重組精胺酸脫亞胺酶及其含穿胞胜肽HBHAc-HE修飾劑型分別於乳腺癌2D 與3D細胞培養模型的效果研究

Study on the effects of recombinant arginine deiminase and its modified form with a cell-penetrating peptide HBHAc-HE, in 2D and 3D cell cultures, respectively, of breast cancer cells

張博勛(Po-Hsun Chang)

指導教授 ： 沈麗娟

國立臺灣大學/醫學院/藥學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900219

摘要

由於腫瘤細胞的異型性使單層細胞模型與真實人體腫瘤的差異性增大，因此目前的2D細胞培養模型並不足以用來預測人體的生理狀況。為了縮小體外試驗與活體試驗的差異性，發展與人體具有生理相似性的體外3D細胞培養模型在近年來受到了愈來愈多的關注。本研究以人類乳腺癌MCF-7與MDA-MB-231細胞，建立體外3D細胞培養模型，並利用兩株乳腺癌細胞不同的特性，比較其在3D和2D細胞培養模型對所製備之蛋白質藥物ADI與HBHAc-HE-ADI的敏感性差異。期望能以更接近人體內腫瘤真實狀況的體外3D細胞培養模型，了解所製備藥物之發展與應用的可行性。在藥物製備部分，以轉型作用將ADI及HBHAc-HE-ADI之質體送入E.coli BL21，藉由定序確認基因序列正確性後，以2X YT medium培養並加入IPTG使菌體大量表現蛋白，離心後將菌體打破以取得內涵體並溶出蛋白，經重新摺疊、濃縮、純化並透析等步驟後獲得ADI及HBHAc-HE-ADI，並以SDS-PAGE及Western Blots確認其正確性，再經Bradford protein assay定量，並以ADI activity assay測得其活性分別可達259及177 mU/mg。在2D細胞培養模型試驗部分，設計不同pH及氧氣濃度之差異，並以MCF-7及MDA-MB-231此二細胞株相互比較，結果顯示：MDA-MB-231 與MCF-7相比，不論在不同pH或氧氣濃度下皆對ADI及HBHAc-HE-ADI更具敏感性；而HBHAc-HE-ADI與ADI相比，在不同pH及不同氧氣濃度下對兩細胞株之效果皆無顯著差異。在建立3D細胞培養模型部分，以非貼附型培養法並比較不同加入胞外基質方式，培養出均質的MCF-7及MDA-MB-231腫瘤球；接著藉由測量不同細胞密度所形成之腫瘤球的直徑大小，選定適合之起始細胞濃度，並測量不同天之代謝活性，綜合比較腫瘤球的外型、直徑與代謝活性後選定最適合之給藥日。在3D細胞培養模型試驗部分，MDA-MB-231及MCF-7腫瘤球對ADI及HBHAc-HE-ADI皆具有好的抗性，在0.001至10 mU/mL劑量間的細胞存活率皆無顯著差異，測量腫瘤球在給藥期間的直徑大小亦無變化，顯示出3D與2D細胞培養模型對藥物之敏感性具有一定程度之差異。本研究所建立起之3D細胞培養模型，在未來亦可應用於其它藥物之篩選開發，作為藥物由2D細胞培養模型試驗進入活體試驗前的橋樑。

關鍵字

精胺酸去亞胺酶 ； 3D細胞培養 ； 腫瘤球 ； 乳腺癌 ； 細胞穿透胜肽

並列摘要

The differences between a monolayer cell culture and spheroids in human come from the heterogeneity of spheroids. Thus, current 2D cell culture is not enough to be an ideal model for predicting physiological conditions in human bodies. To make the difference between in vitro and in vivo assays smaller, the development of 3D in vitro cell culture which is similar to human body has drawn more and more attentions in recent years. This study built up 3D in vitro cell cultures of MCF-7 and MDA-MB-231 breast cancer cells and compared the differences of sensitivity to the protein drugs we prepared, ADI and HBHAc-HE-ADI, between 2D and 3D cell cultures. We would like to know the possibility of development and application of the drugs prepared in this study by a 3D in vitro cell culture which is more similar to spheroids in human body. On the part of drug preparation, we transformed the plasmid of ADI and HBHAc-HE-ADI into E.coli BL21. After confirming the sequences were correct, we cultivated the bacteria by 2X YT medium and added IPTG to make them express the protein in a large amount. The pellets were centrifuged and lysed to get the inclusion bodies. Then the proteins were soluted out and went through the steps of refolding, condensing, purification and dialysis. Finally, we got the ADI and HBHAc-HE-ADI. We confirmed the correctness of them by SDS-PAGE and western blots, and used Bradford protein assay to quantify the amount of proteins. With ADI activity assay, we knew the unit activity of ADI and HBHAc-HE-ADI were 259 and 177 mU/mg, respectively. On the part of 2D cell culture assays, we design different pH and oxygen concentrations in each group and compared the differences between MCF-7 and MDA-MB-231. The results showed that MDA-MB-231 was more sensitive to ADI and HBHAc-HE-ADI than MCF-7 either at different pH or oxygen concentrations, and no significant differences between the effects of HBHAc-HE-ADI and ADI on two cell cultures were found at different pH and oxygen concentrations. On the part of building up the 3D cell cultures, non-adherent culture was used and different methods of adding excellular matrix were compared in order to form up homogeneous MCF-7 and MDA-MB-231 spheroids. After measuring the diameter of spheroids formed by different seeding cells, the most appropriate seeding cell density was chosen and the metabolic activity of them at different days were measured. Finally, the dosing day was chosen after taking the morphology, diameter, and metabolic activity of spheroids into account. On the part of 3D cell culture assays, both MDA-MB-231 and MCF-7 spheroids showed resistance to ADI and HBHAc-HE-ADI that their cell viability had no significant differences between the doses ranged from 0.001 to 10 mU/mL. Also, the diameter of spheroids didn’t change during drug administration. These results showed that the sensitivity to drugs was different between 3D and 2D cell cultures. The 3D cell culture models built up in this study can be applied to the screening and development of other drugs, which would be a link between 2D cell culture assays and in vivo assays.

並列關鍵字

arginine deiminase ； 3D cell culture ； spheroids ； breast cancer ； cell-penetrating peptide

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