摘要
目的探讨甲状腺滤泡细胞自噬对慢性淋巴细胞性甲状腺炎(CLT)的影响及炎性因子干扰素⁃γ(IFN⁃γ)对甲状腺滤泡细胞自噬参与CLT的作用。方法2018年1—7月,选取新疆喀什地区第二人民医院24例CLT甲状腺切除的病人作为CLT组,并选择同期入院的24例单纯甲状腺肿(SG)甲状腺切除病人作为SG组;对人甲状腺滤泡细胞分离、培养、鉴定;流式细胞技术检测甲状腺组织中CD3+T细胞占比;放射性免疫法检测甲状腺滤泡细胞三碘甲状腺原氨酸(T3)、甲状腺素(T4)、甲状腺球蛋白(Tg)的水平,酶联免疫吸附试验(ELISA)试剂盒检测IFN⁃γ、环磷酸腺苷(cAMP)水平;蛋白质印迹法检测自噬相关蛋白Be⁃cline、微管相关蛋白1轻链3⁃Ⅱ(LC3B⁃Ⅱ)、哺乳动物雷帕霉素靶蛋白(mTOR)及不同浓度IFN⁃γ对Becline、LC3B⁃Ⅱ、mTOR蛋白的影响。结果成功分离出人甲状腺滤泡上皮细胞,倒置显微镜下观察,可见人甲状腺滤泡细胞在贴壁之前呈圆形或椭圆形或不规则形状,CLT病人组织切片可见多个淋巴生发中心,大量淋巴细胞浸润,甲状腺滤泡细胞萎缩。SG病人甲状腺组织甲状腺滤泡细胞未见明显炎症浸润。流式细胞分析结果显示,CLT组甲状腺滤泡上皮细胞内浸润的CD3+淋巴细胞数所占百分比(25.76±4.05)%明显高于SG组(10.35±4.21)%,两组间比较差异有统计学意义(n=24,P<0.001)。与SG组比较,CLT组中T3、T4、Tg、INF⁃γ及cAMP水平[甲状腺组织:(6.70±0.32)nmol/L,(44.05±0.39)nmol/L,(0.65±0.01)μg/L,(15.84±1.43)μg/L,(3.680±0.046)μmol/L;血清:(9.89±0.32)nmol/L,(39.25±0.27)nmol/L,(1.86±0.42)μg/L,(16.32±0.31)μg/L,(4.325±0.012)μmol/L]均升高,差异有统计学意义。CLT组织与SG组织相比自噬水平低下,表现为Becline,LC3B⁃Ⅱ蛋白低表达[Becline蛋白:(7.16±0.15);LC3B⁃Ⅱ蛋白:(0.82±0.01)],mTOR蛋白高表达(12.26±0.16)。IFN⁃γ干预后,甲状腺滤泡细胞中自噬蛋白Be⁃cline,mTOR,LC3B⁃Ⅱ被诱导,表现为Becline,LC3B⁃Ⅱ水平高表达[250 U/mL组:(7.80±0.07),(1.51±0.05);500 U/mL组:(8.50±0.10),(1.82±0.04);1000 U/mL:(9.16±0.14),(2.47±0.09)],mTOR水平低表达[250 U/mL组:(12.26±0.16);500 U/mL组:(0.84±0.13);1000 U/mL组:(9.66±0.07)],随着IFN⁃γ浓度的增加,诱导作用愈明显。结论甲状腺滤泡细胞自噬参与CLT的发展,且作用机制可能与炎性因子IFN⁃γ有关。
Objective To investigate the effect of autophagy of thyroid follicular cells on chronic lymphocyti thyroiditis and the impact of IFN⁃γon thyroid follicular autophagy involved in chronic lymphocytic thyroiditis.Methods From January 2018 to July 2018,24 patients with CLT thyroidectomy in Second People’s Hospital of Kashgar were selected as CLT group,and 24 patients with simple goiter(SG)thyroidectomy admitted in the same period were selected as SG group.The thyroid follicular cells were isolated,cultured and identified.The proportion of CD3+T cells in thyroid tissue was measured by flow cytometry;the levels of T3,T4 and Tg were detected in thyroid follicular cells by radioimmunoassay,and the levels of IFN⁃γ,cAMP were detected by ELISA kit;the expressions of Becline,LC3B⁃Ⅱ,mTOR protein were detected by western blot analysis and different expression Becline,LC3B⁃Ⅱ,mTOR protein by different concentrations of IFN⁃γ.Results Human thyroid follicular epithelial cells were successfully isolated.Under the inverted microscope,human thyroid follicular cells were found to be round or oval or irregular before adhering to the wall.Multiple lymphogerminal centers,a large number of lymphocytes infiltration and thyroid follicular cells atrophy were found in the tis⁃sue sections of CLT patients.There was no obvious inflammatory infiltration in thyroid follicular cells of SG patients.The results of flow cytometry showed that the percentage of CD3+lymphocytes infiltrated into thyroid follicular epithelial cells in CLT group(25.76±4.05)%was significantly higher than that in SG group(10.35±4.21)%,the difference between the two groups was statistically significant(n=24,P<0.001).Compared with SG group,T3,T4,Tg,IFN⁃γand cAMP levels in CLT group[thyroid tissue:(6.70±0.32)nmol/L,(44.05±0.39)nmol/L,(0.65±0.01)g/L,(15.84±1.43)g/L,(3.680±0.046)mol/L;serum:(9.89±0.32)nmol/L,(39.25±0.27)nmol/L,(1.86±0.42)g/L,(16.32±0.31)g/L,(4.325±0.012)mol/L]were all increased,the differences were statistically significant.The autophagy level of CLT tissue was lower than that of SG tissue,characterized by lower Becline,LC3BⅡexpression[Becline protein:(7.16±0.15);LC3BⅡprotein:(0.82±0.01)],and higher mTOR protein(12.26±0.16).After IFN⁃γintervention,the autophagic proteins becline,mTOR and LC3BⅡin thyroid follicular cells were induced,showing high expression of becline and LC3BⅡ[250 U/mL group:(7.80±0.07),(1.51±0.05);500 U/mL group:(8.50±0.10),(1.82±0.04);1000 U/mL(9.16±0.14)Densi⁃ty value,(2.47±0.09)],mTOR level low expression[250 U/mL group:(12.26±0.16);500 U/mL group:(0.84±0.13);1000 U/mL group:(9.66±0.07)],with the increase of IFN⁃γconcentration,the induction effect became more obvious.Conclusion Autophagy of thyroid follicular cells participates in the development of Hashimoto thyroiditis,and its mechanism may be related to IFN⁃γ.
作者
李北宁
于娟
LI Beining;YU Juan(Department of Endocrinology,Second People’s Hospital of Kashgar,Kashgar,Xinjiang Uygur Autonomous Region 844000,China)
出处
《安徽医药》
CAS
2020年第3期560-564,共5页
Anhui Medical and Pharmaceutical Journal
