發(fā)布時(shí)間：2018-06-03 12:58

  本文選題：CXCR4 + CXCL12　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：目的：美國(guó)最新癌癥統(tǒng)計(jì)數(shù)據(jù)表明,白血病尤其是急性髓系白血病(AML)每年的發(fā)病率和死亡率依然很高,患者很難達(dá)到完全緩解和長(zhǎng)期無(wú)病生存,其主要原因是白血病細(xì)胞在體內(nèi)的浸潤(rùn)、耐藥、微量殘留和復(fù)發(fā)。大量研究顯示,骨髓內(nèi)的基質(zhì)細(xì)胞通過(guò)大量分泌基質(zhì)細(xì)胞衍生因子1(CXCL12,亦稱為SDF-1)激活白血病細(xì)胞表面高表達(dá)的特異性受體CXCR4,介導(dǎo)白血病細(xì)胞向其遷移、粘附并獲取促增殖、抗凋亡、耐藥信號(hào),從而形成保護(hù)微環(huán)境,導(dǎo)致患者化療后的白血病細(xì)胞微量殘留,為其之后的復(fù)發(fā)埋下隱患。因此,拮抗CXCR4/CXCL12生物軸對(duì)于白血病治愈具有重要意義�？紤]到人工設(shè)計(jì)的小分子多肽具有免疫原性低、成本低廉、設(shè)計(jì)靈活性高等優(yōu)越性,本研究根據(jù)CXCR4受體序列特征設(shè)計(jì)多肽,篩選出能有效抑制CXCR4/CXCL12生物軸介導(dǎo)的AML細(xì)胞遷移、粘附的拮抗多肽,并將其與多種化療藥物聯(lián)合使用,在體外(細(xì)胞水平)、體內(nèi)(AML小鼠異種移植模型水平)評(píng)價(jià)其治療效果和研究其作用機(jī)理。利用健康小鼠評(píng)價(jià)多肽的體內(nèi)安全性。方法：以人AML細(xì)胞系HL-60、NB4、THP-1、U937及小鼠骨髓基質(zhì)細(xì)胞MS-5、人臍靜脈內(nèi)皮細(xì)胞ea.hy926為模型,通過(guò)流式細(xì)胞術(shù)分析多肽與白血病細(xì)胞的結(jié)合力；利用流式細(xì)胞術(shù)和western blot檢測(cè)Annexin V-PI的結(jié)合和caspase-3的激活,評(píng)價(jià)多肽對(duì)AML細(xì)胞和非惡性細(xì)胞MS-5、ea.hy926凋亡的影響；利用transwell細(xì)胞遷移小室以及細(xì)胞共培養(yǎng)方法研究該多肽對(duì)CXCR4/CXCL12軸介導(dǎo)的AML細(xì)胞遷移、粘附的作用；并利用激光共聚焦顯微鏡觀察細(xì)胞骨架微絲蛋白重排,western blot檢鋇CXCR4下游Akt、Erk、p38信號(hào)通路的變化,探討多肽可能的分子作用機(jī)制。利用Annexin V-PI雙染法和臺(tái)盼藍(lán)染色法檢測(cè)多肽聯(lián)合化療藥(長(zhǎng)春新堿、順鉑、十羥基喜樹堿)對(duì)與骨髓基質(zhì)細(xì)胞MS-5共培養(yǎng)的AML細(xì)胞凋亡和活力的影響。建立AML小鼠異種移植模型,分別于皮下注射多肽前后通過(guò)尾靜脈采集外周血,利用CD33標(biāo)記和流式細(xì)胞術(shù)研究多肽對(duì)AML細(xì)胞的動(dòng)員效果。將多肽與化療藥(長(zhǎng)春新堿和環(huán)磷酰胺)聯(lián)用對(duì)AML小鼠進(jìn)行治療,統(tǒng)計(jì)小鼠生存期,并利用流式細(xì)胞術(shù)和組織切片觀察評(píng)價(jià)小鼠骨髓、脾臟、外周血中的AML細(xì)胞負(fù)荷以及脾臟、肝臟內(nèi)AML細(xì)胞的浸潤(rùn)情況。對(duì)健康BALB/c小鼠皮下注射多肽,通過(guò)觀察組織切片和檢測(cè)肝、腎功主要血生化指標(biāo)評(píng)價(jià)多肽的體內(nèi)安全性。結(jié)果：(1)篩選出的多肽(命名為E5)與四株AML細(xì)胞均有較迅速而穩(wěn)定的結(jié)合;(2)E5有效抑制CXCL12及分泌CXCL12的骨髓基質(zhì)細(xì)胞MS-5誘導(dǎo)的AML細(xì)胞定向遷移,并顯著減少AML細(xì)胞與MS-5之間的粘附,隨著E5濃度在O.1～10μM范圍內(nèi)逐漸提高抑制效果增強(qiáng),E5對(duì)AML細(xì)胞遷移、粘附的抑制具有濃度依賴性；(3)濃度高于20 μM時(shí),E5直接誘導(dǎo)AML細(xì)胞凋亡,但對(duì)非惡性細(xì)胞(MS-5和ea.hy926)的活力沒(méi)有顯著影響；(4)E5干擾了CXCL12誘導(dǎo)的AML細(xì)胞微絲骨架定向聚集,使微絲分布彌散,并抑制了CXCL12誘導(dǎo)的CXCR4下游Akt、Erk、p38三種蛋白的磷酸化,拮抗了CXCR4/CXCL12軸介導(dǎo)的信號(hào)通路；(5)E5與化療藥長(zhǎng)春新堿、順鉑、十羥基喜樹堿聯(lián)合使用,48 h后顯著提高了化療藥誘導(dǎo)的AML細(xì)胞凋亡率,10天內(nèi)基質(zhì)細(xì)胞共培養(yǎng)體系中的AML細(xì)胞活力持續(xù)地降低,E5顯著逆轉(zhuǎn)了MS-5介導(dǎo)的AML細(xì)胞耐藥性；(6)皮下注射E54h后,AML小鼠外周血中AML細(xì)胞比例提升約1.84倍,E5動(dòng)員了滯留在骨髓微環(huán)境中的AML細(xì)胞進(jìn)入外周血；(7)將E5與環(huán)磷酰胺、長(zhǎng)春新堿聯(lián)合治療AML小鼠,與單用化療藥組相比,聯(lián)合用藥組小鼠骨髓、脾臟、外周血中AML細(xì)胞比例顯著降低,脾腫大現(xiàn)象得到緩解,AML細(xì)胞對(duì)脾臟、肝臟的侵襲、浸潤(rùn)顯著減少,小鼠的生存期也得到延長(zhǎng),E5顯著提高了兩種常規(guī)化療藥的藥效；(8)對(duì)健康BALB/c小鼠隔天皮下注射高劑量E5多肽,30天后E5沒(méi)有引起小鼠各器官壞死、炎癥等異常病理性改變,組織形態(tài)完整,各器官重量沒(méi)有顯著變化,并且肝功、腎功主要血生化指標(biāo)均正常。結(jié)論：多肽E5通過(guò)拮抗CXCR4/CXCL12生物軸的作用,抑制了骨髓基質(zhì)細(xì)胞誘導(dǎo)的AML細(xì)胞遷移、粘附效應(yīng),減少AML細(xì)胞向體內(nèi)臟器的浸潤(rùn),并動(dòng)員AML細(xì)胞脫離受保護(hù)的骨髓基質(zhì)微環(huán)境進(jìn)入外周血,從而提高了多種化療藥的治療效果。同時(shí)E5多肽具有良好的體內(nèi)外安全性。因此,多肽E5在調(diào)節(jié)腫瘤微環(huán)境和治療白血病中具有潛在的應(yīng)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.71

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