Gfi1基因点突变所致中性粒细胞缺失对动脉粥样硬化形成的影响

Atherosclerosis is a chronic inflammatory disease of the wall of large- and medium-sized arteries, which remains the leading cause of death and disability in many countries. In the past decades tremendous efforts have been made to understand the molecular and cellular basis for the development of atherosclerosis. However, neutrophils as key component of the inflammatory response have long been neglected in such studies, as a result of lack in genetic model with neutrophil deficiency. The current neutrophil deficient models are not possible for application of studying atherosclerosis which requires a long term investigation.Currently the only approach to neutrophil deficiency is based on anti-PMN treatment which leads to dramatic but transient depletion of these cells in periphery and the full recovery of neutrophils occurs two weeks after antibody injection. Moreover neutrophil depletion in this manner also affects the other immune cells such as lymphocytes,monocytes. The genetic model, Gfi1-/- mouse can only survive for 2-3 months after birth in SPF condition with antibiotic treatment. Therefore it is not possible to model atherogenesis which requires months by feeding the ApoE deficient mice with western diet, in these Gfi1-/- mice. Very recently we obtained a Gfi1 point mutation mouse model, designated as Genista mice which are quite different from Gfi1 knockout mice. Genista mice are highly specific in neutrophil deficiency and keep an intact adaptive immunity. More importantly the mice are as fertile and viable as the wild type mice. Based on this special model, we will develop a new model deficient both in neutrophil and ApoE, to investigate atherosclerosis development in the absence of neutrophils. We will also a surgical regression model by transplant of diseased artery to Genista mice to address the critical question whether neutrophils are required for the plaque regression in atherosclerotic hosts. By means of analyzing the immune cells with 10 color flow cytometry and histological analysis, we will elucidate the role of neutrophils in development and regression of atherosclerosis, which could be very important information for the treatment of patients with arthrosclerosis.

动脉粥样硬化是严重威胁人类健康的慢性炎症性疾病。但由于缺乏合适的动物模型，中性粒细胞在动脉粥样硬化形成中的作用一直被忽视，而后者是炎症反应的主要细胞群。研究动脉粥样硬化，需要对动物模型长时间跟踪，而目前已有的中性粒细胞缺失小鼠只能存活数周时间，且需要在SPF环境下加抗生素喂养。本课题为了明确动脉粥样硬化形成过程是否需要中性粒细胞，及中性粒细胞是否参与动脉粥样硬化的修复两个问题，首次将Gfi1点突变引入到ApoE敲除小鼠基因组，构建中性粒细胞缺失的动脉粥样硬化小鼠遗传模型。Gfi1点突变小鼠是目前世界上唯一能长期存活的中性粒细胞缺失小鼠模型。利用10-荧光流式细胞仪分析技术，动脉粥样硬化组织切片病理分析，及小鼠病变动脉移植实验，本课题将首次明确中性粒细胞对动脉粥样硬化形成与消退的影响。研究结果将揭开中性粒细胞对动脉粥样硬化的形成与修复的作用，对动脉粥样硬化诊断治疗具有十分重要的潜在价值。

本研究首先获得了ApoE–/–Gfi1C318Y/C318Y纯合子小鼠（即为ApoE基因敲除和Gfi1点突变小鼠），检测结果表明新获得的小鼠ApoE基因缺失，而且小鼠无法发育获得成熟的中性粒细胞。对ApoE–/–小鼠、ApoE–/–Gfi1C318Y/C318Y小鼠连续饲喂高脂饲料12周以后，测定小鼠血清生化指标、获取主动脉进行油红O染色、获取心脏组织进行冰冻切片然后对切片进行油红O和H&E染色，结果表明，中性粒细胞缺失会显著降低高脂饲料饲喂后ApoE–/–小鼠的动脉粥样硬化病变程度。进一步，为了揭示Gfi1基因是通过何种信号通路来调控动脉粥样硬化疾病形成过程，获取小鼠腹腔原代巨噬细胞使用流式细胞术对清道夫受体CD36表达进行检测和oxLDL吸收能力测定，结果表明，点突变小鼠腹腔原代巨噬细胞中CD36的表达有显著降低、对oxLDL的吸收能力显著降低，这也解释了Gfi1C318Y/C318Y点突变小鼠动脉粥样硬化病变程度减轻的原因。为了验证Gfi1基因是通过调控CD36信号通路来控制动脉粥样硬化病变程度，在小鼠巨噬细胞系RAW264.7中使用CRISPR/Cas9基因组编辑系统对Gfi1基因进行打靶并成功获得Gfi1基因敲除单克隆细胞株，结果表明其CD36表达量也有显著降低、对oxLDL的吸收能力也变弱。此结果与小鼠腹腔原代巨噬细胞结果完全相符合。为了揭示Gfi1基因是通过何种途径来调控CD36的表达，通过生物信息学预测，发现转录抑制子Gfi1可以与过氧化物酶体增殖物激活受体γ（PPARγ）结合，而PPARγ是调控CD36表达的最关键的转录因子，在Gfi1C318Y/C318Y点突变小鼠腹腔原代巨噬细胞中，检测发现PPARγ表达量在RNA水平和蛋白水平都有显著降低，而PPARγ基因表达量的降低也会进一步使CD36的表达受到抑制，导致动脉粥样硬化病变程度的减轻。在Gfi1基因敲除的小鼠巨噬细胞系RAW264.7中过表达PPARγ基因后，发现CD36的表达得到很好的恢复，而且其对oxLDL的吸收能力也可以得到挽救，以上结果表明，Gfi1基因通过调控PPARγ基因的表达从而影响CD36信号通路的正常传递，导致巨噬细胞对oxLDL的吸收减少，最终动脉粥样硬化症状减轻。通过本项目研究发现的全新的PPARγ基因表达调控位点可以作为临床针对心血管疾病治疗中潜在的靶标。