Screening of genes those are related to dynamic transition of myofiber of vascular smooth muscle cell upon phenotypic modulation.

筛选与表型调节时血管平滑肌细胞肌纤维动态转变相关的基因。

• 批准号: 17590218
• 负责人: OKAGAKI Tsuyoshi
• 金额: $ 2.3万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590218/
• 关键词: blood vessel; vascular smooth muscle cell; atherosclerosis; S100 family; 形質変換; ミオシン; サブトラクション; 収縮型細胞; 合成型細胞

Vascular smooth muscle cell (VSMC) shows two typical phenotype, contraction-type and synthetic-type upon various situations. We developed model system of phenotypic modulation by using cultured VSMC, P53LMAC01. Upon addition of PDGF, the VSMC changed to synthetic-type phenotype, and it changed to typical contraction-type phenotype in the presence of sodium butyrate (Na-B), which induce growth suppression by inhibiting acetylation of DNA. We isolated mRNA from these typical two types of cell and constructed subtraction cDNA library. We sequenced inserts of the library, and screened 230 clones from PDGF-specific library, and 160 clones from NaB-specific library. Further inserts of each clone were spotted to nylon membrane and examined by putative Northern hybridization to confirm the specificity in each types of cell. Four sets of membrane was hybridized with four sets of probes, two subtracted cDNAs and two original cDNAs from PDGF-and NaB-treated cells. As a result, 15 clones from PDGF-specific library and 19 clones from NaB-specific library were confirmed in the specificity of the expression. To quantify the expression level of these clones in PDGF-and NaB-treated cell, we made primer sets for each clone and examined with real time PCR. Clones showing 2-fold difference of expression among these two types of cell were S 100A4, S100A6, α-actin and others. We isolated the full-length cDNA of S 100A4 and S 100A6 by RT-PCR, and expressed these genes in bacteria. Using these gene products we affinity purified three proteins that interact with S 1004 and S100A6 proteins from the VSMC.

血管平滑肌细胞(VSMC)在不同的情况下表现为两种典型的表型：收缩型和合成型。我们利用培养的VSMC P53LMAC01建立了表型调控模型系统。加入PDGF后，VSMC转变为合成型表型，在丁酸钠(Na-B)存在下，VSMC转变为典型的收缩表型，这通过抑制DNA的乙酰化而导致生长抑制。我们从这两种典型的细胞中提取了mRNA，并构建了消减文库。我们对文库的插入片段进行了测序，从PDGF特异性文库中筛选出230个克隆，从NAB特异性文库中筛选出160个克隆。进一步将每个克隆的插入片段斑点到尼龙膜上，并通过假定的Northern杂交进行检测，以确认在每种类型的细胞中的特异性。四组膜与四组探针、两个从PDGF和NAB处理的细胞中的消减cDNA和两个原始cDNA杂交。结果，从PDGF特异性文库中筛选出15个克隆，从NAB特异性文库中筛选出19个克隆，证实了其表达的特异性。为了量化这些克隆在PDGF和NAB处理的细胞中的表达水平，我们为每个克隆设置了引物集，并用实时荧光定量PCR进行了检测。两种细胞表达差异2倍的克隆是S 100A4、S100A6、α-肌动蛋白等。利用RT-PCR法分离了S 100A4和S 100A6的全长基因，并在细菌中进行了表达。利用这些基因产物，我们从血管平滑肌细胞中亲和纯化了三个与S 1004和S100A6蛋白相互作用的蛋白。

项目成果

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle.

• DOI: 10.1093/jb/mvi121
• 发表时间: 2005-09
• 期刊: Journal of biochemistry
• 影响因子: 2.7
• 作者: T. Okagaki;Masaki Takami;K. Hosokawa;M. Yano;S. Higashi-Fujime;A. Ooi

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

海水鱼普通肌球蛋白和暗肌肌球蛋白的机械化学特性

• 发表时间: 2007
• 期刊: Fish. Sci 73
• 作者: Okagaki;Yang;Ooi
• 通讯作者: Ooi

Intracellular signal transduction for migration and actin remodeling in ascular smooth muscle cells after sphingosylphosphorylcholine stimulation

鞘氨醇磷酰胆碱刺激后血管平滑肌细胞迁移和肌动蛋白重塑的细胞内信号转导

• 发表时间: 2006
• 期刊: Am. J. Physiol. Heart Circ. Physiol 291
• 作者: Li;Tanaka;Wang;Yoshiyama;Kumagai;Nakamura;Brawn;Thatcher;Wright;Kohama.
• 通讯作者: Kohama.

Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

• DOI: 10.1152/ajpheart.00901.2005
• 发表时间: 2006-09-01
• 期刊: AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
• 影响因子: 4.8
• 作者: Li, Sheng;Tanaka, Hideyuki;Kohama, Kazuhiro
• 通讯作者: Kohama, Kazuhiro

Calcium inhibition of Physarum myosin as examined by the recombinant heavy mero-myosin.

• DOI: 10.1007/978-4-431-38453-3_22
• 发表时间: 2007
• 期刊: Advances in experimental medicine and biology
• 作者: H. Kawamichi;Ying Zhang;Mizuki Hino;A. Nakamura;Hideyuki Tanaka;L. Farkas;L. Nyitray;K. Kohama