2015: Effects of fluoride on TH - Zebrafish

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199. 薛文娟 氟对斑马鱼下丘脑—垂体—甲状腺轴干扰效应的研究 《山西农业大学》 2015年收藏 | 手机打开
http://cdmd.cnki.com.cn/Article/CDMD-10 ... 623693.htm
Xue Wenjuan
"Effect of fluoride on the zebrafish hypothalamic-pituitary-thyroid axis"

【摘要】：目前已有大量的研究表明高氟会抑制鱼类的生长发育,但对其影响机制还尚无定论。内分泌的调节对机体的生长发育至关重要,下丘脑-垂体-甲状腺(HPT)轴是内分泌系统的成员之一。为探究氟是否通过影响鱼类HPT轴影响鱼类正常生长发育,本试验选用两月龄健康斑马鱼,随机分为3组,分别为对照组(0mg/L),40mg/L和80 mg/L氟暴露组。分别在氟暴露45 d和90 d时,对斑马鱼的生长发育状况进行记录,并通过HE染色对斑马鱼甲状腺进行组织病理学观察,用酶联免疫ELISA检测鱼体内T3和T4激素水平,最后对斑马鱼头部和肝脏组织提取总RNA,应用实时荧光定量PCR方法检测HPT轴相关基因的表达。试验结果如下：1.对生长发育分析结果显示,氟暴露45 d时,雌雄斑马鱼体重、增重率、特定增重率及肥满度均表现为40 mg/L促进80 mg/L抑制,体长及增长率均表现为随着浓度的升高生长抑制作用越大。氟暴露90天时除雄鱼肥满度外,其他各性别各指标均随着浓度的上升而下降。2.甲状腺组织病理学结果显示,氟暴露可造成斑马鱼甲状腺滤泡的病变,包括滤泡肿大、上皮细胞呈扁平状、增生和内褶,胶质萎缩或呈泡沫状等。且随着暴露浓度增大和时间的延长,甲状腺滤泡的病变越严重；肝脏组织病理学结果显示,40 mg/L氟暴露组在45 d时就发生了脂肪样变、血栓等病变,且暴露浓度越高越严重。在氟暴露90d时,雌鱼肝脏脂肪样变、血栓情况加深,雄鱼80 mg/L组肝细胞大量溶解,细胞核聚集,肝组织严重受到损伤。3.甲状腺激素T3和T4 ELISA结果显示,氟暴露可影响斑马鱼体内甲状腺激素T3和T4的水平及T3/T4的比值,且影响程度随暴露时间、浓度及性别不同而不同。总体上分析,暴露45 d时,氟可提高T3和T4水平,但T3/T4比值降低。氟暴露90 d时,除80mg/L组雌鱼T3水平和T3/T4比值降低外,其他各组T3水平和T3/T4比值均升高,但T4水平均降低。4.对氟暴露后斑马鱼HPT轴CRH、TSH、TG、NIS, TTR、DIO1、DIO2、TRα、TRβ和iUGT1ab等10个基因的表达量结果分析显示：氟暴露45 d时,雌斑马鱼40 mg/L组除CRH与TRRα外,其他8个基因表达均显著下调,80 mg/L组除TG、NIS、TTR及TRα外,其他6个基因表达均显著下调；雄鱼氟暴露45 d时,40mg/L组除CRH, NIS及D1O1外，其他7个基因表达均显著上调,80 mg/L组除UGT1ab与TTR基因外,其他8个基因表达均显著上调。氟暴露90 d时,雌鱼40 mg/L组DIO1、DIO2 mRNA和80 mg/L组TG、DIO1、 DIO2、TRα、TRβ mRNA表达显著上调,40 mg/L组TTR、TRα和80 mg/L组UGT1ab mRNA表达显著下调,其余都未有显著变化；雄鱼40 mg/L组TSH、TG、DIO1、TTR、TRα和80mg/L TG、DIO1、TRα mRNA表达均显著上调,40mg/L组CRH和80mg/L组CRH、DIO2、TTR、UGT1ab mRNA表达显著下调。试验结果表明,氟暴露可通过影响斑马鱼甲状腺和肝脏组织形态、甲状腺激素水平影响其生长发育,且随着暴露浓度的增加及暴露时间的延长影响逐渐增加。氟对斑马鱼HPT轴10个相关基因的表达均有影响,且影响的大小与暴露浓度、时间和性别有关,提示氟可通过影响斑马鱼HPT轴中基因的表达来干扰机体内甲状腺激素的含量,进而影响机体的生长发育。本试验研究结果为氟影响斑马鱼生长发育机制提供基础依据。
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S917.4

Xue Wenjuan - "Effect of fluoride on the zebrafish hypothalamic-pituitary-thyroid (HPT) axis" (Shanxi Agricultural University, 2015)

Abstract:
A large number of studies have shown that high fluoride levels can inhibit the growth and development of fish, but the underlying mechanisms remain unclear. Endocrine regulation is essential for organismal growth and development, and the hypothalamic-pituitary-thyroid (HPT) axis is a key component of the endocrine system. To investigate whether fluoride affects normal fish development by interfering with the HPT axis, healthy 2-month-old zebrafish were randomly divided into three groups: a control group (0 mg/L fluoride), and two fluoride exposure groups (40 mg/L and 80 mg/L).

At 45 and 90 days of fluoride exposure, the growth and development of the zebrafish were recorded. Histopathological examination of the thyroid gland was performed using HE staining. ELISA was used to measure T3 and T4 hormone levels. Total RNA was extracted from the heads and livers of the fish, and real-time quantitative PCR was used to assess expression of HPT axis-related genes.

Results:

Growth and Development:

After 45 days, in both male and female zebrafish, 40 mg/L fluoride exposure promoted body weight, weight gain rate, specific growth rate, and condition factor, while 80 mg/L inhibited these indices. Body length and growth rate decreased with increasing fluoride concentration. After 90 days, all indices (except male condition factor) decreased with increasing fluoride concentration.

Histopathology:

Fluoride exposure caused pathological changes in thyroid follicles, including follicular enlargement, epithelial flattening, hyperplasia, infolding, colloid atrophy, and foamy colloid. Severity increased with exposure time and concentration. In the liver, 40 mg/L fluoride exposure for 45 days caused fatty degeneration and thrombosis. At 90 days, female fish had worsened liver changes, while in males exposed to 80 mg/L, massive hepatocyte lysis, nuclear aggregation, and severe tissue damage were observed.

Thyroid Hormones (T3 and T4):

Fluoride exposure affected T3 and T4 levels and the T3/T4 ratio, with differences based on time, concentration, and sex. After 45 days, T3 and T4 levels were elevated, but the T3/T4 ratio declined. After 90 days, T4 levels decreased overall; T3 levels and the T3/T4 ratio increased in most groups, except in 80 mg/L females where both decreased.

Gene Expression (HPT axis):

Ten genes were assessed: CRH, TSH, TG, NIS, TTR, DIO1, DIO2, TRα, TRβ, and UGT1ab.

At 45 days:

Female fish: in the 40 mg/L group, 8 of 10 genes were significantly downregulated (except CRH and TRα). In the 80 mg/L group, 6 genes were downregulated (except TG, NIS, TTR, TRα).

Male fish: in the 40 mg/L group, 7 genes were significantly upregulated (except CRH, NIS, and DIO1). In the 80 mg/L group, 8 genes were upregulated (except UGT1ab and TTR).

At 90 days:

Female fish: DIO1 and DIO2 mRNA in the 40 mg/L group and TG, DIO1, DIO2, TRα, TRβ mRNA in the 80 mg/L group were significantly upregulated. TTR and TRα (40 mg/L), and UGT1ab (80 mg/L) were downregulated.

Male fish: TSH, TG, DIO1, TTR, TRα (40 mg/L) and TG, DIO1, TRα (80 mg/L) were upregulated. CRH (40 mg/L), and CRH, DIO2, TTR, UGT1ab (80 mg/L) were downregulated.

Conclusion:

Fluoride exposure affects zebrafish growth and development by altering thyroid and liver morphology and thyroid hormone levels. The impact increases with concentration and duration. Fluoride influences the expression of HPT axis-related genes in a sex-, dose-, and time-dependent manner. These findings suggest fluoride can disrupt thyroid hormone homeostasis via interference with the HPT axis, thereby impairing growth and development in zebrafish. This study provides foundational data for understanding the mechanism of fluoride’s effects on zebrafish development.

Degree-granting institution: Shanxi Agricultural University
Degree level: Master's
Year: 2015
Classification code: S917.4