HBx基因稳定转染对SUDHL-4细胞增殖和转移影响

Effects of HBx gene stable transfection on proliferation and metastasis of SUDHL-4 cells

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 malignant [ymphoma patients are also infected with hepatitis B virus. The relationship between hepatitis B virus infection and malignant lymphoma is becoming a hot topic. Our experiment was to investigate the effects of HBx stable transfection on proliferation and metastasis in lymphoma line SUDHL-4.METHODS The reconstituted plasmid pcDNA3.1-x and empty vector pcDNA3.1 were transfected into SUDHL-4 cells by lipid-mediated transfection, which were named SUDHL-4 HBx and SUDHL-4-con,and the stable transfection cell line SUDHL-HBx was screened by G418. Untransfected SUDHL-4 and SUDHL-4-con were used as controls. Cell reproduc- tive ability was detected by CCK8 in 24, 48, 72 and 96 h. Cell cycle and cell apoptosis were detected by flow cytometer. The adhesion ability of the ceils were tested by modified MTT assay. The cells migration and invasion in vitro were detec- ted by Transwell assay. RESULTS The expressions of HBx mRNA and protein were comfirmed in the stable transfec- tion cell line SUDHL-4-HBx. The results showed that the optical absorbance of SUDHL-4-HBx group were 0. 36 ± 0. 011,0.76±0. 009,1. 55±0. 042 and 1.58±0. 057, the SUDHL-4 group were 0. 29±0. 003,0.46±0. 126,0.84±0. 026 and 0.90 ±0.015, the SUDHL-4-con group were 0.22 ±0.001,0.38±0. 008,0.83±0.035 and 1.01±0.054 in 24,48,72 and 96 h, respectively. Compared with SUDHL-4 and SUDHL-4-con group, the proliferative capacity of cells in SUDHL-4-HBx group was markedly enhanced, and there was a synergistic interaction between HBx and time （P〈0.05）. The cell cycle of SUDHL-4-HBx group did not changed significantly compared with the control group. The apop- toms rate of SUDHL-4, SUDHL-4-con and SUDHL-4-HBx group were （14.9±0.18） %, （10.1±0.31） % and （4.84±0.26） %, respectively. Compared with the control group, the apoptosis rate of SUDHL-4-HBx group was significantly decreased （P〈0. 05）. The modified MTT assay showed the adhesion abilities of SUDHL-4, SUDHL-4-con and SUDHL-4-HBx group respectively were 0.70±0.14, 0. 63±0.21 and 1. 23±0.43. Compared with the control group, the adhesion abilities of SUDHL-4-HBx group was significantly increased （P〈0.05）. Transwell assay showed that the numbers of migrating cells in SUDHL-4-HBx group was （73 ± 5） cells/400 X field, significantly higher than the （58 ± 4） ceils and （564-5） cells/400X field in control group （P〈0.05）. The numbers of invasion ceils of SUDHL 4, SUDHL-4- con and SUDHL-4-HBx group were （64±5） cells, （63±6） cells and （81±5） cells/400X field, respectively, showing the invasion of SUDHL-4-HBx group was significantly increased （P〈0.05）. CONCLUSION HBx stable transfection pro- motes SUDHL-4 cell proliferation, adhesion, migration and invasion.