酶促产生的25-OHC对血管壁巨噬细胞增殖凋亡及动脉粥样硬化进程的影响和ORP4L介导作用机制的研究

巨噬细胞增殖和凋亡是动脉粥样硬化（AS）进程的主要事件。体外低浓度氧化固醇促细胞增殖，高浓度则诱导细胞凋亡。但25-hydroxylase (CH25H)催化产生的25-氧化固醇(25-OHC)对巨噬细胞增殖和凋亡的影响未见报道。我们发现ORP4L调控钙信号并对抗25-OHC诱导的细胞凋亡。培养细胞中过表达CH25H提高其增殖，但敲低ORP4L则降低这个效率；CH25H过表达同时Oxysterol-7α-hydroxylase (CYP7B1)沉默提高细胞凋亡敏感性，但ORP4L过表达消减该现象。因此，我们提出假说:CH25H催化的25-OHC通过ORP4L介导Ca2+信号,促进巨噬细胞增殖；其浓度过高则抑制ORP4L及Ca2+信号,致巨噬细胞凋亡。本课题将在分子、细胞、动物水平等多层次揭示酶促产生的25-OHC在巨噬细胞命运及AS进程中的作用和ORP4L的介导机制，为AS防治提供证据。

Macrophage apoptosis and proliferation are the centrol events in atherosclerosis. Oxysterols enhance cell proliferation but the extra increased oxysterols mediate cell apoptosis.So far, no reports are found in the study that the effects of 25-hydroxylase catalyzed 25-OHC in cell proliferation and apoptosis. We have reported that oxysterol binding protein-related protein-4 (ORP4L) regulates calcium signaling, sustains Bcl-XL expression and counteracts the cytotoxicity of exogenous 25-hydroxycholesterol (25-OHC) to protect macrophage from apoptosis (Circ Res. 2016 Dec9; 119(12):1296-1312). Based on the findings above, the editorial suggested that the impact of endogenous 25-OHC on macrophage apoptosis and proliferation should been assessed, which will offer the evidences that how lipids deposited in the subendothelial layer and cell debris cannot be cleaned efficiently by macrophages（Circ Res. 2016;119:1258-1261. In cultured cells, we found that CH25H overexpression promoted cell proliferation whereas attenuated by ORP4L knockdown. Overexpression of CH25H as well as knockdown of oxysterol-7α- hydroxylase (CYP7B1) significantly increased the sensitivity of cells to apoptosis. However, this phenomenon can be abolished by ORP4L overexpression. Thus,we speculate that in the blood vessel wall the enzyme catalyzed 25-oxysterol may enhance macrophages proliferation via ORP4L mediaed-Ca2+ signaling, but the extra increased oxysterol may inactive ORP4L function, resulting cell apoptosis. In this study , we will produce macrophage specific TgCH25H and CYP7B1-/- mice, which will be crossed with ORP4L-/+/LDLr-/- or TgORP4L/LDLr-/-mice to get TgCH25H /ORP4L-/+/LDLr-/- and TgCH25H /TgORP4L/LDLr-/-,or TgCH25H / CYP7B1-/-/ORP4L-/+/LDLr-/- and TgCH25H / CYP7B1-/-/TgORP4L/LDLr-/- ,respectively. Combined the analysis of molecule and cell biology in vitro, we aim to reveal in vivo the effects of the interaction of endogenous enzyme catalyzed 25-OHC and ORP4L in apoptosis and proliferation of macrophages and atherosclerosis. We hope to find out prevention and therapeutic approaches for the direct intervention of atherosclerosis.