【摘要】：目的:1.構建PCB118持續(xù)低濃度損害特點的大鼠甲狀腺模型;2.觀察PCB118對大鼠甲狀腺形態(tài)及功能的影響;3.探討PCB118誘導甲狀腺細胞功能障礙的可能機制。方法:1.將PCB118溶于玉米油,配成相應濃度的PCB118玉米油溶液,用于大鼠腹腔注射(給藥容積0.5m L/kg)。選取40只清潔級健康成年雄性Wistar大鼠,體重(200±10)g,按PCB118給予劑量隨機分為溶劑對照組(0μg/kg/d)、低劑量組(10μg/kg/d)、中劑量組(100μg/kg/d)、高劑量組(1000μg/kg/d)4組,每組各10只,每周連續(xù)給藥5天(每天1次),每日稱重后繪制大鼠周齡體重生長曲線,造模13周后心臟采血處死,立即取出甲狀腺組織,按實驗要求分別保存。血樣經(jīng)4℃離心后分離血清,按需分裝后于-70℃凍存。2.取大鼠一側甲狀腺組織經(jīng)4%多聚甲醛固定后石蠟包埋,連續(xù)切片后進行HE染色,光鏡下觀察甲狀腺病理結構變化并拍照。3.取1mm3大小甲狀腺組織,用5%戊二醛-1%鋨酸雙重固定,經(jīng)塊染、脫水、浸漬、包埋、聚合后,超薄切片,透射電鏡下觀察大鼠甲狀腺超微結構變化并拍照。4.采用放射性免疫法測定大鼠血清FT3、FT4、TSH、TG、TGAb及TPOAb水平,參照相應RIA分析試劑盒說明書進行。5.提取甲狀腺組織總RNA后,RT-q PCR方法檢測大鼠甲狀腺組織NIS、TG、TPO、AKT、Fox O3a m RNA表達變化。6.提取甲狀腺組織總蛋白后,Western blotting方法檢測大鼠甲狀腺組織AKT、p-AKT及p-Fox O3a蛋白表達變化。結果:1.腹腔PCB118注射給藥后,實驗組與對照組比較,大鼠行為無明顯異常改變,正常攝食、飲水,無明顯腹瀉。各組間體重無明顯差異(P0.05)。2.光鏡下對照組大鼠甲狀腺濾泡腔完整,為單層立方上皮,腔內(nèi)充滿均勻膠質。各實驗組均可見甲狀腺組織增生、濾泡擴張、濾泡腔塌陷、上皮脫落、膠質缺失、間質纖維化、小血管增生及纖維素樣壞死等,損傷程度隨著PCB118劑量的增加而明顯加重。3.透射電鏡下對照組甲狀腺細胞頂端膜微絨毛豐富、胞質中內(nèi)質網(wǎng)排列整齊、緊密,線粒體嵴清晰可見,形態(tài)正常;各實驗組甲狀腺濾泡上皮細胞核周間隙增加,內(nèi)質網(wǎng)擴張伴空泡化,線粒體腫脹,部分伴空泡化、線粒體嵴消失、密度下降,損傷程度亦隨PCB118劑量的增加而明顯加重。4.隨著PCB118劑量增加,大鼠血清FT3、FT4、TSH、TG濃度均逐漸降低,高劑量組FT3濃度明顯低于對照組和低劑量組(P0.05);各實驗組血清FT4、TSH濃度均明顯低于對照組(P0.05);中、高劑量組血清TG水平明顯低于對照組(P0.05)。與對照組比較,低、中劑量組血清TGAb、TPOAb濃度顯著升高(P0.05);5.與對照組比較,甲狀腺組織NIS和TG m RNA表達水平在中、高劑量組明顯減低(P0.05),但在低劑量組則無明顯改變。與低劑量組比較,中、高劑量組TG m RNA明顯減低,差異有統(tǒng)計學意義(P0.05)。與對照組比較,中劑量組甲狀腺組織TPO m RNA表達水平明顯上升(P0.05),高劑量組TPO m RNA表達水平明顯低于中劑量組(P0.05)。6.(1)與對照組比較,甲狀腺組織AKT m RNA表達水平在高劑量組明顯升高(P0.05),但在低、中劑量組則無明顯改變。各實驗組甲狀腺組織Fox O3a m RNA表達水平與對照組比較無明顯改變。(2)與對照組比較,甲狀腺組織AKT蛋白表達水平在中、高劑量組明顯升高(P0.05),但在低劑量組則無明顯改變;p-AKT蛋白表達僅在高劑量組明顯升高(P0.05);各實驗組甲狀腺組織p-Fox O3a蛋白表達與對照組比較均明顯升高(P0.05)。結論:1.慢性低濃度PCB118持續(xù)暴露可造成大鼠甲狀腺組織形態(tài)結構異常及細胞功能障礙,并存在一定劑量效應關系;2.慢性低濃度PCB118可誘導甲狀腺自身免疫反應,導致血清甲狀腺自身抗體水平增加;3.慢性低濃度PCB118可引起甲狀腺激素重要功能基因NIS及TG m RNA表達減低,亦存在一定劑量效應關系;4.慢性低濃度PCB118可以激活PI3K/AKT通路,促使Fox O3a磷酸化水平增加。
[Abstract]:Objective:1. To construct a rat thyroid model with the characteristics of persistent low-concentration damage of PCB118; The effect of PCB 118 on the thyroid morphology and function of rats was observed. To explore the possible mechanism of PCB118 to induce the dysfunction of thyroid cell. Method:1. The PCB 118 is dissolved in corn oil to prepare a corresponding concentration of the PCB 118 corn oil solution for intraperitoneal injection of the rat (administration volume of 0.5 m L/ kg). 40 healthy adult male Wistar rats, weighing (200 to 10) g, were randomly divided into a solvent control group (0. mu.g/ kg/ day), a low-dose group (10. mu.g/ kg/ day), a medium-dose group (100. mu.g/ kg/ day), a high-dose group (1000. mu.g/ kg/ d) of 4 groups, each group of 10 rats, The body weight growth curve of the rat was drawn daily for 5 days (once a day), and the body weight growth curve of the rat was drawn after daily weighing. After the model was made for 13 weeks, the heart was sacrificed and the thyroid tissue was taken out immediately and stored according to the requirements of the experiment. The blood samples were centrifuged at 4 & deg; C and the serum was isolated and stored at -70 鈩，

本文編號：2503158