NAADP/TPC2/Ca2+信号通路调节细胞自噬在低氧性肺动脉高压中的作用及机制研究

课题组在上一个国家自然基金的资助下发现低氧时双孔通道2（two pore channel 2, TPC2）在人和大鼠肺动脉平滑肌细胞溶酶体上高表达，且受到烟酸腺嘌呤二核苷酸磷酸（Nicotinic acid adenine dinucleotide phosphate, NAADP）调控，通过诱导“钙火花”促进肺动脉平滑肌细胞（PASMCs）增殖。但其高表达在调控溶酶体介导的细胞自噬中的作用尚无研究。本课题拟通过透射电镜和免疫荧光方法观察低氧时PASMCs自噬水平的动态变化；通过荧光共聚焦、RNA-seq测序及Label free等分析NAADP/TPC2是否通过Ca2+、CaMKK β、AMPKα1、TSC2、mTORc1、ULK1途径影响低氧诱导的PASMCs增殖、肺动脉高压的发生。阐明 NAADP/TPC2和自噬对低氧性肺动脉高压发病的作用，为寻找新的防治靶点提供理论依据。

With the support of our previous National Natural Science Foundation of China, we found that two pore channels 2 (TPC2) was highly expressed on lysosomes of human and rat pulmonary artery smooth muscle cells（PASMCs）under hypoxic condition, and was mediated by Nicotinic acid adenine dinucleotide phosphate (NAADP), promotes PASMCs proliferation of by inducing "Ca2+ spark". However, the role and mechanism of NAADP/TPC2 in regulating lysosomal-mediated autophagy have not been studied. This project intends to observe the dynamic changes of autophagy in PASMCs under hypoxia by means of transmission electron microscopy and immunofluorescence; Using confocal microscopy to analyze whether NAADP / TPC2 regulates autophagy through Ca2+ release; Exploreing whether NAADP / TPC2 regulates autophagy through Ca2+, CaMKK β, AMPKα1, TSC2, mTORc1, ULK1 signaling pathway , by Fluorescence confocal, RNA-seq transcriptase sequencing and Label free methods; We would like to clarify the effect of NAADP / TPC2 and cell autophagy on the pathogenesis of hypoxia pulmonary hypertension and provide a theoretical basis for finding new prevention and treatment targets.