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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,P<0.05。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,P<0.05。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,P<0.05。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,P<0.05;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,P<0.05。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。
Objective Clinically, many patients with malignant lymphoma are found to be infected with hepatitis B virus. The relationship between the two is gaining more and more attention. The purpose of this study was to investigate the effects of stable transfection of HBx gene on the proliferation and metastasis of lymphoma SUDHL-4 cells. Methods SUDHL-4-HBx was transfected into SUDHL-4 cells by Lipofectamine 2000 transfection method. SUDHL-4-HBx cell line stably expressing HBx was selected by G418. SUDHL-4 cells and SUDHL cells transfected with empty vector -4-con were used as control. Cell proliferation kit was detected by CCK8 at 24, 48, 72 and 96 hours. Flow cytometry was used to detect the cell cycle and apoptosis Cell adhesion was measured by MTT assay. Transwell assay was used to detect cell migration and invasion. Results HBx was transfected into SUDHL-4 cells, and the expression of HBx mRNA and protein was detected in the selected SUDHL-4-HBx cells. The results of cell proliferation assay showed that the absorbance (A) values at 24, 48, 72 and 96h were 0.36 ± 0.011, 0.76 ± 0.009, 1.55 ± 0.042 and 1.58 ± 0.057 respectively in SUDHL-4-HBx cell line stably. SUDHL- The A values of 24, 48, 72 and 96 h were 0.29 ± 0.003, 0.46 ± 0.126, 0.84 ± 0.026 and 0.90 ± 0.015, respectively. The A values at 24, 48, 72 and 96 h of SUDHL-4-con cells were 0.22 ± 0.001, 0.38 ± 0.008, 0.83 ± 0.035 and 1.01 ± 0.054. The proliferation of SUDHL-4-HBx cell line was significantly increased compared with SUDHL-4 cells and SUDHL-4-con cells after 48h treatment, and there was a synergistic interaction between HBx and time (P <0.05). No significant changes in cell cycle. The apoptotic rates in SUDHL-4, SUDHL-4-con and SUDHL-4-HBx groups were (14.9 ± 0.18)%, (10.1 ± 0.31)% and Apoptosis was significantly reduced compared with the other two groups was statistically significant, P <0.05. The adhesion of SUDHL-4, SUDHL-4-con and SUDHL-4-HBx groups were 0.70 ± 0.14, 0.63 ± 0.21 and 1.23 ± 0.43 respectively by modified MTT assay. Compared with the other two groups, the adhesion of SUDHL- The adhesion of 4-HBx group was significantly increased, P <0.05. The results of Transwell chamber showed that the numbers of migrating cells in SUDHL-4 and SUDHL-4-con groups were (58 ± 4) / 400 times and (56 ± 5) / 400 times, SUDHL-4-HBx group migrated The number of cells in the SUDHL-4, SUDHL-4-con and SUDHL-4-HBx groups was (64 ± SD), and the number of cells was (73 ± 5) / 400 times higher than that in the control group (P <0.05). The invasive capacity of SUDHL-4-HBx group was significantly higher than that of SUDHL-4-HBx group (P <0.05). Conclusion The stable expression of SUDHL-4-HBx and HBx in successfully constructed HBx-overexpressing HBx can inhibit the apoptosis of human lymphoma cell line SUDHL-4 and promote cell proliferation and cell adhesion, migration and invasion.