可溶性转化生长因子β_1Ⅱ型受体逆转录病毒表达载体对人肝细胞凋亡的抑制作用

Effect of expression vector containing TβRⅡ-IgG1 Fc genes on human hepatocytes apoptosis

目的构建表达人转化生长因子-β1(TGF-β1)Ⅱ型受体细胞外结合区和人IgG1Fc的融合蛋白逆转录病毒载体,并研究重组质粒pL(TβRⅡ-IgG1Fc)SN是否拮抗外源性TGF-β1诱发的肝细胞凋亡,从而为该载体对于肝纤维化的临床应用提供理论依据。方法(1)以RT-PCR法扩增目的基因TβRⅡ-IgG1Fc,扩增产物纯化后克隆至测序载体pGEM-T-Easy,挑取阳性克隆酶切鉴定后测序;利用重组DNA技术,将TβRⅡ-IgG1Fc基因亚克隆至逆转录病毒载体pLXSN中,重组质粒pL(TβRⅡ-IgG1Fc)SN在脂质体介导下转染PA317包装细胞,G418筛选,直至出现抗性克隆,扩大培养,测定病毒滴度。(2)以人正常肝细胞HL-7702为靶细胞,将细胞分为空白对照组、TGF-β1组、载体组、TGF-β1+载体组。用TUNEL原位标记法和流式细胞仪对各组细胞凋亡率进行检测。结果(1)经测序、限制性酶切分析及PCR方法鉴定,载体插入基因序列、大小、位置均正确无误,并用PA317细胞进行包装、病毒滴度测定、筛选,建立具有较高滴度的感染性重组病毒产生细胞系。(2)TGF-β1组TUMEL阳性细胞明显多于其他各组,而TGF-β1+载体组和空白对照、载体组细胞相比,差异无统计学意义(P>0.05)。流式细胞仪分析结果显示,与其他各组细胞相比,TGF-β1组细胞碎片占细胞总数的百分比明显增加,而加入载体后细胞周期的比例、凋亡率和正常对照组相比没有明显变化。结论成功构建的重组质粒pL(TβRⅡ-IgG1Fc)SN可有效的阻断外源性TGF-β1诱导肝细胞凋亡的作用,提示该载体可望为肝纤维化的基因治疗提供有效途径。

Objective To construct recombinant human retroviral vector, which carries soluble TGFβ1 type Ⅱ receptor （TβR Ⅱ） gene and IgG Fc in vitro stud）, on its inhibiting effects of apoptosis on human hepatocytes and evaluate its anti-fibrotic effects. Methods The TβR Ⅱ -IgG1 Fc gene was amplified by RT-PCR and the purified PCR products was ligated into the sequencing vector pGEM-T-Easy. The positive clones were selected and the recombinant plasmid was purified, which was further identified by restriction endonucleases and sequenced . TβR Ⅱ-IgG1 Fc gene was subcloned into retroviral vector pLXSN by recombinant DNA technology, pL（ TβR Ⅱ-IgG1 Fc）SN was packaged with PA317 and screened by G418 to obtain the positive clones, which was able to produce retrovirus steadily. The integration and expression of TβR Ⅱ -IgG1 Fc gene in PA317 cells were identified by RT-PCR. Then we examined the anti-fibrotic effects of the retroviral vector on human hepatocyte line HL-7702. The cell apoptosis was determined by means of TUNEL and FCM. Results The retroviral vector pL（TβR Ⅱ-IgG1 Fc）SN was constructed successfully. PCR demonstrated that TβR Ⅱ-IgG1 Fc gene was integrated into the cells genome and expressed at the mRNA level. The ratio of apoptosis in HL-7702/TGF-β cells was higher than the HL-7702/ TβRⅡ-IgG1 Fc and HL-7702 cells in vitro. There was no obvious difference in the HL-7702/TβR Ⅱ-IgG1 Fc and HL-7702 groups. Conclusion TGFβ1 is a key mediator in establishing liver fibrosis. Therefore, TGFβ as a causative agent may serve as a primal- target for antifibrotic gene therapy approaches. We have shown that the retroviral delivery of pL（ TβR Ⅱ -IgG1 Fc）SN reduced the availability of active TGFβ1 in hepatocyte. It suggests that pL（TβR Ⅱ-IgG1 Fc）SN has certain reverse effects on liver fibrosis and can be used as a potantial candidate for gene therapy.