Molecular biological study of cytotoxicity due to leukotoxins and their metabolites

白细胞毒素及其代谢物细胞毒性的分子生物学研究

• 批准号: 12470135
• 负责人: ISHIZAKI Takeshi
• 金额: $ 7.17万
• 依托单位: Fukui Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470135/
• 关键词: Leukotoxin; Epoxide; Linoleate; Cell-cycle; Cyclin-dependent kinase; cyclin dependent kinase inhibitor; cell cycle; P16 INK4a; senescence; ディオール; cyclin dependent kinase; セルサイクル

We continued to study the effect of Leukotoxin (Lx), a linoleate epoxide, on the cell cycle. By using cultured human pulmonary arterial cells (HPEAC), we assessed the effect of antisense p16 INK4aS-oligo in terms of cell growth and used senescence-associated(SA)- β galactosidase staying for evaluating the degree of cell senescence in the presence/absence of Lx.Results : 1) Cell cycle of the HPEAC was arrested at the phase of G1 in the presence of Lx. The capability of DNA synthesis recovered when Lx was excluded or when pretreated with antisense p16INK4aS-oligo. The expression of mRNA and protein of p16INK4a was increased 3-fold at 48hr after addition of Lx and 4-fold at 96hr, respectively. However, either antisense p16 INK4aS-oligo or washout of Lx suppressed such an effect of Lx.2) When HPEAC passed PD50 the capability of growth of HPEAC was loosed and positive stained cells with SA-β galactosidase were increased. Whereas Lx suppressed the increase of SA-β galactosidase cells. The simultaneous presence of Lx and antisense p16INK4aS-oligo raised the cell number of positive SA-β galactosidase stain.Our current experiment suggests that Lx caused cell growth suppression and furthermore cell senescence suppression.

我们继续研究环氧化亚油酸亚油酸酯对细胞周期的影响。采用体外培养的人肺动脉细胞，观察反义p16INK4aS-OGO对细胞生长的影响，并用衰老相关(SA)-β半乳糖苷酶滞留来评价细胞在Lx作用下的衰老程度。结果：1)Lx作用后，细胞周期停滞于G1期。当LX被排除或反义p16INK4aS-OGO处理后，DNA合成能力恢复。加入LX后48h，p16INK4a的mRNA和蛋白表达分别增加3倍和4倍。但反义p16INK4aS寡核苷酸和Lx的洗脱均可抑制Lx的这种作用。2)当HPEAC通过PD50时，HPEAC的生长能力减弱，SA-β半乳糖苷酶染色阳性的细胞增多。而LX则抑制SA-β半乳糖苷酶细胞的增加。LX和反义p16INK4aS-OGO同时存在时，SA-β半乳糖苷酶染色阳性的细胞数增加。我们目前的实验表明，LX引起细胞生长抑制，进而抑制细胞衰老。

项目成果

若林聖伸 他: "リノール酸及びそのエポキシッド化合物に対する肺動脈血管内皮細胞の応答"生化学. 72. 732 (2000)

Seinobu Wakabayashi 等人：“肺动脉内皮细胞对亚油酸及其环氧化合物的反应”生物化学 72. 732 (2000)。

Wakabayashi M, Ishizaki T, Demura Y, Ameshima S, Miyamori I, Matsukawa S: "A linoleate epoxide, leukotoxin, 9, 10-epoxy-12-octadecenoate causes premature senescence-like effect on pulmonary arterial endothelial cells"Am.J.Respir.Crit.Care.Med. 165(8). A37

Wakabayashi M、Ishizaki T、Demura Y、Ameshima S、Miyamori I、Matsukawa S：“亚油酸酯环氧化物、白细胞毒素、9, 10-环氧-12-十八烯酸酯对肺动脉内皮细胞造成过早衰老样作用”Am.J.

Nakanishi M, Ishizaki T, Demura Y, Okamura S, Ameshima S, Sakaki F, Matsukawa S, Miyamori I: "Leukotoxin, 9, 10-epoxy-12-octadecenoate causes pulmonary vasodilation via stimulation of vascular eNOS and iNOS"Lung. 178(3). 137-148 (2000)

Nakanishi M、Ishizaki T、Demura Y、Okamura S、Ameshima S、Sakaki F、Matsukawa S、Miyamori I：“白细胞毒素，9, 10-环氧-12-十八碳烯酸酯通过刺激血管 eNOS 和 iNOS 引起肺血管舒张”肺。

若林聖伸, 松川茂, 石崎武志他: "肺動脈血管内皮細胞におけるリノール酸エポキシド (Leukotoxin : Lx)による細胞周期停止機構"生化学. 73(8). 840 (2001)

Seinobu Wakabayashi、Shigeru Matsukawa、Takeshi Ishizaki 等：“肺动脉内皮细胞中亚油酸环氧化物（白细胞毒素：Lx）诱导的细胞周期停滞机制”生物化学 73(8)。

松川茂, 若林聖伸, 石崎武志: "リノール酸エポキシド(9, 10-epoxyoctadecenoic acid : Lx)のproteasome活性抑制作用と解毒"生化学. 73(8). 840 (2001)

Shigeru Matsukawa、Seinobu Wakabayashi、Takeshi Ishizaki：“亚油酸环氧化物（9, 10-环氧十八碳烯酸：Lx）抑制蛋白酶体活性和解毒”生物化学 73(8)。