长期低硒、低碘对仔代发育期大鼠甲状腺形态结构及PCNA、TTF-1表达的影响

发布时间：2018-07-31 07:42

【摘要】： 背景与目的碘缺乏引起甲状腺形态结构以及功能的改变在人和动物的体内得到充分证实。在这一过程中微量元素硒也参与其中。其机制研究目前主要集中在硒防御超氧化物损伤以及硒影响甲状腺激素的代谢过程。以往这方面的动物研究大多以原代成年动物为研究对象，而流行病学调查显示世代繁衍生息在低硒低碘地区的人们，其仔代的甲状腺往往受到更严重的损伤。目前对低硒低碘两因素在仔代不同发育期甲状腺中有什么样的作用还未见有报道。本实验在课题组前期已经成功建立的动物模型基础上，选定长期持续低硒低碘喂养繁殖的仔三代三个不同发育时间点(4日龄、21日龄、成年)的大鼠甲状腺，对其进行组织形态学观察、体视学测量以及检测其甲状腺上皮细胞的增殖细胞核抗原(PCNA)与甲状腺转录因子1(TTF-1)的表达情况，从滤泡形态、细胞增殖情况以及细胞的功能状态等几方面来进行研究，探讨低硒低碘对仔代不同发育期大鼠甲状腺形态和功能的作用。方法选取本课题组前期成功建立的人工低硒，低碘饲料喂养繁殖的SD大鼠动物模型仔三代胎鼠(F3E)、4日龄(F3P4)、21日龄(F3P21)、成年(F3A)四个发育时间点的大鼠的甲状腺组织做为研究对象，每发育时间点大鼠分四组：①对照组(control)、②低硒组(Se~-)、③低碘组(I~-)、④低硒低碘组(Se~-I~-)。分别进行以下实验研究： 1．HE染色后组织学观察F3E、F3P4、F3P21、F3A各个组别的甲状腺形态结构变化。 2．HE染色后体视学测量F3P4、F3P21、F3A各个组别甲状腺的平均滤泡数、平均滤泡腔面积，平均滤泡上皮细胞高度。 3．免疫组织化学染色检测F3P4、F3P21、F3A各个组别甲状腺滤泡上皮细胞的增殖细胞核抗原(PCNA)的表达。 4．免疫组织化学染色检测F3P4、F3P21、F3A各个组别甲状腺滤泡上皮细胞的甲状腺转录因子1(TTF-1)的表达。结果 1．组织形态观察结果：F3A、F3P21、F3P4三个发育时间点：Se~-组大鼠甲状腺与对照组相比较结构相似，，其中F3ASe~-组中部分大鼠甲状腺滤泡有坏死纤维化改变。I~-组与Se~-I~-组甲状腺形态结构改变相似，都为小滤泡增生改变，腔小甚至无腔，胶质稀薄，上皮细胞立方至高柱状。F3E甲状腺与其他日龄相比呈现明显的幼稚状态，细胞密集，典型的滤泡少。 2．体视学测量结果：①平均滤泡数：F3A平均滤泡数Se~-组与对照组无差别(P＞0.05)，I~-组与Se~-I~-组滤泡数多于对照组与Se~-组(P＜0.01)。F3P21平均滤泡数Se~-组与对照组无差别(P＞0.05)，I~-组与Se~-I~-组滤泡数多于对照组与Se~-组(P＜0.01)。F3P4平均滤泡数Se~-组、I~-组与对照组无差别(P＞0.05)，Se~-I~-组滤泡数多于其他各组(P＜0.05)。②平均滤泡腔面积：F3A Se~-组与对照组无差别(P＞0.05)，I~-组与Se~-I~-组滤泡腔面积明显缩小(P＜0.01)，其中Se~-I~-组缩小最明显。F3P21对照组与Se~-组以及I~-组与Se~-I~-组之间没有差别(P＞0.05)。I~-组与Se~-I~-组的滤泡腔面积明显小于对照组与Se~-组(P＜0.01)。F3P4 Se~-组与Se~-I~-组之间有差别(P＜0.05)，其他各组之间均无差别(P＞0.05)。其中I~-组与Se~-I~-组的滤泡腔面积最小。③平均上皮细胞高度：F3A Se~-组与对照组之间无差异(P＞0.05)。I~-组与Se~-I~-组细胞高度增加(P＜0.01)，其中Se~-I~-组滤泡上皮细胞最高。F3P21各实验组均与对照组有显著差别(P＜0.01)，其中Se~-组细胞最低，Se~-I~-组细胞最高。F3P4对照组与Se~-组以及I~-组与Se~-I~-组之间没有差别(P＞0.05)。I~-组与Se~-I~-组细胞高度比对照组明显增加(P＜0.01)，其中Se~-I~-组细胞最高。 3．PCNA表达结果：F3A、F3P21、F3P4大鼠甲状腺滤泡上皮细胞的表达为从对照组到Se~-I~-组表达逐渐增强。对照组与Se~-组的表达多为阴性或弱阳性，I~-组与Se~-I~-组的表达明显增强(多集中在+++～++++)。四组的强阳性(++++)表达百分比逐渐增加：F3A各组分别为Control组0％，Se~-组6.3％，I~-组28.6％，Se~-I~-组50％。F3P21各组分别为Control组0％，Se~-组0％，I~-组16.7％，Se~-I~-组21.4％。F3P4各组分别为Control组0％，Se~-组0％，I~-组42.9％，Se~-I~-组33.3％。 4．TTF-1表达结果：F3A对照组的表达全部集中在阴性或弱阳性，Se~-组、I~-组与Se~-I~-组的表达明显增强(多集中在++～++++)。四组强阳性(++++)表达百分比分别为Control组0％，Se~-组23.5％，I~-组12.5％，Se~-I~-组53.8％。F3P21四组的表达强度多集中在(+++～++++)，四组的强阳性表达百分率达到了Control组61.5％，Se~-组55.6％，I~-组90％，Se~-I~-组85.3％。F3P4四组的表达之间没有差异。都无强阳性表达。结论 1．单纯的低硒可导致仔三代不同发育期大鼠的甲状腺形态结构发生改变，使其滤泡数量增多，体积缩小，上皮细胞增生。但作用的程度小于低碘。并且低硒低碘的作用有叠加效应。低硒的这种作用对各个发育日龄的影响程度不同，是随着个体发育的成熟而逐渐明显。 2．低硒与低碘都可使仔三代大鼠甲状腺滤泡上皮细胞的增殖细胞核抗原(PCNA)表达增强，低硒的作用弱于低碘，并且低硒低碘的作用有叠加效应。低硒增强PCNA表达的作用在4日龄时不明显，21日龄时有表现并保持到成年，是一个随着个体的发育成熟而逐渐明显的过程。 3．低硒与低碘都可使仔三代大鼠甲状腺滤泡上皮细胞的甲状腺转录因子1(TTF-1)表达增强，低硒的作用弱于低碘，并且低硒低碘的作用有叠加效应。低硒增强TTF-1表达的作用在4日龄时没有表现，21日龄时有表现并且在成年时表现最明显，是一个随着个体的发育成熟而逐渐明显的过程。
[Abstract]:Background and purpose
The changes in thyroid morphology and function caused by iodine deficiency are fully confirmed in human and animal bodies. In this process, trace element selenium is also involved. The mechanism research is mainly focused on selenium defense superoxide damage and selenium affecting the metabolic process of thyroid hormone.
In the past, most of the animal studies in this field are based on primary adult animals, and epidemiological surveys show that generations of people who reproduce in low selenium and iodine areas often suffer more serious damage to their offspring's thyroid gland. At present, there is no effect on the role of the two factors of low selenium and low iodine in the thyroid gland of the offspring of the offspring. In this experiment, on the basis of the animal model that has been successfully established in the earlier period of the project, the thyroid gland of the three generation of the offspring of the offspring (4 days of age, 21 days of age, adult) was selected for a long period of long-term sustained low selenium and low iodine feeding, and the histomorphology observation, stereological measurement and detection of the proliferation of thyroid epithelial cells were carried out. The expression of nuclear antigen (PCNA) and thyroid transcription factor 1 (TTF-1), from follicular morphology, cell proliferation and cell functional status were studied to explore the effect of low selenium and iodine on the thyroid morphology and function of rats in different developmental stages of the offspring.
Method
The SD rat model of artificial low selenium and low iodine feed was successfully established in the group of three generation fetal mice (F3E), 4 days of age (F3P4), 21 days of age (F3P21) and four adult (F3A) developmental time points of the rat's thyroid tissue as the research object, and the rats were divided into four groups at each time point: (control), and second group (2). Se~-), low iodine group (I~-), low selenium low iodine group (Se~-I~-).
1.HE staining was used to observe the morphological changes of thyroid tissue in F3E, F3P4, F3P21 and F3A groups.
2. After HE staining, the average follicular number, average follicular cavity area and average follicular epithelial cell height were measured.
3. The expression of proliferating cell nuclear antigen (PCNA) in thyroid follicular epithelial cells of F3P4, F3P21 and F3A groups was detected by immunohistochemical staining.
4. The expression of thyroid transcription factor-1 (TTF-1) in thyroid follicular epithelial cells of F3P4, F3P21 and F3A groups was detected by immunohistochemical staining.
Result
1. tissue morphology observation: F3A, F3P21, F3P4 three developmental time points: the thyroid gland of group Se~- was similar to that of control group. In group F3ASe~-, the thyroid follicular fibrosis changed in group.I~- and the morphological changes of thyroid gland in group Se~-I~-, which were small follicular hyperplasia, small cavity or even cavity without cavity. The F3E thyroid gland is markedly immature, densely packed, and typically has fewer follicles.
2. the results of stereological measurement: (1) mean follicular number: F3A average follicular number Se~- group and control group no difference (P > 0.05), the number of follicles in group I~- and Se~-I~- group is more than that of control group and Se~- group (P < 0.01).F3P21 average follicular number Se~- group and control group no difference (P > 0.05), I~- group and Se~ group follicular number more than the control group and group (0.01) The average follicular number in group Se~- was no difference between group I~- and control group (P > 0.05). The number of follicles in group Se~-I~- was more than that of other groups (P < 0.05). The mean follicular area of group F3A Se~- group was not different from that of control group (P > 0.05), and the area of follicle cavity in I~- group and Se~-I~- group was significantly reduced (P < 0.01). There was no difference between group Se~-I~- and group Se~-I~- (P > 0.05) the area of follicular cavity in group.I~- and group Se~-I~- was significantly smaller than that between the control group and Se~- group (P < 0.01), there was no difference between the.F3P4 Se~- group and the Se~-I~- group (P < 0.05), and there was no difference between the other groups (P > 0.05). There was no difference between the 3A Se~- group and the control group (P > 0.05) the cell height increased in the.I~- group and the Se~-I~- group (P < 0.01). The highest.F3P21 in the follicle epithelial cells in the Se~-I~- group was significantly different from the control group (P < 0.01), and the Se~- group was the lowest. There was no difference (P > 0.05). The cell height of group.I~- and group Se~-I~- was significantly higher than that of control group (P < 0.01), and Se~-I~- group had the highest cell height.
The expression of 3.PCNA expression: the expression of thyroid follicle epithelial cells in F3A, F3P21, F3P4 rats increased gradually from the control group to the Se~-I~- group. The expression of the control group and the Se~- group was mostly negative or weak positive, the expression of the I~- group and the Se~-I~- group was obviously enhanced (mostly in + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +) in the control group and the Se~-I~- group (+ + + + + + +). 3A group was 0%, group Se~- 6.3%, group I~- 28.6%, Se~-I~- group 50%.F3P21 group 0%, Se~- group 0%, I~- group 16.7%, 21.4%.F3P4 group of Se~-I~- group were Control group 0%, 0% group, 42.9% group.
4.TTF-1 expression results: the expression of F3A control group was all negative or weak positive, Se~- group, group I~- and Se~-I~- group were obviously enhanced (mostly + + + + + +). Four groups of strong positive (+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +). + + + + + + + +), the strong positive expression percentage of four groups reached 61.5% in group Control, 55.6% in group Se~-, 90% in group I~-, and no difference in the expression of 85.3%.F3P4 four groups in group Se~-I~-. No strong positive expression was found.
conclusion
1. simple selenium can lead to the change of thyroid morphologic structure in the three generation of the offspring, which makes the number of follicular follicles increased, the size of the cells and the epithelial cells proliferate. But the effect is less than the low iodine. And the effect of low selenium and iodine is superimposed. The maturity of the ontogeny is gradually apparent.
2. low selenium and low iodine can enhance the expression of proliferating cell nuclear antigen (PCNA) in the thyroid follicular epithelial cells of the three generation rats. The effect of low selenium is weaker than low iodine, and the effect of low selenium and iodine is superimposed. The effect of low selenium and low iodine on the expression of selenium is not obvious at 4 days of age. At the age of 21 days, it is shown and kept to adulthood at the age of 21. The process of mature and gradual development.
3. low selenium and low iodine can enhance the expression of thyroid transcription factor 1 (TTF-1) in the thyroid follicular epithelial cells of the three generation rats. The effect of low selenium is weaker than that of low iodine, and the effect of low selenium and iodine is superimposed. The effect of low selenium on the expression of TTF-1 is not manifested at 4 days of age. At the age of 21 days, the expression of low selenium is the most obvious and most obvious in adulthood. It is a gradual process with the development and maturity of individuals.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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