人工设计的CXCR4靶向肽应用于AML治疗的基础研究

发布时间：2018-06-03 12:58

本文选题：CXCR4 + CXCL12　；参考：《北京协和医学院》2015年博士论文

【摘要】：目的：美国最新癌症统计数据表明,白血病尤其是急性髓系白血病(AML)每年的发病率和死亡率依然很高,患者很难达到完全缓解和长期无病生存,其主要原因是白血病细胞在体内的浸润、耐药、微量残留和复发。大量研究显示,骨髓内的基质细胞通过大量分泌基质细胞衍生因子1(CXCL12,亦称为SDF-1)激活白血病细胞表面高表达的特异性受体CXCR4,介导白血病细胞向其迁移、粘附并获取促增殖、抗凋亡、耐药信号,从而形成保护微环境,导致患者化疗后的白血病细胞微量残留,为其之后的复发埋下隐患。因此,拮抗CXCR4/CXCL12生物轴对于白血病治愈具有重要意义。考虑到人工设计的小分子多肽具有免疫原性低、成本低廉、设计灵活性高等优越性,本研究根据CXCR4受体序列特征设计多肽,筛选出能有效抑制CXCR4/CXCL12生物轴介导的AML细胞迁移、粘附的拮抗多肽,并将其与多种化疗药物联合使用,在体外(细胞水平)、体内(AML小鼠异种移植模型水平)评价其治疗效果和研究其作用机理。利用健康小鼠评价多肽的体内安全性。方法：以人AML细胞系HL-60、NB4、THP-1、U937及小鼠骨髓基质细胞MS-5、人脐静脉内皮细胞ea.hy926为模型,通过流式细胞术分析多肽与白血病细胞的结合力；利用流式细胞术和western blot检测Annexin V-PI的结合和caspase-3的激活,评价多肽对AML细胞和非恶性细胞MS-5、ea.hy926凋亡的影响；利用transwell细胞迁移小室以及细胞共培养方法研究该多肽对CXCR4/CXCL12轴介导的AML细胞迁移、粘附的作用；并利用激光共聚焦显微镜观察细胞骨架微丝蛋白重排,western blot检钡CXCR4下游Akt、Erk、p38信号通路的变化,探讨多肽可能的分子作用机制。利用Annexin V-PI双染法和台盼蓝染色法检测多肽联合化疗药(长春新碱、顺铂、十羟基喜树碱)对与骨髓基质细胞MS-5共培养的AML细胞凋亡和活力的影响。建立AML小鼠异种移植模型,分别于皮下注射多肽前后通过尾静脉采集外周血,利用CD33标记和流式细胞术研究多肽对AML细胞的动员效果。将多肽与化疗药(长春新碱和环磷酰胺)联用对AML小鼠进行治疗,统计小鼠生存期,并利用流式细胞术和组织切片观察评价小鼠骨髓、脾脏、外周血中的AML细胞负荷以及脾脏、肝脏内AML细胞的浸润情况。对健康BALB/c小鼠皮下注射多肽,通过观察组织切片和检测肝、肾功主要血生化指标评价多肽的体内安全性。结果：(1)筛选出的多肽(命名为E5)与四株AML细胞均有较迅速而稳定的结合;(2)E5有效抑制CXCL12及分泌CXCL12的骨髓基质细胞MS-5诱导的AML细胞定向迁移,并显著减少AML细胞与MS-5之间的粘附,随着E5浓度在O.1～10μM范围内逐渐提高抑制效果增强,E5对AML细胞迁移、粘附的抑制具有浓度依赖性；(3)浓度高于20 μM时,E5直接诱导AML细胞凋亡,但对非恶性细胞(MS-5和ea.hy926)的活力没有显著影响；(4)E5干扰了CXCL12诱导的AML细胞微丝骨架定向聚集,使微丝分布弥散,并抑制了CXCL12诱导的CXCR4下游Akt、Erk、p38三种蛋白的磷酸化,拮抗了CXCR4/CXCL12轴介导的信号通路；(5)E5与化疗药长春新碱、顺铂、十羟基喜树碱联合使用,48 h后显著提高了化疗药诱导的AML细胞凋亡率,10天内基质细胞共培养体系中的AML细胞活力持续地降低,E5显著逆转了MS-5介导的AML细胞耐药性；(6)皮下注射E54h后,AML小鼠外周血中AML细胞比例提升约1.84倍,E5动员了滞留在骨髓微环境中的AML细胞进入外周血；(7)将E5与环磷酰胺、长春新碱联合治疗AML小鼠,与单用化疗药组相比,联合用药组小鼠骨髓、脾脏、外周血中AML细胞比例显著降低,脾肿大现象得到缓解,AML细胞对脾脏、肝脏的侵袭、浸润显著减少,小鼠的生存期也得到延长,E5显著提高了两种常规化疗药的药效；(8)对健康BALB/c小鼠隔天皮下注射高剂量E5多肽,30天后E5没有引起小鼠各器官坏死、炎症等异常病理性改变,组织形态完整,各器官重量没有显著变化,并且肝功、肾功主要血生化指标均正常。结论：多肽E5通过拮抗CXCR4/CXCL12生物轴的作用,抑制了骨髓基质细胞诱导的AML细胞迁移、粘附效应,减少AML细胞向体内脏器的浸润,并动员AML细胞脱离受保护的骨髓基质微环境进入外周血,从而提高了多种化疗药的治疗效果。同时E5多肽具有良好的体内外安全性。因此,多肽E5在调节肿瘤微环境和治疗白血病中具有潜在的应用前景。
[Abstract]:Objective: the latest American cancer statistics show that the incidence and mortality of leukemia, especially acute myeloid leukemia (AML), are still high annually, and it is difficult for patients to achieve complete remission and long-term disease free survival, mainly due to the infiltration, resistance, trace residue and recurrence of leukemic cells in the body. A large number of studies show that the bone marrow is in the bone marrow. Stromal cells activate the specific receptor CXCR4 of high expression on the surface of leukemic cells by producing a large number of matrix cell derived factor 1 (CXCL12, also called SDF-1), which mediates the migration, adhesion and proliferation, anti apoptosis, and drug resistance signals of leukemic cells, thus forming a protective microenvironment, resulting in a trace residue of leukemia cells after chemotherapy. Therefore, the antagonism of the CXCR4/CXCL12 biological axis is of great significance to the cure of leukemia. Considering the low immunogenicity, low cost and high flexibility in designing the artificially designed small molecular peptides, this study designs peptides based on the characteristics of CXCR4 receptor sequence and can effectively suppress CXCR4/CXCL. 12 biological axis mediated AML cells migrated and adhered to antagonistic peptides, and combined with a variety of chemotherapeutic drugs, in vitro (cell level), in vivo (AML mice xenotransplantation model level) to evaluate the therapeutic effect and study the mechanism of its action. The safety of polypeptides was evaluated by healthy mice. Methods: Human AML cell line HL-60, NB4, T HP-1, U937 and mouse bone marrow stromal cells, MS-5, human umbilical vein endothelial cells ea.hy926, were used to analyze the binding force of peptide to leukemic cells by flow cytometry; the apoptosis of AML cells and non malignant cells was evaluated by flow cytometry and Western blot detection of Annexin V-PI and caspase-3 activation. The effect of Transwell cell migration chamber and cell co culture method was used to study the effect of the polypeptide on the migration and adhesion of AML cells mediated by CXCR4/CXCL12 axis, and to observe the rearrangement of the cytoskeleton microfilament protein by laser confocal microscopy, and the changes of Akt, Erk and p38 signaling pathways downstream of barium CXCR4 in Western blot, and to explore the possibility of polypeptide. The mechanism of molecular action. Annexin V-PI double staining and trypan blue staining were used to detect the effect of polypeptide combined with chemotherapeutic drugs (vincristine, cisplatin, ten hydroxycamptothecin) on the apoptosis and activity of AML cells co cultured with bone marrow stromal cells (MS-5). A xenotransplantation model of AML mice was established and collected from the tail vein before and after subcutaneous injection of polypeptide. CD33 markers and flow cytometry were used to study the effect of polypeptide on the mobilization of AML cells. The peptides were combined with chemotherapeutic drugs (vincristine and cyclophosphamide) to treat AML mice. The survival period of mice was statistically analyzed. The bone marrow, spleen, AML cell load in peripheral blood and spleen were evaluated by flow cytometry and tissue section observation. The infiltration of AML cells in the liver. By subcutaneous injection of polypeptide in healthy BALB/c mice, the safety of peptide in vivo was evaluated by observing tissue section and detecting liver, and the main blood biochemical indexes of kidney work. Results: (1) the selected polypeptide (named E5) had a fast and stable combination with four AML cells; (2) E5 effectively suppressed CXCL12 and secreted CXC. MS-5 induced migration of AML cells induced by L12 bone marrow stromal cells, and significantly decreased the adhesion between AML cells and MS-5. With the concentration of E5 in O.1 to 10 mu M, the inhibition effect was enhanced gradually. E5 to AML cells migrated and the inhibition of adhesion was dependent on concentration. (3) when the concentration was higher than 20 mu M, E5 directly induced apoptosis, but to non The activity of malignant cells (MS-5 and ea.hy926) was not significantly affected; (4) E5 interfered with CXCL12 induced AML cell microfilament skeleton directed aggregation, dispersed the microfilament distribution, and inhibited the phosphorylation of CXCL12 induced CXCR4 downstream Akt, Erk, p38 three proteins, antagonized the signal pathway mediated by CXCR4/ CXCL12 axis; (5) Combined use of platinum and ten hydroxycamptothecin, after 48 h, the apoptosis rate of AML cells induced by chemotherapeutic drugs was significantly increased. The activity of AML cells in the co culture system of stromal cells decreased continuously in 10 days, and E5 significantly reversed the MS-5 mediated AML cell resistance. (6) the proportion of AML cells in peripheral blood of AML mice increased by about 1.84 times, E5 movement after E54h. The AML cells in the bone marrow microenvironment entered the peripheral blood, and (7) the combination of E5 and cyclophosphamide and vincristine in the treatment of AML mice, compared with the single chemotherapy group, the proportion of AML cells in the bone marrow, spleen and peripheral blood of the combined group of mice decreased significantly, the splenomegaly phenomenon was relieved, the invasion of the spleen and the liver, and the infiltration of the AML cells were significant. The survival time of mice was also prolonged, and E5 significantly improved the efficacy of two kinds of conventional chemotherapy drugs. (8) the healthy BALB/c mice were subcutaneously injected with high dose of E5 polypeptide in the other day. After 30 days, E5 did not cause abnormal pathological changes such as necrosis of organs, inflammation and other organs, and the weight of various organs did not change significantly, and the liver function and renal function were not changed. The main blood biochemical indexes are normal. Conclusion: polypeptide E5 inhibits the migration of AML cells induced by bone marrow stromal cells by antagonizing the effect of CXCR4/CXCL12 biological axis, and reduces the adhesion effect of the bone marrow stromal cells, reduces the infiltration of AML cells into the organs of the body, and mobilizes the AML cells to enter the peripheral blood from the protected bone marrow stroma microenvironment, thus improving a variety of chemotherapeutic drugs. At the same time, E5 polypeptide has good in vitro and in vivo safety. Therefore, polypeptide E5 has potential application prospect in regulating tumor microenvironment and treating leukemia.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.71

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