使用TRIzol提取FRTL-5细胞总RNA;逆转录试剂盒合成c DNA。应用Real-Time PCR检测各组细胞Fas m RNA的表达水平,基因表达量用2-ΔΔCt相对量表示。4.采用FACSVerse流式细胞仪,分析在不同浓度IFN-γ干预条件下FRTL-5细胞Fas蛋白表达。结果1.正常甲状腺组织内IFN-γ表达阴性,Fas阳性表达甲状腺滤泡细胞不明显。在被检的HT患者组织切片中,均见到浸润的淋巴细胞IFN-γ表达阳性,甲状腺滤泡细胞Fas表达阳性,且Fas蛋白阳性甲状腺滤泡细胞常见于淋巴细胞浸润区,无淋巴细胞浸润的区域,较少见到甲状腺滤泡细胞Fas表达阳性。2.IFN-γ上调FRTL-5细胞Fas m RNA表达,且存在时间-剂量依赖关系。FRTL-5细胞Fas m RNA的表达量与IFN-γ刺激时间相关,24h、48h、72h组皆显著高于对照组,差异有统计学意义(`x±s分别为6.39±2.02,7.45±4.33,11.40±5.05,P0.01);FRTL-5细胞Fas m RNA的表达量与IFN-γ刺激浓度相关,20ng/ml、40ng/ml、80ng/ml组皆显著高于对照组,差异有统计学意义(`x±s分别为4.01±1.48,10.53±1.54,3.62±2.22,P0.01)。3.流式细胞术检测示未经IFN-γ刺激的FRTL-5细胞Fas蛋白阳性表达率为10.1%,随着IFN-γ的剂量的增加,FRTL-5细胞Fas蛋白阳性表达率逐渐增高,20ng/ml的IFN-γ刺激组FRTL-5细胞Fas蛋白阳性表达率为28.3%,40ng/ml刺激组Fas蛋白阳性表达率为30.0%,80ng/ml刺激组Fas蛋白阳性表达率为38.9%。结论:桥本甲状腺炎甲状腺组织浸润的淋巴细胞分泌的IFN-γ通过上调甲状腺细胞Fas的表达,导致甲状腺组织的凋亡,与HT患者的甲状腺功能减退可能相关。
[Abstract]:Objective 1.To investigate the relationship between Th1 cytokine interferon- 纬 (IFN- 纬) and apoptotic protein (Fas) in thyroid tissue of Hashimoto thyroiditis (HT).To investigate the effect of Th1 cytokine IFN- 纬 on the expression of FAS in rat thyroid cell line FRTL-5.Method 1.The thyroid tissue of Hashimoto's thyroiditis originated from the pathological section of thyroid gland and the normal thyroid tissue adjacent to thyroid adenoma was collected as the normal control group.Immunohistochemical method was used to detect the expression and distribution of IFN- 纬 and Fas in HT and normal thyroid tissues.FRTL-5 cells cultured in vitro were treated with IFN- 纬 at the final concentration of 40 ng/ml for 72 h, and FRTL-5 cells without IFN- 纬 were treated with IFN- 纬 for 72 h.After 48 hours of culture, the cells collected were treated with IFN- 纬 as IFN- 纬 stimulation group and FRTL-5 cells without IFN- 纬 as control group. After 48 hours of culture, the cells were collected for later detection.Total RNAs of FRTL-5 cells were extracted by TRIzol and c DNA was synthesized by reverse transcription kit.The expression level of Fas m RNA was detected by Real-Time PCR, and the expression of Fas m RNA was expressed by the relative amount of 2- 螖 Ct.FACSVerse flow cytometry was used to analyze the expression of Fas protein in FRTL-5 cells treated with different concentrations of IFN- 纬.Result 1.The positive expression of IFN- 纬 in thyroid follicular cells was not obvious.The positive expression of IFN- 纬 in infiltrated lymphocytes and Fas expression in thyroid follicular cells were found in the tissue sections of HT patients. The positive expression of Fas protein in thyroid follicular cells was found in the lymphocytic infiltrating area, without lymphocytic infiltration.The positive expression of Fas in thyroid follicular cells. 2. IFN- 纬 upregulated the expression of Fas m RNA in FRTL-5 cells, and the expression of Fas m RNA in FRTL-5 cells was significantly higher than that in the control group for 72 h, and the expression of Fas m RNA in FRTL-5 cells was significantly higher than that in the control group.The expression of Fas m RNA in FRTL-5 cells was significantly higher than that in the control group ('x 卤s 6.39 卤2.02 卤7.45 卤4.33 卤11.40 卤5.05 卤5.05), respectively. The expression of Fas m RNA in FRTL-5 cells was significantly higher than that in the control group (`x 卤s = 4.01 卤1.48U 10.53 卤1.543.62 卤2.22g / ml P 0.01g / ml), and was significantly higher than that in the control group (`x 卤s = 4.01 卤1.48U 10.53 卤1.543.62 卤2.22g / ml P 0.01g / ml).Flow cytometry showed that the positive expression rate of Fas protein in FRTL-5 cells without IFN- 纬 stimulation was 10.1. With the increase of the dose of IFN- 纬, the positive expression rate of Fas protein in FRTL-5 cells gradually increased with 20 ng / ml IFN- 纬 stimulation. The positive expression rate of Fas protein in FRTL-5 cells was 28.3ng / ml.The positive expression rate of Fas protein in the stimulation group was 30.0% and the positive expression rate of Fas protein was 38.9% in the 80ng / ml stimulation group.Conclusion: IFN- 纬 secreted by infiltrated lymphocytes in Hashimoto thyroiditis leads to thyroid apoptosis by up-regulating the expression of Fas in thyroid cells, which may be related to hypothyroidism in HT patients.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R581.4

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