本文關(guān)鍵詞： HBx基因 脂質(zhì)體轉(zhuǎn)染 淋巴瘤 腫瘤增殖 腫瘤轉(zhuǎn)移 腫瘤浸潤　出處：《中華腫瘤防治雜志》2017年15期 　論文類型：期刊論文

【摘要】：目的臨床上發(fā)現(xiàn)許多惡性淋巴瘤患者伴有乙型肝炎病毒的感染,兩者的關(guān)系日益受到關(guān)注。本研究旨在研究HBx基因穩(wěn)定轉(zhuǎn)染對淋巴瘤細(xì)胞SUDHL-4增殖和轉(zhuǎn)移的影響。方法利用脂質(zhì)體轉(zhuǎn)染法將pcDNA3.1-x轉(zhuǎn)入SUDHL-4細(xì)胞中,經(jīng)G418篩選出穩(wěn)定表達(dá)HBx的細(xì)胞株SUDHL-4-HBx,以SUDHL-4及轉(zhuǎn)染空載體的細(xì)胞SUDHL-4-con作對照,采用細(xì)胞計(jì)數(shù)盒8(cell counting kit 8,CCK8)法檢測各組細(xì)胞24、48、72和96h的增殖活性;利用流式細(xì)胞儀檢測3組細(xì)胞的細(xì)胞周期和凋亡變化情況;采用改良四甲基偶氮唑藍(lán)(methyl thiazolyl tetrazolium,MTT)法檢測細(xì)胞黏附力;用Transwell小室實(shí)驗(yàn)檢測細(xì)胞遷移和侵襲力。結(jié)果將HBx轉(zhuǎn)入SUDHL-4細(xì)胞中,經(jīng)篩選的SUDHL-4-HBx細(xì)胞有HBx mRNA及蛋白質(zhì)的表達(dá)。細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示,穩(wěn)定構(gòu)建的SUDHL-4-HBx細(xì)胞株24、48、72和96h吸光度(A)值分別為0.36±0.011、0.76±0.009、1.55±0.042和1.58±0.057,對照組SUDHL-4細(xì)胞24、48、72和96h的A值分別為0.29±0.003、0.46±0.126、0.84±0.026和0.90±0.015,SUDHL-4-con細(xì)胞24、48、72和96h的A值分別為0.22±0.001、0.38±0.008、0.83±0.035和1.01±0.054。穩(wěn)定構(gòu)建的SUDHL-4-HBx細(xì)胞株相比于對照組SUDHL-4細(xì)胞和SUDHL-4-con細(xì)胞48h后增殖顯著加快,HBx與時間之間存在協(xié)同的交互作用,P0.05。細(xì)胞周期無明顯變化。SUDHL-4、SUDHL-4-con和SUDHL-4-HBx組細(xì)胞的凋亡率分別為(14.9±0.18)%、(10.1±0.31)%和(4.8±0.26)%,SUDHL-4-HBx組細(xì)胞凋亡明顯減少,與其他兩組比較差異有統(tǒng)計(jì)學(xué)意義,P0.05。改良MTT法測得SUDHL-4組、SUDHL-4-con組和SUDHL-4-HBx組細(xì)胞的黏附力分別為0.70±0.14、0.63±0.21和1.23±0.43,與其他兩組相比,SUDHL-4-HBx組細(xì)胞的黏附力顯著增加,P0.05。Transwell小室實(shí)驗(yàn)結(jié)果顯示,SUDHL-4和SUDHL-4-con組遷移細(xì)胞數(shù)分別為(58±4)個/400倍視野和(56±5)個/400倍視野,SUDHL-4-HBx組遷移細(xì)胞數(shù)為(73±5)個/400倍視野,與對照組相比顯著增加,P0.05;SUDHL-4、SUDHL-4-con和SUDHL-4-HBx組侵襲細(xì)胞數(shù)分別為(64±5)個/400倍視野、(63±6)個/400倍視野和(81±5)個/400倍視野,SUDHL-4-HBx組細(xì)胞侵襲能力顯著增加,P0.05。結(jié)論成功構(gòu)建穩(wěn)定轉(zhuǎn)染HBx的淋巴瘤細(xì)胞SUDHL-4-HBx,HBx的穩(wěn)定表達(dá)可抑制人淋巴瘤細(xì)胞SUDHL-4的凋亡,促進(jìn)細(xì)胞增殖,提高細(xì)胞的黏附、遷移、侵襲能力。
[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.

【作者單位】： 河南省(鄭州大學(xué))醫(yī)藥科學(xué)研究院河南省肝病藥理重點(diǎn)實(shí)驗(yàn)室;鄭州大學(xué)藥學(xué)院;鄭州市中心醫(yī)院藥學(xué)部;
【基金】：河南省重點(diǎn)科技攻關(guān)計(jì)劃(142102310085)
【分類號】：R512.62;R733.1

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