【作者】 颜彬；

【导师】 冯作化；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 中性粒细胞作为天然免疫系统的重要组成部分,广泛存在于各种人类癌症中,其存在与肿瘤的发生、发展密切相关。通常认为中性粒细胞具有抑制肿瘤生长的潜能,然而近年来越来越多的研究表明其具有促进肿瘤生长的作用。是什么样的因素导致了中性粒细胞的抑瘤潜能无法发挥而转变为促进肿瘤生长的功能目前仍没有明确的结论。我们应用荷瘤小鼠动物模型,分离获取小鼠骨髓中性粒细胞细胞、肿瘤组织中性粒细胞以及腹腔中性粒细胞,分别研究了受到肿瘤分泌的各种细胞因子调控的骨髓PMN、完成趋化过程但没有与肿瘤微环境相互作用的PMN以及既完成趋化过程又受到肿瘤微环境刺激的PMN的脱颗粒相关的基因表达、功能以及信号通路的变化。在本研究中我们发现,小鼠中性粒细胞的功能转变发生在骨髓,这种转变需要IL-6和G-CSF的联合作用。IL-6和G-CSF联合作用于小鼠PMN,能够在其进入肿瘤微环境之前使其发生功能改变,使其抑瘤潜能转变为促瘤作用,从而有利于肿瘤的生长。导致这种改变的一个重要原因是PMN的脱颗粒功能受损,在大量释放时具有杀伤肿瘤活性的细胞毒性物质不能有效释放,在低剂量释放时表现出了相反的促进肿瘤生长的活性。IL-6和G-CSF联合作用使得TRAIL的合成和释放均发生障碍,而MPO、NE等主要是通过不能有效释使得PMN的功能发生改变。颗粒物质不能有效释放的一个重要原因是由于在G-CSF作用下P13K通路和p38MAPK通路不能充分激活,这使得PMN的诱导脱颗粒能力受损。另一方面,在IL-6和G-CSF的联合作用下激活了STAT3信号通路,使得Rab27a的表达下调,抑制了PMN的自发脱颗粒能力,这使得PMN的自发释放能力明显下降。诱导和自发脱颗粒的能力同时受损使得细胞产生促瘤潜能。体内转染表达IFN-β能够恢复PMN中Rab27a的表达从而恢复PMN的自发脱颗粒能力,使得PMN的促瘤作用消失,但是不能恢复其抑瘤作用。综上所述,IL-6联合G-CSF能够调控中性粒细胞中信号通路的激活,改变细胞内颗粒物质的合成以及释放量,从而有利于肿瘤的生长。目的：探讨正常以及荷瘤小鼠骨髓中性粒细胞对肿瘤生长的影响,以及造成这种影响的调控因素。方法：(1)用密度梯度离心的方法分离小鼠骨髓中的中性粒细胞,建立肿瘤细胞与PMN混合接种动物模型,观察正常组骨髓PMN和荷瘤鼠骨髓PMN对肿瘤生长的影响；(2)构建IL-6、G-CSF以及IL-6和G-CSF受体的真核表达载体,观察在肿瘤细胞与PMN混合接种动物模型中,在不荷瘤的情况下表达这两种细胞因子以及在荷瘤情况下阻断这两种细胞因子时PMN对肿瘤生长的影响；(3)用肌肉部位转染表达趋化因子真核表达载体的方法,建立体内趋化PMN至肿瘤部位模型,观察不同处理组PMN对肿瘤生长的影响；(4)在小鼠腹腔转染表达趋化因子,将PMN募集到腹腔,Percoll分离腹腔PMN,获得完成趋化过程但尚未进入肿瘤微环境与之发生反应的PMN,与肿瘤细胞混合接种,观察正常小鼠、荷瘤小鼠以及不荷瘤但在肌肉局部表达IL-6和G-CSF的小鼠骨髓细胞对肿瘤生长的影响。结果：(1)正常小鼠骨髓PMN抑制肿瘤生长,而荷瘤小鼠骨髓PMN促进肿瘤生长；(2)IL-6联合G-CSF能够改变小鼠骨髓PMN的抑瘤作用,使其具有促进肿瘤生长的作用,在荷瘤小鼠中同时阻断IL-6和G-CSF的表达可以消除荷瘤小鼠骨髓PMN的促进肿瘤生长作用；(3)这种改变不受体内趋化过程的影响,在PMN进入肿瘤微环境之前得以保持；(4)趋化至腹腔的荷瘤小鼠PMN保持了这种促瘤功能,局部转染表达了IL-6和G-CSF真核表达载体的小鼠腹腔PMN同样也能促进肿瘤生长。证明G-CSF和IL-6的确可在PMN进入肿瘤微环境之前其抑瘤功能转变成为促瘤功能。结论：肿瘤对于PMN的作用开始于骨髓PMN细胞,在肿瘤细胞分泌的众多细胞因子中,IL-6联合G-CSF作用于小鼠骨髓中PMN,在进入肿瘤微环境之前就调控了PMN的功能,使其抑瘤功能转变成为促瘤功能。目的：为了进一步研究在骨髓中已经发生了改变的PMN在进入肿瘤微环境中后是否在功能上有相应的改变,我们进一步研究了肿瘤中PMN的相关功能。方法：(1)用CXC1趋化不同处理组的PMN到肿瘤组织中,分离肿瘤组织中的PMN, real-time PCR方法检测Trail、Mpo, NE的基因表达水平；(2)化学发光的方法检测肿瘤微环境中MPO、NE的活性；(3)western-blot检测肿瘤组织间液中TRAIL的蛋白水平；(4)不同处理组的PC-PMN与H22细胞体外共培养,体外杀伤实验检测PMN的体外杀瘤活性。结果：(1)和对照组小鼠PMN相比,荷瘤小鼠和联合表达了IL-6和G-CSF小鼠的肿瘤组织中PMN的NE的mRNA水平没有变化,而MPO的mRNA水平在组织中的PMN中检测不到；(2)和对照小鼠PMN相比,来源于转染了pG-CSF和pIL-6质粒的小鼠TRAIL的mRNA水平降低,胞内TRAIL蛋白水平降低,胞内MPO和NE的蛋白水平稍有升高,和对照比较,组织中MPO和NE的活性在pG/pI6小鼠中明显降低；(3)肿瘤组织中的TRAIL, MPO, and NE的检测结果显示,对照组可以检测到胞外的TRAIL(可溶性TRAIL),而在联合表达了IL-6和G-CSF质粒的小鼠中检测不到胞外的TRAIL;(4)和对照组相比,荷瘤小鼠以及联合转染表达了IL-6和G-CSF质粒小鼠的PC-PMN的杀瘤活性明显降低。结论：IL-6和G-CSF的联合作用对TRAIL的表达和释放均产生抑制影响,而对MPO和NE的影响主要表现在释放上,减少了两者的释放。和对照组小鼠PMN相比,荷瘤小鼠和联合表达了IL-6和G-CSF小鼠的腹腔PMN对肿瘤细胞的毒性显著下降。目的：探讨中性粒细胞脱颗粒能力减弱的相关机制。方法：(1)获取正常小鼠PC-PMN,用P13K信号通路抑制剂wortmannin或p38MAPK信号通路抑制剂SB203580预处理细胞后用Ts-Ms刺激,检测PMN释放MPO的能力；(2)用Western Blot的方法检测了正常小鼠、荷瘤小鼠以及IL-6和G-CSF联合处理小鼠的PMN的PI3K, p38MAPK信号通路的激活程度；(3)用MPO的释放率代表PMN嗜天青颗粒的释放能力,在体内转染表达IL-6和G-CSF的真核表达载体,研究了IL-6和G-CSF在P13K和p38MAPK信号通路激活中以及在脱颗粒受损中起到的作用；(4) Real time PCR的方法和Western Blot的方法检测了和脱颗粒相关的Rac2和Rab27a在正常、荷瘤以及联合转染表达IL-6和G-CSF小鼠PMN中的表达；(5)用信号通路抑制剂研究了STAT3、 ERK和P13K三条信号通路对Rab27a的调控；(6)在体内转染表达IFN-β,用表达CXCL1的肝脏细胞趋化PMN至腹腔,用Ts-Ms刺激检测PC-PMN的脱颗粒能力以及混合接种实验中对肿瘤生长的影响。结果：(1)P13K和p38MAPK通路参与了Ts-Ms诱导的PMN嗜天青颗粒的脱颗粒过程；(2)Ts-Ms能够激活PC-PMN中的P13K和p38MAPK通路,但是来源于荷瘤小鼠和pG/pI6小鼠的PMN细胞的Akt和p38的激活程度要明显低于来自正常小鼠的腹腔PMN细胞；(3)在体内表达G-CSF而不是IL-6,能够减弱P13K和p38MAPK通路的激活,并且降低嗜天青颗粒的释放能力；(4)荷瘤以及体内联合转染IL-6和G-CSF质粒能够通过STAT3激活信号通路,使Rab27a明显下降,导致了PMN的自发脱颗粒能力的下降,在体外实验中,用IL-6和G-CSF联合刺激正常小鼠骨髓PMN时Rab27a也下调；(5)STAT3抑制Rab27a的表达,P13K则拮抗G-CSF和IL-6的抑制作用；(6)荷瘤以及体内联合转染IL-6和G-CSF质粒对骨髓以及腹腔PMN的Rac2并不下调,而Rab27a明显下降。与之相对应的是,在体外实验中,用IL-6和G-CSF联合刺激正常小鼠骨髓PMN时Rab27a下调。肝脏转染表达了IFN-β的荷瘤小鼠pG/pI6小鼠的骨髓PMN的Rab27a得以恢复,相应的其脱颗粒能力部分恢复,自发释放能力恢复,但是诱导脱颗粒能力不能恢复。肝脏转染表达了IFN-β的荷瘤小鼠pG/pI6小鼠的骨髓PMN混合接种结果显示,体内表达IFN-β,可以消除PMN的促瘤功能,但不能完全恢复其抑瘤功能。结论：P13K和p38信号通路不能充分激活导致了PMN的诱导脱颗粒能力受损。IL-6联合G-CSF激活STAT3信号通路,从而下调Rab27a,使PMN的自发脱颗粒下降。IFN-β能够通过降低STAT3的激活恢复PMN的自发脱颗粒能力,但是诱导脱颗粒能力不能恢复,其促瘤功能消失但是抑瘤功能没有恢复。更多还原

【Abstract】 Polymorphonuclear leukocytes (PMNs or neutrophils) make up a significant portion of the inflammatory cell infiltrate found in a wide variety of human cancers and murine models. They are known to have anti-tumor potential. However, in recent years the tumor-promoting effect of neutrophils has been well demonstrated. So far it remains unclear what causes the conversion of neutrophil function from tumor suppressive to tumor promotional. Here we report that the conversion of murine neutrophil function occurs in bone marrow, and that IL-6cooperation with G-CSF is required for causing this conversion. IL-6cooperated with G-CSF to modulate neutrophils in bone marrow, altering the activation potential of signaling pathways in neutrophils resulting in the attenuating of degranulationg ability of PMN. Co-stimulation with G-CSF and IL-6attenuated the activation of and increased STAT3expression in neutrophils. In one hand, the attenuating of the PI3K and p38MAPK pathways caused inefficient inducing degranulation of TRAIL, MPO and NE. On the other hand, the heigher activation of STAT3pathway downregulated the Rab27a resulting in the decreasing of spontaneous degranulation. Taken together, these results demonstrate that G-CSF and IL-6, despite their well-known physiological functions, could modulate the activation potential of signaling pathways in neutrophils, resulting in the production or release of the above-mentioned factors in a way that favors tumor growth. Objective:To explore whether the function of neutrophils could be modulated before they entered tumor milieu and what factors cause this convertion.Methods:we first tested the effect of bone marrow neutrophils on tumor growth in co-inoculation test. To clarify the roles of G-CSF and IL-6, we evaluated their effect on the function of bone marrow neutrophils by expressing soluble extracellular domains of G-CSFR, IL-6R, and gp130in local tissues to block tumor-derived G-CSF and IL-6, or expressing G-CSF and/or IL-6in vivo in absence of tumor. We recruited neutrophils to the tissues at inoculation site by expressing CXCL1in H22hepatocarcinoma model to investigate whether the function of neutrophils could be maintained in blood and in the process of chemotaxis. We recruited neutrophils to peritoneal cavity of mice by using CXCL1-expressing hepatocytes, and isolated the neutrophils from peritoneal cavity for further proof.Results:The neutrophils from bone marrow of naive mice (N-BM) suppressed tumor growth, whereas the neutrophils from bone marrow of tumor-bearing mice (T-BM) promoted tumor growth. G-CSF and IL-6cooperated to modulate bone marrow neutrophils, converting their function from tumor suppressive to tumor promotional. The recruitment of neutrophils suppressed the growth of tumor in control mice, but promoted tumor growth in pG/pI6-mice, suggesting that the function of neutrophils was maintained in blood and in the process of chemotaxis. In co-inoculation test, the neutrophils from peritoneal cavity of naive mice (N-PC) suppressed tumor growth, whereas the neutrophils from that of pG/pI6-mice (pG/pI6-PC) and tumor-bearing mice (T-PC) promoted tumor growth.Conclusion:The above results showed that G-CSF and IL-6modulated the function of neutrophils in bone marrow, and that the function of neutrophils was maintained in blood and in the process of chemotaxis. Objective:To further prove that pre-modulation by G-CSF and IL-6altered the function of neutrophils, we analysis the function of neutrophils in tumor envirenment.Methods:To further prove that pre-modulation by G-CSF and IL-6altered the function of neutrophils, we analyzed the neutrophils recruited to the tissues at inoculation sites.The function of these neutrophils might represent the final function of the neutrophils in tumor milieu, since these neutrophils not only finished the process of chemotaxis, but also were stimulated by complex stimuli in the tissue. To compare the effect of G-CSF/IL-6with that of tumor on neutrophils, we used another model in which the mice received secondary inoculation with tumor cells after the removal of primary tumor. The effect of primary tumor on neutrophils was analyzed by recruiting neutrophils to the tissues at the sites of secondary inoculation.Results:Compared with the neutrophils from control mice, the neutrophils from pG/pI6-mice expressed higher levels of Mmp9and Bv8mRNAs, but lower level of Trail mRNA. The mRNA of Mpo or NE was undetectable or unchanged respectively in the neutrophils isolated from the tissues (data not shown). The neutrophils from pG/pI6-mice contained more intracellular MPO and NE, but much less intracellular TRAIL. We then detected the extracellular TRAIL (soluble TRAIL), MPO, and NE in the tissues. Extracellular TRAIL was detectable in the tissues of control mice, but not pG/pI6-mice. The activities of MPO and NE in local tissues of pG/pI6-mice were significantly lower than those in control mice. For the secondary inoculation model, the results showed that the modulatory effect of G-CSF and IL-6on neutrophils was similar to that of primary tumor. In the test of the function of neutrophils isolated from peritoneal cavity of mice, the neutrophils from N-PC had cytotoxicity to tumor cells in absence or presence of the mixture of soluble molecules from tumor (T-sMs), which might represent complex stimuli in tumor milieu. The cytotoxicity of the neutrophils from pG/pI6-PC and T-PC was significantly lower than that of the neutrophils from N-PC.Conclusion:These data suggested that G-CSF and IL-6could alter the expressions of Trail gene in neutrophils, and attenuate neutrophil degranulation to decrease the release of MPO and NE. The function of the neutrophils was indeed altered by G-CSF and IL-6. Objective:To explore the mechanism involved in the attenuation of neutrophil degranulation, we next analyzed the activation of PI3K and p38MAPK pathways in neutrophils. We further investigated neutrophil degranulation-related protein Rab27a and Rac2. Methods:For this purpose, we stimulated the neutrophils from peritoneal cavity with T-sMs. We analyzed the expressions of Rac2and Rab27a using real-time PCR and Western Blot. WeResults:T-sMs could activate PI3K and p38MAPK pathways in neutrophils. The phosphorylation levels of Akt and p38MAPK in neutrophils from pG/pI6-PC and T-PC were significantly lower than those in neutrophils from N-PC. In vivo expression of G-CSF, but not IL-6, resulted in the inefficient activation of PI3K and p38MAPK pathways. Consistently, in vivo expression of G-CSF attenuated T-sMs-induced degranulation of neutrophils, evaluated by the release of MPO. However, both spontaneous degranulation and induced degranulation of neutrophils from pG/pI6-mice were significantly decreased. In pG/pI6-mice and tumor-bearing mice, the expression of Rab27a gene was decreased in bone marrow neutrophils and the neutrophils recruited to peritoneal cavity. Consistently, the expression of Rab2la gene in neutrophils from N-BM could be down-regulated by co-stimulation with G-CSF and IL-6in vitro. The effect of G-CSF and IL-6was abolished by inhibiting STAT3, but enhanced by inhibiting PI3K. In vivo expression of IFN-P in pG/pI6-mice and tumor-bearing mice increased Rab27a expression in bone marrow neutrophils and the neutrophils recruited to peritoneal cavity. When neutrophils from peritoneal cavity were stimulated with T-sMs, both spontaneous degranulation and induced degranulation of neutrophils from pG/pI6-mice and tumor-bearing mice were significantly decreased, In vivo expression of IFN-β recovered spontaneous degranulation of neutrophils, but failed to fully recover the induced degranulation, since the activation of PI3K and p38MAPK pathways was still inefficient (data not shown). Moreover, IFN-β abrogated tumor-promoting potential of bone marrow neutrophils.Conclusion:These data suggested that the attenuated degranulation was partially attributed to the inefficient activation of PI3K and p38MAPK pathways, and the augmented STAT3activation in neutrophils was another reason for attenuated neutrophil degranulation.更多还原