摘要
目的构建并观察靶向β-连环蛋白(β-catenin)的shRNA慢病毒对儿童髓母细胞瘤(MB)细胞株生物学行为的影响。方法 (1)设计靶向β-catenin的shRNA 1、2、3和阴性对照(NC),分别插入p CH-CMV-MCS-EF1-cop GFP质粒中,通过慢病毒包装后获得相应的重组慢病毒。(2)培养儿童MB细胞株DOAY并分为Lentivirus(LV)-shRNA 1、2、3、NC组和空白对照组(Blank),前4组感染相应的重组慢病毒,Blank组未感染病毒,感染72 h后在荧光显微镜下观察绿色荧光蛋白(GFP)表达,通过RT-PCR和Western Blot检测各组细胞中β-catenin的表达情况,选取对β-catenin抑制效果最佳的LVshRNA,用嘌呤霉素分别筛选稳定感染该LV-shRNA和LV-NC的DOAY细胞系。(3)设3组,LV-shRNA组和LV-NC组为筛选得到的稳定感染的细胞系,Blank组为未感染病毒的细胞,采用MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡情况,Transwell实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力。结果 (1)LV-shRNA 1、2、3组β-catenin的mRNA和蛋白表达均低于LV-NC组和Blank组,LV-shRNA 1组低于LV-shRNA 2、3组,差异均有统计学意义(P<0.05),以4μg·m L-1嘌呤霉素筛选得到稳定表达LV-shRNA 1和LV-NC的DOAY细胞株。(2)与LV-NC和Blank组比较,LV-shRNA1组MTT实验第12、24、36、48、72 h时的OD值较低,细胞总凋亡率较高,穿膜细胞数目较少,细胞迁移距离较短,差异均有统计学意义(P<0.05)。结论慢病毒介导shRNA抑制β-catenin后,能显著抑制DOAY细胞的增殖能力,降低其侵袭和迁移能力,促进其凋亡。
Objective To construct lentivirus-mediated shRNA targetingβ-catenin to infect DOAY cells,to observe the expression ofβ-catenin and the biological behavior of cells.Methods3kinds of shRNA targetingβ-catenin were designed and inserted into the pCH-CMV-MCS-EF1-copGFP,the recombinant pCHD-shRNA was obtained.The pCHD-shRNA was transfected into HEK293T cells,and the lentivirus-shRNA was obtained.DOAY cells were infected by lentivirus-shRNA and divided into5groups:Lentivirus-shRNA1group(LV-shRNA1),Lentivirus-shRNA2group(LV-shRNA2),Lentivirus-shRNA3group(LV-shRNA3),Lentivirus-NC group(LV-NC)and blank group(Blank).After72h,the expression of green fluorescent protein(GFP)was observed,the expression ofβ-catenin was detected by reverse transcription PCR and Western Blot,theβ-catenin gene stable suppression cells were screened out by puromycin.Effects of silencingβ-catenin expression on cell biological behavior were observed.There were3groups:LV-shRNA1,LV-NC and Blank.The proliferation and apoptosis of cells were detected by MTT assay and flow cytometry,the invasion and migration of the cells were detected by Transwell and cell scratch test.Results The lentivirus-mediated shRNA targetingβ-catenin was constructed successfully.72h after injection,the expression of GFP was not found in the blank group,the infection efficiency of LV-shRNA1,LV-shRNA2,LV-shRNA3and LV-NC was more than89%(P>0.05).The expression ofβ-catenin mRNA(0.24±0.09)and protein(0.15±0.07)in LV-shRNA1group was significantly lower than that in the other4groups,and the difference was statistically significant(P<0.05).Compared with LV-NC and blank group,the OD(570nm)values of LV-shRNA1group at24,36,48,72h were significantly decreased,the total apoptosis rate(31.28%)was significantly increased,the numbers of crossed cells(8.41±21.27)and migration distance(497.33±45.28)μm were significantly reduced,the difference was statistically significant(P<0.05).Conclusion Lentivirus-mediated shRNA targetingβ-catenin was successfully constructed.LV-shRNA1could signi
作者
万江
张海燕
刘义红
李新涛
涂胜英
吴丽敏
WAN Jiang;ZHANG Hai-yan;LIU Yi-hong;LI Xin-tao;TU Sheng-ying;WU Li-min(Department of Pediatrics, Xiaogan Hospital affiliated to Wuhan University of Science and Technology, Xiaogan 432000, China)
出处
《中国循证儿科杂志》
CSCD
北大核心
2017年第6期463-467,共5页
Chinese Journal of Evidence Based Pediatrics
