HBx基因稳定转染对SUDHL-4细胞增殖和转移影响

发布时间：2018-02-22 04:01

本文关键词： HBx基因脂质体转染淋巴瘤肿瘤增殖肿瘤转移肿瘤浸润　出处：《中华肿瘤防治杂志》2017年15期 　论文类型：期刊论文

【摘要】：目的临床上发现许多恶性淋巴瘤患者伴有乙型肝炎病毒的感染,两者的关系日益受到关注。本研究旨在研究HBx基因稳定转染对淋巴瘤细胞SUDHL-4增殖和转移的影响。方法利用脂质体转染法将pcDNA3.1-x转入SUDHL-4细胞中,经G418筛选出稳定表达HBx的细胞株SUDHL-4-HBx,以SUDHL-4及转染空载体的细胞SUDHL-4-con作对照,采用细胞计数盒8(cell counting kit 8,CCK8)法检测各组细胞24、48、72和96h的增殖活性;利用流式细胞仪检测3组细胞的细胞周期和凋亡变化情况;采用改良四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞黏附力;用Transwell小室实验检测细胞迁移和侵袭力。结果将HBx转入SUDHL-4细胞中,经筛选的SUDHL-4-HBx细胞有HBx mRNA及蛋白质的表达。细胞增殖实验结果显示,稳定构建的SUDHL-4-HBx细胞株24、48、72和96h吸光度(A)值分别为0.36±0.011、0.76±0.009、1.55±0.042和1.58±0.057,对照组SUDHL-4细胞24、48、72和96h的A值分别为0.29±0.003、0.46±0.126、0.84±0.026和0.90±0.015,SUDHL-4-con细胞24、48、72和96h的A值分别为0.22±0.001、0.38±0.008、0.83±0.035和1.01±0.054。稳定构建的SUDHL-4-HBx细胞株相比于对照组SUDHL-4细胞和SUDHL-4-con细胞48h后增殖显著加快,HBx与时间之间存在协同的交互作用,P0.05。细胞周期无明显变化。SUDHL-4、SUDHL-4-con和SUDHL-4-HBx组细胞的凋亡率分别为(14.9±0.18)%、(10.1±0.31)%和(4.8±0.26)%,SUDHL-4-HBx组细胞凋亡明显减少,与其他两组比较差异有统计学意义,P0.05。改良MTT法测得SUDHL-4组、SUDHL-4-con组和SUDHL-4-HBx组细胞的黏附力分别为0.70±0.14、0.63±0.21和1.23±0.43,与其他两组相比,SUDHL-4-HBx组细胞的黏附力显著增加,P0.05。Transwell小室实验结果显示,SUDHL-4和SUDHL-4-con组迁移细胞数分别为(58±4)个/400倍视野和(56±5)个/400倍视野,SUDHL-4-HBx组迁移细胞数为(73±5)个/400倍视野,与对照组相比显著增加,P0.05;SUDHL-4、SUDHL-4-con和SUDHL-4-HBx组侵袭细胞数分别为(64±5)个/400倍视野、(63±6)个/400倍视野和(81±5)个/400倍视野,SUDHL-4-HBx组细胞侵袭能力显著增加,P0.05。结论成功构建稳定转染HBx的淋巴瘤细胞SUDHL-4-HBx,HBx的稳定表达可抑制人淋巴瘤细胞SUDHL-4的凋亡,促进细胞增殖,提高细胞的黏附、迁移、侵袭能力。
[Abstract]:A number of malignant lymphoma patients with hepatitis B virus infection found on clinic, the relationship between the two is paid more and more attention. This research aims to study the effects of HBx gene transfection on proliferation and metastasis of lymphoma SUDHL-4 cells. Methods the liposome is used to transfect pcDNA3.1-x into SUDHL-4 cells and screened with G418 stable expression HBx cell line SUDHL-4-HBx based on SUDHL-4, and empty vector transfected SUDHL-4-con cells as control, using cell 8 (cell counting 8 kit, CCK8) were detected by 24,48,72 and 96h cell proliferation; cell cycle and apoptosis by flow cytometry in 3 groups of cells; the modified four methyl thiazolyl tetrazolium (methyl thiazolyl tetrazolium, MTT) were detected by cell adhesion assay; Transwell cell migration and invasion. The results will be HBx into SUDHL-4 cells, the sieve The expression of HBx mRNA and protein in SUDHL-4-HBx cells. The cell proliferation experiment results show that the stability of the constructed SUDHL-4-HBx cell line 24,48,72 and 96h absorbance (A) = 0.36 + 0.011,0.76 + 0.009,1.55 + 0.042 and 1.58 + 0.057, the control group of SUDHL-4 cells 24,48,72 and 96h: A = 0.29 + 0.003,0.46 + 0.126,0.84 + 0.026 and 0.90 + 0.015, SUDHL-4-con 24,48,72 and 96h A cells were SUDHL-4-HBx cells 0.22 + 0.001,0.38 + 0.008,0.83 + 0.035 and 1.01 + 0.054. stable construct compared with the control group, SUDHL-4 cells and SUDHL-4-con cells 48h proliferation significantly accelerated, there is interaction between HBx and collaborative time, no significant changes in cell cycle of P0.05. cells.SUDHL-4, SUDHL-4-con and apoptosis cells in SUDHL-4-HBx group were (14.9 + 0.18)% and (10.1 + 0.31)% and (4.8 + 0.26)%, SUDHL-4-HBx group cell apoptosis was significantly reduced, and There is statistical significance difference between the two groups, SUDHL-4 group P0.05. improved MTT method, the adhesion of SUDHL-4-con and SUDHL-4-HBx groups were 0.70 + 0.14,0.63 + 0.21 and 1.23 + 0.43, compared with the other two groups, adhesion of cells in SUDHL-4-HBx group increased significantly, P0.05.Transwell chamber experiment results show that the SUDHL-4 and SUDHL-4-con group the number of migrated cells were (58 + 4) /400 double vision and (56 + 5) /400 double vision, SUDHL-4-HBx group migration cell number was (73 + 5) /400 power field, compared with the control group increased significantly, P0.05; SUDHL-4, SUDHL-4-con and SUDHL-4-HBx group respectively (64 invasive cell number. 5) a /400 magnification, (63 + 6) /400 double vision and (81 + 5) /400 power field, the invasion ability of SUDHL-4-HBx cells was significantly increased in P0.05. group. Conclusion the successful construction of stably transfected HBx lymphoma cell SUDHL-4-HBx, the expression of HBx can inhibit The apoptosis of lymphoma cell SUDHL-4 promotes cell proliferation and increases cell adhesion, migration and invasion.

【作者单位】：河南省(郑州大学)医药科学研究院河南省肝病药理重点实验室;郑州大学药学院;郑州市中心医院药学部;
【基金】：河南省重点科技攻关计划(142102310085)
【分类号】：R512.62;R733.1

【相似文献】