髓源抑制性细胞与肥大细胞向肿瘤趋化的分子机制与促瘤效应
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摘要
目的：炎症是肿瘤微环境的重要特征之一,在肿瘤组织中存在多种大量的炎性细胞,它们在肿瘤发生、生长、侵袭、转移与免疫抑制等过程中发挥了重要作用。本研究将探讨髓源抑制性细胞与肥大细胞向肿瘤组织趋化的分子机制以及其到达肿瘤部位后能够发挥促瘤效应。
方法：(1) RT-PCR与Western-Blot检测乳腺癌、胃癌与卵巢癌患者以及小鼠黑色素瘤与肝癌组织中趋化因子CCL2的表达,并检测相应的髓源抑制性细胞中CCL2受体CCR2的表达。(2) Transwell实验在体外检测肿瘤组织对相应髓源抑制性细胞的趋化作用。(3)流式细胞术检测CCR2基因敲除荷瘤小鼠肿瘤组织与脾脏中髓源抑制性细胞的比例。(4)肿瘤生长实验检测髓源抑制性细胞对小鼠体内肿瘤生长的影响。(5) RT-PCR与Western-Blot检测7种人类肿瘤细胞与肿瘤组织以及5种小鼠肿瘤细胞与肿瘤组织中SCF的表达。(6) Transwell实验与体内迁移实验检测肿瘤组织对肥大细胞的趋化作用。(7)肿瘤生长实验检测肥大细胞对小鼠体内肿瘤生长的影响。(8) Real-time PCR检测肥大细胞对肿瘤组织中炎性细胞因子表达的影响。(9)流式细胞术检测肥大细胞对肿瘤组织中Treg和Th17的影响。
结果：(1)乳腺癌、胃癌与卵巢癌患者以及小鼠黑色素瘤与肝癌组织中均有CCL2的表达,相应的髓源抑制性细胞有CCR2的表达。(2)髓源抑制性细胞可以被肿瘤组织所趋化,CCL2与CCR2的中和抗体可以抑制这种趋化作用。(3)CCR2基因敲除荷瘤小鼠肿瘤组织与脾脏中髓源抑制性细胞的比例降低。(4)CCR2基因敲除小鼠来源的髓源抑制性细胞对小鼠体内肿瘤生长的促进作用明显减弱。(5)被检测的7种人类肿瘤细胞与肿瘤组织以及5种小鼠肿瘤细胞与肿瘤组织中均有SCF的表达。(6)体内外肿瘤组织对肥大细胞均有趋化作用,SCF和c-Kit的中和抗体以及肿瘤细胞表达的SCF被siRNA沉默后这种趋化作用可以被抑制。(7)在小鼠体内肥大细胞可以促进肿瘤生长,并且可以被SCF和c-Kit的中和抗体以及SCF的siRNA所抑制。(8)肥大细胞可以促进肿瘤组织中IL-10与TGF-p的表达而抑制IL-2的表达。(9)肥大细胞可以使肿瘤组织中Treg和Thl7的数量增加。
结论：CCL2/CCR2通路介导髓源抑制性细胞向肿瘤组织趋化并促进肿瘤生长,SCF/c-Kit通路介导肥大细胞向肿瘤组织趋化并抑制免疫促进肿瘤生长。
Objective:Inflammation is an important feature of the tumor microenvironment, there are many kinds and a large number of inflammatory cells in the tumor tissue, they played an important role in tumorigenesis, tumor growth, invasion, metastasis and immune suppression. This study will to explore the molecular mechanisms of the myeloid-derived suppressor cells and mast cells chemotaxising to the tumor tissues and their pro-tumor effects.
Methods:(1) RT-PCR and Western-Blot assays to detect the expression of chemokine CCL2 in the tumor tissues from breast cancer, gastric cancer, ovarian cancer patients as well as mice melanoma and liver cancer tissues, at the same time to detect the expression of CCR2 which is the receptor of CCL2 in corresponding myeloid-derived suppressor cells. (2) Transwell experiment to detect the chemotaxis effects on myeloid-derived suppressor cells from the tumor tissues in vitro. (3) Flow cytometry assay to detect the ratio of myeloid-derived suppressor cells in the tumor tissues and spleen from CCR2 knockout tumor bearing mice. (4) Tumor growth assay to detect the influence of myeloid-derived suppressor cells on tumor growth in mice. (5) RT-PCR and Western-Blot assays to detect the expression of SCF in seven kinds of human tumor cell lines and tissues as well as five kinds of mouse tumor cell lines and tissues. (6) Transwell experiment and migration assay in vivo to detect the chemotaxis effects on mast cells from the tumor tissues. (7) Tumor growth assay to detect the influence of mast cells on tumor growth in mice. (8) Real-time PCR experiment to detect the influence of mast cells on the expression of inflammatory cytokines in tumor tissue. (9) Flow cytometry assay to detect the influence of mast cells on the number of Treg and Th17 in tumor tissue.
Results:(1) Tumor tissues from the breast cancer, gastric cancer, ovarian cancer patients as well as mice melanoma and liver cancer all expressed CCL2, at the same time the corresponding myeloid-derived suppressor cells expressed CCR2 which is the receptor of CCL2. (2) The myeloid-derived suppressor cells could be chemotaxised by tumor tissues, and this effect could be inhibited by CCL2 or CCR2 neutralizing antibody. (3) The ratio of myeloid-derived suppressor cells in tumor tissue and spleen from the CCR2 knockout tumor bearing mice reduced compared to the wide type tumor bearing mice. (4) The pro-tumor effect of the myeloid-derived suppressor cells from CCR2 knockout mice was significantly decreased. (5) The seven kinds of human tumor cell lines and tissues as well as five kinds of mouse tumor cell lines and tissues detected all expressed SCF. (6) The tumor tissues had the chemotactic effects on mast cells in vitro and in vivo, and this role could be inhibited by SCF or c-Kit neutralizing antibody or siRNA silenced the SCF expression in tumor cells. (7) Mast cells could promote tumor growth in vivo, and this effect could be inhibited by SCF or c-Kit neutralizing antibody or SCF siRNA. (8) Mast cells could promote the expression of IL-10 and TGF-βbut inhibit the expression of IL-2 in tumor tissue. (9) Mast cells could increase the number of Treg and Th17 in tumor tissue.
Conclusions:The CCL2/CCR2 pathway mediated the myeloid-derived suppressor cells chemotaxising to tumor tissues and promoted tumor growth, The SCF/c-Kit pathway mediated mast cells chemotaxising to tumor tissues, promoted tumor growth and immune suppression.
方法：(1) RT-PCR与Western-Blot检测乳腺癌、胃癌与卵巢癌患者以及小鼠黑色素瘤与肝癌组织中趋化因子CCL2的表达,并检测相应的髓源抑制性细胞中CCL2受体CCR2的表达。(2) Transwell实验在体外检测肿瘤组织对相应髓源抑制性细胞的趋化作用。(3)流式细胞术检测CCR2基因敲除荷瘤小鼠肿瘤组织与脾脏中髓源抑制性细胞的比例。(4)肿瘤生长实验检测髓源抑制性细胞对小鼠体内肿瘤生长的影响。(5) RT-PCR与Western-Blot检测7种人类肿瘤细胞与肿瘤组织以及5种小鼠肿瘤细胞与肿瘤组织中SCF的表达。(6) Transwell实验与体内迁移实验检测肿瘤组织对肥大细胞的趋化作用。(7)肿瘤生长实验检测肥大细胞对小鼠体内肿瘤生长的影响。(8) Real-time PCR检测肥大细胞对肿瘤组织中炎性细胞因子表达的影响。(9)流式细胞术检测肥大细胞对肿瘤组织中Treg和Th17的影响。
结果：(1)乳腺癌、胃癌与卵巢癌患者以及小鼠黑色素瘤与肝癌组织中均有CCL2的表达,相应的髓源抑制性细胞有CCR2的表达。(2)髓源抑制性细胞可以被肿瘤组织所趋化,CCL2与CCR2的中和抗体可以抑制这种趋化作用。(3)CCR2基因敲除荷瘤小鼠肿瘤组织与脾脏中髓源抑制性细胞的比例降低。(4)CCR2基因敲除小鼠来源的髓源抑制性细胞对小鼠体内肿瘤生长的促进作用明显减弱。(5)被检测的7种人类肿瘤细胞与肿瘤组织以及5种小鼠肿瘤细胞与肿瘤组织中均有SCF的表达。(6)体内外肿瘤组织对肥大细胞均有趋化作用,SCF和c-Kit的中和抗体以及肿瘤细胞表达的SCF被siRNA沉默后这种趋化作用可以被抑制。(7)在小鼠体内肥大细胞可以促进肿瘤生长,并且可以被SCF和c-Kit的中和抗体以及SCF的siRNA所抑制。(8)肥大细胞可以促进肿瘤组织中IL-10与TGF-p的表达而抑制IL-2的表达。(9)肥大细胞可以使肿瘤组织中Treg和Thl7的数量增加。
结论：CCL2/CCR2通路介导髓源抑制性细胞向肿瘤组织趋化并促进肿瘤生长,SCF/c-Kit通路介导肥大细胞向肿瘤组织趋化并抑制免疫促进肿瘤生长。
Objective:Inflammation is an important feature of the tumor microenvironment, there are many kinds and a large number of inflammatory cells in the tumor tissue, they played an important role in tumorigenesis, tumor growth, invasion, metastasis and immune suppression. This study will to explore the molecular mechanisms of the myeloid-derived suppressor cells and mast cells chemotaxising to the tumor tissues and their pro-tumor effects.
Methods:(1) RT-PCR and Western-Blot assays to detect the expression of chemokine CCL2 in the tumor tissues from breast cancer, gastric cancer, ovarian cancer patients as well as mice melanoma and liver cancer tissues, at the same time to detect the expression of CCR2 which is the receptor of CCL2 in corresponding myeloid-derived suppressor cells. (2) Transwell experiment to detect the chemotaxis effects on myeloid-derived suppressor cells from the tumor tissues in vitro. (3) Flow cytometry assay to detect the ratio of myeloid-derived suppressor cells in the tumor tissues and spleen from CCR2 knockout tumor bearing mice. (4) Tumor growth assay to detect the influence of myeloid-derived suppressor cells on tumor growth in mice. (5) RT-PCR and Western-Blot assays to detect the expression of SCF in seven kinds of human tumor cell lines and tissues as well as five kinds of mouse tumor cell lines and tissues. (6) Transwell experiment and migration assay in vivo to detect the chemotaxis effects on mast cells from the tumor tissues. (7) Tumor growth assay to detect the influence of mast cells on tumor growth in mice. (8) Real-time PCR experiment to detect the influence of mast cells on the expression of inflammatory cytokines in tumor tissue. (9) Flow cytometry assay to detect the influence of mast cells on the number of Treg and Th17 in tumor tissue.
Results:(1) Tumor tissues from the breast cancer, gastric cancer, ovarian cancer patients as well as mice melanoma and liver cancer all expressed CCL2, at the same time the corresponding myeloid-derived suppressor cells expressed CCR2 which is the receptor of CCL2. (2) The myeloid-derived suppressor cells could be chemotaxised by tumor tissues, and this effect could be inhibited by CCL2 or CCR2 neutralizing antibody. (3) The ratio of myeloid-derived suppressor cells in tumor tissue and spleen from the CCR2 knockout tumor bearing mice reduced compared to the wide type tumor bearing mice. (4) The pro-tumor effect of the myeloid-derived suppressor cells from CCR2 knockout mice was significantly decreased. (5) The seven kinds of human tumor cell lines and tissues as well as five kinds of mouse tumor cell lines and tissues detected all expressed SCF. (6) The tumor tissues had the chemotactic effects on mast cells in vitro and in vivo, and this role could be inhibited by SCF or c-Kit neutralizing antibody or siRNA silenced the SCF expression in tumor cells. (7) Mast cells could promote tumor growth in vivo, and this effect could be inhibited by SCF or c-Kit neutralizing antibody or SCF siRNA. (8) Mast cells could promote the expression of IL-10 and TGF-βbut inhibit the expression of IL-2 in tumor tissue. (9) Mast cells could increase the number of Treg and Th17 in tumor tissue.
Conclusions:The CCL2/CCR2 pathway mediated the myeloid-derived suppressor cells chemotaxising to tumor tissues and promoted tumor growth, The SCF/c-Kit pathway mediated mast cells chemotaxising to tumor tissues, promoted tumor growth and immune suppression.
引文
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