肿瘤相关巨噬细胞对胃癌MGC-803细胞恶性生物学行为的影响
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摘要
目的:探讨肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)对胃癌MGC-803细胞增殖、迁移、侵袭、凋亡的影响及其可能的作用机制。方法:体外培养人单核细胞株THP-1,加入佛波酯(PMA)和IL-4后,用ELISA法检测细胞培养上清液中IL-12、IL-10的水平。取对数生长期MGC-803细胞和M2型TAM,根据培养方式不同分为单独细胞培养组、非接触共培养组和接触共培养组,用MTT法、Transwell小室法分别检测MGC-803细胞的增殖、迁移和侵袭能力,用AnnexinV-FITC/PI双染流式细胞术检测MGC-803细胞的凋亡和细胞周期变化,用qPCR、Western blotting分别检测MGC-803细胞中MMP-9、MMP-2 mRNA和蛋白的表达水平。结果:与PMA组相比,PMA+IL-4组细胞上清液中IL-12水平显著降低、IL-10水平显著升高(均P<0.05),成功将THP-1细胞诱导分化为M2型TAM。与单独细胞培养组相比,非接触共培养组、接触共培养组(:1)MGC-803细胞的增殖率显著升高(均P<0.05);(2)细胞的迁移、侵袭数量升高(均P<0.05);(3)细胞凋亡率显著降低(均P<0.05);(4)S、G2期细胞的数量升高、G1期数量降低(均P<0.05)(;5)细胞中MMP-9、MMP-2 mRNA和蛋白的表达水平均显著升高(均P<0.05)。结论:TAM可促进胃癌MGC-803细胞增殖、迁移和侵袭,解除细胞G1期阻滞,减少细胞凋亡,其机制可能与上调胃癌细胞MMP-9、MMP-2表达有关。
Objective: To investigate the effects of tumor-associated macrophages(TAM) on proliferation, migration, invation and apoptosis of gastric cancer MGC-803 cells and the possible mechanisms. Methods: Human monocyte THP-1 was cultured in vitro. After being added with PMA and IL-4, the levels of interleukin-12(IL-12) and interleukin-10(IL-10) in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). MGC-803 cells at logarithmic phase and M2-type TAM cells were divided into single cell culture group, non-contact co-culture group and contact co-culture group according to different culture methods. MTT assay was used to detect the proliferation of MGC-803 cells, Transwell assay was used to detect cell migration and invasion, and Annexin V-FITC/PI staining flow cytometry was used to examine the apoptosis and cell cycle changes of MGC-803 cells; In addition, the mRNA and protein expressions of matrix metalloproteinase-9(MMP-9) and MMP-2 were detected by Real-time fluorescence quantitative PCR(qPCR) and Western blotting. Results: Compared with PMA group, the level of IL-12 in cell supernatant of PMA+IL-4 group decreased significantly while the level of IL-10 increased significantly(all P<0.05), indicating THP-1 cells were successfully induced to differentiate into M2-type TAM. Compared with the single cell culture group, the non-contact co-culture group and the contact co-culture group exhibited:(1) significantly increased proliferation rate of MGC-803 cells(P<0.05);(2) increased number of migrated and invaded cells(all P < 0.05);(3) significantly decreased apoptotic rate(P<0.05);(4) increased proportion of S, G2 phase cells and decreased proportion of G1 phase cells(all P<0.05);and(5) significantly increased mRNA and protein expressions of MMP-9 and MMP-2(all P<0.05).Conclusion: TAM can promote the proliferation, migration and invasion of gastric cancer MGC-803 cells, relieve G1 phase arrest and reduce cell apoptosis, which may be related to the up-regulation of MMP-9 and MMP-2 expression in gastric cancer cells.
Objective: To investigate the effects of tumor-associated macrophages(TAM) on proliferation, migration, invation and apoptosis of gastric cancer MGC-803 cells and the possible mechanisms. Methods: Human monocyte THP-1 was cultured in vitro. After being added with PMA and IL-4, the levels of interleukin-12(IL-12) and interleukin-10(IL-10) in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). MGC-803 cells at logarithmic phase and M2-type TAM cells were divided into single cell culture group, non-contact co-culture group and contact co-culture group according to different culture methods. MTT assay was used to detect the proliferation of MGC-803 cells, Transwell assay was used to detect cell migration and invasion, and Annexin V-FITC/PI staining flow cytometry was used to examine the apoptosis and cell cycle changes of MGC-803 cells; In addition, the mRNA and protein expressions of matrix metalloproteinase-9(MMP-9) and MMP-2 were detected by Real-time fluorescence quantitative PCR(qPCR) and Western blotting. Results: Compared with PMA group, the level of IL-12 in cell supernatant of PMA+IL-4 group decreased significantly while the level of IL-10 increased significantly(all P<0.05), indicating THP-1 cells were successfully induced to differentiate into M2-type TAM. Compared with the single cell culture group, the non-contact co-culture group and the contact co-culture group exhibited:(1) significantly increased proliferation rate of MGC-803 cells(P<0.05);(2) increased number of migrated and invaded cells(all P < 0.05);(3) significantly decreased apoptotic rate(P<0.05);(4) increased proportion of S, G2 phase cells and decreased proportion of G1 phase cells(all P<0.05);and(5) significantly increased mRNA and protein expressions of MMP-9 and MMP-2(all P<0.05).Conclusion: TAM can promote the proliferation, migration and invasion of gastric cancer MGC-803 cells, relieve G1 phase arrest and reduce cell apoptosis, which may be related to the up-regulation of MMP-9 and MMP-2 expression in gastric cancer cells.
引文
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[14]杜恒,戴德坚,郭晓雷,等. HBX蛋白与M2巨噬细胞介导肝癌细胞上皮间质转化的实验研究[J].中华普通外科杂志, 2016, 31(6):497-500. DOI:10.3760/cma.j.issn.1007-631X.2016.06.018.
[15]杨闯,柯星,张淑平,等. THP-1源性巨噬细胞对卵巢癌SKOV3细胞Toll样受体mRNA表达的影响[J].临床检验杂志, 2014, 32(4):277-280. DOI:10.13602/j.cnki.jcls.2014.04.10.
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[18]郑海燕,王兴芬,潘彦珞,等.胃癌组织中MCP-1、MMP-9的表达与间质中TAMs浸润的关系[J].临床肿瘤学杂志, 2010, 15(3):218-221. DOI:10.3969/j.issn.1009-0460.2010.03.007.
[19]冯稳,陈奎生,于庆凯,等.肿瘤相关巨噬细胞、基质金属蛋白酶-9与食管鳞癌浸润、转移的关系[J].实用医学杂志, 2016, 32(2):235-238. DOI:10.3969/j.issn.1006-5725.2016.02.020.
[2]张子龙,曾放,庞典付,等.胃癌术后化疗联合自体肿瘤细胞抗原致敏DC-CIK细胞治疗的疗效[J].中国肿瘤生物治疗杂志, 2017,24(6):660-664. DOI:10.3872/j.issn.1007-385X.2017.06.015.
[3]郭秋均,李杰.肿瘤相关巨噬细胞在重塑肿瘤免疫微环境中的作用[J].肿瘤, 2013, 33(10):128-130. DOI:10.3781/j. issn. 1000-7431.2013.10.014.
[4]齐蕾,许欣,巩翠珂,等.肿瘤相关巨噬细胞促进胃癌上皮-间质转化的机制[J].临床与实验病理学杂志,2018,34(4):241-243. DOI:10.13315/j.cnki.cjcep.2018.04.006.
[5]刘婕,唐世孝. EBV相关性胃癌发病机制的研究进展[J].重庆医学,2013, 42(31):3836-3838. DOI:3969/j.issn.1671-8348.2013.31.044.
[6]李育刚,韩扬,史阳,等.非小细胞肺癌中M2型巨噬细胞对临床预后的影响[J].肿瘤, 2014, 34(4):349-356. DOI:10.3781/j.issn.1000-7431.2014.04.009.
[7]章述军,雷青松,李麟,等.两种PMA诱导方案对THP-1巨噬细胞M1和M2亚型相关基因表达的影响[J].中国细胞生物学学报,2017, 39(6):765-771. DOI:10.11844/cjcb.2017.06.0372.
[8]韩宇,王化泉,付蓉,等.骨髓增生异常综合征患者单个核细胞诱导的巨噬细胞的特征及功能[J].中华血液学杂志, 2017, 38(8):258-260. DOI:10.3760/cma.j.issn.0253-2727.2017.08.011.
[9]陈汜尘,陈广洁,陈红. M1/M2型巨噬细胞与心血管疾病关系的研究进展[J].现代免疫学, 2015, 35(6):507-510. DOI:1001-2478.2015.06
[10] RUFFELL B, CHANG-STRACHAN D, CHAN V, et al. Macrophage IL-10 blocks CD8(+)T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells[J]. Cancer Cell, 2014, 26(5):623-637. DOI:10.1016/j. ccell.2014.09.006.
[11] BEI Z, BAILEY W M, BRAUN K J, et al. Age decreases macrophage IL-10 expression:implications for functional recovery and tissue repair in spinal cord injury[J/OL]. Exp Neurol, 2015, 273:83-91[2018-09-02]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644435/. DOI:10.1016/j.expneurol.2015.08.001.
[12] MAKITA N, HIZUKURI Y, YAMASHIRO K, et al. IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration[J]. Int Immunol, 2015, 27(3):131-141. DOI:10.1093/intimm/dxu090.
[13]李亚,陈思成,王静,等.肿瘤相关M2型巨噬细胞通过上调VEGFR3的表达促进肺腺癌A549细胞的迁移和侵袭[J].肿瘤, 2016,36(8):846-856. DOI:10.3781/j.issn.1000-7431.2016.11.319.
[14]杜恒,戴德坚,郭晓雷,等. HBX蛋白与M2巨噬细胞介导肝癌细胞上皮间质转化的实验研究[J].中华普通外科杂志, 2016, 31(6):497-500. DOI:10.3760/cma.j.issn.1007-631X.2016.06.018.
[15]杨闯,柯星,张淑平,等. THP-1源性巨噬细胞对卵巢癌SKOV3细胞Toll样受体mRNA表达的影响[J].临床检验杂志, 2014, 32(4):277-280. DOI:10.13602/j.cnki.jcls.2014.04.10.
[16]傅余相. M1型和M2型巨噬细胞对胃癌SGC-7901不同作用的实验研究[D].青岛大学, 2012.
[17]花晨. miR-16在M2型巨噬细胞选择性激活中的作用研究[D].扬州大学, 2014.
[18]郑海燕,王兴芬,潘彦珞,等.胃癌组织中MCP-1、MMP-9的表达与间质中TAMs浸润的关系[J].临床肿瘤学杂志, 2010, 15(3):218-221. DOI:10.3969/j.issn.1009-0460.2010.03.007.
[19]冯稳,陈奎生,于庆凯,等.肿瘤相关巨噬细胞、基质金属蛋白酶-9与食管鳞癌浸润、转移的关系[J].实用医学杂志, 2016, 32(2):235-238. DOI:10.3969/j.issn.1006-5725.2016.02.020.