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研究生:蘇婉佩
研究生(外文):Wan-PeiSu
論文名稱:鋅手指樣蛋白對特定癌症誘發免疫反應以達抗癌效果
論文名稱(外文):Self-polymerizing Zfra peptides elicit immune response for targeting cancer
指導教授:張南山
指導教授(外文):Nan-Shan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:63
中文關鍵詞:鋅手指樣蛋白含雙色胺酸功能區氧化還原酶玻尿酸酶-2細胞凋亡抗癌胜肽神經膠質瘤乳癌神經母細胞瘤皮膚癌
外文關鍵詞:ZfraWOX1Hyal-2apoptosisanti-cancer peptidegliomaneuroblastomabreast cancerskin cancer
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Zfra(Zinc finger-like protein that regulates apoptosis) 參與在TNF訊息傳導路徑中,並且與腫瘤抑制蛋白WOX1有交互作用。Zfra可於活體外模式下引起細胞凋亡,當Zfra的Ser8改變成Gly時則會破壞這項能力。最近我們證實了,部分蛋白質一旦與Zfra共價結合,被Zfra化(Zfration)的蛋白質將會透過非泛素/蛋白酶體(proteasome/ubiquitination)的路徑快速降解。Zfra可抑制WOX1引起的細胞凋亡,但Zfra影響WOX1的機制尚不清楚,在此,我們證實了Zfra與WOX1間可能是透過共價鍵鍵結,此外也證實了Zfra中的Ser6和Ser7未參與在調控Zfra所引起的細胞凋亡中。在活體實驗中,預先於裸鼠尾靜脈注射Zfra1-31或截斷型的Zfra4-10可抑制人類神經膠質瘤U87-MG,乳癌細胞MDA-MB-468,神經母細胞瘤SH-SY5Y以及皮膚癌細胞的生長。將施打過Zfra的裸鼠的脾臟細胞注射到NOD SCID老鼠血液中,這些NOD SCID對於植入的腫瘤也具有抗性,推測存在有一群非T細胞的免疫細胞參與在限制腫瘤生長中。在Zfra注射前,預先注射辨認B或T細胞的抗體,我們發現CD19+、CD27+或IL2R-α+的細胞可能為Zfra作用的標的細胞或者是參與在調控Zfra於活體內預防癌症的能力。此外,我們也發現了一個具有成為Zfra受器的蛋白,Hyal-2 (Hyaluronidase type 2),Hyal-2抗體可於裸鼠、NOD-SCID以及BALB/c中抑制皮膚癌生的生長。我們的結果推測Zfra可能是透過活化Hyal-2+的免疫細胞以達到抗癌效果。
Zfra (Zinc finger-like protein that regulates apoptosis), which is involved in TNF signaling, physically interacts with a candidate tumor suppressor WOX1. Zfra induces cancer cell apoptosis in vitro, while alteration Ser8 in Zfra to Gly would abolishes the apoptotic effects. Most recently, we demonstrated that once covalently interacted with Zfra (zfration), the zfrated proteins undergo rapid degradation through the proteasome/ubiquitination-indepen- dent pathway. Zfra abolishes the apoptosis induced by WOX1. How Zfra affects the WOX1 apoptotic functions is not clear. Here, we demonstrated that Zfra may covalently bind to WOX1. On the other hand, Ser6 and Ser7 are not involved in Zfra-mediated cell death. Pre-injection of the full length Zfral-31, or truncated Zfra4-10, via tail vein protects nude mice from growing human glioma U87-MG, breast cancer MDA-MB-468, neuroblastoma SH-SY5Y and skin cancers. When spleen cells from Zfra-treated nude mice were transferred to NOD SCID mice, these mice became resistant to the growth of implanted cancer cells, suggesting that non-T “memory” cells are involved in blocking cancer growth. Pre-treating nude mice with antibodies which against B or T-cells before Zfra stimulation, we found CD19+, CD27+ or IL2R-α+ cells may be the Zfra target cells or participating in regulate Zfra prevention in vivo. Furthermore, we found a potential Zfra receptor, Hyal-2 (Hyaluronidase type 2). Hyal-2 antiserum inhibits skin cancer growth in nude mice, NOD SCID and BAB/c. Our results suggest that Zfra may activate Hyal-2+ immune cells for targeting cancer.
中文摘要 I
Abstract II
誌謝 III
Table of contents IV
Index of figures VI
Abbreviation VII
Introduction 1
Goals of This Study 1
Zinc Finger-like Protein that Regulates Apoptosis, Zfra 2
WW Domain-Containing Oxidoreductase 6
Hyaluronic Acid (Hyaluronan, HA) 12
Materials and Methods 18
Cell Lines 18
cDNA Constructs and Transient Gene Expression 18
Site-Directed Mutagenesis 19
Cell Cycle Analysis 19
Co-immunoprecipitation, Immunoblotting with Antibodies 20
Zfra Peptide Preparation, Antibodies, and Tumor Xenografts 21
Statistical Analysis 22
Results 23
Zfra may Covalently Bind WOX1 23
Ser6 and Ser7 Are Not Involved in Zfra-Mediated Cell Death 23
Zfra Suppresses the Growth of Glioma U87-MG, Neuroblastoma, and Breast Cancer 24
Zfra Primes Spleen Cells for Cancer Suppression 24
Zfra Suppresses Melanoma B16F10 Metastasis 25
Zfra Inhibits Cancer Cells Growth through Activating a Novel Population of Spleen Cells 26
Hyal-2 Is a Potential Receptor of Zfra 27
Hyal-2+ cell Are Responsible for Preventing Cancer Growth 27
Ultrasonicated Hyaluronan But Not Wild Type Prevent Melanoma Cell Growth In Vivo 28
Discussion 30
Self-polymerization and Anti-cancer Function of Zfra 30
Zfra in Cancer Prevention 31
Zfra Activates Novel Spleen Cells for Targeting cancer 31
Hyal-2+ Cells in Cancer Prevention 32
Activation of Hyal-2+ Cells 33
References 34
Figures 48
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