哺乳期安赛蜜暴露影响成年小鼠甜味偏好机制初步研究
发布时间：2018-08-27 06:38
【摘要】：甜味偏好具有可塑性，发育早期的味觉经历可改变后期的甜味偏好。而该可塑性的形成机制目前尚不清楚。本课题在建立甜味偏好可塑性动物模型的基础上，对甜味偏好可塑性的外周味觉分子机制进行了深入研究。我们以ICR小鼠作为实验对象，在哺乳期（P4-P21）给予母鼠不同浓度的安赛蜜（acesulfame-K，AK）饮水暴露，其子代小鼠通过母乳接受AK暴露。随后，对成年子代小鼠进行双瓶喜好（Two-bottle-preference, TBP）测试，观察其对不同甜味剂的偏好阈值和偏好率变化，寻求合适的AK暴露剂量，建立一个稳定的甜味偏好可塑性动物模型。在此基础上，采用qRT-PCR、Western blot、免疫组化和ELISA等实验方法检测小鼠在暴露刚结束时（P21）及TBP实验即将开始前（P49），其外周味觉系统舌面菌状味蕾和软腭味蕾形态变化和味细胞内甜味信号传导相关分子的变化，探讨小鼠甜味偏好可塑性变化的细胞和分子生物学机制。 母鼠哺乳期接受不同浓度AK暴露，对其成年子代小鼠进行TBP测试发现：生后早期0.15mM、1.5mM暴露17天（P4-P21）和5mM暴露10天（P4-P14）对成年子代小鼠AK偏好无影响。而5mM、12.5mM、25mM和50mM暴露17天（P4-P21）均不同程度提高了成年小鼠AK偏好阈值，降低AK糖偏好率：对照组小鼠偏好阈值为0.13mM，而5mM和12.5mM暴露组偏好阈值升高至0.42mM,25mM和50mM暴露组偏好阈值为1.33mM；与对照组相比，暴露组小鼠成年时对偏好阈值附近浓度的AK糖偏好率降低，分别为：5mM AK暴露显著降低小鼠对0.42mM AK偏好率；12.5mM、25mM、50mM暴露组偏好率显著降低的浓度分别为1.33mM和13.28mM、0.42mM和1.33mM、0.13mM和0.42mM。对小鼠的体重监测（P4-P49）发现：25mM和50mM AK暴露显著降低了小鼠在成年时的体重，可能影响小鼠的生理健康；而5mM和12.5mMAK暴露对小鼠体重无影响。所以我们选择以5mM和12.5mM暴露17天（P4-P21）的方式来构建动物模型。我们还发现，早期AK暴露组小鼠对其他甜味剂（蔗糖和糖精）的偏好阈值也提高，偏好率发生变化：5mM AK暴露组小鼠对蔗糖偏好阈值由对照组的12mM升高至38mM；对糖精的偏好阈值由0.13mM升高至0.43mM。同时5mM AK暴露降低了小鼠对1400mM蔗糖的偏好率，提高了小鼠对430mM糖精的厌恶程度。由此可见，哺乳期AK暴露改变了小鼠成年时期的甜味偏好阈值和偏好率。 对外周味觉系统舌面和软腭味蕾研究显示，与对照组相比，AK暴露组小鼠的舌或软腭组织中，，trpm5以及其它甜味转导相关的重要分子（包括t1r2、t1r3、Gα-gust、cb1和ob-rb）的mRNA在P21或P49时，均没有显著改变。免疫印迹实验和形态学研究结果发现：5mM AK暴露刚结束时（P21），小鼠舌面和软腭中甜味受体T1R2和T1R3、Gα-gustducin和TRPM5的蛋白表达，与对照组相比没有显著改变。而调控甜味信号的激素分子受体发生变化：舌面味蕾中甜味正调控分子CB1受体表达增加了1.3倍，而软腭上甜味负调控分子leptin受体Ob-Rb表达降低了约30%。在小鼠成年时（P49），暴露组小鼠舌面上甜味受体T1R2表达升高约90%，单个味蕾中Gα-gustducin阳性味细胞数目增加约25%；而软腭上Gα-gustducin表达降低了约80%，Ob-Rb蛋白水平升高了1.7倍。同时软腭味蕾最大横截面积减小约20%，舌面味乳头数目和味蕾形态无显著改变。 此外，对早期AK暴露小鼠在青少年和老年时期的甜味偏好研究发现：哺乳期AK暴露，降低子代小鼠在青少年期的蔗糖偏好阈值和偏好率；提高小鼠老年期的AK偏好阈值。同时哺乳期AK暴露可能抑制小鼠衰老过程中偏好率下降的趋势。 综上，哺乳期AK糖暴露提高成年小鼠甜味偏好阈值，偏好率也发生一定改变。同时，小鼠外周味觉系统中发生可塑性变化，舌面和软腭味蕾中甜味信号转导分子和调控甜味信号激素分子受体的蛋白表达发生改变，这些变化可能是甜味偏好发生可塑性变化的外周机制。此外，早期的甜味剂经历对青年时期和老年时期甜味偏好具有不同程度的影响。
[Abstract]:Sweet preference has plasticity, and taste experience in early development can change sweet preference in later period. The mechanism of plasticity formation is still unclear. Based on the establishment of animal model of sweet preference plasticity, the molecular mechanism of sweet preference plasticity was studied in detail. ICR mice were used as the real object. Subjects were exposed to different concentrations of acesulfame-K (AK) during lactation (P4-P21), and their offspring were exposed to AK through breast milk. Then, two-bottle preference (TBP) test was carried out on the offspring of adult mice to observe their preference thresholds and preference rates for different sweeteners, and to find suitable AK. A stable sweet preference plasticity animal model was established by exposure dose. On this basis, qRT-PCR, Western blot, immunohistochemistry and ELISA were used to detect the morphological changes of lingual fungus taste buds and soft palate taste buds in mice at the end of exposure (P21) and before the beginning of TBP (P49). The changes of sweet signal transduction related molecules were studied to explore the cellular and molecular biological mechanisms of the plasticity of sweet preference in mice.
TBP test of adult offspring mice exposed to different concentrations of AK during lactation showed that early postnatal exposure to 0.15mM, 1.5mM for 17 days (P4-P21) and 5mM for 10 days (P4-P14) had no effect on AK preference of adult offspring mice. K-glucose preference rate: the control mice had a preference threshold of 0.13 mM, while the 5 and 12.5 mM exposure groups had a preference threshold of 0.42 mM, and the 25 and 50 mM exposure groups had a preference threshold of 1.33 mM. Preference rate; 12.5mM, 25mM, 50mM exposure group significantly decreased the concentration of preference rate were 1.33mM and 13.28mM, 0.42mM and 1.33mM, 0.13mM and 0.42mM, respectively. Weight monitoring in mice (P4-P49) found that 25mM and 50mmAK exposure significantly reduced the body weight of mice in adulthood, possibly affecting the physiological health of mice; 5mM and 12.5mMAK exposure in mice. We also found that the preference thresholds for other sweeteners (sucrose and saccharin) were increased and the preference rates were changed in the early AK exposure group: the sucrose preference thresholds of the mice exposed to 5mM AK increased from 12 mM to 38 mM in the control group; The preference threshold for saccharin increased from 0.13 mM to 0.43 mM.At the same time, 5 mAK exposure decreased the preference rate for 1 400 mM sucrose and increased the aversion to 430 mM saccharin in mice.
Compared with the control group, the expression of trpm5 and other important sweetness transduction-related molecules (including t1r2, t1r3, Galpha-gust, CB1 and ob-rb) in the tongue or soft palate of AK exposed mice did not change significantly at P21 or P49. At the end of 5 mm AK exposure (P21), the expression of sweet taste receptors T1R2 and T1R3, Galpha-gustducin and TRPM5 in the tongue and soft palate of mice did not change significantly compared with the control group. The expression of leptin receptor Ob-Rb decreased by about 30%. In adult mice (P49), the expression of sweet receptor T1R2 increased by about 90%. The number of Ga-gustducin positive taste cells in a single taste bud increased by about 25%. The expression of Ga-gustducin on the soft palate decreased by about 80% and the level of Ob-Rb protein increased by 1.7 times. The maximum cross sectional area decreased by about 20%, and the number of tongue papilla and taste bud morphology did not change significantly.
In addition, the sweet taste preference of mice exposed to early AK in adolescence and old age was studied. AK exposure during lactation decreased sucrose preference thresholds and preference rates of offspring mice in adolescence, increased AK preference thresholds in old age mice, and AK exposure during lactation may inhibit the declining trend of preference rates in the aging process of mice.
In conclusion, lactation exposure to AK sugar increased the sweet preference threshold and changed the rate of sweet preference in adult mice. At the same time, plasticity changes occurred in the peripheral taste system, sweet signal transduction molecules in the tongue and soft palate taste buds, and the expression of sweet signal hormone receptors were altered. These changes may be sweet preference. In addition, early sweetener experiences have different degrees of influence on sweetness preference in youth and old age.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
本文编号：2206436
[Abstract]:Sweet preference has plasticity, and taste experience in early development can change sweet preference in later period. The mechanism of plasticity formation is still unclear. Based on the establishment of animal model of sweet preference plasticity, the molecular mechanism of sweet preference plasticity was studied in detail. ICR mice were used as the real object. Subjects were exposed to different concentrations of acesulfame-K (AK) during lactation (P4-P21), and their offspring were exposed to AK through breast milk. Then, two-bottle preference (TBP) test was carried out on the offspring of adult mice to observe their preference thresholds and preference rates for different sweeteners, and to find suitable AK. A stable sweet preference plasticity animal model was established by exposure dose. On this basis, qRT-PCR, Western blot, immunohistochemistry and ELISA were used to detect the morphological changes of lingual fungus taste buds and soft palate taste buds in mice at the end of exposure (P21) and before the beginning of TBP (P49). The changes of sweet signal transduction related molecules were studied to explore the cellular and molecular biological mechanisms of the plasticity of sweet preference in mice.
TBP test of adult offspring mice exposed to different concentrations of AK during lactation showed that early postnatal exposure to 0.15mM, 1.5mM for 17 days (P4-P21) and 5mM for 10 days (P4-P14) had no effect on AK preference of adult offspring mice. K-glucose preference rate: the control mice had a preference threshold of 0.13 mM, while the 5 and 12.5 mM exposure groups had a preference threshold of 0.42 mM, and the 25 and 50 mM exposure groups had a preference threshold of 1.33 mM. Preference rate; 12.5mM, 25mM, 50mM exposure group significantly decreased the concentration of preference rate were 1.33mM and 13.28mM, 0.42mM and 1.33mM, 0.13mM and 0.42mM, respectively. Weight monitoring in mice (P4-P49) found that 25mM and 50mmAK exposure significantly reduced the body weight of mice in adulthood, possibly affecting the physiological health of mice; 5mM and 12.5mMAK exposure in mice. We also found that the preference thresholds for other sweeteners (sucrose and saccharin) were increased and the preference rates were changed in the early AK exposure group: the sucrose preference thresholds of the mice exposed to 5mM AK increased from 12 mM to 38 mM in the control group; The preference threshold for saccharin increased from 0.13 mM to 0.43 mM.At the same time, 5 mAK exposure decreased the preference rate for 1 400 mM sucrose and increased the aversion to 430 mM saccharin in mice.
Compared with the control group, the expression of trpm5 and other important sweetness transduction-related molecules (including t1r2, t1r3, Galpha-gust, CB1 and ob-rb) in the tongue or soft palate of AK exposed mice did not change significantly at P21 or P49. At the end of 5 mm AK exposure (P21), the expression of sweet taste receptors T1R2 and T1R3, Galpha-gustducin and TRPM5 in the tongue and soft palate of mice did not change significantly compared with the control group. The expression of leptin receptor Ob-Rb decreased by about 30%. In adult mice (P49), the expression of sweet receptor T1R2 increased by about 90%. The number of Ga-gustducin positive taste cells in a single taste bud increased by about 25%. The expression of Ga-gustducin on the soft palate decreased by about 80% and the level of Ob-Rb protein increased by 1.7 times. The maximum cross sectional area decreased by about 20%, and the number of tongue papilla and taste bud morphology did not change significantly.
In addition, the sweet taste preference of mice exposed to early AK in adolescence and old age was studied. AK exposure during lactation decreased sucrose preference thresholds and preference rates of offspring mice in adolescence, increased AK preference thresholds in old age mice, and AK exposure during lactation may inhibit the declining trend of preference rates in the aging process of mice.
In conclusion, lactation exposure to AK sugar increased the sweet preference threshold and changed the rate of sweet preference in adult mice. At the same time, plasticity changes occurred in the peripheral taste system, sweet signal transduction molecules in the tongue and soft palate taste buds, and the expression of sweet signal hormone receptors were altered. These changes may be sweet preference. In addition, early sweetener experiences have different degrees of influence on sweetness preference in youth and old age.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【参考文献】
相关博士学位论文 前1条
1 张根华;甜味偏好可塑性的味觉细胞学基础[D];浙江工商大学;2007年
本文编号：2206436
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