Gene Transfer of Endostatin cDNA to Bone Marrow Cells of Mice with Hepatocellular Carcinoma

肝细胞癌是一种多血管肿瘤。在本研究中，我们探讨了抗血管生成因子基因治疗对肝细胞癌是否有效。Endostatin和Kringle1-5是两种有效的抗血管生成因子，在体外可以抑制内皮祖细胞的增殖。我们利用逆转录病毒感染系统将内皮抑素和Kringle1-5cDNA导入骨髓细胞。Kringle1-5基因已成功导入。但未见内皮抑素基因的表达。然后，我们将脂质体-kringle1-5cDNA复合体静脉注射到裸鼠体内，移植到肝癌细胞系的肝脏内，研究肿瘤的生长、肝内转移和生存。Kringle1-5基因转移抑制了三种肝癌细胞系的肿瘤生长。Kringle1-5基因转移可延长小鼠的生存期，减少肝内转移。接下来，我们用Kringle1-5基因转染的骨髓细胞对裸鼠进行了骨髓转染。然后，对裸鼠进行肝内肝癌细胞移植。骨髓转染4周后处死小鼠。在肿瘤组织中，约10%的血管内皮细胞由骨髓来源的内皮祖细胞组成。在来源于内皮祖细胞的血管内皮细胞中，约有20%的细胞表达Kringle1-5蛋白。但是，Kringle1-5基因在骨髓中的表达对肿瘤体积和肿瘤组织中的血管密度没有明显的抑制作用，这可能是由于逆转录病毒系统的转染率较低，导致Kringle1-5蛋白在肿瘤组织中的表达太弱，不能抑制肿瘤的发展。

Hepatocellular carcinoma is a hypervascular tumor. In the present study, we investigated whether gene therapy with anti-angiogenic factor cDNA could be effective or not for hepatocellular carcinoma.Endostatin and Kringle1-5, which were potent anti-angiogenic factors, suppressed proliferation of endothelial progenitor cells in vitro. We transfected endostatin and kringle1-5 cDNAs to bone marrow cells with retrovirus fransfection system. Kringle1-5 cDNA was successfully transfected. However, endostatin was not transfected. Then we injected intravenously liposome-kringle1-5 cDNA complexes in nude mice implanted into the liver with hepatoma cell lines to study tumor growth, intrahepatic metastasis and survival. Gene transfer of kringle1-5 suppressed tumor growth of the three hepatoma cell lines. kringle1-5 gene transfer prolonged the survival period, reduced the number of intrahepatic metastases. Next, we performed bone marrow transfection to nude mice with kringle1-5 cDNA transfected bone marrow cells. Then, the nude mice were received hepatoma cell transplantation in the liver. After 4 weeks of the bone marrow transfection, the mice were sacrificed. In tumor tissues, about 10% of vascular endothelial cells were consisted with bone marrow derived endothelial progenitor cells. About 20% of vascular endothelial cells derived from endothelial progenitor cells produced kringle 1-5 protein. However, tumor volume and vascular density in tumor tissues were not suppressed by bone marrow transfection with kringle1-5 cDNA at all.These results seems that the expression of kringle1-5 protein in tumor tissues was too weak to suppress the tumor development due to low transfection efficacy of retrovirus system.