2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/686177摘要：早衰症(Hutchinson-Gilford progeria syndrome, HGPS)為造成個體提前衰老之罕見疾病，此病症 由細胞核中突變的A 型合纖層蛋白(Lamin A)所形成的早衰蛋白(Progerin)引起。帶有早衰蛋白 的細胞，其明顯病徵為細胞核型態異變、細胞分裂緩慢、活性氧物種(Reactive oxygen species) 累積及促使細胞進入細胞凋亡(Apoptosis)。水解磷酸脂(Lysophosphatidic acid, LPA)為一透過其 六個受器於體內廣泛地調控生理現象之脂質生長因子，於本實驗室研究中發現帶有早衰蛋白 的HEK-293 細胞，大量表現第二及第三型水解磷酸脂受器，因此，我們在此細胞中利用水解 磷酸脂第二及第三型受器之促進劑活化兩型受器和短髮夾RNA(short hairpin RNA)抑制兩受 器表現，並透過CM-H2DCHDA 染劑和&szlig;-gal 染劑分別偵測活性氧物種的產生，以及細胞的老 化程度，於其中我們發現第二型水解磷酸脂受器加速早衰細胞的老化，而第三型水解磷酸脂 受器則延緩此老化的過程。因此，於上述研究結果顯示，第二型及第三型水解磷酸脂受器極 有可能在早衰症致病機轉中造成相反的調控結果。在本三年期研究計畫的首年，我們預期釐 清各水解磷酸脂受器在老化過程中的的調控機轉，其中包括水解磷酸脂透過何種酵素造成活 性氧化物的產生，進而影響早衰細胞中的DNA 斷裂。此外，接下來的兩年，我們預期於斑馬 魚系統中建立研究早衰症的模式動物，將第二型或第三型水解磷酸師受器剔除之斑馬魚與早 衰症斑馬魚配種，用以研究其受器與早衰症在動物中的老化機制。最後，我們預期篩選專一 的水解磷酸脂受器之受體，並在動物體內測試其延緩老化的可能性，以應用於未來臨床使用。<br> Abstract: Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare laminopathy, which results in premature aging by producing a mutant form of pre-lamin A known as progerin. HGPS cells show morphological abnormalities of the nuclear membrane, reduced rate of cell proliferation, accumulation of reactive oxygen species (ROS), enhanced apoptosis and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates multiple physiological functions via activation of six LPA GPCR. Using quantitative real-time PCR method, we showed that Lpar2 and Lpar3 transcripts were up-regulated in HEK-293 transfected with the mutant progerin from HGPS. We applied CM-H2DCHDA-stain for detection of generation of ROS and SA-β-gal staining as indication of the degrees of cellular senescence. The effects of LPA2 and LPA3 were monitored using either chemical agonists or shRNA for both receptors in HGPS HEK-293. Activation of LPA2 accelerated the aging process of HGPS cells, whereas LPA3 reduced it. Our preliminary results demonstrated that activation of LPA2 promotes the aging progress in HGPS cells, while LPA3 alleviates it. Thus, our preliminary data from HGPS cell model suggests an opposite role of LPA2 and LPA3 in HGPS. In the first year of this 3 years grant, we propose to clarify the detailed regulatory mechanism of LPA receptors in cell senescence, including the relationship among LPA, ROS induction and DNA damage in the HGPS cell model. In the following two years, we propose to establish HGPS animal models to study the systematic effects of LPA receptors on aging process, such a zebrafish. LPA2 or LPA3 knockout animals will mate with HGPS individuals to investigate the roles of these receptors during the premature aging process. At the last year, we proposed to select specific ligands for LPA receptors. The potential effects of LPA receptors agonists or antagonists to prevent aging process will be determined in HGPS animals for future clinical application.水解磷酸脂水解磷酸脂受器早衰症A 型合纖層蛋白早衰蛋白活性氧物種Lysophosphatidic acidLysophosphatidic acid receptorHGPSLamin AProgerinROS,