Imatinib抑制肿瘤相关巨噬细胞M2型极化发挥抗肿瘤转移的作用研究

发布时间：2017-12-27 11:45

本文关键词：Imatinib抑制肿瘤相关巨噬细胞M2型极化发挥抗肿瘤转移的作用研究　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的：近年来,大量的临床与实验数据表明,M2型的肿瘤相关巨噬细胞(TAM)在肿瘤的转移,尤其是肿瘤转移至靶器官形成转移灶的过程中发挥着关键的促进作用。故而,有针对性的发展干预TAM M2型极化的化合物是极具应用前景的抗肿瘤转移新策略。但当前临床上干预巨噬细胞M2型极化的药物仍是空白,因此从小分子化合物中筛选出有效抑制巨噬细胞M2型极化的化合物显得尤为重要与迫切。本文通过巨噬细胞M2型极化体外模型,筛选到了能有效抑制巨噬细胞M2型极化的小分子化合物Imatinib,并进一步评价了Imatinib对巨噬细胞极化的影响,研究其在肿瘤转移至靶器官形成转移灶的过程中的作用,以及Imatinib抑制巨噬细胞M2型极化的分子机制,我们的研究提出了Imatinib抗肿瘤转移的新机制,为Imatinib治疗肿瘤转移提供新的思路与实验依据,丰富了小分子药物通过干预巨噬细胞M2型极化发挥抗肿瘤转移的策略的可行性。方法：(1)采用SRB法,考察Imatinib对巨噬细胞增殖的影响以及条件培养基对LLC细胞的影响。(2)采用流式细胞术手段,检测M2型巨噬细胞细胞表面标志性蛋白CD206、M1型巨噬细胞细胞表面标志性蛋白CD86以及F4/80巨噬细胞细胞表面标志性蛋白的影响；(3)RT-PCR方法检测巨噬细胞M2型或M1型标志基因的mRNA水平；(4)收集IL-13与Imatinib单独或共同孵育巨噬细胞的条件培养基,将其作用于LLC细胞,通过Transwell小室细胞迁移实验考察其运动迁移能力,用同样的方式考察IL-13与Imatinib单独或共同作用对LLC细胞运动能力的影响；(5)建立LLC细胞肺转移模型,检测Imatinib的体内抗肿瘤转移作用；(6)HE染色法或肺表面转移灶计数的方式考察肿瘤肺转移的情况；(7)采用流式细胞术或组织免疫荧光或免疫组化方法检测小鼠原发部位的肿瘤和转移靶器官肺脏中的巨噬细胞M2型极化情况；(8)用western blot技术考察相关蛋白的变化情况；(9)采用细胞免疫荧光方法检测STAT6在细胞内的分布情况。结果：(1) Imatinib能抑制巨噬细胞M2型极化而对M1极化无影响我们采用流式细胞术检测IL-13或IL-4诱导的RAW264.7或BMDM,考察巨噬细胞M2型表面标志性蛋白CD206的变化情况,发现IL-13能显著增加CD206阳性细胞的比例,从1.51±0.97%至32.65±3.79%(p0.01)。而同时作用最高浓度的Imatinib能将其下调至9.78±0.55%(p0.01)。而在IL-4诱导模型中也有相似的情况。采用Real-time PCR检测M2型巨噬细胞的标志性基因Arg1,Mgl2, Mrc1,CDHl以及CCL2的表达,结果显示Imatinib能显著抑制由IL-13或IL-4上调的M2型巨噬细胞的标志性基因的转录。以Argl基因为例,IL-13可诱导Argl的mRNA至对照组的71.7±13.1倍,而同时作用Imatinib可下调至4.6±2.2倍,具有显著性差异(p0.001)。另一方面我们采用LPS和IFNγ共同刺激RAW264.7,构建巨噬细胞M1型极化模型,通过流式细胞术考察巨噬细胞表面M1型标志性蛋白水平,结果表明LPS和IFNγ能显著上调CD86阳性细胞比例,从2.29±0.09%至43.56±4.4%(p0.01),而同时作用Imatinib则对其无显著影响(p0.05)。我们也通过Real-time PCR技术考察M1型巨噬细胞的标志性基因iNOS、CCL5、TNFα的表达,发现IFNγ和LPS能显著上调iNOS、CCL5、TNFα分别至对照组的6.1±1.6、1.7±0.6、2.0±0.3,而同时作用Imatinib则无显著变化(p0.05)。(2) Imatinib通过干预巨噬细胞M2型极化发挥抗肿瘤转移的作用在Transwell小室细胞迁移实验中,我们发现IL-13诱导的条件培养基显著增加LLC细胞迁移至小室下方,从每个视野118±14至211±27个(p0.01)；相比IL-13组条件培养基,Imatinib和IL-13共同孵育BMDM所得的条件培养基能显著抑制LLC细胞的迁移至120±17个(p0.01)。而IL-13和Imatinib单独或共同的直接作用对LLC细胞的迁移运动能力没有明显影响(p0.05)。在小鼠LLC腋下接种模型和尾静脉接种模型中,我们发现Imatinib能显著减少小鼠肺脏肿瘤转移灶的数量,分别从平均1.3个减少至平均0.3个(p0.05)以及从11.8±4.6个减少至6.0±4.3个(p0.05)。在LLC腋下接种模型中,我们考察了肿瘤原发部位的巨噬细胞极化情况,发现Imatinib组小鼠其中的M2-like TAM的比例(28.2±12.1%)显著少于对照组(48.7±19.2%)(p0.01)；在尾静脉接种模型中,我们考察了实验进行第7天以及实验终点的小鼠肿瘤转移的靶器官肺脏中M2-like TAM的比例。实验第7天时,免疫荧光结果表明Imatinib组小鼠肺脏中M2-like TAM在巨噬细胞中的比例(25.7±4.5%)明显少于对照组(64.5±2.3%)(p0.01),流式细胞术方法考察也得到了相似的结果；而在实验的终点(实验第24天),免疫组化结果显示Imatinib明显减少小鼠肺脏转移灶处的M2-like TAM数量。以上结果表明,Imatinib可有效减少肿瘤细胞在肺脏的转移灶处的M2-like巨噬细胞比例,抑制肿瘤细胞在肺脏中的进一步增殖。(3) Imatinib干预巨噬细胞M2型极化的机制探究我们通过western blot技术考察Imatinib对IL-13诱导的巨噬细胞M2型极化过程中STAT6蛋白的影响,发现Imatinib能显著抑制STAT6的磷酸化。之后我们又通过免疫荧光考察巨噬细胞中STAT6蛋白的分布,发现Imatinib能显著抑制IL-13诱导的STAT6入核的现象。说明Imatinib通过抑制STAT6的磷酸化以及入核作用发挥干预IL-13诱导的巨噬细胞M2型极化。结论：(1) Imatinib能抑制巨噬细胞M2型极化而对M1型极化无明显影响。(2) Imatinib通过干预巨噬细胞M2型极化发挥抗肿瘤转移的作用。(3) Imatinib通过抑制STAT6的磷酸化以及入核,干预巨噬细胞M2型极化。
[Abstract]:Objective: in recent years, a large number of clinical and experimental data show that M2 type tumor associated macrophages (TAM) play a key role in tumor metastasis, especially in the process of tumor metastasis to target organ formation and metastasis. Therefore, the targeted development of TAM M2 polarization compounds is a promising new strategy for anti tumor metastasis. However, the drugs that interfere with macrophage M2 polarization are still blank. Therefore, it is particularly important and urgent to screen compounds that effectively inhibit the M2 polarization of macrophages. The M2 type macrophage polarization model in vitro, screened can effectively inhibit macrophage M2 polarization of small molecule compounds Imatinib, and further evaluated the effects of Imatinib on macrophage polarization, study the formation process of tumor metastasis to target organ metastases in the role of Imatinib and inhibition of macrophage M2 polarization molecular mechanism our study provides a new mechanism for Imatinib tumor metastasis, provide ideas and experimental basis for the new treatment of Imatinib tumor metastasis, abundant small molecule drugs by macrophage M2 polarization play feasibility of anti metastasis strategies. Methods: (1) the effect of Imatinib on the proliferation of macrophages and the effects of conditioned medium on LLC cells were investigated by SRB. (2) by flow cytometry method, detection of M2 type macrophage cell surface marker protein of CD206, M1 type macrophage cell surface marker protein CD86 and F4/80 macrophage cell surface marker protein effect; (3) RT-PCR method for detection of macrophage M2 type or M1 type marker gene mRNA level; (4) to collect IL-13 with Imatinib alone or co incubated with macrophage conditioned medium, its effect on LLC cell migration experiment investigated the movement ability by Transwell cell in the same way, effects of IL-13 and Imatinib alone or combined effect on the migration ability of LLC cells; (5) to establish the model of LLC cell lung metastasis detection Imatinib in vivo anti metastasis effect; (6) HE staining foci counting method or the surface of the lung metastasis way of lung metastasis; (7) by flow cytometry and immunofluorescence M2 type macrophage polarization or immunohistochemical method in the primary site of the tumor and metastasis of target organ in the lungs; (8) to investigate the changes of related proteins with Western blot technology; (9) using immunofluorescence assay of STAT6 in intracellular distribution. Results: (1) Imatinib can inhibit macrophage M2 polarization and M1 polarization has no effect we detected by IL-13 or IL-4 by flow cytometry RAW264.7 or BMDM, investigate the change of CD206 protein in macrophages M2 surface markers, found that IL-13 significantly increased the CD206 positive cell ratio from 0.97% to 1.51. 32.65 + 3.79% (P0.01). At the same time, the highest concentration of Imatinib can be reduced to 9.78 + 0.55% (P0.01). There is also a similar situation in the IL-4 induced model. Real-time PCR was used to detect the expression of Arg1, Mgl2, Mrc1, CDHl and CCL2 in M2 macrophages. Results showed that Imatinib could significantly inhibit transcription of the marker genes which were up regulated by IL-13 or IL-4. Taking Argl gene as an example, IL-13 can induce mRNA of Argl to 71.7 + 13.1 times to control group, while Imatinib can be reduced to 4.6 + 2.2 times, with significant difference (p0.001). On the other hand, we use LPS and IFN gamma costimulates RAW264.7, construction of M1 type macrophage polarization model, through the study of M1 type macrophage surface marker protein level by flow cytometry, the results showed that LPS and IFN gamma can significantly increase the proportion of CD86 positive cells, from 2.29 + 0.09% to 43.56 + 4.4% (P0.01), and at the same time effect the Imatinib had no significant effect (P0.05). We also through the expression of marker genes iNOS, Real-time PCR CCL5, M1 macrophages of TNF alpha, IFN gamma and LPS can significantly increase iNOS, CCL5 and TNF respectively to a control group of 6.1 + 1.6, 1.7 + 0.6, 2 + 0.3, while Imatinib had no significant changes (P0.05). (2) Imatinib by macrophage M2 polarization play anti metastasis effect in Transwell cell migration experiments, we found that IL-13 induced by conditioned medium significantly increased LLC cell migration into the chamber below, from 118 to 211 in each field + 14 + 27 (P0.01); compared with group IL-13 conditioned medium, Imatinib BMDM and IL-13 were incubated with the conditioned medium could significantly inhibit the migration of LLC cells to 120 + 17 (P0.01). The direct effect of IL-13 and Imatinib alone or in common on the mobility of LLC cells was not significantly affected (P0.05). In the mouse LLC axillary inoculation model and the tail vein inoculation model, we found that Imatinib significantly reduced the number of metastatic tumors in lung tumors from 1.3 to 0.3 on average (P0.05), and decreased from 11.8 + 4.6 to 6 + 4.3 (P0.05). In LLC inoculated with the model, we investigated macrophage polarization of the primary tumor, M2-like TAM found the percentage in Imatinib group (28.2 + 12.1%) was significantly lower than the control group (48.7 + 19.2%) (P0.01); the intravenous inoculation model, we examine the experimental target organ seventh day and experimental mouse tumor metastasis end point in M2-like TAM ratio. The seventh day of the experiment, the immunofluorescence results showed that Imatinib group mice lungs M2-like TAM in macrophages in the proportion of (25.7 + 4.5%) was significantly lower than the control group (64.5 + 2.3%) (P0.01), flow cytometry analysis also yielded similar results; and in the end point (the twenty-fourth day), immune group results show that Imatinib significantly reduced the number of lung metastases in mice at M2-like TAM. These results suggest that Imatinib can effectively reduce the tumor cells in the lung
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【相似文献】