色素上皮衍生因子抑制人肝癌裸鼠移植瘤生长

Pigment epithelium-derived factor gene therapy inhibits the growth of transplanted human hepatocellular carcinoma in nude mice

目的观察人色素上皮衍生因子（PEDF）对血管生成的抑制作用，探讨PEDF在肝癌基因治疗中的应用前景。方法构建色素上皮衍生因子慢病毒表达质粒pLentiPEDF，经293T细胞包装，收集病毒上清液，感染肝癌细胞株HepG2。收集各组细胞的条件培养基，通过Western blot分析各组细胞PEDF的表达情况，并通过人脐静脉血管内皮细胞增殖及迁移试验研究其体外生物学活性。人肝癌细胞HepG2移植到裸鼠皮下，研究Lenti-PEDF病毒对肝癌移植瘤生长的抑制作用，逆转录聚合酶链反应检测肿瘤组织中PEDF mRNA表达。多组间均数比较采用单因素方差分析，组间两两比较采用LSD法。结果限制性内切酶酶切分析和测序结果均表明成功构建了PEDF慢病毒表达载体，以293T细胞包装的重组慢病毒能够高效感染肝癌细胞，肝癌细胞感染重组慢病毒后可高效表达PEDF并分泌到培养上清液中。体外研究结果表明，重组PEDF可明显抑制人脐静脉血管内皮细胞的增殖及迁移，抑制率分别达29％和48％，与空白对照组相比，差异有统计学意义（P值均〈0．01）。Lenti-PEDF病毒能明显抑制人肝癌裸鼠皮下移植瘤的生长（P〈0．01）。瘤内注射Lenti-PEDF病毒21d后，逆转录聚合酶链反应在肿瘤组织检测到PEDFmRNA的过表达。结论本研究初步提示PEDF能有效遏制肿瘤的血管生成和肿瘤生长，为肝癌的基因治疗提供了一条新的思路。

Objectives To evaluate the antiangiogenic property of pigment epithelium-derived factor （PEDF） in heptocarcinoma cell lines and explore its possible application in the gene therapy of human hepatocellular carcinoma （HCC）. Methods The gene encoding human PEDF was subcloned into lentiviral vector to generate the recombinant plasmid pLenti-PEDE The plasmid pLenti-PEDF and two other packaging plasmids were cotransfected to 293T cells by calcium phosphate. Then HepG2 was infected with recombinant lentivirns and the expression efficiency of PEDF was analyzed by western blot. Proliferation and migration assay of human umbilical vein endothelial cells （HUVEC） was used to evaluate the biological activity of PEDF in vitro. Murine subcutaneous tumor model was established to investigate the therapeutic effects of Lenti-PEDF on HCC, and the expression of PEDF mRNA in tumor tissues was analyzed by RT- PCR. Results Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plas- mid pLenti-PEDF was constructed successfully. HepG2 secreted PEDF in the media effectively after infected with the recombinant lentivirus and this protein exhibited strong inhibitory effects on proliferation and migration of human umbilical vein endothelial cells （P 〈 0.01）. Intratumoral injection of Lenti-PEDF caused significant inhibition of tumor growth （P 〈 0.01）, and high level expression of PEDF mRNA was detected in tumor tissues by RT-PCR. Conclusions Our data suggest that PEDF may exert an inhibitory effect on tumor angiogenesis and PEDF gene therapy may provide a new approach for the treatment of HCC.