趋化因子5介导胶质瘤相关间充质干细胞促肿瘤增殖和侵袭的机制

Chemokine ligand 5 mediates the mechanism of glioma-associated mesenchymal stem cells promoting tumor proliferation and invasion

目的探讨趋化因子配体5(CCL5)及受体在脑胶质瘤微环境中的表达,及其介导脑胶质瘤相关间充质干细胞(gaMSCs)促肿瘤细胞的增殖和侵袭机制。方法提取并培养gbMSCs(取自15例2017年1月至10月华中科技大学同济医学院附属协和医院神经外科手术切除人脑胶质瘤新鲜肿瘤组织标本,WHOⅣ)。使用实时荧光定量聚合酶链反应法(qPCR)检测U87、gbMSCs、BM-MSC和4例外伤脑组织中CCL5表达。使用蛋白质印迹法(Western blot)法、qPCR和免疫细胞染色检测脑胶质瘤细胞株U87和GBM细胞中CCL5受体CCR1、CCR3、CCR5的表达。使用细胞计数法、细胞增殖/毒性检测试剂盒-8(CCK-8)实验、磺酰罗丹明B比色(SRB)法和3D肿瘤球体细胞侵袭实验检测gaMSCs对U87和GBM的增殖和侵袭作用。使用细胞计数法、磺酰罗丹明B比色(SRB)法和3D肿瘤球体细胞侵袭实验检测使用不同浓度CCL5及其抗体对gaMSCs促脑胶质瘤细胞增殖和侵袭的具体机制。多组的比较采用单因素方差分析,进一步实验组或对照组两两比较采用LSD-t检验,Western blot实验结果采用t检验。结果gaMSCs可促进脑胶质瘤细胞增殖和侵袭,CCK-8法检测gaMSC1,gaMSC2组及Control组培养胶质瘤细胞U87增殖能力,细胞活性为1.320±0.063、1.250±0.087和0.700±0.012(F=42.270,P<0.05)。3D肿瘤球体细胞侵袭实验检测U87在Control、gaMSCs1、gaMSCs2组的细胞侵袭性定量分别为10.23±0.64、29.25±0.87和24.79±0.92(F=37.610,P<0.05)。gaMSCs和骨髓间充质干细胞(BMSCs)一样高表达CCL5,qPCR检测CCL5 mRNA在BMSCs和gaMSCs1、gaMSCs2、gaMSCs3中的表达,分别为0.62±0.05、0.61±0.03、0.62±0.04、0.59±0.07(F=5.290,P>0.05)。进一步比较GBM中和Control外伤脑组织的CCL5 mRNA表达,分别为0.55±0.03、0.22±0.05,两两比较,差异有统计学意义(F=34.750,P<0.05)。脑胶质瘤组织中持续表达CCR3(14/15的阳性表达),部分表达CCR5(11/15的阳性表达),而CCR1几乎不表达(0/15的阳性表达),差异有统计学意义(F=86.010,P<0.05)。CCL5 Ab可以明显抑制gaMSCs条件培养液促胶质瘤细胞的增殖和侵袭的能力,不同浓度CCL5(20μg/L和50μg/L)均能促U87更快的增殖和侵袭。SRB实验:U87细胞的Control组、gaMSCs组和gaMSCs+CCL5 Ab(20μg/L)组细胞存活率分别为0.480±0.030、0.831±0.010和0.571±0.160,比较与对照组,CCL5Ab(20μg/L)明显抑制gaMSCs条件培养液促U87细胞的增殖,差异有统计学意义(F=57.930,P<0.05)。含0、25、50μg/L CCL5的无血清培养液,培养U87细胞4 d后,平均细胞计数分别为18.1×10^4个/孔、21.9×10^4个/孔、26.6×10^4个/孔,细胞增殖数依次增高(F=42.830,P<0.05)。3D肿瘤球体细胞侵袭实验:U87细胞球在Control、gaMSCs、gaMSCs+CCL5 Ab(25μg/L)培养液,细胞侵袭性定量分别为10.53±0.04、26.78±0.61和11.37±0.23,差异有统计学意义(F=54.270,P<0.05)。gaMSCs条件培养液中加入CCL5 Ab(25μg/L)明显抑制gaMSCs条件培养液促U87细胞侵袭能力;U87细胞球在含0、10、20μg/L CCL5的无血清培养液中,细胞侵袭性定量分别为9.97±0.82、15.25±0.43和24.52±0.21,细胞球侵袭面积依次增大,两两比较,差异有统计学意义(F=74.360,P<0.05)。结论脑胶质瘤相关间充质干细胞促进脑胶质瘤增殖和侵袭,CCL5在该过程起关键作用。

Objective To investigate the expression of CC chemokine ligand 5(CCL5)and its receptors in the glioma microenvironment,and the mechanism of glioblastoma-associated mesenchymal stem cells(gaMSCs)regulating the proliferation and invasion of glioma cells.Methods GaMSCs were extracted and cultured(those were obtained from 15 fresh tumor samples of human glioma resected by Department of Neurosurgery,Union Hospital,Tongji Medical College from January to October 2017,WHOⅣ).Real-time Quantitative polymerase chain reaction(qPCR)was performed to detect the expression of CCL5 in U87 cells,gbMSCs,BM-MSCs and 4 cases of traumatic brain tissues.Western blotting,qPCR and immunohistochemical staining were used to detect the expression of CCL5 receptors(CCR)1/CCR3/CCR5 in U87 and GBM cells.Cell counting,Cell Counting Kit-8(CCK-8),sulforhodamine B(SRB)assay,and 3D Tumor Spheroid Invasion Assay were conducted to detect the effects of gaMSCs on the proliferation and invasion of U87 and GBM cells.Cell counting,SRB assay,and 3D Tumor Spheroid Invasion Assay were conducted to detect the specific mechanism of different concentrations of CCL5 and CCL5 antibody on the proliferation and invasion of glioma cells induced by gaMSCs.Single factor analysis of variance was used for the comparison of multiple groups.LSD-t test was used for the further comparison between the experimental group and the Control group,and t-test was used for the results of Western blotting.Results GaMSCs could promote the proliferation and invasion of glioma cells.For CCK-8 assay,the proliferation ability of U87 glioma cells in gaMSC1,gaMSC2 groups and Control group was detected:the cell activity was 1.320±0.063,1.250±0.087 and 0.700±0.012 respectively(F=42.270,P<0.05).For 3D Tumor Spheroid Invasion Assay,the quantitative invasiveness of U87 cells in Control,gaMSCs1 and gaMSCs2 groups was 10.23±0.64,29.25±0.87 and 24.79±0.92 respectively(F=37.610,P<0.05).The expression of CCL5 in gaMSCs was as high as that in bone marrow mesenchymal stem cells(BMSCs).For qPCR detection,the expression of CCL5 mRNA in BMSCs and gaMSCs1,gaMSCs2,gaMSCs3 was 0.62±0.05,0.61±0.03,0.62±0.04 and 0.59±0.07 respectively(F=5.290,P>0.05).Furthermore,the expression of CCL5 mRNA in traumatic brain tissue of GBM and Control groups was 0.55±0.03 and 0.22±0.05 respectively(F=34.750,P<0.05).In gliomas,CCR3 was continuously expressed(positive expression of 14/15),CCR5 was partially expressed(positive expression of 11/15),but almost no expression of CCR1 was detected(positive expression of 0/15)(F=86.010,P<0.05).Different concentrations of CCL5(20μg/L and 50μg/L)could promote the proliferation and invasion of U87 faster,but CCL5 antibody(20μg/L,25μg/L/CCL5 antibody)can significantly inhibit the proliferation and invasion of glioma cells stimulated by gaMSCs conditioned medium.For SRB assay,the cell survival rates of U87 cells in Control group,gaMSCs group and gaMSCs+CCL5 antibody(20μg/L)group were 0.48±0.03,0.831±0.01 and 0.57±0.16 respectively.As compared with the Control group,CCL5 antibody(20μg/L)significantly inhibited the proliferation of U87 cells stimulated by gaMSCs conditioned medium(F=57.930,P<0.05).For cell counting,after 4 days of culture in serum-free medium containing 0,25 and 50μg/L CCL5,the average cell count of U87 cells was 18.9×10^4/well,21.1×10^4/well and 26.6×10^4/well respectively,and the number of proliferative cells increased successively(F=42.830,P<0.05).For 3D Tumor Spheroid Invasion Assay,the quantitative invasiveness of U87 cell spheres in Control,gaMSCs and gaMSCs+CCL5 antibody(25μg/L)groups was 10.53±0.04,26.78±0.61 and 11.37±0.23 respectively(F=54.270,P<0.05).The addition of CCL5 antibody(25μg/L)significantly inhibited the invasion of U87 cells in gaMSCs conditioned medium.The invasiveness of U87 cell spheres in serum-free medium containing 0,10 and 20μg/L CCL5 was 9.97±0.82,15.25±0.43 and 24.52±0.21 respectively(F=74.360,P<0.05).Conclusion GaMSCs can promote the proliferation and invasion of glioma cells,and CCL5 plays a key role in this process.