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

我们建立了一种缺乏编码维生素D Ia-羟化酶基因的动物模型（1αOHase-/-），该酶是激活维生素D的关键酶。表型分析显示，与维生素D相似。受体基因敲除小鼠（VDR-/-）中，该动物表现出生长迟缓、软骨过度增殖、低钙血症、甲状旁腺功能亢进和骨形成受损。由于这些异常主要源于钙营养不良，因此通过饲喂高钙/高乳糖饮食几乎完全挽救了动物。我们目前正在尝试产生（1αOHase-/-）/（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。除了上述突变体动物，我们已经产生了人成骨细胞MG-63 VDR敲低细胞的稳定克隆，并显示该细胞倾向于比其正常VDR表达细胞更快和更显著地增殖和分化， ...更多信息 提示VDR对成骨细胞的增殖和分化起负调节作用。VDR敲除细胞与其亲本细胞（正常MG-63细胞）相比表现出更快和更强的矿化。我们目前正在使用LLC-GFP肺癌细胞进行VDR敲低对癌症转移和癌症诱导的血管生成的影响的分析。我们开发了一种新的活性维生素D类似物α（2α-氟-19-去甲-22-氧杂-1 α，25-二羟基维生素D_3：2αF-22-oxa-，25-D_3）具有与非钙离子化作用相关的关键结构基序，25-D_3α能更有效地抑制LLC-GFP细胞的成瘤和血管生成，在LLC-GFP诱导的癌症动物模型中，与其它活性维生素D3类似物相比，维生素D3的活性依赖性更高。此外，在高钙/高乳糖饮食喂养的VDR-/-小鼠中发现2αF-22-oxa-1，25-D_3的抗肿瘤发生和抗血管生成作用，表明2αF-22-oxa-1，25-D_3直接抑制LLC-GFP细胞的肿瘤发生和血管生成，而不改变钙代谢。本研究为评价活性维生素D类似物对肿瘤发生和肿瘤血管生成的预防和治疗作用提供了一种有效的动物模型，也为抗癌药物的开发提供了一条新的途径。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