利用人類腎臟近曲小管表皮細胞建立三維細胞培養模型

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范毓婷(Yu-Ting Fan) 查詢紙本館藏

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論文名稱

利用人類腎臟近曲小管表皮細胞建立三維細胞培養模型
(Establish a three-dimensional in vitro model with human proximal tubule epithelial cell)

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至系統瀏覽論文 (2026-1-27以後開放)

摘要(中)

近年來在台灣接受血液透析及腎功能衰竭的患者數量迅速增加，除了主要的風險因素例如糖尿病和高血壓外，許多人還患有不明病因的慢性腎臟疾病（CKDu）。藥物濫用、飲食習慣或環境暴露（如食品安全問題和空氣、水污染問題），可能都是導致CKDu的潛在風險因素。目前對於確切肇成CKDu的了解有限，因此建立有效的腎細胞模型來預測其生物學反應和腎臟腎毒性至關重要。
HK-2細胞是來自人類腎臟近曲腎小管上皮細胞的永生化細胞株，表達許多轉運蛋白，其中多數與腎臟近曲小管對於藥物分泌至尿液或再吸收回血液有重大的相關性。因此HK-2細胞時常被用於腎毒性研究，然而二維細胞培養因環境過於單純，不易反應體內結構和微環境的複雜性。本研究利用臨床級明膠海綿作為支架來建立HK-2細胞的三維細胞培養模型。並進一步比較二維和三維細胞培養之間增殖的差異、表現形態和轉運蛋白等的基因表達，期為CKDu潛在因子建立一個有效的腎毒性測試平台。研究結果顯示在型態上三維培養的細胞呈現管狀分化，並比二維培養細胞表現較多的運輸蛋白。

摘要(英)

In recent years, the number of patients receiving hemodialysis or even kidney failure has increased rapidly in Taiwan. In addition to predominant risk factors, such as diabetics and hypertension, many are suffered from chronic kidney disease of unknown etiology (CKDu). Drug-induced nephrotoxicity due to our medication consumption habit and environmental exposure such as worsen issues of food safety and air pollution can all be potential risk factors leading to CKDu. Given limited information about CKDu in Taiwan, it is crucial to establish an effective model to predict biological responses and renal damage to nephrotoxicity.
HK-2 cell is an immortalized cell line from human renal proximal tubule epithelial cells, which expresses many transporters for renal drug secretion and urine compound reabsorption. The HK-2 cell line has been extensively used for nephrotoxicity study. However, the simplicity of the two-dimensional cell culture made it challenging to recapitulate the complexity of the in vivo architecture and microenviroment. Here, we developed a three-dimensional model for HK-2 cells using clinical grade-gelatin sponge as our scaffolds. We compared the cell viability, proliferation, morphology, and the gene expression of transporter markers between 2D and 3D culture. Our results showed that 3D cells displayed a tubule-like morphology and expressed more key transporters than 2D cells did. With this, we hope that we have established a more effective testing platform for nephrotoxicity and underline mechanism investigation of CKDu.

關鍵字(中)

★ 近曲小管表皮細胞
★ 三維培養
★ 腎毒性
★ 體外模型

關鍵字(英)

★ proximal tubule epithelial cell
★ 3D culture
★ Nephrotoxicity
★ in vitro model

論文目次

目錄　　　　　　
中文摘要…………………………………………………………………………………… ii
英文摘要…………………………………………………………………………………… iii
誌謝………………………………………………………………………………………… iv
目錄………………………………………………………………………………………… v
圖目錄……………………………………………………………………………………… vii
表目錄……………………………………………………………………………………… vii
中英文簡稱對照表………………………………………………………………………… viii
一、前言………………………………………………………………………………… 1
1.1 三維立體培養系統（3D culture）………………………………………… 3
1.1.1 三維培養系統…………………………………………………………………… 3
1.1.2 2D培養及3D培養之比較 …………………………………………………… 4
1.2 腎臟細胞及研究………………………………………………………………… 6
1.2.1 腎臟型態、功能與運作方式…………………………………………… 6
1.2.2 近曲小管細胞（Human renal proximal tubule cells （RPTCs））… 7
1.2.3 HK-2細胞（human kidney-2 cell）……………………………………………… 10
二、實驗材料與方法………………………………………………………………… 14
2.1 細胞繼代………………………………………………………………………… 14
2.2 平面明膠塗層及細胞種植…………………………………………………… 15
2.3 立體細胞前處理與種植……………………………………………………… 16
2.4 細胞增生測試(WST-1 assay)………………………………………………… 17
2.5 Live and dead staining ………………………………………………………… 17
2.6 細胞免疫螢光染色 …………………………………………………………… 18
2.7 RNA萃取………………………………………………………………………… 19
2.8 RNA膠體電泳 ………………………………………………………………… 20
2.9 Complementary DNA (cDNA)合成………………………………………… 21
2.10 定量即時聚合酶鏈鎖反應(Quantitative real-time PCR)……………… 21
2.11 DNA膠體電泳………………………………………………………………… 23
三、結果………………………………………………………………………………… 24
3.1 建立HK-2細胞3D培養模式……………………………………………… 24
3.1.1 利用2D培養計算HK-2細胞增殖時間(generation time)…………… 24
3.1.2 利用WST-1進行細胞增殖測驗…………………………………………… 25
3.1.3 利用Live and dead staining觀察細胞生長與凋亡……………………… 33
3.2 比較HK-2細胞在2D與3D培養的基因表現………………………… 35
3.2.1 RNA濃度………………………………………………………………………… 35
3.2.2 以qRT-PCR檢測不同培養方式及時間點的基因表現………………… 36
3.2.3 抗體染色(immunocytochemistry) ………………………………………… 45
四、討論 ……………………………………………………………………………… 49
4.1 建立2D及3D細胞培養之技術討論…………………………………… 49
4.1.1 過去文獻與本次研究所使用之細胞材料比較…………………………… 49
4.1.2 HK-2細胞無法貼附於有明膠塗層的low-attach孔盤………………… 50
4.1.3 明膠海綿泡製時間過長導致影響3D細胞培養………………………… 51
4.1.4 WST-1無法反應3D環境中細胞的生長數目…………………………… 52
4.1.5 Live and dead staining確認種植細胞數目及增殖與型態……………… 53
4.2 基因表現的測定………………………………………………………………… 54
4.2.1 qPCR 的測定技術探討……………………………………………………… 54
4.3 2D、2.5D 與3D細胞培養的比較…………………………………………… 56
4.3.1 3D培養的細胞生長穩定，並近似近曲小管型態………………………… 56
4.3.2 3D培養細胞的基因表現較穩定，並表現較多量的的功能相關蛋白…………………………………………………………………………………
56
4.3.3 近曲小管transporter對於藥物代謝之重要性………………………… 58
五、結語………………………………………………………………………………… 61
六、參考文獻………………………………………………………………………… 62
七、附件………………………………………………………………………………… 69
7.1 live and dead cell count number …………………………………………… 69
7.2 RNA concentration …………………………………………………………… 70
7.3 qRT-PCR raw data ……………………………………………………………… 72
7.4 qRT-PCR P-value matrix ……………………………………………………… 84

圖目錄
圖1.1 2D（A）與3D（B-D）細胞培養示意圖。…………………………………… 4
圖1.2 細胞在二維（左）及三維（右）環境下，藥物、氧氣、廢棄物等代謝之差
異。………………………………………………………………………………
5
圖1.3 人體腎臟與腎元的型態………………………………………………………… 7
圖1.4 近曲小管的構造………………………………………………………………… 8
圖1.5 近曲小管運作主要的三個途徑：胞吞作用、藉由轉運蛋白主導的再吸收，及藉由轉運蛋白主導的分泌………………………………………………………
9
圖2.1 2D、2.5D 及3D 細胞培養之建立方式………………………………………… 17
圖3.1 利用WST-1 偵測細胞增生…………………………………………………… 30
圖3.2 比較細胞培養於一般培養皿及gelatin coating 於玻片並置放在低貼附培養皿中兩者細胞生長結果……………………………………………………………
31
圖3.3 細胞種植於gelatin 塗層的玻璃片上（2.5D），培養較長時間仍有聚集、堆疊及dome 之型態，特別在玻璃片的邊緣…………………………………………
32
圖3.4 Live and dead assay，綠螢光為活細胞，紅色螢光為死細胞…………………… 34
圖3.5 以Live and dead assay 比較細胞存活/死亡的計數…………………………… 35
圖3.6 RNA 萃取濃度比較……………………………………………………………… 36
圖3.7 近曲小管在胞吞作用及轉運蛋白再吸收的基因表現………………………… 38
圖3.8 頂層轉運蛋白分泌在2D、2.5D、3D 環境中不同天數的表現，主要分為三類 43
圖3.9 底層分泌功能的轉運蛋白在2D、2.5D、3D 環境中不同天數的表現，主要分為2 種……………………………………………………………………………
44
圖3.10 cytokeratin 8/18/19 在不同時間點中，2.5D 及3D 之間的表現……………… 46
圖3.11 GGT-1 在不同時間點中，2.5D 及3D 之間的表現…………………………… 47
圖3.12 OAT4 在不同時間點中，2.5D 及3D 之間的表現…………………………… 48
圖4.1 WST-1 原理……………………………………………………………………… 52
圖4.2 本次研究中，2D 及3D 細胞培養的基因表現之比較………………………… 58

表目錄
表2.1 本實驗使用抗體………………………………………………………………… 19
表2.2 qRT-PCR 引子…………………………………………………………………… 22
表3.1 細胞培養繼代計數……………………………………………………………… 24
表3.2 細胞培養測試條件及結果……………………………………………………… 29
表4.1 近曲小管中轉運蛋白位置及功能……………………………………………… 50

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指導教授

粘仲毅(Chung-Yi Nien)

審核日期

2021-1-27

推文