【摘要】：甜味偏好具有可塑性，發(fā)育早期的味覺(jué)經(jīng)歷可改變后期的甜味偏好。而該可塑性的形成機(jī)制目前尚不清楚。本課題在建立甜味偏好可塑性動(dòng)物模型的基礎(chǔ)上，對(duì)甜味偏好可塑性的外周味覺(jué)分子機(jī)制進(jìn)行了深入研究。我們以ICR小鼠作為實(shí)驗(yàn)對(duì)象，在哺乳期（P4-P21）給予母鼠不同濃度的安賽蜜（acesulfame-K，AK）飲水暴露，其子代小鼠通過(guò)母乳接受AK暴露。隨后，對(duì)成年子代小鼠進(jìn)行雙瓶喜好（Two-bottle-preference, TBP）測(cè)試，觀察其對(duì)不同甜味劑的偏好閾值和偏好率變化，尋求合適的AK暴露劑量，建立一個(gè)穩(wěn)定的甜味偏好可塑性動(dòng)物模型。在此基礎(chǔ)上，采用qRT-PCR、Western blot、免疫組化和ELISA等實(shí)驗(yàn)方法檢測(cè)小鼠在暴露剛結(jié)束時(shí)（P21）及TBP實(shí)驗(yàn)即將開(kāi)始前（P49），其外周味覺(jué)系統(tǒng)舌面菌狀味蕾和軟腭味蕾形態(tài)變化和味細(xì)胞內(nèi)甜味信號(hào)傳導(dǎo)相關(guān)分子的變化，探討小鼠甜味偏好可塑性變化的細(xì)胞和分子生物學(xué)機(jī)制。 母鼠哺乳期接受不同濃度AK暴露，對(duì)其成年子代小鼠進(jìn)行TBP測(cè)試發(fā)現(xiàn)：生后早期0.15mM、1.5mM暴露17天（P4-P21）和5mM暴露10天（P4-P14）對(duì)成年子代小鼠AK偏好無(wú)影響。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偏好閾值，降低AK糖偏好率：對(duì)照組小鼠偏好閾值為0.13mM，而5mM和12.5mM暴露組偏好閾值升高至0.42mM,25mM和50mM暴露組偏好閾值為1.33mM；與對(duì)照組相比，暴露組小鼠成年時(shí)對(duì)偏好閾值附近濃度的AK糖偏好率降低，分別為：5mM AK暴露顯著降低小鼠對(duì)0.42mM AK偏好率；12.5mM、25mM、50mM暴露組偏好率顯著降低的濃度分別為1.33mM和13.28mM、0.42mM和1.33mM、0.13mM和0.42mM。對(duì)小鼠的體重監(jiān)測(cè)（P4-P49）發(fā)現(xiàn)：25mM和50mM AK暴露顯著降低了小鼠在成年時(shí)的體重，可能影響小鼠的生理健康；而5mM和12.5mMAK暴露對(duì)小鼠體重?zé)o影響。所以我們選擇以5mM和12.5mM暴露17天（P4-P21）的方式來(lái)構(gòu)建動(dòng)物模型。我們還發(fā)現(xiàn)，早期AK暴露組小鼠對(duì)其他甜味劑（蔗糖和糖精）的偏好閾值也提高，偏好率發(fā)生變化：5mM AK暴露組小鼠對(duì)蔗糖偏好閾值由對(duì)照組的12mM升高至38mM；對(duì)糖精的偏好閾值由0.13mM升高至0.43mM。同時(shí)5mM AK暴露降低了小鼠對(duì)1400mM蔗糖的偏好率，提高了小鼠對(duì)430mM糖精的厭惡程度。由此可見(jiàn)，哺乳期AK暴露改變了小鼠成年時(shí)期的甜味偏好閾值和偏好率。 對(duì)外周味覺(jué)系統(tǒng)舌面和軟腭味蕾研究顯示，與對(duì)照組相比，AK暴露組小鼠的舌或軟腭組織中，，trpm5以及其它甜味轉(zhuǎn)導(dǎo)相關(guān)的重要分子（包括t1r2、t1r3、Gα-gust、cb1和ob-rb）的mRNA在P21或P49時(shí)，均沒(méi)有顯著改變。免疫印跡實(shí)驗(yàn)和形態(tài)學(xué)研究結(jié)果發(fā)現(xiàn)：5mM AK暴露剛結(jié)束時(shí)（P21），小鼠舌面和軟腭中甜味受體T1R2和T1R3、Gα-gustducin和TRPM5的蛋白表達(dá)，與對(duì)照組相比沒(méi)有顯著改變。而調(diào)控甜味信號(hào)的激素分子受體發(fā)生變化：舌面味蕾中甜味正調(diào)控分子CB1受體表達(dá)增加了1.3倍，而軟腭上甜味負(fù)調(diào)控分子leptin受體Ob-Rb表達(dá)降低了約30%。在小鼠成年時(shí)（P49），暴露組小鼠舌面上甜味受體T1R2表達(dá)升高約90%，單個(gè)味蕾中Gα-gustducin陽(yáng)性味細(xì)胞數(shù)目增加約25%；而軟腭上Gα-gustducin表達(dá)降低了約80%，Ob-Rb蛋白水平升高了1.7倍。同時(shí)軟腭味蕾最大橫截面積減小約20%，舌面味乳頭數(shù)目和味蕾形態(tài)無(wú)顯著改變。 此外，對(duì)早期AK暴露小鼠在青少年和老年時(shí)期的甜味偏好研究發(fā)現(xiàn)：哺乳期AK暴露，降低子代小鼠在青少年期的蔗糖偏好閾值和偏好率；提高小鼠老年期的AK偏好閾值。同時(shí)哺乳期AK暴露可能抑制小鼠衰老過(guò)程中偏好率下降的趨勢(shì)。 綜上，哺乳期AK糖暴露提高成年小鼠甜味偏好閾值，偏好率也發(fā)生一定改變。同時(shí)，小鼠外周味覺(jué)系統(tǒng)中發(fā)生可塑性變化，舌面和軟腭味蕾中甜味信號(hào)轉(zhuǎn)導(dǎo)分子和調(diào)控甜味信號(hào)激素分子受體的蛋白表達(dá)發(fā)生改變，這些變化可能是甜味偏好發(fā)生可塑性變化的外周機(jī)制。此外，早期的甜味劑經(jīng)歷對(duì)青年時(shí)期和老年時(shí)期甜味偏好具有不同程度的影響。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R114

【參考文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 張根華;甜味偏好可塑性的味覺(jué)細(xì)胞學(xué)基礎(chǔ)[D];浙江工商大學(xué);2007年

本文編號(hào)：2206436