超级白细胞介素-6重组慢病毒的构建及其对肝细胞的促增殖作用

The influence of the recombinant lentiviral vectors encoding Hyper-IL-6 on the proliferation of human hepatic cell

目的观察超级白细胞介素-6重组慢病毒对人正常肝细胞L—02的促增殖作用。方法利用慢病毒表达质粒及其包装系统构建超级白细胞介素-6重组慢病毒（FIV—Hyper—IL-6）、IL-6重组慢病毒（FIV-IL-6）和空慢病毒载体（FIV），用嘌呤霉素筛选的方法测定其病毒滴度。将人肝细胞L02分为4组：FIV-Hyper—IL-6组、FIV-IL-6组、FIV组及未感染组，各组分别感染其相应的重组慢病毒，采用四甲基偶氮唑盐法检测其对L-02细胞生长状态的影响；并用RT—PCR法检测重组慢病毒感染L02细胞后产生的急性时相蛋白结合珠蛋白mRNA表达量的变化。应用方差分析进行统计学分析。结果构建出的各重组慢病毒能成功感染靶细胞，嘌呤霉素筛选法测得的各重组慢病毒的病毒滴度均为10^3pfu／ml。病毒颗粒感染L02细胞后48h，细胞的促增殖作用（4值）FIV—Hyper—IL-6组为0．6267±0．0256，FIV—IL-6组、FIV组及未感染组分别为0．5563±0．0112、0．5040±0．0078、0．4790±0．0201，F=41．09，P值均〈0．01，且随着时间的延长，对细胞的促增殖作用有所增加，并于感染后72h达到最高，为0．8000±0．0166。FIV—Hyper—IL-6组细胞结合珠蛋白mRNA吸光度值为0．7030±0．0106，FIV—IL-6组、FIV组及未感染组分别为0．3355±0．0093、0．1143±0．0153、0．1145±0．0076，q=57．5007，P值均〈0．01。结论构建的Hyper-IL-6重组慢病毒能够显著刺激L-02细胞表达结合珠蛋白，对L-02细胞有较强的促增殖作用。

Objective To investigate the effect of Hyper-IL-6 on the proliferation of L-02 cells. Methods The recombinant lentiviral vectors encoding Hyper-IL-6 （FIV-Hyper-IL-6）, IL-6 （FIV-IL-6） and the lentiviral （FIV） were prepared using the FIV-based lentiviral vectors and the packaging system. The titer of FIV-Hyper-IL-6, FIV-IL-6 and FIV was tested with puromycin. L-02 cells were infected with FIV-Hyper- IL-6, FIV-IL-6, FIV, or mock-infected. The growth rate of L02 cells was analyzed with MTT at different time points after infection. The changes of Haptoglobin in L-02 cells were analyzed with RT-PCR. Results FIV- Hyper-IL-6, FIV-IL-6 and FIV were successfully constructed, and the titer was 107 pfu/ml. 48 hours after infection, the absorbance of the cells infected with FIV-Hyper-IL-6 was 0.6267 ± 0.0256, and the absorbances of FIV-IL-6 infected cells, FIV infected cells and mock-infected cells were 0.5563 ±0.0112, 0.5040 ± 0.0078 and 0.4790 ±0.0201, respectively. There were significant differences between the FIV-Hyper-IL-6 group and the other groups （F = 41.09, P 〈 0.01）. The ratio of the absorbance between haptoglobin mRNA and beta-actin was 0.7030 ± 0.0106, 0.3355 ± 0.0093, 0.1145± 0.0076 and 0.1143 ±0.0153, respectively,in FIV-Hyper-IL-6 infected cells, FIV-IL-6 infected cells, FIV infected cells and mock-infected cells. There were significant differences between the FIV-Hyper-IL-6 group and the others （q = 57.5007, P 〈 0.01）. Condusion Compared with IL-6, Hyper-IL-6 is more potent to stimulate proliferation and induce the expression of Haptoglobin in L-02 cells.