Identifying new aggrecanase and producing antibody and gene-targeting mouse

鉴定新的聚集蛋白聚糖酶并产生抗体和基因靶向小鼠

• 批准号: 13470312
• 负责人: HIROHATA Satoshi
• 金额: $ 8.96万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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/en/grant/KAKENHI-PROJECT-13470312/
• 关键词: aggrecanase; extracellular matrix; rheumatoid arthritis; aggecan; matrix metalloproteinase; ADAMTS; メタロプロテアーゼ

To investigate new aggrecanase, we have done the following experiments and got some data.1) Various ADAMTS-specific primers were designed and RT-PCR was performed. We cultured human chondrosarcoma cell lines OUMS-27 (Okayama University Medical School-27) and stimulated with interleukine-1 beta (IL-1β). To determine gene expression quantitatively, we employed real-time RT-PCR method. Briefly, total RNA was extracted and then DNAse treatmeat was done to eliminate contaminating genomic DNA. After reverse transcribed with random primers and enzymes, cDNA was served as a template for RT-PCR. GAPDH was used for the internal control.2) The expression and gene regulation by IL-1β was different amonf the ADAMTS-1,4, and -5, which were reported to have aggrecan cleaving property in vitro. We also investigated other ADAMTS gene expressions.3) We identified another up-regulating ADAMTS gene by IL-1β in OUMS-27 cells. We raised polyclonal antibody against this new ADAMTS gene using peptide sequence of this ADAMTS.4) We also started to making knock-out mouse for this gene. We screened mouse genomic library and identified several clones including this new ADAMTS gene. Under the collaboration with Dr. Apte's lab in the USA, we started to put our clones to ES cells.

为了研究新的聚集蛋白聚糖酶，我们进行了以下实验，并获得了一些数据：1）设计ADAMTS特异性引物，进行RT-PCR。我们培养人软骨肉瘤细胞系OUMS-27（冈山大学医学院-27）并用白细胞介素-1 β（IL-1β）刺激。为了定量测定基因表达，我们采用了实时RT-PCR方法。简言之，提取总RNA，然后进行DNA酶处理以消除污染的基因组DNA。用随机引物和酶进行逆转录后，将cDNA作为RT-PCR的模板。2）ADAMTS-1、4和-5在体外表达及IL-1β的基因调控作用不同，ADAMTS-1、4和-5在体外表达及IL-1β的基因调控作用不同。3）我们在OUMS-27细胞中发现了另一个被IL-1β上调的ADAMTS基因。利用ADAMTS的肽段序列制备了抗ADAMTS基因的多克隆抗体。4）建立了ADAMTS基因敲除小鼠模型。我们筛选了小鼠基因组文库，并鉴定了几个克隆，包括这个新的ADAMTS基因。在与美国Apte博士实验室的合作下，我们开始将我们的克隆细胞植入ES细胞。

项目成果

Hirohata S: "Punctin, a novel ADAMTS-like molecule (ADAMTSL-1) in extracellular matrix"J Biol Chem. 277(14). 12182-12189 (2002)

Hirohata S：“Punctin，细胞外基质中的一种新型 ADAMTS 样分子 (ADAMTSL-1)”J Biol Chem。

Komata T, Kondo Y, Kanzawa T, Hirohata S, Koga S, Sumiyoshi H, Srinivasula SM, Barna BP, Germano IM, Takakura M, Inoue M, Alnemri ES, Shay JW, Kyo S, Kondo S.: "Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using

Komata T，Kondo Y，Kanzawa T，Hirohata S，Koga S，Sumiyoshi H，Srinivasula SM，Barna BP，Germano IM，Takakura M，Inoue M，Alnemri ES，Shay JW，Kyo S，Kondo S.：“恶性肿瘤的治疗

Takemoto S: "Increased expression of dermatopontin mRNA in the infarct zone of experimentally induced myocardial infarction in rats : comparison with decorin and type I collagen mRNAs"Basic Research in Cardiology. 97(6). 461-468 (2002)

Takemoto S：“实验诱导大鼠心肌梗塞梗塞区皮桥蛋白 mRNA 表达增加：与核心蛋白聚糖和 I 型胶原 mRNA 比较”心脏病学基础研究。

Komata T, Kondo Y, Kanzawa T, Ito H, Hirohata S, Koga S, Sumiyoshi H, Takakura M, Inoue M, Bama BP, Germano IM, Kyo S, Kondo S.: "Caspase-8 gene therapy using the human telomerase reverse transcriptase promoter for malignant glioma cells"Hum Gene Ther.. 1

Komata T、Kondo Y、Kanzawa T、Ito H、Hirohata S、Koga S、Sumiyoshi H、Takakura M、Inoue M、Bama BP、Germano IM、Kyo S、Kondo S.：“使用人类端粒酶的 Caspase-8 基因治疗

Fernandes RJ: "Procollagen II Amino Propeptide Processing by ADAMTS-3. INSIGHTS ON DERMATOSPARAXIS"J Biol Chem. 276(34). 31502-31509 (2001)

Fernandes RJ：“ADAMTS-3 处理原胶原 II 氨基前肽。关于皮肤性皮肤病的见解”J Biol Chem。