targeting of iNOS with antisense DNA plasmid prevents cytokine-induced inhibition of osteoblastic differentiation

用反义 DNA 质粒靶向 iNOS 可防止细胞因子诱导的成骨细胞分化抑制

• 批准号: 13672082
• 负责人: HIKIJI Hisako
• 金额: $ 2.24万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672082/
• 关键词: iNOS; antisenae; gene targeting; gene therapy; bone destruction; 遺伝子治療; 骨破壊病変

Purpose :Proinflammatry cytokines, tumor necrosis factor (TNF) -α combined with interleukin-1 (IL-1) -β, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite, peroxynitrite (ONOO^-) via indueible nitric oxide synthase (iNOS) in mouse osteoblasts. In this investigation, to estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid targeting the iNOS gene for long-lasting inhibition The identifications of the transformed antisense cell lines were 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NOS activity and 4) the level of NO production. These cell lines targeting which showed decreased production of both NO and ONOO^-, prevented the inhibition iNOS, of osteoblastic differentiation as was assayed by the mRNA expression of alkaline phosphatase (ALPase) in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to prevent the cytokine-induced reduction of osteoblastic activity.

目的：促炎细胞因子肿瘤坏死因子（TNF）-α和白细胞介素1（IL-1）-β通过诱导型一氧化氮合酶（iNOS）诱导小鼠成骨细胞产生一氧化氮（NO）及其代谢产物过氧亚硝基阴离子（ONOO^-）。本研究以iNOS基因为靶点，构建反义质粒转化成骨细胞系，观察iNOS反义DNA对成骨细胞活性的影响。结果表明，反义质粒转染的成骨细胞系中，iNOS基因表达明显减弱，NOS活性降低，NO生成量减少。这些细胞系显示NO和ONOO^-的产生减少，阻止了成骨细胞分化的iNOS抑制，如通过在促炎细胞因子存在下碱性磷酸酶（ALP 3）的mRNA表达所测定的。目前的结果表明，iNOS的反义DNA质粒是有效的，以防止苦参碱诱导的成骨细胞活性的降低。

项目成果

高戸毅, 引地尚子: "骨延長と再生医療"Clinical Calcium. 12(2). 29(207)-33(211) (2002)

Takeshi Takato、Naoko Hikichi：“骨延长和再生医学”临床钙 12(207)-33(211) (2002)。

Naohiko Sakai: "Sequence Analysis of Fibroblast Growth Factor Recepter 2 (FGFR2) in Japanese Patients with Craniosynostosis"The Jouranl of Craniofacail Surgery. 12. 580-585 (2001)

Naohiko Sakai：“日本颅缝早闭患者中成纤维细胞生长因子受体 2 (FGFR2) 的序列分析”《颅面外科杂志》。

引地尚子, 綿谷早苗, 高戸毅, 江口智明, 富塚健: "口唇形成術前外鼻矯正のための改良型口蓋床の使用経験"形成外科. 45(3)(in press). (2002)

Naoko Hikichi、Sanae Wataya、Tsuyoshi Takato、Tomoaki Eguchi、Ken Tomizuka：“阴唇整形术前使用改进的腭底进行外鼻矫正的经验”整形外科 45(3)（出版中）。

Jinhong Huang: "Differential Regulation of Cell Migration, Actin Stress Fiber Organization, and Cell Transformation by Functional Domains of Crk-associated Substrate"Journal of Biological Chemistry. 277. 27265-27272 (2002)

Jinhong Huang：“Crk相关底物功能域对细胞迁移、肌动蛋白应力纤维组织和细胞转化的差异调节”生物化学杂志。

Naohiko Sakai: "A Case of a Japanese Patient with Cleidocranial Dyspalasia Possessing a Mutation of CBFA1 Gene"The Journal of Craniofacial Surgery. 13. 31-34 (2002)

Naohiko Sakai：“一例患有 CBFA1 基因突变的锁骨颅骨发育不良的日本患者”《颅面外科杂志》。