生醫材料於嗅覺上皮再生之應用

The application of biomaterials on the regeneration of olfactory neuroepithelium

李聖典(Sheng-Tien Li)

指導教授 ： 楊台鴻

共同指導教授 ： 黃琮瑋(Tsung-Wei Huang)

國立臺灣大學/工學院/醫學工程學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800257

摘要

嗅覺失能 (Olfactory dysfunction) 將嚴重影響著病患各方便的生活品質，包含食而無味、食慾不振及減少對環境中氣味改變的察覺能力，再者影響病患之身心理狀態。近二十年來，全球嗅覺功能低落之發生率，從3%上升至20%，然而目前在臨床上仍無有效的治療方法且效果差異甚大，因而發展一個有效之治療方法是有其必要性的。其中，嗅覺上皮 (olfactory neuroepithelium, ON) 位於中上鼻甲，為氣味分子啟動訊號的起始位置；此外，嗅覺上皮細胞群 (olfactory neuroepithelial cells, ONCs) 因含有終其一身可自我再生的幹細胞 (basal cells)，其可進一步分化成負責偵測氣味的嗅覺受體神經 (olfactory receptor neurons, ORNs) 和調節黏膜離子平衡的支持細胞 (sustentacular cells, SCs)，因此是周邊神經中唯一能進行神經再生的細胞。而利用體外培養分析系統 (in vitro) 尋找適合其維持原有特性、協助生長，甚至是嗅覺上皮重建之生醫材料是本研究之目的。
首先，評估水溶性之幾丁聚醣 (chitosan) 對嗅覺上皮細胞，是否有助於ORNs之分化。嗅覺上皮細胞取自懷孕大鼠 (wistar rats) 之第17天胚胎，並以有無培養在含有幾丁聚醣 (0.1 mg/mL) 之培養基中作為實驗組及控制組。藉由西方墨點法分析培養3、6、9天之細胞分化程度之變化，並藉由免疫螢光染色相互印證細胞分化之狀態。在實驗結果中，添加幾丁聚醣培養的嗅覺上皮細胞，呈現著不對稱的雙極軸突之神經型態。此外，隨著培養天期的增加，未成熟ORNs之標記蛋白 (βIII tubulin) 表現量逐漸地減少，但成熟ORNs之嗅覺標記蛋白 (olfactory marker protein, OMP) 及不可或缺的功能性訊號傳導要素，嗅覺神經特異G蛋白 (olfactory neuron specific-G protein, Golf) 及腺苷酸環化酶3 (adenylate cyclase 3, ADCY3) 之表現趨勢皆隨之增加，且螢光表現皆於同一細胞上。因而，進一步利用3-甲基吲哚 (3-methylindole, 3-MI) 誘發嗅覺失能之動物模式，以驗證水溶性之幾丁聚醣是否可經由鼻腔途徑治療，進而促進嗅覺功能的恢復。使用的評估方法如下：食物搜尋之動物行為測試、免疫組織化學染色及西方墨點法。實驗結果顯示，誘發嗅覺低落前，老鼠搜尋食物之歷程約80秒；一旦經3-MI破壞嗅覺上皮後，搜尋食物的歷程將超過5分鐘，但在適當時間以後，開始經由鼻腔給予幾丁聚醣治療，為期三周且每周兩次之頻率後，搜尋歷程將隨之下降。從組織切片及西方墨點法顯示，經幾丁聚醣治療之組別，嗅覺上皮厚度及成熟ORNs皆有顯著性增加，且伴隨著未成熟ORNs及SCs之表現量下降。再者，利用溴化去氧尿苷 (Bromodeoxyuridine, BrdU) 以追蹤24小時間所新生的細胞族群。在治療組中，表現BrdU的細胞，多分布於嗅覺上皮之中下層位置，並大多表現βIII tubulin。相較下，未治療之組別，表現BrdU的細胞，則是散落分布於嗅覺上皮中，且同時表現未成熟ORNs或SCs之標記蛋白 (cadherin-1, CDH1)。意味著，幾丁聚醣提供ORNs成熟化之引導線索及調節嗅覺上皮細胞群之動態平衡。故，幾丁聚醣溶液極具有作為嗅覺功能低落與重建之有潛力的生醫材料，未來應用於臨床病人上必將有所裨益。
進一步利用乙烯醇-乙烯共聚物 (poly (ethylene-co-vinyl alcohol), EVAL) 為基材，建立人類嗅覺上皮細胞 (human olfactory neuroepithelial cells, HONCs) 之體外培養系統，並探討基材與細胞間之可能調節機制。HONCs來源為進行鼻竇手術之患者，並取得其同意後獲取。接著於分離培養後，利用免疫螢光染色、晶片 (microarray)、即時定量聚合酶反應、酵素免疫分析法及西方墨點之方法進行評估。從實驗結果中顯示，控制組之細胞型態多為上皮狀且表現SCs標記(CDH1及cytokeratin 18, CK18)；然而，在EVAL之實驗組，則呈現著不對稱的雙極軸突之神經型態，並表現成熟ORNs之OMP標記。而在發現實驗組與控制組之差異後，進一步利用晶片分析可能的調節因子。結果呈現，實驗組中之神經肽Y (neuropeptide Y, NPY) 及雙調蛋白 (amphiregulin, AREG)，相較於控制組，分別在神經投射發育類別 (neuron projection development) 中，呈現調升及調降最多之表現，而後也經由即時定量聚合酶反應及酵素免疫分析法證實此趨勢。最後，分別添加不同濃度之NPY、NPY受體之抑制劑 (BIBP3226)、AREG或AREG受體抑制劑 (AG1478) 於控制組或實驗組中，觀察成熟ORNs及SCs之表現。在控制組中，隨著NPY濃度之增加，OMP表現上升；然而隨著AG1478濃度之增加，的確使SCs隨之下降。相對地，在實驗組中，隨著BIBP3226濃度之增加，OMP表現下降；但隨著AREG濃度之增加，卻對SCs無濃度上之效應。更進一步分析發現，其原因為實驗組中，AREG的受體 (上皮生長因子受體 (epidermal growth factor receptor, EGFR) 在EVAL上培養初期，其磷酸化程度即已受到抑制。因此，此生醫材料-EVAL或許將是一個潛能性的基材，以用做嗅覺相關之研究模型。

關鍵字

嗅覺失能 ； 嗅覺上皮 ； 嗅覺受體神經 ； 支持細胞 ； 幾丁聚醣 ； 乙烯醇-乙烯共聚物 ； 神經肽Y ； 雙調蛋白 ； 上皮生長因子受體

並列摘要

Olfaction not only contributes to enjoyments of food, but also provides a clue to escape from dangerous environmental hazards. Although loss of smell is not a lethal disease, it is thought to affect 90% of neurodegenerative patients as an early marker. Additionally, olfactory dysfunction is commonly progressive and its prevalence during this two decades continually increases from 3% to 20% in the world and 12.3% in Taiwan. Currently, topical or systemic application of glucocorticoids is a general modality, but the therapeutic effect is controversy. In this study, we apply biomaterials to establish in vitro culture systems from rat olfactory neuroepithelial cells (rONCs) and human olfactory neuroepithelial cells (HONCs) since olfactory neuroepithelium (ON) has an ability to continuously regenerate through adulthood, comprising neural-lineage cells, olfactory receptor neurons (ORNs) to detect odors, and non-neural-lineage cells, sustentacular cells (SCs) to regulate ionic balance of olfactory mucus.
In Part A, we use soluble chitosan to develop a nasal spray modality and start with an in vitro culture system from rONCs. The morphological analysis indicated that rONCs treated with chitosan exhibited an asymmetric bipolar shape. Also, the number of mature ORNs, expressing olfactory marker protein (OMP), significantly rose at day 9 in chitosan-treated groups, accompanied with a lower number of immature ORNs, expressing βIII tubulin (p<0.05). In addition, olfactory neuron specific-G protein (Golf) and adenylate cyclase 3 (ADCY3), the essential components for odorant receptors, showed a similar trend to the OMP expression. Next, an anosmic animal model was employed to further verify this strategy. 16 rats underwent 3-methylindole (3-MI) injection to destroy the ON, except the normal group. The effect of chitosan treatment was assessed by behavior test (food-finding test), immunohistochemistry and western blots at specific time points. Our results demonstrated that the duration of finding food after chitosan treatment decreased from over 5 minutes to around 80 seconds on day 28 following 3-MI injection. Histological images showed that no matter the thickness of the ON or the expression of OMP, they gradually restored after chitosan treatment at appropriate time point, whereas those without chitosan treatment may pause at the stage of immature ORNs owing to the highest βIII tubulin expression among groups and co-localization of 5-Bromo-2´-Deoxyuridine (BrdU) and βIII tubulin, meaning chitosan may serve as a clue for immature ORNs to mature. Additionally, the other BrdU expressing cells in the sham group were co-expressed cadherin-1 (CDH1), a marker of SCs, which is more than those in chitosan-treated group (3.7%±0.5% vs. 0.3%±0.06%), implying chitosan prevented hyperplasia of SCs from affecting the ORN growth during regenerative processes. Consequently, chitosan may balance olfactory neuronal homeostasis and serve as a promising clinical application for restorative ON in the future.
In Part B, we used poly (ethylene-co-vinyl alcohol) (EVAL) as an substrate to establish an in vitro culture system for HONCs, as well as to study the regulatory pathway between substrate and responsive cells. The effects of treatment were assessed using immunocytochemistry, microarray analysis, quantitative PCR, ELISA and western blots following culturing. The results showed that most of the cell morphology on controls was epithelial, and expresses markers of SCs, CDH1 and cytokeratin18 (CK18), whereas the main population on EVAL exhibited morphology with an asymmetric bipolar shape, expressing OMP. Microarray analyses revealed that neuropeptide Y (NPY) and amphiregulin (AREG) are the two important regulating factors on EVAL films. HONCs cultured on EVAL films enhanced the development of mature ORNs through NPY signaling, while AREG showed insignificant differences with increasing AREG, instead of lowering the growth of SCs by blocking epidermal growth factor receptor (EGFR) activation, which is a receptor of AREG. Therefore, EVAL would be a potential substrate as a human equivalent of olfactory in the future.

並列關鍵字

Olfactory dysfunction ； olfactory neuroepithelium ； olfactory receptor neuron ； sustentacular cells ； chitosan ； poly (ethylene-co-vinyl alcohol) ； neuropeptide Y ； amphiregulin ； epidermal growth factor receptor.

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