摘  要

目的：胰腺癌是一种恶性程度极高的消化系统肿瘤，预后差，致死率高，5年相对生存率仅为13.3%，被称为“癌症之王”。近年来，胰腺癌的发病率和死亡率都在持续增加。胰腺癌治疗效果不佳的主要原因之一在于基质细胞构成致密基质屏障和免疫耐受，形成了特殊的肿瘤微环境。细胞因子与其受体的相互作用是胰腺癌微环境形成的关键。基质细胞分泌大量的趋化因子CXCL12，与胰腺癌细胞表面高表达的CXCR4相互作用，激活细胞内PI3K、MAPK等多条核心通路，诱导胰腺癌细胞的迁移、粘附、增殖以及耐药，同时抑制杀伤性T淋巴细胞的浸润，诱导免疫耐受，从而促进胰腺癌的进展，使疾病难治和复发。因此，拮抗CXCR4/CXCL12生物轴有望抑制肿瘤转移和耐药，改善免疫耐受，从而提高抗胰腺癌的治疗疗效。基于此，本论文计划筛选并确定具有拮抗胰腺癌CXCR4作用的多肽，研究其抑制胰腺癌细胞与基质细胞的作用；建立原位胰腺癌小鼠模型，研究拮抗多肽的治疗作用及体内应用安全性。

方法：利用流式细胞术和激光共聚焦显微镜观察筛选出多肽P12，检测其与胰腺癌细胞、成纤维细胞、巨噬细胞的结合情况；利用CCK-8试剂检测多肽P12对细胞活性的影响；利用Transwell迁移小室检测多肽P12对CXCL12介导的胰腺癌细胞迁移的影响；利用荧光显微镜和酶标仪检测多肽P12对成纤维细胞或巨噬细胞与胰腺癌细胞粘附的影响；利用Western blot检测多肽P12对胰腺癌细胞的CXCR4下游Erk和p38信号蛋白磷酸化的影响。

建立小鼠原位胰腺癌模型，给予多肽P12、吉西他滨、多肽P12与吉西他滨联合治疗。在实验结束时处死小鼠，分离胰腺并称重肿瘤组织；将肿瘤组织固定后，利用H&E和免疫荧光染色检测肿瘤组织中CXCR4、Ki67、Caspase-3、F4/80、CD206、CD8的表达，分析各治疗组中小鼠肿瘤组织微环境病理改变；分离脾脏淋巴细胞，利用CCK-8检测淋巴细胞对胰腺癌细胞的杀伤作用。腹腔注射FITC-P12到健康小鼠体内，检测多肽随时间的变化情况，分析P12在小鼠体内的分布规律。

结果：多肽P12以浓度依赖方式与CXCR4阳性的胰腺癌细细胞、成纤维细胞以及巨噬细胞相结合，在0~40 μM范围内对细胞活性无明显影响。多肽P12能够有效抑制CXCL12介导的胰腺癌细胞的迁移，抑制胰腺癌细胞与成纤维细胞和巨噬细胞的粘附。多肽P12能够显著抑制CXCR4下游信号蛋白Erk和p38的磷酸化。

通过免疫荧光染色结果可以看出，腹腔注射P12能够显著降低肿瘤组织中CXCR4的表达，降低细胞增殖标志物Ki67的表达，同时上调凋亡关键蛋白Caspase-3的表达，表明P12可通过拮抗CXCR4，抑制肿瘤细胞增殖，并促进其凋亡。同时，P12可显著降低M2型巨噬细胞标志物CD206的表达水平，增加T淋巴细胞在肿瘤组织中的浸润，增强了T淋巴细胞对胰腺癌细胞的杀伤作用。腹腔注射P12能够迅速入血并代谢，注射P12对小鼠外周血中的红细胞和血小板数量没有显著影响。

结论：新型CXCR4拮抗多肽P12可有效拮抗CXCR4的作用，抑制CXCL12诱导的胰腺癌细胞的迁移、基质细胞的粘附以及CXCR4相关下游信号通路蛋白活化。P12通过改善肿瘤免疫耐受，增加肿瘤组织中T淋巴细胞的浸润，抑制肿瘤细胞的增殖，促进肿瘤细胞凋亡。多肽P12有望为胰腺癌治疗提供新的CXCR4拮抗药物。

Abstract

Objective: Pancreatic cancer is a highly malignant digestive tumor with poor prognosis and high mortality rate. The 5-year survival rate of pancreatic cancer is only 13.3%, which is titled as the "king of cancers". The incidence and mortality rates of pancreatic cancer have continued to increase recently. One of the main reasons for the poor therapeutic effect of pancreatic cancer is that stromal cells constitute a dense matrix barrier and immune tolerance, forming a special tumor microenvironment. The interaction between cytokines and their receptors is critical for the formation of pancreatic cancer microenvironment. Stromal cells secrete large amounts of chemokine CXCL12 interacting with CXCR4, which is highly expressed on the surface of pancreatic cancer cells. The CXCL12-CXCR4 axis can activate several core pathways such as PI3K and MAPK, induce migration, adhesion, proliferation and drug resistance of pancreatic cancer cells, and inhibit the infiltration of killer T lymphocytes and immune tolerance. Thus, CXCR4 plays a critical role in promoting the progression, disease refractory and relapse of pancreatic cancer. Therefore, antagonizing CXCR4 is expected to inhibit tumor metastasis and drug resistance, improve immune tolerance, and thus improve the therapeutic efficacy of pancreatic cancer. Based on this, the purpose of this work is to screen and identify the pancreatic cancer CXCR4 antagonizing peptides. An orthotopic pancreatic cancer mouse model was established to study the therapeutic effect and safety of the antagonistic peptide in vivo.

Methods: Peptide P12 was screened by flow cytometry and confocal laser microscopy observation to detect the binding affinity to pancreatic cancer cells, fibroblasts and macrophages. The cytotoxicity of P12 on the cells was detected by using cell-counting kit-8 (CCK-8) assay. Transwell migration chamber was used to detect the effect of P12 on CXCL12-mediated migration of pancreatic cancer cells. The adhesion of fibroblasts or macrophages to the pancreatic cancer cells were analyzed by fluorescence microscopy and microplate reader. The CXCR4 related intracellular signal protein activation of phosphorylation of Erk and p38 protein on pancreatic cancer cells were detected by Western blot.

An orthotopic pancreatic cancer mouse model was established by injecting mouse pancreatic cancer cell pan02 into pancreas. Peptide P12, gemcitabine (GEM) as well as peptide P12 combined with GEM were intraperitoneally injected into the mice. At the end of the experiment, the mice were sacrificed, the pancreas was isolated and the tumor tissue was weighed. After the tumor tissue was fixed, the expressions of CXCR4, Ki67, Caspase-3, F4/80, CD206 and CD8 in the tumor tissue were analyzed by H&E and immunofluorescence staining respectively. The spleen lymphocytes were isolated and the CCK-8 assay was applied to analyze the killing effect of lymphocytes on the pancreatic cancer cells. FITC-P12 was intraperitoneally injected into healthy mice to analyze the distribution and metabolism of the peptide.

Results: The peptide P12 bound to pancreatic cancer cells, fibroblasts and macrophages in a concentration dependent manner, and did not display significant cytotoxicity to cell viability in the range of 0~40 μM. The peptide P12 effectively inhibited the CXCL12-induced pancreatic cancer cells migration, as well as the adhesion of fibroblasts and macrophages to pancreatic cancer cells. Additionally, the  peptide P12 significantly inhibited the CXCR4 downstream phosphorylation of Erk and p38 proteins.

According to the results of immunofluorescence staining, peptide P12 significantly reduced the expression of CXCR4 in the tumor tissues, reduced the expression of the cell proliferation marker Ki67, and up-regulation of apoptotic protein Caspase-3, indicated that P12 could inhibit the proliferation of tumor cells and promote their apoptosis by antagonizing CXCR4. At the same time, peptide P12 significantly reduced the expression level of M2-type macrophage marker CD206, increased the T lymphocytes infiltration in the tumor tissues, and enhance the killing effect of splenic lymphocytes on pancreatic cancer cells. The intraperitoneally injected P12 quickly entered the blood and metabolized. The number of red blood cells and platelets in peripheral blood of mice was not significantly affected by the injection of P12.

Conclusion: A novel CXCR4 antagonistic peptide P12 were identified, which can effectively antagonize CXCR4, inhibiting CXCL12-induced migration of pancreatic cancer cells, adhesion of stromal cells and activation of CXCR4-related downstream signaling pathway. Furthermore, P12 can rectify tumor immune tolerance in some degree, with the infiltration of T lymphocytes in tumor tissue, the proliferation of tumor cells inhibition, and the apoptosis of tumor cells promotion. Peptide P12 is expected to provide as a new CXCR4 antagonists for the treatment of pancreatic cancer.