Construction of gene targeting animal models for evaluating preventive and therapeutic anti-cancer effectiveness of active vitamin D analogues

我们建立了一个缺乏编码维生素D -羟化酶基因的动物模型（1α oase -/-），维生素D -羟化酶是激活维生素D的关键酶。表型分析显示，与维生素D受体基因敲除小鼠（VDR-/-）相似，动物表现出生长迟缓、软骨增生、低钙血症、甲状旁腺功能亢进和骨形成受损。由于这些异常主要是由钙营养不良引起的，因此通过喂食高钙/高乳糖饮食，动物几乎完全获救。我们目前正在尝试生成(1α oase -/-)/(VDR-/-)双敲除小鼠，以阐明维生素D在骨形成和软骨生长方面的生理功能。除了上述突变动物外，我们还克隆了一个稳定的人成骨细胞MG-63 VDR敲低细胞，结果表明，与正常表达VDR的细胞相比，VDR敲低细胞的增殖和分化速度更快、更显著，更说明VDR在成骨细胞的增殖和分化中起负调节作用。与母细胞（正常MG-63细胞）相比，VDR敲除细胞表现出更快、更强的矿化。我们目前正在用LLC-GFP肺癌细胞分析VDR敲除对癌症转移和癌症诱导血管生成的影响。我们开发了一种新的活性维生素D类似物α(2α-氟-19-no -22-oxa-lα， 25-二羟基维生素D_3: 2αF-22-oxa-， 25-D_3)，具有与非钙化作用相关的关键结构基元，在lc - gfp诱导的癌症动物模型中发现，2αF-22-oxa-1,25-D_3α比其他活性维生素D3类似物更有效和剂量依赖性地抑制肿瘤发生和血管生成。2α f -22-oxa-1,25- d_3在高钙/高乳糖饮食的VDR-/-小鼠中具有抗肿瘤和抗血管生成作用，提示2α f -22-oxa-1,25- d_3在不改变钙代谢的情况下直接抑制LLC-GFP细胞的肿瘤发生和血管生成。通过这些研究，我们可以为评价活性维生素D类似物对癌细胞发生和血管生成的预防和治疗效果提供一个有用的动物模型，并为抗癌药物的开发提供新的途径。少

We have generated a line of animal model (1αOHase-/-) lacking the gene encoding vitamin D la-hydroxylase that is a key enzyme for activation of vitamin D. Phenotypic analysis revealed that similar to vitamin D. receptor gene knockout mice (VDR-/-), the animal exhibited growth retardation, hyperproliferation of cartilage, hypocalcemia, hyperparathyroidism, and impaired bone formation. As these abnormalities mainly derived from calcium malnutrition, the animals were almost completely rescued by feeding a high calcium/high lactose diet. We have been currently trying to generate (1αOHase-/-)/(VDR-/-) double knockout mice in order to clarify physiological function of vitamin D in terms of bone formation and growth of cartilage. In addition to the above mutant animals, we have generated a stable clone of human osteoblast MG-63 VDR knockdown cells and revealed that the cells tended to proliferate and differentiate much more rapidly and remarkably than their normal VDR expressing cells, sugges … More ting that VDR acts as a negative regulator in proliferation and differentiation of osteoblast. The VDR knockdown cells exhibited more rapid and stronger mineralization compared to their parent cells (normal MG-63 cells). We are currently conducting analysis of VDR knockdown effects on cancer metastasis and cancer induced angiogenesis using LLC-GFP lung carcinoma cells. We have developed a novel active vitamin D analogueα(2α-fluom-19-nor-22-oxa-lα, 25-dihydroxyvitamin D_3: 2αF-22-oxa-, 25-D_3) having a key structural motif related to non-calcemic action and it was found that 2αF-22-oxa-1,25-D_3αsuppressed tumorigenesis and angiogenesis of LLC-GFP cells more effectively and dose-dependently than the other active vitamin D3 analogues in a LLC-GFP induced cancer animal model. Furthermore, the anti-tumorigenetic and anti-angiogenetic effects of 2αF-22-oxa-1,25-D_3 were found in a VDR-/-mice fed a high calcium/high lactose diet, suggesting that 2αF-22-oxa-1,25-D_3 directly suppress tumorigenesis and angiogenesis of LLC-GFP cells without changing calcium metabolism.Through these studies, we could provide a useful animal model for evaluating preventive and therapeutic effectiveness of active vitamin D analogues on the tumorigenesis and angiogenesis of cancer cells, and provide a new approach for the development of anti-cancer drugs. Less