1999 - Effects of fluoride on fetal thyroid - Full paper

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贵州省氟病区胎儿甲状腺体视学参数测定
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数理医药学杂志 1999年第1期第0卷 临床科研分析

作者：于燕妮　刘家骝

单位：贵阳医学院病理教研室　贵州550004

关键词：氟中毒；胎儿；体视学；甲状腺滤泡上皮细胞

　　摘　要　对贵州省氟病区胎儿甲状腺滤泡上皮细胞进行体视学参数测定。结果发现，氟病区胎儿骨氟含量明显升高，与非氟病区胎儿相比，差异有显著性(P＜0.05)。体视学参数测定结果发现，甲状腺滤泡上皮细胞线粒体平均周长、平均截面积、平均体密度及平均体积均明显增大，与非病区胎儿相比，差异有显著性(P＜0.05)。

　　氟中毒对甲状腺有损害作用，表现甲状腺吸131I率升高，血中T3、T4、TSH含量变化，甲状腺滤泡上皮细胞出现形态学改变［1～4］。但氟中毒对甲状腺超微结构体视学影响的研究报道极少。本文拟对氟病区胎儿甲状腺超微结构进行体视学观测，以期提供氟中毒时甲状腺损伤的超微结构形态学定量测定参数的实验依据。

1　材料与方法

　　1.1　材料

　　1.1.1　病区胎儿　10例氟病区胎儿均取自贵州省织金县氟病发病区孕妇，其长期居住于高氟区，食用高氟粮食，患有不同程度氟斑牙，水囊引产后按孕妇停经史及胎儿顶臀长计算胎龄，胎龄均为5～7月。

　　1.1.2　非病区胎儿　10例非氟病区胎儿均取自贵州省贵阳市非氟病流行区。孕妇无氟中毒表现。胎儿取材及胎龄计算方法同上。胎龄均为5～7月。

　　1.2　检查项目与方法

　　1.2.1　孕妇尿及血清氟含量　取孕妇清晨空腹静脉血及晨尿。用氟离子选择电极法测是血清及尿氟含量。

　　1.2.2　胎儿骨组织氟含量　取胎儿右股骨剔去软组织，烘干、炭化及灰化，用上述方法测定骨氟含量。

　　1.2.3　胎儿甲状腺超微结构观测　水囊引产之胎儿娩出后，即取右侧甲状腺组织1mm3，固定于3%戌二醛中，常规电镜标本制片后观察。体视学参数测定氟病区与非氟病区胎儿各3例。每例组织块切片后捞两个铜网，电镜下择用结构清晰、反差较明显的铜网，在15，000放大倍数下，每网随机摄取甲状腺滤泡上皮细胞，每组照片30张，底片进行光学放大，放大倍数为两部，即共放大30，000倍。用单方测试格手工点计数法，测试格点距为5mm，每张照片计486点，每组30张照片共计14，580点。以测试格所覆盖的单位胞浆为参照系，计算公式参照郑富盛主编《细胞形态立体计量学》［6］。测定参数有：细胞质体密度(VVC);细胞核体密度(VVN)、比表面(δN)、表面积密度(SVN)、截面平均周长()、平均截面积()、平均体积()；线粒体体密度(VVM)、比表面(δM)、截面平均周长()、平均截面积()、数密度(NVM)、平均体积()。所得数据采用t检验或t检验统计处理。

2　结果

　　2.1　孕妇血清、尿氟含量和胎儿骨氟含量

　　表1显示，氟病区孕妇尿氟含量高于非病区孕妇，差异有显著性(P＜0.025)。氟病区胎儿骨氟含量高于非病区胎儿，差异非常显著(P＜0.01)。

表1　孕妇血清、尿氟和胎儿骨氟含量　(±S)

组别 例数 孕　　　妇 胎　儿
尿氟(μg/ml) 血清氟(μg/ml) 骨氟(μg/g)
非病区 10 1.67±0.82 0.41±0.15 2.50±0.11
氟病区 10 4.37±2.94 0.55±0.21 2.77±0.25
P值 　 ＜0.025 ＞0.05 ＜0.01
　　2.2　电镜观察

　　电镜发现，氟病区胎儿甲状腺滤泡上皮细胞微绒毛断裂、减少，甚至消失，胞质中线粒体明显肿胀、增大，电子致密度降低，线粒体嵴断裂，甚至消失。

　　2.3　甲状腺滤泡上皮细胞体视学参数

表2　氟病区与非病区胎儿甲状腺滤泡上皮细胞体视学参数比较(±S)

参数 单位 非病区 氟病区 P值
VVC μm0 0.25±0.08 0.60±0.11 ＞0.05
VVN μm0 0.18±0.06 0.16±0.06 ＞0.05
δN μm-1 0.63±0.15 0.58±0.09 ＞0.05
SVN μm-1 0.09±0.02 0.07±0.02 ＞0.05
μm 8.42±0.30 8.21±0.26 ＞0.05
μm2 16.15±0.51 15.49±1.31 ＞0.05
μm3 48.62±10.39 62.11±11.63 ＜0.05
表2显示，与非病区相比，氟病区胎儿甲状腺滤泡上皮细胞核平均体积增大，差异有显著性(P＜0.05)，余参数两组间均无显著性。
表3　氟病区与非病区胎儿甲状腺滤泡上皮线粒体体视学参数比较(±S)

参数 单位 非病区 氟病区 P值
VVM μm0 0.09±0.07 0.15±0.08 ＜0.01
δM μm-1 4.68±0.75 5.07±0.76 ＞0.05
μm 23.53±0.95 34.52±0.51 ＜0.05
μm2 5.07±0.71 9.08±0.87 ＜0.05
NVM μm-3 0.15±0.03 0.08±0.02 ＜0.05
μm3 24.36±4.10 51.37±6.29 ＜0.05
　　表3显示，氟病区胎儿甲状腺滤泡上皮细胞线粒体体密度、平均周长、平均截面积及平均体积均明显高于非病区胎儿，有显著性差异(P＜0.05)；数密度则氟病区明显低于非病区(P＜0.05)。

3　讨论

　　本实验收集的胎儿均来自贵州省氟病区孕妇，这些孕妇表现氟斑牙、尿氟明显升高(P＜0.05)，说明其体内有过量氟蓄积。过量氟经胎盘进入胎儿体内，对甲状腺发生作用。

　　我们以往的实验发现［1］，体内过量的氟可作用于甲状腺滤泡上皮细胞，引起血中T3、T4降低，TSH升高，吸131I率明显升高，并出现滤泡上皮细胞变性等形态学变化。本实验进一步用体视学方法检测甲状腺滤泡上皮细胞的超微结构，发现氟病区胎儿甲状腺滤泡上皮细胞线粒体平均周长、平均截面积、体密度及平均体积增大，表明线粒体发生了肿胀，由于线粒体肿胀，严重者崩解，所以导致线粒体数密度减少。

　　有资料表明［5］，氟的主要亚细胞靶结果是线粒体，通过对线粒体结构造成损伤，致使线粒体功能受到影响，从而破坏整个细胞的结构与功能。本文的超微结构形态计量学参数测定进一步证实了这一点。至于氟对线粒体结构的损伤机理有待深入研究。

　　从本实验结果看出，氟损伤甲状腺滤泡上皮细胞，出现超微结构变化，可推测过量氟可影响人胎甲状腺功能。因此，作者认为，应注重对氟病区婴幼儿甲状腺功能进行检查，及时发现婴幼儿甲状腺功能受损情况，并从中发现氟病区智力低下的病儿，以便有效而及时地防治氟病及其对神经系统发育的影响。

参考文献

　[1]董仲，刘万兴，刘家骝.慢性氟中毒对甲状腺影响的实验研究.中华病理学杂志，1989，18(3)：226～227.

　　[2]卢传华，边建朝，叶平，等.过量氟和硒对大鼠垂体、甲状腺、甲状旁腺的影响.中国地方病防治杂志，1996，11(1)：18～20.

　　[3]谭郁彬.氟对机体代谢及各系统的影响.中国地方病防治杂志，1994，9(4)：230～232.

　　[4]许德顺，王艳玲，李野.氟化物对实验大鼠甲腺125碘分布影响的研究.中国地方病防治杂志，1994，9(4)：218～220.

　　[5]于燕妮，刘家骝，董仲，等.地方性氟病发病区胎儿肝细胞体视学参数的观测.中华物理医学杂志，1996，18(1)：33～35.

　　[6]郑富盛主编.细胞形态立体计量学.北京：北京医科大学.中国协和医科大学联合出版社，1990.


Fetal thyroid fluorosis areas of Guizhou Province stereological parameters measured

Journal of Mathematical Medicine 1999 an analysis of clinical research Volume 0

Author: Jenny Liu at Monkey

Unit: Department of Pathology, Medical College, Guiyang, Guizhou 550004

Keywords: fluorosis; fetus; stereological; thyroid follicular epithelial cells

　　Abstract　 Guizhou fluorosis areas on fetal thyroid follicular epithelial cells stereological parameters measured. The results showed that fetal bone fluorosis areas fluorine content was significantly increased, compared with non-fluorosis areas fetus, the difference was statistically significant (P <0.05). Stereological parameters measured results showed that thyroid follicular epithelial cells mitochondria mean circumference, the average cross-sectional area, the average bulk density and average size were significantly increased compared with the non-endemic fetus, the difference was statistically significant (P <0.05) .

　　Fluorosis has damaging effects on the thyroid, thyroid uptake performance 131 I rate increased, blood T 3 , T 4 , TSH content change, thyroid follicular epithelial cells showed morphological changes [1-4] . But fluorosis on thyroid ultrastructure stereological effects reported rarely. This paper on the ultrastructure of fetal thyroid fluorosis areas stereological observations, in order to provide fluoride poisoning ultrastructural morphology of thyroid damage experimental basis for quantitative determination of parameters.

1 Materials and methods

　　1.1 Materials

　　1.1.1 Ward fetus　10 cases were taken from fetuses fluorosis areas Zhijin County, Guizhou Province District fluorine disease pregnant women, the long-term living in high fluoride areas, edible food fluoride, with varying degrees of dental fluorosis, bladders cited Postpartum maternal history of menopause by fetal crown-rump length and calculate gestational age, gestational age was 5 to 7 months.

　　1.1.2 Non-Ward fetus　10 cases were taken from non-fluorosis areas fetus Guiyang non-endemic fluorosis endemic area. Pregnant fluorine poisoning. Gestational age of the fetus and the calculation methods drawn above. Gestational age was 5 to 7 months.

　　1.2 Inspection Items and Methods

　　1.2.1 and serum fluoride content in urine of pregnant women　pregnant women to take the morning fasting blood and urine. With fluoride ion selective electrode measurement of serum and urinary fluoride content.

　　1.2.2 Fetal bone fluoride content　to take the fetal right femur Tiqu soft tissue, drying, charring and ashing methods described above bone fluoride content.

　　1.2.3 ultrastructural observations of fetal thyroid　cysts of the fetus after abortion, that is to take the right thyroid tissue 1mm 3 , fixed at 3% Xu dialdehyde, the conventional electron microscope specimens observed after production. Stereological parameters measured endemic fluorosis areas and non-fluorine all three cases the fetus. After the biopsy tissue blocks per case two copper fishing nets, the electron microscope choose to use a clear structure, the more obvious contrast copper mesh, at 15,000 magnification, each net uptake of thyroid follicular epithelial cells randomly, each photo 30, film for optical zoom magnification of two, ie a total of 30,000 times magnification. Hand with unilateral test grid point counting method, the test grid dot pitch 5mm, total 486 points each photo, each 30 photos total 14,580 points. To test the units covered by the cytoplasm of cells as the reference, calculated with reference to Zheng Fusheng editor of "three-dimensional morphology metrology" [6] . Parameters measured are: cytoplasmic density (V VC ); nuclear density (V VN ), surface area (delta N ), surface area density (S VN ), cross-sectional average length ( ), the average cross-sectional area ( ), mean volume ( ); mitochondrial density (V VM ), surface area (delta M ), cross-sectional average length ( ), the average cross-sectional area ( ), the number density (N VM ), average volume ( ). The data obtained using the t test or t  test statistic processing.

2 Results

　　2.1 maternal serum, urine fluoride content and fetal bone fluoride content

　　Table 1 shows that urinary fluoride levels fluorosis areas than non-pregnant women, pregnant women wards, the difference was statistically significant (P <0.025). Fetal bone fluorosis areas fluorine content than non-endemic fetus, the difference was significant (P <0.01).

Table 1 maternal serum, urine fluoride and fetal bone fluoride content ( ± S)

Category The number of cases Pregnant woman Fetus
Urinary fluoride (μg / ml) Serum fluoride (μg / ml) Bone fluoride (μg / g)
Non-endemic area 10 1.67 ± 0.82 0.41 ± 0.15 2.50 ± 0.11
Fluorine Ward 10 4.37 ± 2.94 0.55 ± 0.21 2.77 ± 0.25
P-value 　 <0.025 > 0.05 <0.01
　　2.2 electron microscopy

　　Electron microscopy found that fluoride Ward fetal thyroid follicular cell microvilli fracture, reduce, or even disappear, significant swelling of mitochondria in the cytoplasm, increased electron density decreases mitochondrial cristae, or even disappear.

　　2.3 thyroid follicular epithelial cells stereological parameters

Table 2 and non-endemic fluorosis areas of fetal thyroid follicular epithelial cells stereological parameters between ( ± S)

Parameter Unit Non-endemic area Fluorine Ward P-value
V VC m 0 0.25 ± 0.08 0.60 ± 0.11 > 0.05
V VN m 0 0.18 ± 0.06 0.16 ± 0.06 > 0.05
delta N m -1 0.63 ± 0.15 0.58 ± 0.09 > 0.05
S VN m -1 0.09 ± 0.02 0.07 ± 0.02 > 0.05
μm 8.42 ± 0.30 8.21 ± 0.26 > 0.05
m 2 16.15 ± 0.51 15.49 ± 1.31 > 0.05
m 3 48.62 ± 10.39 62.11 ± 11.63 <0.05
Table 2 shows that, compared with the non-endemic area, fluorine Ward fetal thyroid follicular epithelial cells average volume increases, the difference was statistically significant (P <0.05), I parameters between the two groups were not statistically significant.
Table 3 endemic fluorosis areas and non-fetal thyroid follicular epithelium mitochondrial stereological parameters between ( ± S)

Parameter Unit Non-endemic area Fluorine Ward P-value
V VM m 0 0.09 ± 0.07 0.15 ± 0.08 <0.01
delta M m -1 4.68 ± 0.75 5.07 ± 0.76 > 0.05
μm 23.53 ± 0.95 34.52 ± 0.51 <0.05
m 2 5.07 ± 0.71 9.08 ± 0.87 <0.05
N VM m -3 0.15 ± 0.03 0.08 ± 0.02 <0.05
m 3 24.36 ± 4.10 51.37 ± 6.29 <0.05
　　Table 3 shows that fluorine Ward fetal thyroid follicular cell mitochondrial density, mean circumference, the average cross-sectional area and mean volume were significantly higher than non-endemic fetus, there is a significant difference (P <0.05); number density of the fluorine Ward was significantly lower than non-endemic area (P <0.05).

3 Discussion

　　In this study, fetuses were collected from pregnant women fluorosis areas of Guizhou Province, the performance of these women dental fluorosis, fluoride in urine was significantly higher (P <0.05), stating the body has excess fluoride accumulation. Excess fluoride through the placenta into the fetus, the thyroid gland taking effect.

　　We found that previous experiments [1] , the body of excess fluoride can act on the thyroid follicular epithelial cells, causing blood T 3 , T 4 decreased, TSH elevated, suction 131 I was significantly increased, and the emergence of follicular epithelial cells degeneration and other morphological changes. This experiment further with stereological methods detect thyroid follicular cell ultrastructure and found fluorine Ward fetal thyroid follicular cell mitochondria mean circumference, the average cross-sectional area, density and average volume increases, suggesting that mitochondria occurred swelling due to mitochondrial swelling, severe disintegration, resulting in mitochondrial density decreases.

　　Some data indicate that [5] , fluoride is mainly the result of subcellular targets mitochondria, mitochondrial structure through damage, resulting in mitochondrial function is affected, thereby undermining the entire cell structure and function. This ultrastructural morphometry parameters measured further confirms this. As for the structure of fluoride on mitochondrial damage mechanism remains to be elucidated.

　　From the experimental results shows that the fluorine damage thyroid follicular epithelial cells, ultrastructural changes occur, can be speculated that excessive fluoride may affect human fetal thyroid function. Therefore, the authors believe, should focus on infants fluorosis areas to check thyroid function, thyroid function in infants and young children to discover the damage, and discover fluorosis areas of mental retardation sick children, in order to effectively and timely prevention and treatment of endemic fluorosis and Development of the nervous system.

References

　[1] Dong Zhong, Liu Wanxing, Liu monkeys. Chronic fluoride poisoning on the thyroid in rats. Journal of Pathology, 1989,18 (3): 226 ~ 227.

　　[2] Lu Zhuanhua, Bian Jianxin North Korea, Ye Ping, et al. Excessive fluoride and selenium on rat pituitary, thyroid, parathyroid impact. Chinese Endemiology, 1996,11 (1): 18-20.

　　[3] Tan Yubin. Fluoride on metabolism and the system. Chinese Journal of Prevention and Treatment, 1994,9 (4): 230 ~ 232.

　　[4] Xu Deshun Wang Yanling, Li Ye. Fluoride on Rat Thyroid 125 iodine distribution of. Chinese Journal of Prevention and Treatment, 1994,9 (4): 218 ~ 220.

　　[5] on Jenny, Liu Ka-Dong Chung, et disease endemic fluorosis areas of fetal liver cells stereological parameters observed Journal of Physical Medicine, 1996,18 (1): 33 ~ 35.

　　[6] Zheng Fusheng editor of three-dimensional morphology Metrology, Beijing: Beijing Medical University, China Union Medical College Press, 1990.