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目的:探讨在慢病毒构建的稳定表达酪氨酸相关激酶(fyn-related kinase,FRK)的细胞系中,过表达FRK对人脑胶质瘤细胞增殖、侵袭及迁移能力的影响。方法:1.将FRK慢病毒质粒与辅助质粒共转染293T细胞进行慢病毒包装,然后用包装好的慢病毒感染胶质瘤U251、U87细胞,建立稳定表达FRK的细胞系,同时运用Western blotting(WB)技术检测FRK在U251、U87细胞系中的过表达情况;2.细胞划痕实验与Transwell迁移实验检测过表达FRK对脑胶质瘤细胞迁移能力的影响;3Transwell侵袭实验检测过表达FRK对脑胶质瘤细胞侵袭能力的影响;4.Ed U实验与平板集落形成实验检测过表达FRK对脑胶质瘤细胞增殖能力的影响。结果:1.荧光显微镜下可见慢病毒感染U251、U87细胞的效率达到90%以上,WB结果显示外源性FRK蛋白在U251、U87细胞系中充分表达;2.细胞划痕实验显示:与空载体组相比,过表达FRK组U251细胞迁移的细胞数在24 h和48 h分别减少了(50.00±1.04)%和(44.14±7.05)%,差异有统计学意义(P<0.01);而过表达FRK组U87细胞迁移的细胞数在24 h和48 h分别减少了(58.50±4.12)%和(44.67±3.82)%(P<0.001);3.Transwell迁移实验显示:与空载体组相比,过表达FRK组U251、U87细胞穿过滤膜的细胞数分别减少了(67.76±4.88)%和(50.76±4.54)%(P<0.001);4.Transwell侵袭实验显示:与空载体组相比,过表达FRK组U251、U87细胞穿过Matrigel基质胶及滤膜的细胞数分别减少了(50.94±4.83)%和(57.92±6.19)%(P<0.001);5.Ed U实验显示:与空载体组相比,过表达FRK组U251、U87细胞的Ed U阳性细胞率分别下降了(28.06±4.31)%和(33.51±7.26)%(P<0.01);6.平板集落形成实验结果显示:与空载体组相比,过表达FRK组U251、U87细胞集落形成分别减少了(35.94±6.76)%与(52.33±7.42)%(P<0.01)。结论:过表达FRK可以抑制脑胶质瘤细胞的增殖,并且可以抑制胶质瘤细胞的侵袭和迁移能力。
OBJECTIVE: To investigate the effect of overexpression of FRK on the proliferation, invasion and migration of human glioma cells in lentivirus-stable cell lines stably expressing the tyrosine-related kinase (FRK). 293 cells were cotransfected with lentivirus and co-transfected with FRK lentiviral plasmid and helper plasmids, and then glioma U251 and U87 cells were infected with the packaged lentivirus to establish a cell line stably expressing FRK. Western blotting (WB) was used to detect the overexpression of FRK in U251 and U87 cell lines.2. The cell scratch assay and Transwell migration assay were used to detect the effect of overexpression FRK on the migration ability of glioma cells; 3Transwell invasion assay was used to detect the expression of FRK On glioma cell invasion ability; 4.EdU experiment and colony forming assay to detect the effect of overexpression FRK on the proliferation of glioma cells. Fluorescence microscopy showed that the efficiency of lentivirus-infected U251 and U87 cells was over 90%. The results of WB showed that exogenous FRK protein was fully expressed in U251 and U87 cell lines.2. Compared with the vector group, the numbers of cells migrating in U251 cells over-expressing FRK group decreased by (50.00 ± 1.04)% and (44.14 ± 7.05)% at 24 h and 48 h, respectively, with statistical significance (P <0.01) The number of U87 cells in FRK group decreased by 58.50 ± 4.12% and 44.67 ± 3.82%, respectively (P <0.001) at 24 h and 48 h after transfection; 3.Transwell migration assay showed that compared with blank vector group (67.76 ± 4.88)% and (50.76 ± 4.54)%, respectively (P <0.001). The results of Transwell invasion assay showed that the number of U251 and U87 cells that were overexpressed in FRK group decreased significantly (50.94 ± 4.83)% and (57.92 ± 6.19)%, respectively (P <0.001). The number of cells in U251 and U87 cells that were overexpressed in FRK group decreased by 50.94 ± 4.83% Compared with the blank vector group, the positive rate of Ed U-positive cells in U251 and U87 cells decreased by (28.06 ± 4.31)% and (33.51 ± 7.26)%, respectively (P <0.01) Display: empty (35.94 ± 6.76)% and (52.33 ± 7.42)% (P <0.01), respectively, in U251 and U87 cells overexpressing FRK group. Conclusion: Overexpression of FRK can inhibit the proliferation of glioma cells and inhibit the invasion and migration of glioma cells.