IL-6体外诱导巨噬细胞M2样分化的机制研究

发布时间：2018-08-18 14:24

【摘要】：研究背景巨噬细胞是人体免疫系统中一种具有多种特性的细胞群体,在机体防御细菌、病毒和寄生虫感染方面发挥着重要作用。随着研究的深入,巨噬细胞在肿瘤发生发展的作用也越来越被重视。浸润在肿瘤微环境中的巨噬细胞常被称为肿瘤相关巨噬细胞(TAMs)。TAM是外周循环的单核细胞在局部CCL2、巨噬细胞集落刺激因子(M-CSF)和血管内皮生长因子(VEGF)等细胞因子的刺激下聚集分化形成的。巨噬细胞主要有两个表型即M1和M2型。M1型巨噬细胞可由LPS或IFN-γ诱导分化产生;IL-4和IL-13可促进巨噬细胞向M2型极化。M1型巨噬细胞是强有力的效应细胞,能杀死微生物,产生炎性细胞因子,如肿瘤坏死因子(TNF-α)和IL-12,并在清除细菌、病毒和真菌感染中起重要作用。相反,M2型巨噬细胞通过产生IL-10和转化生长因子(TGF-β)等,抑制炎症反应。M2型巨噬细胞在组织重塑、寄生虫感染、血管生成和肿瘤进展中也发挥着重要作用。白细胞介素-6具有多效性,其可以影响人体多方功能,如血管疾病、脂质代谢、胰岛素抵抗和神经心理行为。IL-6是STAT3信号通路的一个强有力的激活因子,活化的P-STAT3信号蛋白迅速易位到细胞核中,并结合到靶基因如细胞周期蛋白D1、c-myc、和VEGF的启动子的识别序列,从而增加这些靶基因的转录和表达。大量研究发现IL-6引起的STAT3的活化在肿瘤的进展中起着十分重要的作用。在前期研究中,我们发现在胃癌患者的肿瘤组织中IL-6的表达高于非肿瘤组织。同时,在癌组织中M2型巨噬细胞浸润数量也增加。为了探索IL-6是否与M2巨噬细胞的极化有关,我们建立了IL-6诱导巨噬细胞体外分化的细胞培养模型,通过检测IL-6刺激诱导后的巨噬细胞的表型特点,我们发现IL-6能诱导正常巨噬细胞分化为M2样巨噬细胞,主要表现为IL-10和TGF-β的表达升高,IL-12p35的表达降低。为此,进一步对该诱导分化的机制进行了探讨,同时也对诱导的M2样巨噬细胞的功能做了进一步的研究。目的:1.建立IL-6体外诱导巨噬细胞分化的细胞模型,探究IL-6对巨噬细胞分化的影响及诱导分化的具体信号机制。2.探讨IL-6诱导而来的M2样巨噬细胞对胃癌肿瘤细胞的生物学功能的影响。方法:1、采用阳性磁珠分选法分选健康成人外周血(PBMC)中的CD14+单核细胞,流式细胞仪检测CD14+单核的纯度后,在重组人巨噬细胞集落刺激因子(M-CSF)的刺激下诱导分化5天,使其分化为巨噬细胞,再经过IL-6的诱导刺激,使巨噬细胞进一步向M2表型分化。提取RNA,通过实时荧光定量PCR检测M2型巨噬细胞的分子标志(IL-10、TGF-β)。2、IL-6可以激活JAK—STAT3信号通路,活化的STAT3(即p-stat3)可以进入胞核,作用其下游靶基因,参与转录调控。本实验拟采用Westen blot和免疫荧光证实IL-6通过激活STAT3—p-STAT3这一信号通路而介导巨噬细胞的M2型极化。同时,采用小干扰RNA(si RNA)干扰STAT3目的基因,预先沉默巨噬细胞内STAT3的表达,再采取实时荧光定量PCR检测IL-10、TGF-β的表达,进一步探究STAT3在IL-6诱导巨噬细胞分化这一过程的调节作用。3、采用细胞侵袭及细胞增殖实验(CCK8)对诱导而来的巨噬细胞进行生物学功能的研究。采用胃癌细胞(AGS、SGC-7901)与IL-6诱导的M2样巨噬细胞上清液共培养,研究诱导的巨噬细胞对胃癌细胞的侵袭能力及增殖能力的影响。结果:1、经过M-CSF诱导的CD14+单核细胞分化为巨噬细胞后,在IL-6的刺激下,巨噬细胞表现出IL-10表达升高,TGF-β表达升高,IL-12p35表达降低,并且,随着IL-6刺激浓度的提高,巨噬细胞表达的IL-10和TGF-β也随着增高,而IL-12p35的表达量却随IL-6浓度的升高而降低。2、Western blot检测STAT3和p-STAT3的表达量,结果显示在加入IL-6刺激的巨噬细胞组,细胞总STAT3的表达量没有升高,而活化的磷酸化STAT3即p-STAT3表达量显著升高;提高IL-6的刺激浓度,细胞中总STAT3表达量保持不变,而活化的p-STAT3的表达量却随IL-6刺激浓度的升高而增加。免疫荧光检测显示,加入IL-6刺激的巨噬细胞组,细胞内见大量的活化形式的p-STAT3荧光信号,且活化的p-STAT3都分布在细胞核。在si RNA预沉默STAT3表达后,定量PCR检测IL-10、TGF-β和IL-12p35分子的表达量,结果显示,在siRNA沉默STAT3的细胞组中,p-STAT3的表达量的下降,随着p-STAT3的表达量的下降,IL-10、TGF-β的表达水平也较非沉默组巨噬细胞中IL-10、TGF-β的表达水平相应降低。3、实验将M2样巨噬细胞的培养上清分别与两种胃癌细胞系(AGS、SGC-7901)共培养。Transwell细胞迁移结果显示:在两种细胞系中,M2样巨噬细胞上清液培养组穿过基底膜的肿瘤细胞数(AGS 257.6±6.26,SGC 218.6±4.62)都比相应的对照组中穿过基底膜的肿瘤细胞数(AGS187.8±6.09,SGC 152±7.91)显著增多。同样,CCK8增殖实验结果显示,与M2样巨噬细胞上清共培养组的胃癌细胞在72 h时间点的OD450值(AGS 1.25±0.12;SGC 1.33±0.14)显著高于RPMI-1640对照组的OD450值(AGS0.90±0.02;SGC 0.98±0.07)。最后,在M2巨噬细胞的上清液中加入IL-10或TGF-β阻断抗体后,该M2样巨噬细胞上清液的促肿瘤细胞迁移和增殖作用均减弱。结论:1、IL-6诱导巨噬细胞高表达IL-10和TGF-β,而低表达IL-12p35,其诱导的巨噬细胞表型呈M2样表型(IL-10high TGF-βhigh IL-12p35 low),同时,IL-10,TGF-β的表达量随IL-6的刺激浓度升高而升高,诱导效应存在IL-6浓度依赖性。2、IL-6诱导巨噬细胞M2样分化的过程中,JAK/STAT3信号通路被大量激活,活化的p-STAT3进入细胞核,从而促进IL-10和TGF-β的表达,抑制IL-12p35的表达,参与调控IL-6诱导的巨噬细胞M2样极化。3、IL-6诱导的M2样巨噬细胞能促进胃癌细胞的增殖和迁移,进而促进肿瘤的进展。
[Abstract]:BACKGROUND Macrophages are a group of cells with various characteristics in the human immune system, which play an important role in the body's defense against bacterial, viral and parasitic infections. It is a tumor-associated macrophage (TAMs). TAM is the aggregation and differentiation of peripheral circulating monocytes stimulated by local CCL2, macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Macrophages have two main phenotypes, M1 and M2. M1 macrophages can be induced by LPS or IFN-gamma. M1 macrophages are powerful effector cells that kill microorganisms, produce inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and IL-12, and play an important role in eliminating bacterial, viral and fungal infections. Instead, M2 macrophages produce IL-10 and transform growth factors. M2 macrophages also play an important role in tissue remodeling, parasitic infection, angiogenesis and tumor progression. Interleukin-6 is multipotent and can affect many functions of the body, such as vascular disease, lipid metabolism, insulin resistance and neuropsychological behavior. IL-6 is a STAT3 signaling pathway. A powerful activator, activated P-STAT3 signaling protein, translocates rapidly into the nucleus and binds to the recognition sequence of target genes such as cyclin D1, c-myc, and the promoter of VEGF, thereby increasing the transcription and expression of these target genes. In previous studies, we found that the expression of IL-6 in cancer tissues was higher than that in non-tumor tissues. At the same time, the infiltration of M2 macrophages was also increased in cancer tissues. By detecting the phenotypic characteristics of macrophages induced by IL-6 stimulation, we found that IL-6 could induce normal macrophages to differentiate into M2-like macrophages. The expression of IL-10 and TGF-beta increased, while the expression of IL-12p35 decreased. Objective: 1. To establish a cell model of macrophage differentiation induced by IL-6 in vitro and explore the effect of IL-6 on macrophage differentiation and the specific signal mechanism of inducing differentiation. CD14+ monocytes from peripheral blood (PBMC) of healthy adults were selected by sorting method. The purity of CD14+ monocytes was detected by flow cytometry. After 5 days of induction and differentiation by recombinant human macrophage colony-stimulating factor (M-CSF), macrophages were induced to differentiate into macrophages. After stimulation by IL-6, macrophages were further differentiated into M2 phenotypes. Real-time fluorescence quantitative PCR was used to detect the molecular marker (IL-10, TGF-beta). IL-6 could activate JAK-STAT3 signaling pathway. Activated STAT3 (p-stat3) could enter the nucleus, act on its downstream target genes and participate in transcriptional regulation. In this study, Western blot and immunofluorescence were used to confirm that IL-6 could activate STAT3-p-STAT3 signaling pathway. At the same time, small interfering RNA (si RNA) was used to interfere with STAT3 target gene. STAT3 expression in macrophages was silenced in advance. The expression of IL-10 and TGF-beta was detected by real-time fluorescence quantitative PCR. The regulatory role of STAT3 in the process of IL-6 induced macrophage differentiation was further explored. Invasion and cell proliferation assay (CCK8) was used to study the biological function of induced macrophages. Gastric cancer cells (AGS, SGC-7901) were co-cultured with IL-6-induced M2-like macrophage supernatant to study the effects of induced macrophages on the invasion and proliferation of gastric cancer cells. After nuclear cells differentiated into macrophages, the expression of IL-10, TGF-beta and IL-12p35 were increased and decreased by IL-6 stimulation. Moreover, with the increase of IL-6 stimulation concentration, the expression of IL-10 and TGF-beta in macrophages increased, while the expression of IL-12p35 decreased with the increase of IL-6 concentration. The expression of STAT3 and p-STAT3 in macrophages stimulated by IL-6 did not increase, but the expression of activated phosphorylated STAT3, p-STAT3, increased significantly in macrophages stimulated by IL-6. Immunofluorescence assay showed that a large number of activated p-STAT3 fluorescent signals were observed in the macrophages stimulated by IL-6, and the activated p-STAT3 was distributed in the nucleus of the cells. In the cell group, the expression of p-STAT 3 decreased. With the decrease of p-STAT 3 expression, the expression levels of IL-10 and TGF-beta were also lower than those in the non-silent group. 3. The supernatants of M2-like macrophages were co-cultured with two gastric cancer cell lines (AGS, SGC-7901). The results showed that the number of tumor cells passing through basement membrane (AGS 257.6 (+ 6.26) and SGC 218.6 (+ 4.62) in M2-like macrophage supernatant culture group was significantly higher than that in the corresponding control group (AGS 187.8 (+ 6.09) and SGC 152 (+ 7.91). Similarly, CCK8 proliferation test showed that the number of tumor cells passing through basement membrane in M2-like macrophage supernatant culture group was significantly higher than that in the corresponding control group. The OD450 value of gastric cancer cells in co-culture group at 72 h (AGS 1.25+0.12; SGC 1.33+0.14) was significantly higher than that of RPMI-1640 control group (AGS 0.90+0.02; SGC 0.98+0.07). Conclusion: 1. IL-6 induces macrophages to overexpress IL-10 and TGF-beta, whereas IL-12p35 is underexpressed. The induced macrophage phenotype is M2-like (IL-10 high TGF-beta high IL-12p35 low). At the same time, the expression of IL-10 and TGF-beta increases with the increase of IL-6 stimulation concentration, and the induction effect is IL-6 concentration-dependent.2, IL-6 induces macrophage fineness. During M2-like differentiation, JAK/STAT3 signaling pathway is activated, and activated p-STAT3 enters the nucleus, which promotes the expression of IL-10 and TGF-beta, inhibits the expression of IL-12p35, and participates in the regulation of IL-6-induced M2-like polarization of macrophages.3, IL-6-induced M2-like macrophages can promote the proliferation and migration of gastric cancer cells, and thus promote tumor. Progress.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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