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研究生:林泳沖
研究生(外文):Yong-Chong Lin
論文名稱:毛髮生長因子之表現與應用
論文名稱(外文):Expression and application of the hair growth factors
指導教授:林佳靜林佳靜引用關係
指導教授(外文):Chai-Ching Lin
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:95
中文關鍵詞:毛髮生長因子
外文關鍵詞:hair growth factor
相關次數:
  • 被引用被引用:2
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毛囊為人體中相當複雜且神秘的組織,其週期循環與生長受到許多生長因子
所調控,而本研究推論最具影響力的兩種生長因子:類胰島素生長因子第一型
(Insulin- like growth factor-1, IGF-1)與幹細胞因子(Stem cell factor, SCF),
將之命名為〝毛髮生長因子〞。IGF-1 已被證實其可調控毛髮之週期循環(hair
cycling),並刺激毛髮生長;SCF則調控黑色素細胞(melanocyte)之增殖與存
活,進而影響毛髮黑色素(melanin)之形成。於本實驗室先前報告中証實:若將
此兩種毛髮生長因子合併使用,可有效改善毛髮灰化(hair graying)小鼠模式的
白毛現象、並促進小鼠毛囊體外培養的毛髮生長。
是以本實驗目的:選殖與純化人類毛髮生長因子—hIGF-1 與hsSCF,並證實
其在促進毛髮生長之效用。並產製新型的融合生長因子—double SCF (rdhSCF)
及hIGF-1/hsSCF (rhISF),預期簡化製成與提高生物活性。本實驗將所有生長
因子以大腸桿菌系統(E. coli expression system)表現,並以親合性管柱(affinity
column)成功地進行純化。
經由細胞增殖活性証實,所有毛髮生長因子皆具有生理活性。而hIGF-1 與
hsSCF,分別於毛囊體外培養與小鼠皮膚修復(skin healing)試驗中證實,亦具
有顯著促進毛髮生長及皮膚修復的效果(p<0.05)。基於毛髮生長因子於毛髮之
生理功能,其在未來治療禿頭或毛髮異常脫落上,必扮演一重要性且具潛力性
的角色。
The insulin like-growth factor-1 (IGF-1) and stem cell factor (SCF) are major growth factors to participate in the hair growth, cycling and stem cell migration,which are named as the hair growth factors in the study. The function of IGF-1 on the hair follicle is as an important physiologic regulator on the hair cycling and promotes the hair growth. SCF is a pleiotropic cytokine acting mainly on
melanogenesis by promoting the survival and proliferation of melanocyte, and also stimulate the migration of the melanocyte stem cell. Both of them should have a potential synergistic effect on the hair growth to improve the human baldness.
In the study, the hair growth factors of individual hIGF-1 and hsSCF, as well as the novel fusion proteins of double human SCF (rdhSCF) and hIGF-1/hsSCF(rhISF) were cloned, expressed and purified. All of hIGF-1, hsSCF, rdhSCF and rhISF are cloned into the E. coli expression vector pET24a and expressed in BL21Codon Plus® (DE3)-RIPL strain. These expressed recombinant proteins
constitute up to 22%, 28%, 9% and 15% of the total bacterial protein, respectively. All are purified by using only one column of the affinity His-bind, that is modified to become simpler and quicker. Each hair growth factor is able to successfully stimulate a dose-dependent cell proliferation in 3T3 or HBM cells. Furthermore, hIGF-1 and hsSCF significantly promote the hair growth in vitro and skin healing in vivo (p<0.05). Based on the physiological functions of the hair growth factors in hair follicle, it might be a more
potential therapy for the balding disorder.
誌謝.................................................................3
中文摘要.............................................................5
Abstract............................................................6
Figures and tables..................................................7
Introduction.......................................................11
Chapter 1. Literature review
1.1. Regulatory mechanisms of hair growth
1.1.1. The structures and functions of hair........................13
1.1.2. The cycling of hair follicles...............................15
1.1.3. Growth factors for hair follicle ...........................16
1.2. Stem Cell Factor, SCF
1.2.1. The structure and functions of SCF..........................19
1.2.2. Regulatory mechanisms of SCF................................22
1.2.3. Effect of SCF on hair follicle..............................24
1.3. Insulin-like Growth Factor-I, IGF-1
1.3.1. The structure and functions of IGF-1........................26
1.3.2. Regulatory mechanisms of the IGF-1..........................27
1.3.3. The function of IGF-1on hair follicles......................30
Chapter 2. Materials and methods
2.1. Gene construction of the recombinant human hair growth factors
2.1.1. RNA isolation and first-strand cDNA synthesis...............32
2.1.2. Construction of the human SCF expression vector.............32
2.1.3. Construction of the human IGF-1 expression vector...........33
2.1.4. Construction of the recombinant dual human SCF expression vector.............................................................33
2.1.5. Construction of the novel recombinant fusion protein of human SCF and IGF-1 expression vector....................................34
2.2. Expression and purification of the recombinant human hair growth factors
2.2.1. Expression of the recombinant human hair growth factors in E. coli...............................................................35
2.2.2. Isolation of inclusion bodies...............................36
2.2.3. Purification and refolding of recombinant human growth factors............................................................36
2.2.4. Analysis of the expressed recombinant human hair growth factors...................................................37
2.3. Assay of the hair growth factors on the biological function
2.3.1. Assay of cell proliferation in vitro........................37
2.3.2. Assay of the hair growth in vitro...........................38
2.3.3. Assay of skin healing recovery in vivo......................38
Chapter 3. Results.................................................43
Chapter 4. Discussion..............................................83
Chapter 5. Conclusion..............................................87
References.........................................................88
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