先前許多文獻證明IL-8和IL-8R（CXCR1 / 2）與腫瘤侵襲、轉移和腫瘤形成相關，並且被認為是腫瘤微環境的重要組成因素。此外，藥物誘導的CSC（癌症幹細胞）已被證實會賦予癌細胞化學抗藥性。我們先前的研究開發了一種高性能的CXC受體拮抗劑，即IL-8類似物，稱為RP4。使用這種CXC受體拮抗劑（RP4）與IL-8競爭IL-8R（CXCR1 / 2），RP4可阻斷癌症進程和IL-8信號通路。早前的研究表明RP4可能作為一種新型的抗癌藥物。
我們通過RT-PCR檢測了BxPC-3和BxPC-3GR細胞（Gemcitabine抗藥性）的IL-8和IL-8R的表現量，我們發現BxPC-3GR細胞（Gemcitabine抗藥性）的IL-8和IL-8R表現量增加。在實驗當中我們得知RP4可以抑制BxPC-3GR的細胞增殖。因此我們設計了一系列實驗來揭示IL-8R在BxPC-3GR細胞中所扮演的關鍵作用，並證實了RP4抑制細胞侵襲和轉移的能力。由實驗得知與BxPC-3細胞相比，BxPC-3GR細胞中CSC標記物的表現量會上升，使用RP4可有效抑制癌症幹細胞的表現。
我們的數據表明RP4可以發展成為一種新型的抗癌藥物並且治療具有Gemcitabine抗藥性的胰腺癌患者。本篇研究強調了IL-8R（CXCR1 / 2）在胰腺癌症中的關鍵作用，並揭示了小分子多肽藥物RP4的功能。

Many studies had revealed that IL-8 and IL-8R (CXCR1/2) correlate with tumor progression, metastasis and the development of cancer and is considered as an important component of tumor microenvironment. Besides, drug-induced CSC (cancer stem cell) had been demonstrated that confer chemoresistance in cancer cells. In our previous study, we developed a high-performance CXC-receptor antagonist, an IL-8 analogue, called RP4. Use this CXC-receptor antagonist (RP4) to compete for the IL-8R (CXCR1/2) with IL-8 that blocking the IL-8 signaling pathway in cancer progression and development. Moreover, the previous study suggested that RP4 may develop as a novel anticancer drug.
We detected the IL-8 and IL-8R expression to identify the properties of BxPC-3 and BxPC-3GR cell lines (Gemcitabine resistant) by RT-PCR. We found that BxPC-3GR cells (Gemcitabine resistant ) the IL-8and IL-8R expression increased. Here we showed that RP4 could inhibit cell proliferation in BxPC-3GR. Additionally , we designed a serial of an experiment to reveal the crucial role of IL-8R in BxPC-3GR, examined that RP4 can inhibit cell invasion and migration abilities. Besides, the expression of CSC markers was increased in BxPC-3GR comparing with BxPC-3 and RP4 can efficiently inhibit the expression of cancer stem cells.
In conclusion, our data suggested that RP4 may develop as a novel anticancerdrug and overcome the Gemcitabine-resistance for advanced pancreatic cancer patients. This study highlights the crucial role of IL-8R (CXCR1/2) in pancreatic cancer and reveals the functional properties of the small-molecular polypeptide drug, RP4.

Content
摘要......i
Abstract......i
Content......iv
Figure list......vi
Chapter 1 Introduction......1
1.1Pancreatic cancer......1
1.2 Gemcitabine(Gemzar ®)......2
1.3 Cancer stem cells and drug resistance......3
1.4 IL8(CXCL8)......4
1.5 The ELR+-CXC chemokines and their receptors......5
1.6 Design the antagonist of ELR+-CXC receptors......7
Chapter 2 Material and Methods......9
2.1 Chemical and Reagents......9
2.2 Cell lines......9
2.3 Cell viability assay by MTT......10
2.4 Expression of recombinant IL-8 and RP4......10
2.5 Expression and purification of recombinant IL-8 and RP4......11
2.7 Proliferation assay by CCK8......13
2.8 Colony formation......13
2.9 Wound healing assay......13
2.10 Migration assay......14
2.11.Invasion assay......14
Chapter 3 Results......16
3.1Development of Gemcitabine-Resistant BxPC-3 Pancreatic Tumor Cell Lines......16
3.2 Expression mRNA levels of IL-8 and IL-8R (CXCXR1/2) in BxPC-3GR compared with BxPC-3......17
3.3 Dose-dependent effect of RP4 in BxPC-3GR by invasion assay......17
3.4 RP4 can inhibit cell proliferation and cell survival in BxPC-3GR......18
3.5 RP4 effectively inhibited cell invasion in BxPC-3GR......18
3.6 RP4 can attenuate cell migration ability in BxPC-3GR......19
3.7 Comparison of cell morphology between BxPC-3GR parental cells and spheroids derived from BxPC-3GR......19
3.8 RP4 inhibits spheroid-derived cancer stem cell (SDCSC) formation......19
Chapter 4 Discussion......20
Chapter 5 Conclusion......23

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