探討成纖維細胞生長因子-2促進細胞生長之最佳形式以及不同添加劑對其蛋白穩定性之影響

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研究生：	呂雨庭 Lu, Yu-Ting
論文名稱：	探討成纖維細胞生長因子-2促進細胞生長之最佳形式以及不同添加劑對其蛋白穩定性之影響 Investigation of the effects of different forms of fibroblast growth factor 2 on cell growth and various excipients on the protein stability
指導教授：	張晃猷 Chang, Hwan-You
口試委員:	張壯榮 Chang, Chuang-Rung 張晉源 Chang, Chin-Yuan
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2023
畢業學年度：	111
語文別：	中文
論文頁數：	53
中文關鍵詞：	成纖維細胞生長因子-2 、蛋白質療法、DNA 療法、mRNA 療法、藥物賦形劑
外文關鍵詞：	Fibroblast growth factor-2 (FGF-2), Protein therapy, DNA therapy, mRNA therapy, Pharmaceutical excipients
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成纖維細胞生長因子2 (Fibroblast Growth Factor 2, FGF-2)參與許多重要的生物過程，如胚胎發育、細胞增生以及血管新生等。許多研究指出FGF-2可以有效的促進成纖維細胞的增生以及遷移，並幫助傷口的癒合。然而其極度不穩定的特性，使其在製成醫藥產品時極具挑戰。本研究的目的旨在於找出FGF-2作為藥物之最佳給藥方式，並嘗試增加FGF-2蛋白的穩定性。本研究首先利用大腸桿菌重組蛋白的表達系統產生大量FGF-2並建立純化方式，然後將純化的FGF-2蛋白利用小鼠胚胎成纖維細胞BALB/3T3進行測試。結果表明，重組FGF-2蛋白可以促進BALB/3T3細胞的增生以及遷移，確認自行純化之FGF-2具有生物活性。然而蛋白藥物的製備複雜且耗時，因此我們嘗試使用暫時轉染FGF-2 cDNA以及mRNA的方式，以確認其效果是否優於直接使用蛋白。首先建構兩種不同5′UTR的FGF-2表達質體，分別帶有pEGFP-C1的5′UTR以及具有高核醣體結合的5′UTR，並將之轉染至BALB/3T3細胞。細胞增生率的結果顯示出含有pEGFP-C1的5′UTR的FGF-2可以顯著促進BALB/3T3細胞的增生，但是以西方點墨法分析兩種不同 5′UTR的FGF-2轉染至HEK293T細胞中皆無法明顯增加FGF-2蛋白在HEK293T細胞中的表達量。利用實驗室自行生產的T7 RNA聚合酶以及大腸桿菌多聚腺苷酸聚合酶，成功的在試管中生產出FGF-2 mRNA，也利用商業套組在其上加上5′端帽。但是所建構的FGF-2 mRNA並無法促進BALB/3T3細胞的增生。以上結果顯示出FGF-2暫時轉染的效果不佳，推測可能是由於FGF-2蛋白本身的不穩定性造成。因此我們測試多種可能增加FGF-2蛋白在37℃下穩定性的成分，結果顯示胎牛血清、Triton X-100、胰化蛋白腖、聚磷酸鈉以及羧甲基纖維素可以明顯的增加FGF-2蛋白的穩定性。其中羧甲基纖維素可以促使FGF-2蛋白形成二聚體，並且更加有效的促進BALB/3T3細胞的增生。本研究結果證明了在單純促使細胞生長上，使用FGF-2蛋白質比用cDNA與mRNA的形式要好，並且羧甲基纖維素在FGF-2蛋白藥物傳輸上具有很好之潛力。

Fibroblast growth factor 2 (FGF-2) is a multifunctional growth factor involved in many biological processes, such as embryogenesis, cell proliferation, and angiogenesis. Many studies indicated that FGF-2 could effectively promote fibroblast proliferation and migration than help wound healing. However, FGF-2 is unstable both in vivo and in vitro. This study aims to identify the best FGF-2 form to promote cell proliferation and compounds that can increase the FGF-2 protein stability. This study established an Escherichia coli expression system and a purification scheme to produce FGF-2 recombinant protein. The purified FGF-2 protein was tested on mouse BALB/3T3 fibroblasts, and the results showed that FGF-2 could promote the proliferation and migration of the cells, indicating that the self-established FGF-2 protein has biological activity. This study also tested if transient FGF-2 cDNA and mRNA transfection into the cells could be more effective than the protein treatment. Using FGF-2 plasmids with either the 5′UTR of pEGFP-C1 or the 5′UTR of high-ribosome loading synthetic sequences to transfect BALB/3T3 cells has shown that the FGF-2 encoding plasmid has 5′UTR of pEGFP-C1 can significantly promote the proliferation of mouse fibroblasts. On the other hand, Western blot analysis demonstrated that transfection of FGF-2 plasmids with either 5'UTRs into HEK293T cells could not significantly increase FGF-2 protein synthesis. The in vitro FGF-2 mRNA was synthesized using T7 RNA polymerase and poly(A) polymerase generated in our laboratory and a commercial mRNA capping system. Nevertheless, the FGF-2 mRNA could not promote BALB/3T3 cell proliferation, presumably due to the instability of the FGF-2 protein. This study then tested several compounds for their ability to improve the stability of FGF-2 protein at 37°C. It was noted that the stability of FGF-2 protein could be significantly enhanced in the presence of fetal bovine serum, tryptone, Triton X-100, sodium polyphosphate, or carboxymethyl cellulose. Adding carboxymethyl cellulose can promote the dimerization of FGF-2 protein and the proliferation of BALB/3T3 cells more effectively. In conclusion, this study demonstrated that FGF-2 protein is more effective than FGF-2 cDNA and mRNA in promoting fibroblast proliferation, and carboxymethyl has the potential to be used as a pharmaceutical excipient for FGF-2 protein delivery.

中文摘要............................................II
Abstract...........................................III
致謝.................................................V
目錄...............................................VII
表目錄...............................................X
圖目錄..............................................XI

壹、前言..........................................................1
1 成纖維細胞生長因子-2(Fibroblast growth factor 2, FGF-2)........1
2 蛋白質療法....................................................2
3 DNA療法.......................................................3
4 體外轉錄mRNA(IVT mRNA)療法.....................................4
5 藥物賦形劑.....................................................5
6 研究目的.......................................................6

貳、材料與方法....................................................7
1 大腸桿菌菌株培養與勝任細胞製備..................................7
1.1 大腸桿菌菌株培養.............................................7
1.2 大腸桿菌勝任細胞的製備.......................................7
2 BALB/3T3與HEK293T細胞培養.....................................7
2.1 細胞培養液製備..............................................7
2.2 細胞生長培養液製備..........................................8
2.3 細胞培養條件...............................................8
3 蛋白表達與純化...............................................8
3.1 FGF-2表達與純化............................................8
3.2 T7 RNA聚合酶表達與純化.....................................9
3.3 多聚腺苷酸聚合酶表達與純化.................................10
4 質體建構...................................................10
4.1 引子合成.................................................10
4.2 聚合酶連鎖反應(Polymerase chain reaction, PCR)擴增目標基因片段............................................................11
4.3 目標基因片段(Insert)與載體(Vector)的建構..................11
4.4 連接反應(Ligation).......................................11
4.5 轉型作用(Transformation).................................12
5 mRNA的合成.................................................12
5.1 體外轉錄(In vitro transcription, IVT)合成mRNA.............12
5.2 mRNA 5′端加帽............................................13
5.3 mRNA 3′端加多聚腺苷酸尾(Poly-A Tail)......................13
6 BALB/3T3細胞暫時轉染.......................................14
6.1 質體轉染.................................................14
6.2 mRNA轉染.................................................14
7 BALB/3T3細胞增生測試........................................14
8 BALB/3T3細胞遷移測試........................................15
9 FGF-2蛋白穩定性檢測.........................................15
9.1 FGF-2蛋白於不同條件之DMEM培養基之穩定性檢測.................15
9.2 FGF-2蛋白於不同賦形劑下之穩定性檢測.........................16
10 西方墨點法(Western Blot)檢測................................16
11 Image J 圖像分析與計算......................................17

參、 結果.......................................................18
1 FGF-2蛋白對BALB/3T3細胞的影響................................18
1.1 FGF-2蛋白之表達與純化......................................18
1.2 FGF-2蛋白促進BALB/3T3細胞的增生............................18
1.3 FGF-2蛋白促進BALB/3T3細胞的遷移............................19
2 FGF-2質體暫時轉染對BALB/3T3細胞的影響.........................19
2.1 pcDNA3.1-pEGFP-C1-5′UTR-FGF2質體建構......................19
2.2 pcDNA3.1-5′UTR4-FGF2質體建構..............................20
2.3 轉染帶有pEGFP-C1之5′UTR的FGF-2質體可以促進BALB/3T3 細胞的增生..............................................................20
2.4 以西方點墨法分析轉染FGF-2質體的HEK293T細胞中FGF-2的表達量.....20
3 FGF-2 mRNA暫時轉染對BALB/3T3細胞的影響........................21
3.1 pUC19-ᵠ2.5-pEGFP-C1-UTR質體建構............................21
3.2 體外轉錄mRNA之合成..........................................21
3.3 FGF-2 mRNA轉染無法促進BALB/3T3細胞增生......................22
4 FGF-2蛋白穩定性測試...........................................22
4.1 含有胎牛血清之DMEM培養基可以增加FGF-2蛋白穩定性...............22
4.2 胰化蛋白腖、Triton X-100、聚磷酸鈉以及羧甲基纖維素可以穩定FGF-2蛋白...............................................................23
4.3 不同濃度之Triton X-100、聚磷酸鈉以及羧甲基纖維素對FGF-2蛋白穩定性之影響.............................................................24
4.4 Triton X-100 (0.05% (w/v))、聚磷酸鈉(0.1% (w/v))以及羧甲基纖維素(0.025% (w/v)) 於不同時間點對FGF-2蛋白之穩定性以及二聚體化之影響.....25
4.5 Triton X-100和聚磷酸鈉具有細胞毒性...........................25
4.6 加入羧甲基纖維素的FGF-2蛋白可以促進BALB/3T3細胞的增生.........26

肆、討論.........................................................27
伍、參考資料.....................................................32

                                

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