臺灣博碩士論文加值系統

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。

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.

中文摘要 3
英文摘要 5
一、緒言（Introduction） 7
1.1.舌 7
1.2.味蕾 9
1.3.味蕾的超微結構 9
1.4.味覺的傳導 11
1.5.突變老鼠 12
二、材料與方法（Materials and methods） 15
2.1.動物 15
2.2.組織的製備 15
2.3.組織學研究 15
2.4.免疫組織化學染色 16
2.5.超微結構研究 16
2.6.統計分析 17
三、結果（Results） 19
3.1.組織學觀察 19
3.2.PGP9.5免疫組織化學染色 20
3.3.形態測量與統計分析 21
3.4.超微結構觀察 22
四、討論（Discussion） 26
五、結論與未來研究方向（Conclusion and further work） 31
六、參考文獻（References） 32
七、圖表（Figures & Tables） 36

AhPin P, Ellis S, Arnott C, Kaufman MH. Prenatal development and innervation of the circumvallate papilla in the mouse. Journal of Anatomy 1989; 162:33-42.

Bernier G, De Repentigny Y, Mathieu M, David S, Kothary R. Dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination. Development 1998; 125 (11):2135-2148.

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.

Hamamichi R, Asano-Miyoshi M, Emori Y. Taste bud contains both short-lived and long-lived cell populations. Neuroscience 2006; 141 (4):2129-2138.

Huang YJ, Lu KS. Unilateral innervation of guinea pig vallate taste buds as determined by glossopharyngeal neurectomy and HRP neural tracing. Journal of Anatomy 1996; 189 (Pt 2):315-324.

Huang YJ, Maruyama Y, Dvoryanchikov G, Pereira E, Chaudhari N, Roper SD. The role of pannexin 1 hemichannels in ATP release and cell–cell communication in mouse taste buds. Proceeding of National Academy of Science of the U.S.A. 2007, 104(15):6436-41.

Ichikawa H, De Repentigny Y, Kothary R, Sugimoto T. The survival of vagal and glossopharyngeal sensory neurons is dependent upon dystonin. Neuroscience 2006; 137 (2):531-536.

Ichikawa H, Terayama R, Yamaai T, De Repentigny Y, Kothary R, Sugimoto T. Dystonin deficiency reduces taste buds and fungiform papillae in the anterior part of the tongue. Brain Research 2007; 1129 (1):142-146.

Kawai K, Sugimoto K, Nakashima K, Miura H, Ninomiya Y. Leptin as a modulator of sweet taste sensitivities in mice. Proceedings of the National Academy of Sciences of the United States of America 2000; 97 (20):11044-11049.

Kinnamon JC, Henzler DM, Royer SM. HVEM ultrastructural analysis of mouse fungiform taste buds, cell types, and associated synapses. Microscopy Research and Technique 1993; 26 (2):142-156.

Kinnamon JC, Sherman TA, Roper SD. Ultrastructure of mouse vallate taste buds: III. Patterns of synaptic connectivity. Journal of Comparative Neurology 1988; 270 (1):1-10.

Kinnamon JC, Taylor BJ, Delay RJ, Roper SD. Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses. Journal of Comparative Neurology 1985; 235 (1):48-60.

Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI. Abnormal splicing of the leptin receptor in diabetic mice. Nature 1996; 379 (6566):632-635.

Lopez GF, Krimm RF. Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers. Journal of Neurobiology 2006; 66 (10):1033-1043.

Mistretta CM, Goosens KA, Farinas I, Reichardt LF. Alterations in size, number, and morphology of gustatory papillae and taste buds in BDNF null mutant mice demonstrate neural dependence of developing taste organs. Journal of Comparative Neurology 1999; 409 (1):13-24.

Murray RG. Cellular relations in mouse circumvallate taste buds. Microscopy Research & Technique 1993; 26 (3):209-224.

Ninomiya Y, Imoto T, Yatabe A, Kawamura S, Nakashima K, Katsukawa H. Enhanced responses of the chorda tympani nerve to nonsugar sweeteners in the diabetic db/db mouse. American Journal of Physiology 1998; 274 (5 Pt 2):R1324-1330.

Nosrat IV, Agerman K, Marinescu A, Ernfors P, Nosrat CA. Lingual deficits in neurotrophin double knockout mice. Journal of Neurocytology 2004; 33 (6):607-615.

Oakley B, Brandemihl A, Cooper D, Lau D, Lawton A, Zhang C. The morphogenesis of mouse vallate gustatory epithelium and taste buds requires BDNF-dependent taste neurons. Brain Research Developmental Brain Research 1998; 105 (1):85-96.

Romanov RA, Rogachevskaja OA, Bystrova MF, Jiang P, Margolskee RF, Kolesnikov SS. Afferent neurotransmission mediated by hemichannels in mammalian taste cells. European Molecular Biology Organization Journal 2007; 26 (3):657-667.

Shigemura N, Ohta R, Kusakabe Y, Miura H, Hino A, Koyano K. Leptin modulates behavioral responses to sweet substances by influencing peripheral taste structures. Endocrinology 2004; 145 (2):839-847.

Simon SA, de Araujo IE, Gutierrez R, Nicolelis MA. The neural mechanisms of gustation: a distributed processing code. Nature Reviews Neuroscience 2006; 7 (11):890-901.

State FA. Histological changes following unilateral re-innervation of the circumvallate papilla of rat. Acta Anatomica 1977; 98 (3):343-352.

Tseng KW, Lu KS, Chien CL. A possible cellular mechanism of neuronal loss in the dorsal root ganglia of Dystonia musculorum (dt) mice. Journal of Neuropathology and Experimental Neurology 2006; 65 (4):336-347.

Yang Y, Bauer C, Strasser G et al. Integrators of the cytoskeleton that stabilize microtubules. Cell 1999; 98 (2):229-238.

Yee CL, Jones KR, Finger TE. Brain-derived neurotrophic factor is present in adult mouse taste cells with synapses. Journal of Comparative Neurology 2003; 459 (1):15-24.

Yee CL, Yang R, Bottger B, Finger TE, Kinnamon JC. "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. Journal of Comparative Neurology 2001; 440 (1):97-108.