脂質代謝そして糖代謝を同時に制御する新規蛋白PCSK7の総合的・網羅的機能解析

同时控制脂质和糖代谢的新型蛋白PCSK7的全面综合功能分析

• 批准号: 22K08635
• 负责人: 塚本 和久
• 金额: $ 2.66万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22K08635/
• 关键词: PCSK7; 脂質代謝; 糖代謝

CRISPR/CAS9システムを用いて樹立した遺伝子欠損細胞株、アデノウイルスベクター（ADV）で蛋白を過剰発現/発現抑制した細胞を用いてマイクロアレイ解析を行った。その結果、PCSK7の発現量増加は、内皮細胞系EA hy926細胞においてHDL-C上昇・LDL-C低下に関与するABCA1やLDLRAP1の遺伝子発現を亢進させ、肝細胞系HepG2細胞においてはTG上昇・LDL-C低下・HDL-C低下につながるANGPTL2、FABP5、FABP1、LDLR遺伝子の発現亢進、APOA1遺伝子の発現低下を認めた。HepG2細胞にADVでPCSK7を過剰発現、あるいは発現抑制することで、発現抑制系では細胞LDL受容体蛋白量の減少とアポEおよびアポMの上清中蛋白量の増加（mRNA量は変化なし）、過剰発現系では細胞LDL受容体量の増加とNPC1L1蛋白量の減少（アポEおよびアポMの上清中蛋白量は変化なし）を認めた。PCSK7過剰発現KKAyマウスにおけるTG値上昇、TC値低下、血糖値低下につながる分子メカニズムを、マウス肝臓検体を用いて、核タンパク・代謝関連酵素などの蛋白量やmRNA発現量の検討を行った。核タンパクにおいてはFoxO1低下、CREB低下、LXR上昇などの結果を得るとともに、RT-PCRにてPEPCK低下、GK上昇、LRP低下、などの所見を得た。また、血漿検体を用いて血中リポタンパクの解析も行い、カイロミクロン・VLDL分画が増加することを確認した。そして、正常食C57BL/6マウスおよび代謝疾患モデルである脂肪食負荷C57BL/6マウスにおいても、PCSK7発現が血糖値を低下させることを確認した。さらに脂肪食負荷C57BL/6マウスでは、PCSK7発現が脂肪肝をもたらすことが判明した。

The cell lines of CRISPR/CAS9 and ADV proteins were detected and suppressed. The cell lines were analyzed by scanning electron microscopy. The results showed that the PCSK7 concentration was increased, the endothelial cell line EA hy926 cell cycle showed high levels of LDL-C on HDL-C, and the liver cell line HepG2 cell line showed hyperactivity of LDL-C on HDL-C, while that of liver cell line HepG2 on TG. LDL-C was low on TG, HDL-C was low, ANGPTL2, FABP5, FABP1, LDLR was increased, and APOA1 was low. The results of HepG2 cell ADV PCSK7 assay showed that the protein content in the supernatant of LDL receptor was significantly increased (mRNA), and that of NPC1L1 protein in the supernatant was significantly increased (P < 0.05). PCSK7 showed that KKAy was high in TG, TC was low, blood sugar was low, molecular weight was low, liver fat was used, and nuclear enzyme was used. The amount of linked enzyme protein was measured by mRNA assay. The results showed that the FoxO1 was low, the CREB was low, the LXR was low, the RT-PCR was low, the PEPCK was low, the GK was low, the LRP was low, and the results were negative. The blood samples were divided into two parts, the lines were analyzed, the lines were analyzed, the VLDL was divided into two parts, and the confirmation was confirmed. Normal C57BL/6 diet, normal diet, fat diet, C57BL/6 diet, hypoglycemia, hypoglycemia, hypoglycemia and confirmation. The fat diet contains C57BL/6 and PCSK7. Fatty liver is known as fatty liver.