慢病毒介导高表达OTUD7B对乳腺癌MCF-7细胞生物学行为影响

发布时间：2018-02-04 22:52

本文关键词： MCF-乳腺癌细胞 OTUDB 慢病毒感染增殖迁移　出处：《中华肿瘤防治杂志》2017年09期 　论文类型：期刊论文

【摘要】：目的去泛素化酶OTUD7B与肿瘤的发生、发展密切相关。为了明确OTUD7B在乳腺癌中所发挥的作用,实验利用慢病毒构建高表达OTUD7B载体感染MCF-7乳腺癌细胞后对其生物学行为的影响。方法构建带有绿色荧光蛋白标签的人OTUD7B表达质粒的慢病毒(pEGFP-hOTUD7B)及对照(pEGFP-CI)感染MCF-7乳腺癌细胞;于荧光倒置显微镜下观察病毒转染效果及应用蛋白质印迹法及免疫组化法检测OTUD7B的表达水平;MTS法检测实验组(pEGFP-hOTUD7B)、阴性对照组(pEGFP-CI)和正常对照组对MCF-7乳腺癌细胞增殖能力的影响;应用细胞划痕实验检测细胞迁移能力;流式细胞仪检测细胞凋亡。结果感染病毒后于荧光倒置显微镜下观察病毒感染效率,可见病毒感染成功。应用蛋白质印迹法检测病毒感染率并找出最适病毒感染复数(multiplicity of infection,MOI),当MOI=30时,实验组、阴性对照组和正常对照组灰度值分别为3.81±0.08、2.12±0.078和2.05±0.15,差异有统计学意义,F=402.03,P0.001。应用免疫组化法可见感染OTUD7B表达水平。MTS法结果显示,实验组、阴性对照组和正常对照组细胞24hA值分别为0.36±0.08、0.56±0.25和0.69±0.17,F=11.819,P0.001;48hA值分别为0.65±0.17、1.45±0.48和1.82±0.63,F=23.752,P0.001;在72hA值分别为0.73±0.21、1.58±0.63和1.99±0.27,F=35.563,P0.001。细胞划痕试验显示,24h后实验组组迁移率为(7.7±0.91)%,阴性对照组和正常对照组迁移率分别为(13.4±1.52)%和(12.1±1.32)%,F=49.36,P0.001,48h后实验组迁移率为(12.4±1.29)%,阴性对照组及正常对照组迁移率分别为(32.9±1.71)%和(31.8±1.59)%,F=504.50,P0.001。流式细胞仪检测细胞凋亡结果提示,实验组与阴性对照组及正常对照组相比明显使细胞阻滞在G_0/G_1期,促进其凋亡,F_(G_0/G_1)期=425.102,F_S期=135.063,均P0.001。结论成功构建了能够高表达OTUD7B的慢病毒载体,明显抑制了MCF-7乳腺癌细胞增殖、迁移,并促进了其凋亡。
[Abstract]:Objective to study the role of OTUD7B in breast cancer, which is closely related to the occurrence and development of tumor. The effects of lentivirus on the biological behavior of MCF-7 breast cancer cells infected with high expression OTUD7B vector were studied. Methods Human OTUD7B expression plasmid with green fluorescent protein label was constructed. Lentivirus. MCF-7 breast cancer cells were infected with pEGFP-hOTUD7B and the control group pEGFP-CI. The effect of virus transfection was observed under fluorescence inverted microscope and the expression of OTUD7B was detected by Western blotting and immunohistochemistry. The effects of pEGFP-hOTUD7BX on the proliferation of MCF-7 breast cancer cells were detected by MTS assay. Cell migration ability was detected by cell scratch assay. Results the efficiency of virus infection was observed under fluorescence inverted microscope. The virus infection rate was detected by Western blotting method and the optimum number of viral infection was identified by multiplicity of infective moi). When MOI = 30, the gray values of the experimental group, the negative control group and the normal control group were 3.81 卤0.08, 2.12 卤0.078 and 2.05 卤0.15, respectively. The expression level of OTUD7B was detected by immunohistochemical method. The 24h A values of the negative control group and the normal control group were 0.36 卤0.08 卤0.56 卤0.25 and 0.69 卤0.17 respectively. The 48 h A values were 0.65 卤0.17, 1.45 卤0.48 and 1.82 卤0.63, respectively (P 0.001). At 72 h, the values of A were 0.73 卤0.21 卤1.58 卤0.63 and 1.99 卤0.27 respectively. After 24 hours, the migration rate of the experimental group was 7.7 卤0.91g, that of the negative control group and the normal control group was 13.4 卤1.52% and 12.1 卤1.32%, respectively. After 48 hours, the migration rate of the experimental group was 12.4 卤1.29%. The migration rates of negative control group and normal control group were 32.9 卤1.71% and 31.8 卤1.59 respectively. P0.001.The results of flow cytometry showed that compared with the negative control group and the normal control group, the cells in the experimental group were significantly blocked in the G _ 0 / G _ 1 phase, promoting its apoptosis. Conclusion the lentivirus vector with high expression of OTUD7B was successfully constructed in the number of cases (425.102FS, 135.063, P0.001.Conclusion Lentivirus vectors with high expression of OTUD7B were successfully constructed. The proliferation, migration and apoptosis of MCF-7 breast cancer cells were significantly inhibited.
【作者单位】：河北大学医学院;河北大学附属医院中心实验室;河北大学附属医院乳腺外科;
【分类号】：R737.9
【正文快照】： 中华肿瘤防治杂志,2017,24(9):596-601Chin J Cancer Prev Treat,2017,24(9):596-601去泛素化酶与肿瘤发生、发展密切相关,其中去泛素化酶OTUD7B属于去泛素化酶家族成员之一。研究发现,OTUD7B被沉默后,肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated f

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