Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16743
標題: 動力學研究EGFR-EGF之交互作用與調控
Kineyic Studies on the Interaction and Modulation of EGFR-EGF
作者: 黃邁柏
Huang, Mai-Po
關鍵字: 表皮生長因子受體;EGFR;表面電漿共振;動力學;固定化接受器動態擷取技術;SPR;kinetic;AFTIR
出版社: 化學系所
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摘要: 
EGFR (Epidermal growth factor receptor) 常在人類的癌細胞過量的存在,當其與EGF (Epidermal growth factor) 結合後可引發眾多訊息傳導,導致細胞增生並與血管新生、細胞轉移等生理功能,與癌症難以治療有著相當大的關連性。透過抑制EGFR-EGF交互作用的抗癌藥物,於治療上有相當程度的效果,但亦對正常細胞產生影響,導致嚴重的副作用。進一步地去瞭解EGFR-EGF之交互作用,將有助於抗癌藥物之發展。
本研究之目的在建立EGFR與EGF交互作用的模型,並藉此模型評估小分子調控此交互作用的可行性。首先,選定low-affinity 和high-affinity EGFR分別代表正常細胞與癌細胞之EGFR,分別固定於微流體晶片上,透過SPR (Surface Plasmon Resonance) 偵測器,以動力學參數呈現EGFR-EGF之交互作用。實驗中,探討EGFR的固定量、EGF的濃度以及流速對偵測EGFR-EGF之交互作用動力學常數的影響,尋找能準確測量EGFR-EGF動力學的條件,獲得代表正常細胞與癌細胞的EGFR-EGF之動力學參數。
另一方面,透過此模型分析六十種中草藥中所含小分子對low-affinity EGFR及high-affinity EGFR的影響,評估是否有小分子能有選擇性調low-affinity EGFR與 high-affinity EGFR與EGF之能力?再利用AFTIR技術,分析這些混合物中真正參與調節作用的小分子。
結果顯示,適當控制系統中之EGFR固定量等因素可有效獲得準確之動力學參數,代表正常細胞與癌細胞的EGFR,其親和力(KD)與動力學參數(kd、ka)分別為( KD = 5.49×10-8 M, ka = 8.10×104 M-1s-1, kd = 4.45×10-3 s-1與KD = 8.69×10-9 M , ka = 2.51×105 M-1s-1, kd = 2.18×10-3 s-1)。
針對low-affintiy EGFR與high-affinity EGFR也皆有其中草藥樣品可影響其與EGF結合動力學,其中紅骨蛇 (編號051樣品) 針對high-affinity EGFR可降低其結合能力約3倍,使其親和力降低約100倍。
將紅骨蛇進一步回收後,透過HPLC分析發現其包含二種主要成份 (peak A, peak B),並純化出其中一成份peak B,peak B具有使high-affinity EGFR-EGF之kd值下降之能力。

Over-expression of EGFR (Epidermal Growth Factor Receptor) has always been observed in human cancer cells. As soon as EGF (Epidermal Growth Factor) binds EGFR, it causes EGFR tyrosine kinase domain activation, and downstream signal transductions including cell proliferation, angiogenesis and metastasis. Cancer which hard to cure is known as associated with all of these signal transductions. Some reports showed that EGFR inhibitors induce side effects due to its lower selectivity to cancer cell. To study EGF-EGFR interactions can help to develop anti-cancer drug discovery.
In this study, the goal is to set up a model to investigate EGFR-EGF interactions and by using it to estimate the possibility of small molecular modulation to EGFR-EGF interaction. Two types of EGFR, low-affinity and high-affinity EGFR, represented as normal and cancer cell, are immobilized respectively on a micro-fluidic chip with a SPR (Surface Plasmon Resonance) detector. Kinetic parameters of EGFR-EGF are measured at different conditions. The effect of EGFR density, EGF concentration and flow rate on kinetic parameter measurement is evaluated. It gives a optimal condition for measuring EGFR-EGF kinetic parameters accurately and following screen small molecular modulators.
Besides, by using the model, 60 of TCMs (Traditional Chinese Medicines) are used to pre-treat EGFR. The effect of TCMs on kinetic parameters and selectivity of EGFR modulation are measured as soon. Finally, we can use the AFTIR (After Flowing Through Immobilized Receptor) method to identify the active component.
The result showed that by using SPR detector kinetic constants of EGFR-EGF interaction can be measured accurately for represent normal and cancer cell at a optimal condition of EGFR immobilization density, EGF concentration and flow rates. The kinetic data indicate that low-affinity EGFR with KD of 5.49×10-8 M, ka of 8.10×104 M-1s-1,kd of 4.45×10-3 s-1 and high-affinity EGFR with KD of 8.69×10-9 M , ka of 2.51×105 M-1s-1, kd of 2.18×10-3 s-1.
There are some TCMs can exactly affect the EGF-EGFR kinetic parameter. Kadsurae Radix Cum Caulis (051 sample) causes a 3-fold decrease in high-affinity EGFR-EGF association and also reduces its affinity by apparent 100-fold.
We have collected and purified active compounds in Kadsurae Radix Cum Caulis. Two compounds (as called as peak A and peak B) are identified. Among these, peak B has the ability to reduce dissociation rates constant of high-affinity EGFR-EGF.
URI: http://hdl.handle.net/11455/16743
其他識別: U0005-2108200915442500
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