臺灣博碩士論文加值系統

摘要
傳統之腫瘤治療方式主要有外科手術，化學療法及放射線療法，但治癒率不高而且容易產生副作用及併發症；現今之腫瘤治療以合併治療的方式，比起單獨使用某一方式治療來得有效且可以減低副作用之產生。因此，我們利用一種不同於傳統之腫瘤治療藥物，並合併化療藥物，來進行腫瘤之治療研究，期能找到一種有效且低副作用之腫瘤治療方式。在本研究中，我們將台灣本土之雞母珠毒蛋白(abrin)的毒性A chain基因與愛滋病毒 (human immunodeficiency virus; HIV) 之TAT (transcription activator) 的蛋白傳送區 (protein transduction domain; PTD) 基因進行融合，於大腸桿菌表現與純化後，獲得到TAT-ABR-A 重組蛋白。另外，我們也以限制酶將TAT-ABR-A 之TAT切除，然後表現並純化得ABR-A。 經SDS-PAGE 與西方點漬分析後, 將TAT-ABR-A 或ABR-A等重組蛋白加入肺癌細胞 (Lewis lung carcinoma cell; LLC) 及內皮細胞 (bovine aortic endothelial cell; BAEC) 的培養液中。發現這些重組蛋白均能改變細胞形態，抑制細胞增生，甚至誘發細胞死亡。以Hoechst染色TAT-ABR-A或ABR-A處理之細胞發現細胞核呈現染色體萎縮 (chromosome condensation)或DNA斷裂(DNA fragmentation)，部分細胞有分裂延遲的現象。流式細胞儀分析發現TAT-ABR-A處理明顯增加 sub-G0/G1期之細胞，並改變G0/G1與 S/G2M等細胞週期分布。以西方點漬分析也發現TAT-ABR-A較ABR-A顯著減少bcl-2，cdk-1，及cdk-2 的細胞表現量，由上列研究顯示 TAT-ABR-A 不僅會造成細胞凋亡(apoptosis) 而且會造成細胞分裂延遲。此外，這些結果亦證實TAT-ABR-A抑制細胞生長之能力約為ABR-A之5-10倍強。蛋白合成分析顯示TAT-ABR-A 與 ABR-A 抑制蛋白合成之能力相似。另外，抗氧化劑能緩解TAT-ABR-A 或 ABR-A對細胞增生之抑制作用，顯示兩者造成細胞死亡之機轉均與過氧化物產生有關。利用FITC (fluorescein isothiocyanate) 蛋白結合實驗發現TAT-ABR-A 之FITC 結合蛋白在加入培養液半小時內便能有效被細胞吸收，而加入ABR-A 之FITC 結合蛋白則未發現細胞內顯著螢光，証實TAT-ABR-A較ABR-A更能有效進入細胞。TAT-ABR-A能增加cisplatin 對LLC之生長抑制效應。我們利用動物實驗評估ABR-A，TAT-ABR-A，及化療藥物cisplatin對動物腫瘤模式之單獨或合併治療效果。 發現單獨注射TAT-ABR-A或ABR-A蛋白無法明顯減緩小鼠身上之腫瘤生長。然而，合併TAT-ABR-A 及cisplatin兩種藥物治療之小鼠的腫瘤較其他實驗組明顯縮小。小鼠體重變化指出注射TAT-ABR-A或ABR-A 蛋白未影響化療藥物cisplatin所造成之副作用。總結來說，我們利用基因工程技術生產出具功能性之毒性重組蛋白，並利用這些重組蛋白完成細胞訊息傳導與動物癌症抑制之研究。

Abstract
The major procedures for cancer therapy include surgery, chemotherapy and ionizing radiation therapy. However, none of these approaches were completely effective. Besides, they frequently caused severe side effects and serious complication during clinical application. Therefore, the development of combinatory therapy using chemotherapy drugs and novel anti-cancer agents is highly demanded, which would improve the therapeutic efficacy while minimizing undesired effects. In present study, we produced recombinant cytotoxic A chain of abrin (ABR-A) as well as its fusion form with cell-penetrating peptide TAT (TAT-ABR-A). After expression and purification, the molecular weight of recombinant ABR-A and TAT-ABR-A were determined at 32 and 34 kDa, respectively. ABR-A and TAT-ABR-A exhibited differential abilities in inhibiting the proliferation of Lewis lung carcinoma cells (LLC) and bovine aortic endothelial cell (BAEC). Nucleus staining indicated treatment of cells with TAT-ABR-A resulted in chromosome condensation, DNA fragmentation, and attenuated mitosis. Flow cytometry analysis revealed the TAT-ABR-A increased the number of cells in sub-G0/G1. TAT-ABR-A decreased the levels of bcl-2, cdk-1, and cdk-2 as revealed by western blot analysis. Together, these results indicated the potency of TAT-ABR-A in inhibiting cell proliferation is 5-10 folds higher than ABR-A. In rabbit reticulocyte translation system, the efficacy of TAT-ABR-A in inhibition of protein synthesis is similar to that of ABR-A. Furthermore, pretreatment with antioxidants such as superoxide dismutase- (SOD) or N-acetyl-L-cysteine (NAC) attenuated the cytotoxicity of TAT-ABR-A and ABR-A, indicating generation of reactive oxygen species participated in the cytotoxic mechanism of both proteins. By chemical conjugation with FITC (fluorescein 5-isothiocyanate), cellular uptake of TAT-ABR-A was detected within less than 30 min, whereas there was no significant uptake of ABR-A even after 16 h. These data indicated that fusion with TAT promoted the entry and increased the cytotoxicity of ABR-A. Subsequently, we analyzed the efficacy of cisplatin on LLC proliferation in the presence of TAT-ABR-A or ABR-A, and found that TAT-ABR-A significantly enhanced the cytotoxicity of cisplatin. The combinatory effect of TAT-ABR-A or ABR-A with cisplatin was evaluated in Lewis lung carcinoma grown in C57BL/6 mice, in which the tumors were grown to ~100 mm3 before treatment. Administration of TAT-ABR-A or ABR-A alone had no significant inhibitory effect on tumor growth. However, the combination therapy using TAT-ABR-A and cisplatin significantly reduced the tumor growth. Examination of body weight of tumor-bearing mice indicated that the injection of TAT-ABR-A or ABR-A did not aggravate the side effects of cisplatin. In summary, we demonstrated the feasibility of using genetic engineering techniques to produce biologically functional toxin, ABR-A. Furthermore, the efficacy of this recombinant toxin could be improved through fusion with cell-penetrating peptide. Above all, this improved toxin might be use TAT-ABR-A as adjuvant agents in conjunction with chemotherapy drugs to improve the therapeutic efficacy for treatment of cancer.

Index
Abstract………….…………………………………………………………………………………………………...I
摘要………………………………………………….…………………………………………………………………..…….IV
Acknowledgments…………………………………………………………………………………………….…..VI
Index……………………………………………………………………………………………………………………..…VIII
List of illustrations……………………………………………………………….……………..………………...X
List of tables…………………………………………………………………………………………………….……..XI
Introduction…………………………….…………………………………………………………………..…………..1
Literature review……………………………………………………………………………………………..……4
Endocytosis and Penetration…………………………………………….…………………….……4
Protein Transduction……………………………………………………………………….……….……4
TAT (transactivator of transcription)……….…………………………………..…..…..…....7
PTD (protein transduction domains)……………………………..……………………….…9
ABR-A (abrin subunit A chain)…………………………………………..…………….……..10
Apoptosis (programmed cell death)………………….……………………………….……12
Cancer therapy……………………………………..…………………………….………………………...14
Materials and methods………………………………………………………………………….…..……...15
Expression of TAT-ABR-A and ABR-A in Escherichia coli…….……18
Cell culture……………………………….…………………….………………………………….…….…….19
Proliferation assay………………………..……………………..………………………………….……19
Flow cytometry analysis……………………………………………..……………………….…….20
Hoechst 33258 nuclear staining…………………………………..…………………….……..20
Cell extract and western blot………………………………….…………………………….…...21
MTT assay.…………………………………………………………………..………………………….……..22
Measurement of in vitro protein synthesis inhibition………………….……...22
Fluorescein conjugation……………………………………………………………..……....…..…..23
Mouse studies……………………………………………………………………..………………….……..24
Results………..……………………………………………………………………………………………………..…….25
Construction of E. coli expression plasmids for production
of ABR-A and TAT-ABR-A…….………………………………………………….…..25
Expression and purification of ABR-A and TAT-ABR-A……….………. 25
TAT-ABR-A and ABR-A inhibited the proliferation of
Lewis lung carcinoma cells (LLC) and bovine artery
endothelial cells (BAEC)……………………………………..…………….…….………26
TAT promote entry of ABR-A into cells…………………………….……….……….. 27
TAT-ABR-A and ABR-A induced apoptosis and affected
cell cycle progression in LLC…………………….……………………..…………....27
Reactive oxygen species (ROS) participated in the cytotoxic mechanism of ABR-A and TAT-ABR-A……………………....……..….... 29
TAT-ABR-A and ABR-A inhibited protein synthesis……..…….…...…….30
Combinatory effect of TAT-ABR-A with cisplatin on
proliferation of LLC….…………………….……………………………………….…….…..30
In vivo tumor growth assessment……………….………..…………………………..…..….31
Evaluation of side-effects……………………………….……………………………………...….31
Discussion…………………………………………………………………………….…………………..……………32
Figures and legends………………………………………………………………………………………….. 35
References………….………………………………………………………………………………………..………...55

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