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

肝细胞癌是一种富血供的肿瘤。在本研究中，我们研究了抗血管生成因子cDNA的基因治疗对肝癌是否有效，内皮抑素和Kringle 1 -5，这两种有效的抗血管生成因子，在体外抑制内皮祖细胞的增殖。用逆转录病毒转染系统将内皮抑素和kringle 1 -5基因转染骨髓细胞。Kringle 1 -5 cDNA成功转染。然而，内皮抑制素未被转染。然后，我们静脉注射脂质体kringle 1 -5 cDNA复合物在裸鼠体内植入肝癌细胞系，以研究肿瘤的生长，肝内转移和生存。kringle 1 -5基因转染抑制三种肝癌细胞系的肿瘤生长。kringle 1 -5基因转染可延长生存期，减少肝内转移灶数目。接下来，我们用kringle 1 -5 cDNA转染的骨髓细胞对裸鼠进行骨髓转染。然后，裸鼠接受肝癌细胞肝内移植。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。约20%的内皮祖细胞来源的血管内皮细胞产生Kringle 1-5蛋白。而骨髓转染Kringle 1 -5 cDNA后，肿瘤体积和肿瘤组织血管密度均未受到抑制，这可能是由于逆转录病毒系统的转染效率较低，导致Kringle 1 -5蛋白在肿瘤组织中的表达较弱，无法抑制肿瘤的发生发展。

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.