T细胞免疫球蛋白与黏蛋白1在胶质母细胞瘤的表达及生物学作用

The expression and biological effect of T cell immunoglobulin and mucin 1 in glioblastoma

目的探讨人T细胞免疫球蛋白与黏蛋白1(Tim-1)在胶质母细胞瘤中的表达及对肿瘤细胞生物学功能的影响。方法通过组织芯片免疫组织化学、蛋白质印迹法(Western blot)检测Tim-1在胶质母细胞瘤组织(西安百思达生物科技有限公司,GL805a、GL805c)及细胞株(购自中国科学院上海细胞研究所)中的表达;通过RNA干扰(RNAi)技术构建下调Tim-1表达的胶质母细胞瘤细胞株,采用t检验比较Tim-1敲低组和Tim-1对照组细胞在增殖、侵袭、迁移上的差异。结果Tim-1在胶质母细胞瘤组织中的表达(142.769±37.552)高于正常脑组织(43.501±23.811);在胶质母细胞瘤细胞株(U87、U251)中的表达高于人脑正常胶质细胞株(HEB)(HEB:0.339±0.021,U87:0.919±0.066,t=21.910,P<0.01;U251:1.074±0.085,t=12.559,P<0.01);成功构建下调Tim-1的胶质母细胞瘤细胞株(sh-Tim-1-U87、sh-Tim-1-U251);细胞计数试剂盒(CCK-8)结果显示下调Tim-1后肿瘤细胞活力显著降低(24 h sh-NC:0.379±0.059,sh-U87:0.223±0.046,t=2.767,P<0.05;sh-U251:0.210±0.036,t=5.637,P<0.01。48 h sh-NC:0.677±0.034,sh-U87:0.444±0.052,t=4.968,P<0.01;sh-U251:0.441±0.056,t=8.831,P<0.01。72 h sh-NC:0.801±0.052,sh-U87:0.579±0.060,t=4.487,P<0.01;sh-U251:0.636±0.056,t=8.991,P<0.01);划痕实验结果表明下调Tim-1后肿瘤细胞迁移能力显著降低(sh-NC:74.667±4.510,sh-U87:44.333±4.509,t=11.651,P<0.01;sh-U251:34.003±3.606,t=18.401,P<0.01);Transwell实验显示下调Tim-1后肿瘤细胞侵袭能力显著降低(sh-NC:202.000±10.149,sh-U87:108.333±7.506,t=11.294,P<0.01;sh-U251:137.667±11.061,t=12.763,P<0.01)。结论Tim-1在胶母细胞瘤组织和细胞株中高表达,下调Tim-1可有效抑制胶质母细胞瘤细胞的增殖、迁移和侵袭能力。

Objective To investigate the expression of human T cell immunoglobulin and mucin 1(Tim-1)in glioblastoma and its effect on the biological function of tumor cells.Methods The expression of Tim-1 in glioblastoma tissues(Xi’an Best Biological Technology Co.,LTD.,GL805a,GL805c)and cell lines(purchased from Shanghai Institute of Cell Research,Chinese Academy of Sciences)was detected by immunohistochemistry and Western blot.Glioblastoma cell lines with down-regulated Tim-1 expression were constructed by RNA interference(RNAi),and t-test was used to compare the differences in cell proliferation,invasion and migration between the Tim-1 knockdown group and the Tim-1 control group.Results The expression of Tim-1 in glioblastoma tissues(142.769±37.552)was higher than that in normal brain tissues(43.501±23.811).The expression of glioblastoma cell lines(U87,U251)was higher than that of normal human glioblastoma cell lines(HEB:0.339±0.021,U87:0.919±0.066,t=21.910,P<0.01;U251:1.074±0.085,t=12.559,P<0.01);Glioblastoma cell lines(sh-Tim-1-U87,sh-Tim-1-U251)down-regulating Tim-1 were successfully constructed.Results of cell counting kit-8(CCK-8)showed that tumor cell activity was significantly decreased after Tim-1 was down-regulated(24 h sh-NC:0.379±0.059,sh-U87:0.223±0.046,t=2.767,P<0.05;sh-U251:0.210±0.036,t=5.637,P<0.01;48 h sh-NC:0.677±0.034,sh-U87:0.444±0.052,t=4.968,P<0.01;sh-U251:0.441±0.056,t=8.831,P<0.01.72 h sh-NC:0.801±0.052,sh-U87:0.579±0.060,t=4.487,P<0.01;sh-U251:0.636±0.056,t=8.991,P<0.01);The results of the scratch test showed that tumor cell migration ability was significantly reduced after down-regulation of Tim-1(sh-NC:74.667±4.510,sh-U87:44.333±4.509,t=11.651,P<0.01.sh-U251:34.003±3.606,t=18.401,P<0.01);Transwell assay showed that the invasion ability of tumor cells was significantly reduced after down-regulation of Tim-1(sh-NC:202.000±10.149,sh-U87:108.333±7.506,t=11.294,P<0.01;sh-U251:137.667±11.061,t=12.763,P<0.01).Conclusion Tim-1 is highly expressed in glioblastoma tissues and cell lines,and down-regulation of Tim-1 can effectively inhibit the proliferation,migration and invasion of glioblastoma cells.