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

肝细胞癌是一种高血管肿瘤。在本研究中，我们研究了抗血管生成因子cDNA基因治疗对肝细胞癌是否有效。内皮抑素和Kringle1-5是有效的抗血管生成因子，在体外抑制内皮祖细胞的增殖。我们用逆转录病毒转染系统将内皮抑素和kringle1-5 cdna转染骨髓细胞。成功转染Kringle1-5 cDNA。然而，内皮抑素未转染。然后静脉注射脂质体-kringle1-5 cDNA复合物于肝癌细胞系植入肝脏的裸鼠体内，研究肿瘤的生长、肝内转移和存活。kringle1-5基因转染对三种肝癌细胞系的肿瘤生长均有抑制作用。Kringle1-5基因的转移延长了生存期，减少了肝内转移的数量。接下来，我们用kringle1-5 cDNA转染的骨髓细胞转染裸鼠骨髓。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。约20%来源于内皮祖细胞的血管内皮细胞产生kringle 1-5蛋白。然而，kringle1-5 cDNA转染骨髓后，肿瘤组织的肿瘤体积和血管密度均未受到抑制。这些结果表明，kringle1-5蛋白在肿瘤组织中的表达过弱，无法抑制肿瘤的发展，这是由于逆转录病毒系统转染效率较低所致。

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.