カチオン性リポソーム刺激による細胞内カルシウム動態解析手法の開発

早稲田大学博士(工学)早大学位記番号:新9383doctoral thesi

Development of the observation of membrane fusion with label-free liposomes by calcium imaging

Liposomes are artificial vesicles composed of lipid bilayers that have enabled drugs to be encapsulated and delivered to tumor tissue. Membrane-fusogenic liposomes fuse with the plasma membranes of cells to deliver encapsulated drugs directly to the cytosol, which makes it a promising method for rapid and highly efficient drug delivery. In a previous study, liposomal lipid bilayers were labeled with fluorescent probes, and colocalization of labeled lipids with plasma membrane was observed under a microscope. However, there was concern that fluorescent labeling would affect lipid dynamics and cause liposomes to acquire membrane fusogenic ability. In addition, encapsulation of hydrophilic fluorescent substances in the inner aqueous phase sometimes requires an additional step of removing unencapsulated substances after preparation, and there is a risk of leakage. Herein, we propose a new method to observe cell interaction with liposomes without labeling. Our laboratory has developed two types of liposomes with different cellular internalization pathways, i.e., endocytosis and membrane fusion. We found that cytosolic calcium influx would be triggered following the internalization of cationic liposomes, and different cell entry routes led to different calcium responses. Thus, the correlation between cell entry routes and calcium responses could be utilized to study liposome-cell interactions without fluorescent labeling lipids. Briefly, liposomes were added to phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells, and calcium influx was measured by time-lapse imaging using a fluorescent indicator (Fura 2-AM). Liposomes with high membrane fusogenic ability elicited a strong transient calcium response immediately after adding liposomes, whereas those taken up mainly by endocytosis elicited multiple weak calcium responses. In order to verify the cell entry routes, we also tracked the intracellular distribution of fluorescent-labeled liposomes in PMA-primed THP-1 cells using a confocal laser scanning microscope. It was shown that for fusogenic liposomes, colocalization with plasma membrane occurred at the same time as calcium elevation, whereas for liposomes with a high endocytosis potential, fluorescent dots were observed in the cytoplasm, suggesting the cell internalization by endocytosis. These results suggested that the calcium response patterns correspond to cell entry routes, and membrane fusion can be observed by calcium imaging

Effects of Heat-Not-Burn Cigarette Smoking on the Secretion of Saliva and Its Innate Immune System Components

Saliva and salivary antimicrobial proteins play important roles in the innate immunity, which prevents infections of orally invading bacteria and viruses. In this study, we compared the secretion rates of salivary lactoferrin (Lac) and lysozyme (Lys) in heat-not-burn (HNB) cigarette smokers and non-smokers. The analysis population for this study included 212 members of the fire department, including 32 HNB cigarette smokers, 17 paper cigarette smokers, 14 combined HNB and paper cigarette smokers, and 149 non-smokers. Salivary Lac and Lys concentrations were assessed using enzyme immunoassay. Saliva secretion was significantly lower among HNB cigarette smokers (p p < 0.01). Our findings suggest a possible adverse effect of HNB cigarette on the amount of Lac and Lys released into the oral cavity

Smoking Prevention Education and Smoking Support for University Students - Based on a Survey of Smoking Behavior and Lifestyle of Chubu University Student -

目的:喫煙と生活習慣との間に関連があることを明らかにし、大学生の喫煙防止教育および禁煙支援について検討する。方法:平成23年9月、学部生9,688名を対象にし、無記名自記式調査を行った。対象者を喫煙者と非喫煙者の2群に分けて学生生活、生活習慣について比較検討した。結果:有効回答者5,772名のうち、喫煙者は628名、喫煙率は10.9%であった。喫煙者は、男子学生に多く、「一人暮らしをしている」「サークルに入っていない」という学生生活に有意な差がみられた(p<0.05)。生活習慣では、喫煙者は、「朝食を食べない」「お酒を飲む」に有意差がみられた(p<0.05)。結論:喫煙者の2割は、禁煙を望んでいることから、学生生活の状況を考慮し、食事、飲酒などの生活習慣を踏まえた禁煙支援が必要であることが示唆された。また、新入生対象とした喫煙防止教育は引き続き継続して行うことが重要である