臺灣博碩士論文加值系統

我們發展出一種共培養方法，主要是利用懸浮培養的方式進行共培養，有別於傳統作法，這種方式培養出的細胞會聚合成球，形成立體的構型，而比起其他類似的系統，譬如說懸滴培養法，或是水膠培養法，我們的方法更簡便，而且可以支持長時間的培養。
本研究採用的細胞是纖維母細胞與角質細胞的共培養，纖維母細胞使用的是Hs68細胞系，角質細胞使用的則是HaCaT細胞系。這兩株細胞系皆來自成年男性的包皮，並且也被證實此兩種細胞有交互作用發生。
由於Hs68和HaCaT有懸浮在幾丁聚醣涂佈的細胞培養盤上的特性，我們利用這點發展成一個懸浮共培養方法，在這個系統下，可以很間單的利用曠時攝影的方式觀察細胞的聚集。本研究討論了Hs68和HaCaT於懸浮共培養下細胞的聚集模式，並利用了簡單的模擬去分析。目前在實際操作中的已經有了初步的效果。
而學理上，我們採用了鈣黏蛋白的不同去解釋我們模型的設計，所以，我們也做了有關細胞凝聚力的分析，用來支持我們的模型。

We developed a new co-culture method mainly in co-culturing cells in suspension. Unlike the traditional method, cells cultured by this method will aggregate to a sphere and form a 3D structure, which compared to other systems like the hanging drop method and the hydrogel method, our system is simple, easy and good in long term culture.
Our research used fibroblast and keratinocyte in co-culture, and we adopted Hs68 cell line from fibroblast and HaCaT cell line from keratinocyte. These cells are both from mature male foreskin, and the interaction existing between the two cells have been confirmed.
We used the shared characteristics of Hs68 and HaCaT, that both will suspend in chitosan coated plate, to perform a suspension co-culture method. In this system, we can easily observe how each cells aggregate in time-lapse photography. This paper also discussed the aggregation process in suspension co-culture system, which was analyzed by a simple simulation. We’ve got a first step results in hands-on practice at present.
Academically, we also adopted the theory about cadherin to explain our model design, thus we conducted some experiments in measuring cell-cell cohesion force to support our model.

摘要 i
Abstract ii
目錄 iii
圖目錄 vi
第一章 序論 1
第二章 文獻回顧 2
2.1 共培養系統（co-culture system） 2
2.1.1 trans-well method(構造如圖2-1所示) 2
2.1.2 hydrogel method 2
2.1.3 hanging drop method 3
2.2 纖維母細胞與角質細胞（Fibroblast & Keratinocyte） 3
2.2.1 纖維母細胞（Fibroblast） 3
2.2.2 角質細胞（Keratinocyte） 4
2.2.3 纖維母細胞與角質細胞共培養 4
2.3 細胞聚集因子（Cell aggregation factor） 4
2.4 幾丁聚醣（chitosan） 5
第三章 實驗材料與方法 6
3.1 實驗架構 6
3.2 實驗材料 7
3.3 儀器 9
3.4 試劑配製 14
3.5 實驗方法 16
3.5.1 細胞培養（cell culture）和繼代（passage） 16
3.5.2 幾丁聚醣基材製備 16
3.5.3 細胞活體螢光標定（fluorescent cell tracking） 17
3.5.4 懸浮共培養（suspension Co-culture） 18
3.5.5 共軛焦攝影 (confocal) 19
3.5.6 曠時攝影 (time-lapse) 19
3.5.7 細胞凝聚力測定 (cell cohesion force test) 20
3.5.8 模擬方法 (simulation method) 21
第四章 實驗結果 23
4.1 懸浮共培養形態觀察 23
4.1.1 比例控制 23
4.1.2 種植順序控制 23
4.2 共軛焦攝影 23
4.3 曠時攝影 23
4.4 細胞凝聚力測定 23
4.5 模擬 24
第五章 討論 26
5.1. 懸浮共培養形態觀察 26
5.2. 細胞凝聚力測定 26
5.3. 模擬 27
第六章 結論 30
第七章 參考文獻 31
第八章 圖表 34

圖目錄
圖 2-1 trans-well的構造。 2
圖 2-2 皮膚的構造。 4
圖2-3 幾丁聚醣的合成及結構。 5
圖3-1 實驗流程示意圖。 6
圖3-2 懸浮共培養實驗示意圖。 19
圖4-1不同比例Hs68與HaCaT懸浮共培養，經過三個天期的結果。 34
圖4-2 等比例Hs68與HaCaT懸浮共培養，改變種細胞順序經過三個天期的結果。 35
圖4-3 等比例Hs68與HaCaT懸浮共培養下，三個天期的共軛焦顯微鏡攝影結果。 36
圖4-4 等比例Hs68與HaCaT懸浮共培養下，曠時攝影一日的結果。 37
圖4-5 等比例Hs68與HaCaT懸浮共培養下，曠時攝影二日(第三日、第四日)的結果。 38
圖4-6 等比例Hs68與HaCaT懸浮共培養下，曠時攝影二日(第四日、第五日)的結果。 39
圖4-7 對細胞使用超音波，Hs68與HaCaT細胞團面積的變化。 40
圖4-8 震盪控制分布結果。 41
圖4-9 單種類細胞懸浮培養，本身聚合力之結果。 42
圖4-10 等比例Hs68與HaCaT懸浮共培養下，經一日後，又曠時攝影12小時的結果。 43
圖4-11 模擬方法一，固定居等機率。 44
圖4-12 模擬方法一，變動不定機率。 45
圖4-13 模擬方法二，自由運動模擬。 46

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