M2型巨噬细胞促进A549细胞增殖、迁移及对顺铂的抵抗

发布时间：2018-05-27 15:19

本文选题：肺癌细胞株A549 + M1型巨噬细胞　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:顺铂是治疗非小细胞肺癌的有效药物之一,但临床上发现随着治疗周期次数的增加,癌细胞对顺铂的敏感性逐渐降低,最后出现对顺铂耐受。目前普遍认为肿瘤相关巨噬细胞(tumor associated macrophage,TAM)作为肿瘤微环境的重要基质细胞,不仅直接参与肿瘤血管的形成,而且通过与肿瘤细胞间的相互作用,介导肿瘤细胞的间质转化,促进肿瘤转移。根据巨噬细胞的表型和功能,巨噬细胞分为M1型和M2型;且近期的研究表明M1和M2型巨噬细胞在肿瘤组织中均有分布。但是M1和M2型巨噬细胞对肺癌细胞的增殖、迁移及对顺铂敏感性的作用如何还鲜有报道。本实验采用A549细胞及其荷瘤小鼠为模型,观察了M1和M2型巨噬细胞及其分泌的外泌体在体内外对A549细胞的增殖、迁移及对顺铂敏感性的作用,目的是深入了解不同类型的巨噬细胞对顺铂治疗的影响,为靶向调节巨噬细胞促进顺铂治疗效果提供实验数据。方法:1 M1和M2巨噬细胞的诱导体外培养人单核细胞白血病细胞系THP-1,PMA(15μg/ml)诱导36h,去掉含PMA的培养基并用PBS洗一遍,加入新鲜完全培养基继续培养12h,贴壁的细胞为M0。1.1 M1细胞的诱导:贴壁细胞用含1μg/ml脂多糖(LPS)和20ng/ml人重组干扰素-γ(IFN-γ)的完全培养基培养24h,然后去掉培养上清,PBS洗1次,加入新鲜完全培养基培养24h,收获上清经ELISA测定IL-1β水平,收集细胞提取总蛋白,Western blot分析i NOs(诱导型一氧化碳合酶)、Arg-1(精氨酸酶-1)的表达。1.2 M2细胞的诱导:贴壁细胞用含20ng/ml IL-4的完全培养基培养24h,然后去掉培养上清,PBS洗1次,去掉残存的细胞因子或LPS,加入新鲜完全培养基培养24h,收获上清(ELISA测定IL-1β),收细胞,WB分析i NOs、Arg-1的表达。2外泌体的制备及电镜观察收集M1型和M2型巨噬细胞培养上清,差速离心法分离提取外泌体(M1/exo,M2/exo):2000×g,10min,10,000×g,1h;然后将上清超高速110,000×g离心16 h,以少量PBS重悬所得沉淀。最后用磷钨酸负染外泌体并在透射电镜下观察外泌体的形态,并以Nanodrop进行定量。3观察M1/exo,M2/exo对A549细胞增殖、迁移及对顺铂敏感性的影响3.1 Dil荧光染料标记M2/exo:取11.5mg/ml的M2/exo0.5ml,加Dil荧光染料(50μg/ml),25μg,放培养箱中作用30分钟,取出放入小的超速离心管中,用PBS补满,上超速离心机,2×105g转速,2小时,离心后弃上清,去掉游离的染料,要沉淀外泌体。将荧光标记的外泌体作用于A549细胞24h,荧光显微镜下观察A549对外泌体的摄取。3.2体外培养A549细胞,分别用终浓度50μg/ml的M1/exo或M2/exo刺激A549细胞,Transwell细胞迁移实验、划痕试验分别观察两种不同外泌体对A549迁移能力的影响。3.3用艾森生物实时细胞分析技术(RTCA),即实时无标记细胞分析法,检测M1/exo或M2/exo刺激对A549增殖和对顺铂耐药性的影响。4观察M1/exo或M2/exo对A549细胞的E-Cadherin、N-Cadherin、PTEN、AKT/p-AKT蛋白表达的影响体外培养A549细胞,M1/exo或M2/exo刺激72h,收集细胞并提取总蛋白,Western Blot分析上述蛋白表达。5 M1及M2型巨噬细胞及其分泌的外泌体影响A549荷瘤小鼠肿瘤生长及对顺铂敏感性的观察5.1细胞培养与计数收集体外培养的处于对数生长期A549计数,调整细胞密度为7×107/ml。同1.1和1.2诱导M1型和M2型巨噬细胞,计数后,分别调整细胞密度为7×107/ml。5.2荷瘤小鼠的制备5.2.1实验分组将5-6周的雌性BALB/c裸鼠分为10组,每组5只。第一组:A549荷瘤对照组。将体外培养的A549移植到小鼠右侧腋下,7×106/鼠。第二组:A549+M1组。取等量的A549和M1型细胞(均100μl)混合后,移植到小鼠右侧腋下。第三组:A549+M2组。取等量的A549和M2型细胞(均100μl)混合后,移植到小鼠右侧腋下。第四组:A549+顺铂。右侧腋下移植A549,7×106/鼠,按Q4d×3的方案腹腔注射顺铂(5mg/kg)。第五组:A549+M1+顺铂。同上移植A549和M1,腹腔注射顺铂。第六组:A549+M2+顺铂。同上移植A549和M2,腹腔注射顺铂。第七组:A549+M1/exo。同上移植A549,在移植的当天,在注射肿瘤细胞的部位注射50μg外泌体。隔日一次,连续3周。第八组:A549+M2/exo。同上移植A549和外泌体注射。第九组:A549+M1/exo+顺铂。同上。第十组:A549+M2/exo+顺铂。同上。5.3观察肿瘤瘤重在荷瘤的第四周,解剖荷瘤小鼠,分离肿瘤组织并称重。结果:1 ELASA法检测诱导的M1型巨噬细胞上清液中IL-1β含量低于M2型巨噬细胞组,差异具有统计学意义(P0.05)。M1型巨噬细胞i NOs(诱导型一氧化碳合酶)蛋白表达量明显高于M2型巨噬细胞(P0.05);Arg-1(精氨酸酶-1)蛋白表达量低于M2型巨噬细胞(P0.05)。体外成功诱导M1型巨噬细胞、M2型巨噬细胞。2电镜观察到大小均一的、直径约为100 nm大小的圆形或椭圆形囊泡。3倒置荧光显微镜下观察到A549细胞可以摄取外泌体。4划痕实验观察到,PBS、M1/exo对A549的横向迁移没有明显影响(P0.05);M2/exo可促进A549细胞的横向迁移(P0.05)。Transwell实验显示,M2/exo较对照组及M1/exo组明显促进A549细胞的纵向迁移(P0.05),M1/exo的作用不明显(P0.05)。细胞增殖实验显示M2/exo可促进A549细胞的增殖和降低对顺铂的敏感性。5 Western blot结果显示,与PBS及M1/exo相比较,M2/exo明显上调A549细胞N-Cadherin的表达(P0.05),下调E-Cadherin表达水平(P0.05);表明M2/exo能够促进A549发生间质转化和迁移。A549细胞经M1/exo和M2/exo刺激后,PTEN表达水平在M2/exo刺激组明显低于M1/exo刺激组和对照组(P0.05)。p-AKT(p-AKT/AKT灰度值)活力水平在M2/exo刺激组增高,且显著高于M1/exo刺激组和对照组(P0.05)。6小鼠荷瘤实验结果显示1)A549+M2细胞组荷瘤小鼠的瘤重明显大于A549组和A549+M1细胞组,均有显著差异(均为P0.01)。A549+M1细胞组和A549组瘤重比较无显著差异(P0.05)。以上结果显示M2细胞可以促进A549瘤体增殖。2)A549细胞、A549+M2细胞及A549+M1细胞三组荷瘤小鼠分别经顺铂治疗后,观察到A549+M2细胞组瘤重最大,与其它两组均有显著性差异(均为P0.05)。而M1+A549与A549细胞组的瘤重无统计学差异(P0.05)。3)给A549荷瘤小鼠瘤周注射M2/exo组瘤重最大,与注射M1/exo和PBS组的瘤重相比,均有统计学差异(均为P0.05);A549荷瘤小鼠瘤周注射M1/exo和PBS组的瘤重没有显著差别(P0.05)。4)A549荷瘤小鼠瘤周注射M2/exo后,对顺铂的治疗敏感性较差,该组荷瘤小鼠瘤重与注射M1/exo和PBS组的瘤重相比,均有统计学差异(均为P0.05);而M1/exo和PBS均不影响A549细胞对顺铂的敏感性。结论:1 M2型巨噬细胞可促进A549细胞增殖、迁移,并降低对顺铂敏感性。2 M2型巨噬细胞产生的外泌体可促进A549细胞增殖、迁移,并降低对顺铂敏感性。
[Abstract]:Objective: cisplatin is one of the effective drugs for the treatment of non-small cell lung cancer. However, it is found that the sensitivity of cancer cells to cisplatin gradually decreases with the increase of the number of treatment cycles, and the tumor associated macrophage (TAM) is widely considered as an important matrix of tumor microenvironment. Cells not only directly participate in the formation of tumor vessels, but also mediate interstitial transformation of tumor cells and promote tumor metastasis through interaction with tumor cells. According to the phenotype and function of macrophages, macrophages are divided into M1 type and M2 type. And recent studies have shown that M1 and M2 type macrophages are distributed in tumor tissues. But M1 The effects of M2 type macrophages on proliferation, migration and sensitivity to cisplatin are rarely reported. This experiment uses A549 cells and tumor bearing mice as models to observe the proliferation, migration and sensitivity to cisplatin by M1 and M2 macrophages and the secreted exocrine in vivo and in vivo. To understand the effect of different types of macrophages on cisplatin treatment and to provide experimental data for the targeting of macrophages to promote cisplatin treatment. Methods: 1 M1 and M2 macrophages were induced in vitro to induce human monocytic leukemia cell line THP-1, PMA (15 g/ml) to induce 36h, remove PMA containing medium and wash it with PBS to add fresh finish. The whole culture medium continued to culture 12h, the adherent cells were induced by M0.1.1 M1 cells: adherent cells cultured 24h with 1 mu g/ml lipopolysaccharide (LPS) and 20ng/ml human recombinant interferon gamma (IFN- gamma) complete medium, then removed culture supernatant, PBS washed 1 times, added fresh complete medium to culture 24h, and harvested supernatant to determine IL-1 beta level by ELISA. Cell extraction of total protein, Western blot analysis of I NOs (inducible carbon monoxide synthase), Arg-1 (arginase -1) expression of.1.2 M2 cells induced: adherent cells cultured with 20ng/ml IL-4 containing full medium culture 24h, and then removed the culture supernatant, PBS washed 1 times, remove the remaining cytokine or paste, add fresh complete medium culture, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest, harvest (ELISA determination of IL-1 beta), cell collection, WB analysis of I NOs, Arg-1 expression of.2 exocrine and electron microscope observation to collect M1 and M2 type macrophage culture supernatant, differential centrifugation separation and extraction of exocrine (M1/exo, M2/exo): 2000 * g, 10000 * centrifugation, and then superhigh speed 110000 x centrifugation 16 The morphology of exocrine was observed by negative staining with phosphotungstic acid and under transmission electron microscope. The effects of M1/exo, M2/exo on A549 cell proliferation, migration and cisplatin sensitivity were observed by Nanodrop, and the effect of M2/exo on the M2/exo0.5ml of 11.5mg/ml by 3.1 Dil fluorescent dye M2/exo:, Dil fluorescein (50 mu g/ml), 25 micron g, and 30 minutes in the incubator. The clock was taken out into a small overspeed centrifuge tube, filled with PBS, overspeed centrifuge, 2 x 105g speed, 2 hours, after centrifugation, discarding the supernatant, removing the free dye, and precipitating the exocrine. The fluorescence labeled exocrine was acted on the A549 cell 24h, and the fluorescence microscope observed the uptake of A549 to the exocrine by.3.2 in the culture of A549 cells in vitro. The final concentration was used respectively. 50 g/ml M1/exo or M2/exo stimulates A549 cells, Transwell cell migration experiments, and scratch test to observe the effect of two different exocrine on the migration ability of A549,.3.3 using Eisen biological real-time cell analysis technique (RTCA), that is, real time unlabeled cell analysis, to detect the effect of M1/exo or M2/exo stimulation on A549 proliferation and cisplatin resistance. 4 the effects of M1/exo or M2/exo on the expression of E-Cadherin, N-Cadherin, PTEN, and AKT/p-AKT protein in A549 cells were affected in the culture of A549 cells in vitro. M1/exo or M2/exo stimulated 72h, collecting cells and extracting the total protein. Cisplatin sensitivity observation 5.1 cell culture and count collection in the logarithmic growth period A549 count, adjust cell density to 7 x 107/ml. with 1.1 and 1.2 induction of M1 and M2 type macrophages, after counting, the cell density of 7 * 107/ml.5.2 bearing mice in the preparation of 5.2.1 experiment group for 5-6 weeks female BALB/c nude mice 10 groups, each group of 5. A549 tumor bearing control group. Transplanted A549 in vitro to the right underarm of mice, 7 x 106/ mice. Group second, group A549+M1. The same amount of A549 and M1 cells (100 mu L) were mixed and transplanted into the right arm of the mouse. The third group: A549+M2 group. After mixing the same amount of A549 and M2 cells (100 um L), transplanted into mice. Right axillary subaxillary. Fourth groups: A549+ cisplatin. Right subaxillary transplantation of A549,7 x 106/ mice, intraperitoneal injection of cisplatin (5mg/kg) by Q4d x 3. Group Fifth: A549+M1+ cisplatin. Co transplantation of A549 and M1, intraperitoneal injection of cisplatin. Group sixth: A549+M2+ cisplatin. Co transplantation of A549 and M2, abdominal injection cisplatin. Seventh groups: A549+M1/exo. same transplant recipients, transplanted in transplant On the same day, 50 g exosecrete was injected at the site of the injection of tumor cells. One time every other day for 3 weeks. Group eighth: A549+M2/exo. combined with A549 and exosecrete. Ninth group: A549+M1/exo+ cisplatin. Tenth groups: A549+M2/exo+ cisplatin. Same.5.3 observed tumor weight in the fourth week of the tumor, dissecting tumor bearing mice, separating tumor tissue and weighing Results: results: the content of IL-1 beta in the induction of M1 macrophage supernatant by 1 ELASA method was lower than that of M2 macrophage group, and the difference was statistically significant (P0.05).M1 macrophage I NOs (inducible carbon monoxide synthase) protein expression was significantly higher than that of M2 type macrophage (P0.05), Arg-1 (arginase -1) protein expression was lower than that of macrophage Cell (P0.05). M1 macrophages were successfully induced in vitro. The.2 electron microscopy of M2 type macrophages showed that the size of the size was uniform, and the diameter of the round or oval vesicle with a diameter of 100 nm was observed under the.3 inverted fluorescence microscope, and the A549 cells could be observed in the Exocyst.4 scratch test. PBS, M1/exo had no significant influence on A549 lateral migration (P0.05). Exo could promote the lateral migration of A549 cells (P0.05).Transwell experiment, which showed that M2/exo significantly promoted vertical migration of A549 cells (P0.05) compared with the control group and M1/exo group, and the effect of M1/exo was not obvious (P0.05). Compared with M1/exo, M2/exo obviously up-regulated the expression of N-Cadherin in A549 cells (P0.05), and down regulated the E-Cadherin expression level (P0.05), indicating that M2/exo can promote the transformation of A549 and the migration of.A549 cells after M1/exo and M2/exo stimulation. The activity level was higher in the M2/exo stimulation group and was significantly higher than that in the M1/exo stimulation group and the control group (P0.05).6 mice. The tumor bearing tumor weight of the A549+M2 cell group was significantly greater than that of the A549 group and the A549+M1 cell group. There was significant difference (P0.05 P0.01) between the.A549+M1 cell group and the A549 group (P0.05). The above results showed that M2 cells could promote the proliferation of A549 tumor.2) A549 cells, and the three groups of A549+M2 and A549+M1 cells were treated with cisplatin, respectively. The tumor weight of the A549+M2 cell group was the largest, and the difference between the other two groups was significant (P0.05). There was no statistical difference between the M1+A549 and A549 cell groups (P0.05).3) The tumor weight in the M2/exo group was the largest in the tumor bearing mice. Compared with the injection of M1/exo and PBS, there were statistically significant differences (all P0.05). The tumor weight of A549 mice injected with M1/exo and PBS was not significantly different (P0.05).4) in A549 bearing mice (P0.05).4), the sensitivity to cisplatin was poor in the treatment of cisplatin, and the tumor weight of this group of tumor bearing mice was heavy. Compared with the tumor weight of the M1/exo and PBS groups, there were statistically significant differences (both P0.05), while both M1/exo and PBS did not affect the sensitivity of A549 cells to cisplatin. Conclusion: 1 M2 macrophages can promote the proliferation, migration and decrease of the exoss produced by the cisplatin sensitive.2 M2 type macrophages, which can promote the proliferation, migration, and decrease of A549 cells. Low sensitivity to cisplatin.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36

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