dt/dt與db/db突變老鼠之輪廓乳突及其味蕾的形態變化

Morphology of taste buds in the circumvallate papilla from mutant dystonia (dt/dt) mice and diabetic (db/db) mice

蘇昱人(Yu-Jen Su)

指導教授 ： 盧國賢

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01382

摘要

味覺的感受來自於食物分子刺激味蕾細胞上的各種接受器和離子通道的開關，經過不同的訊息傳遞造成不同的神經衝動，而使大腦產生各種味覺訊息。而某些的疾病可能會對味蕾細胞的種類或形態造成影響，或是改變味蕾細胞上的神經分佈，進而影響對食物的偏好。而在遍佈於舌上的舌乳突當中，輪廓乳突為所含味蕾數量最多的舌乳突，且其味蕾分佈緊密易於觀察，因此為我們所研究的組織構造。我們以dystonia musculorum (dt/dt) 和diabetes (db/db) 兩種不同的突變小鼠作為研究對象，觀察並比較味蕾細胞在形態上有何異同。其中dt/dt小鼠由於基因缺損，缺少一種於神經元表現的連結蛋白dystonin，造成感覺神經的退化，其神經退化可能影響到味蕾的形態改變；而db/db小鼠則為因缺少leptin receptor造成醣類代謝失調而患有先天性糖尿病症的突變種。糖尿病會影響其飲食習慣和對食物的偏好，對味蕾細胞也可能造成影響。
經由HE染色，我們發現dt/dt小鼠與正常小鼠比較其輪廓乳突體積變小（乳突前後徑厚度，330±19.25μm, vs. 240.3±20.07μm；n=3）、輪廓乳突中的味蕾數目也有減少的現象（94.98±3.1, vs. 58.21±5.12個/乳突；n=3），經形態測量及統計分析知其具有顯著差異；db/db小鼠的味蕾形態在輪廓乳突的體積（343±11.43μm；n=3）和輪廓乳突中味蕾數量(98.69±5.06個/乳突；n=3)則無明顯改變。以PGP9.5進行免疫組織化學染色，發現dt/dt小鼠其分佈於味蕾中的神經纖維有明顯減少的現象（味蕾中具有PGP9.5免疫反應的面積/味蕾總面積，47±2.84%, vs. 25±4.02%；n=3），具有統計上的顯著差異；而db/db小鼠味蕾中的神經分佈則無明顯改變（48±8.79%；n=3）。於電子顯微鏡觀察正常小鼠的味蕾可區分第一、二、三型味蕾細胞與基底細胞，細胞間電子密度、細胞核形態、細胞質中胞器分佈各有不同，而dt/dt小鼠的味蕾於電子顯微鏡下我們發現味蕾細胞電子密度與細胞核形態變得不易區分，其第一型味蕾細胞當中的緻密顆粒數量與正常小鼠相比明顯減少，具有統計上的差異，表示dt/dt小鼠的味蕾細胞微環境可能有所改變，而db/db小鼠其超微結構也與正常小鼠無明顯差異。
本研究的推論表示dt/dt小鼠因神經退化所造成的味蕾及其細胞的形態改變較為顯著，而db/db小鼠因代謝失調所引起的味蕾及其細胞形態改變則較不明顯。而味蕾細胞形態上的改變是否會造成味覺訊息傳遞的改變和影響老鼠對食物的偏好，則值得我們未來進一步深入探討。

關鍵字

輪廓乳突 ； 味蕾 ； 神經退化

並列摘要

Recently studies on the taste bud are focused on the neurotransmitters and cell communications and seldom the morphology, of the taste bud. Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse (mutant dt/dt). The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. The db/db mouse has defective leptin receptors and the defects lead to impairments of leptin regulation of food intake and body weight, and result in the expression of diabetic symptoms such as hyperinsulinemia, hyperglicemia, and extreme obesity. In order to determine the effect of hereditary (in dt/dt mice) and metabolic disturbance (in db/db mice) on the morphology and nerve distribution of the vallate taste bud, a comparative study was performed among the wild type, dt/dt, and db/db mice, to examine the morphology at light and electron microscopic level, and distribution and abundance of the PGP-9.5 immunoreactivity in the taste buds of the vallate papilla. Morphometry and quantitative analysis on the distribution of taste buds and their gustatory nerve fibers are also demonstrated.
Compared with wild type mice, the size of vallate papillae is much smaller in dt/dt mice (indicated by the total thickness of frontal sections, 330±19.25μm, vs. 240.3±20.07μm; n=3) and the number of taste buds decreased significantly (94.98±3.1, vs. 58.21±5.12 taste buds/papilla, n=3). However, in db/db mice, the morphology and size of vallate papilla (343±11.43μm, n=3) and the number of its taste buds (98.69±5.06 taste buds/papilla, n=3) are not apparently different from those of the wild type. The results from the PGP9.5 immunohistochemistry showed that the nerve innervation in taste bud cells was significantly reduced in dt/dt mice (25±4.02%, n=3), (indicated by proportion of PGP9.5 immunoreactive cells and fibers of the vallate papilla epithelium containing taste bud), as compared with wild type (47±2.84%, n=3) and db/db (48±8.79%, n=3) mice. Ultrastructural examination reveals four types of taste bud cells, i.e., type I, II, III and baseal cells. All of them express distinctive nuclear morphology, various cytoplasm electron density and characteristic subcellular organelles, in the vallate taste bud from the wild type and db/db mice. However, the cell density and nuclear morphology of the taste cells in dt/dt mice are not so distinct, and it is difficult to differentiate four types of taste bud cells at lower power electron microscopy, however, it is feasible at high power magnification. These findings confirmed our morphological findings at light microscopic level.
We conclude that the sensory deficiency in the mutant dt/dt mice affects the morphology and innervations severely of taste buds, however, the metabolic disturbance of the mutant db/db mice does not interfere the structure and innervation of the taste buds in the vallate lingual papilla.

並列關鍵字

circumvallate ； taste bud ； dystonia ； diabetic

參考文獻

Chandrashekar J, Hoon MA, Ryba NJ, Zuker CS. The receptors and cells for mammalian taste. Nature 2006; 444 (7117):288-294.

Dalpe G, Leclerc N, Vallee A, Messer A, Mathieu M, De Repentigny Y. Dystonin is essential for maintaining neuronal cytoskeleton organization. Molecular and Cellular Neurosciences 1998; 10 (5-6):243-257.

Dalpe G, Mathieu M, Comtois A, Zhu E, Wasiak S, De Repentigny Y. Dystonin-deficient mice exhibit an intrinsic muscle weakness and an instability of skeletal muscle cytoarchitecture. Developmental Biology 1999; 210 (2):367-380.

Delay RJ, Kinnamon JC, Roper SD. Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage. Journal of Comparative Neurology 1986; 253 (2):242-252.

Dowling J, Yang Y, Wollmann R, Reichardt LF, Fuchs E. Developmental expression of BPAG1-n: insights into the spastic ataxia and gross neurologic degeneration in dystonia musculorum mice. Developmental Biology 1997; 187 (2):131-142.