Ufm1对巨噬细胞增殖、凋亡及功能的影响和机制研究

Diabetic macroangiopathy (atherosclerosis) is the leading cause of death and disability in type 2 diabetic patients. Macrophages are centrally important in atherogenesis and may play critical roles in diabetes-accelerated cardiovascular diseases. Ufm1, which has been proved to be associated with proliferation and differentiation of macrophages by previous studies, is a member of the ubiquitin like protein family. We observed the number of peritoneal macrophages decreased in diabetic mice accompnied by cell morphology alteration. Besides this, peritoneal macrophages from diabetic mice showed disfunction in phagocytosis, adhesion and chemotactic migration. With the method of cDNA microarray and realtime PCR, we observed that expression level of Ufm1 gene, as well as some genes related to endoplasmic reticulum stress (ERS), were up-regulated in peritoneal macrophages of diabetic mice. We also founded that the levels of expression and phosphorylation of Ufm1 and ERS related proteins in macrophages increased after treated with chemical inducers of ERS. In present study, we will use technology of gene silencing and gene overexpression mediated by lentivirus to explore the effect of Ufm1 on cell proliferation, apoptosis, phagocytosis, adhesion and chemotaxis of macrophages, and mechanisms for these changes. In vivo study, we will use technology of injecting exogenous macrophages specific knockdown of Ufm1 into the abdominal cavities of zebra fish to observe changes in macrophages phagocytosis of lipid, accumulation in the arterial tissue, migration and expression level of inflammatory cytokines. In vivo and in vitro experiments, our study focuses on the relationship between the Ufm1, ERS and macrophage function to explore the role of Ufm1 in diabetes-related cardiovascular diseases.

糖尿病大血管病变（动脉粥样硬化）是糖尿病患者致残、致死的主要原因。巨噬细胞在动脉粥样硬化的发生和发展中起重要作用。Ufm1属类泛素蛋白，研究发现其与巨噬细胞分化、增殖有关。本课题组发现糖尿病鼠的腹腔巨噬细胞吞噬、粘附及趋化功能明显下降、细胞数量减少、形态改变。我们还发现糖尿病鼠的腹腔巨噬细胞Ufm1基因表达上调，同时上调的还有内质网应激（ERS）相关基因。在化学诱导剂诱导ERS后，Ufm1及ERS相关蛋白表达量及磷酸化水平明显上调。本研究拟运用慢病毒介导的基因沉默及过表达技术，探索Ufm1对巨噬细胞增殖、凋亡、吞噬、粘附、趋化的影响及机制。体内实验运用斑马鱼腹腔内注射外源巨噬细胞技术，观察Ufm1沉默的巨噬细胞吞噬脂质和移行的变化、动脉组织中巨噬细胞聚集及炎症因子表达的变化。本研究着眼于Ufm1/ERS/巨噬细胞功能之间的关系，在体内、外两方面探索Ufm1在糖尿病大血管病变中作用。

糖尿病大血管病变是糖尿病慢性并发症之一，是2型糖尿病患者致残、致死的主要原因， 其主要病理特征是动脉粥样硬化。巨噬细胞具有多种重要功能，参与AS的发生和发展。Ufm1是一种新发现的类泛素蛋白，目前对Ufm1的生物学功能了解较少，而在巨噬细胞中的作用则尚未有人研究。我们的研究表明，db/db小鼠较同窝对照db/m小鼠的腹腔巨噬细胞中，ERS相关分子及Ufm1基因mRNA和蛋白表达水平明显升高；TG或TM诱导Raw264.7细胞发生ERS后，Ufm1基因 mRNA和蛋白表达水平明显升高。用shRNA-Ufm1沉默Ufm1基因后，Raw264.7细胞ERS加重，ERS诱导的凋亡增加;过表达Ufm1后，Raw264.7细胞ERS减轻，ERS诱导的凋亡减少，同时，Raw264.7细胞非特异吞噬能力减弱，趋化侵袭能力增强，粘附能力变化不明显。. 此外，我们进一步研究了Ufm1在巨噬泡沫细胞形成中的作用。发现ApoE-/-小鼠腹腔巨噬细胞中UFM1较野生对照组小鼠高表达。通过构建UFM1过表达和敲低的THP1稳转细胞系，分别给与oxLDL处理，观察巨噬泡沫细胞形成情况，发现UFM1过表达组较对照组胆固醇流出受体ABCA1和ABCG1的表达上调，胆固醇流出量增加，泡沫细胞形成减少；UFM1敲低组较对照组胆固醇流出受体ABCA1和ABCG1的表达下调，胆固醇流出量减少泡沫细胞形成增加。通过对ABCA1和ABCG1上游转录因子LXRα核转位及转录活性的检测，发现UFM1可以促进LXRα入核并增强LXRα的转录活性，利用siRNA干扰LXRα的表达后，UFM1初进ABCA1、ABCG1表达及胆固醇流出的作用受到抑制，说明UFM1缓解oxLDL诱导的巨噬泡沫细胞形成的作用是LXRα依赖的。. 综上所述，我们的研究发现，UFM1具有缓解内质网应激诱导的巨噬细胞凋亡、减弱巨噬细胞非特异性吞噬能力、增强其侵袭能力的作用，同时，UFM1也具有促进巨噬细胞胆固醇流出、抑制oxLDL诱导的巨噬泡沫细胞形成的作用。在后续的研究工作中，我们拟采用免疫共沉淀技术结合液相色谱-质谱串联技术进行高通量的UFM1修饰靶蛋白的鉴定，以期找到UFM1参与巨噬细胞凋亡及巨噬细胞泡沫化进程中的直接靶点，为糖尿病大血管病变的治疗提供新的思路。