高密度脂蛋白糖基化修饰影响细胞胆固醇外排的分子机制

Diabetes can influence the protective effect of high density lipoprotein (HDL) on cardiovascular system by modifying its glycation. Our unpublished research on 26 plasma samples of diabetic demonstrated that the HDL either from type 1 diabetic with a lower HDL-C or type 2 diabetic with a little higher HDL-C exhibit dramatically different effect on [H3]-labeled cholesterol efflux from RAW 264.7 macrophage, as 15 samples significantly decreased cholesterol efflux, 7 smaples showed no impact, and 4 samples markedly increased [H3]-cholesterol efflux. And in vitro non-enzymatic catalyzed HDL glycation by glucose or fructose, the complex also known as advanced glycation end products (AGEs), notably reduced the [H3]-cholesterol efflux. Therefore, we presume that the effect of HDL glycation on cellular cholesterol efflux is not limited on AGEs, the modification of its oligosaccharides on the two major carriers of carbohydrate,apolipoprotein (apo) and gangliosides (GLS), should have important impact on cellular cholesterol efflux, especially for sialic acid on the non-reducing end of the oligosaccharides. Based on our earlier work on sample collection, HDL subfraction separation, release of the O-linked oligosaccharides, monosaccharide analysis of HDL2 and HDL3, apoA-I and apoA-II preparation by anion exchange chromatography (Q-Sepharose Fast Flow) linked to Fast Protein Liquid Chromatography (?KTA-FPLC), total lipid extraction by Folch method, we plan to elucidate the glycation modification difference of HDL between diabetic and healthy people by liquid chromatography tandem mass spectrometry, gas chromatography mass spectrometry, methylation analysis as well as tool enzymes.In order to further elucidate the underlying relationship betweent HDL glycation and cellular cholesterol efflux, we also design to modify the glycation of HDL by cleaving the HDL oligosaccharides by special enzymes (such as neuraminidase, α-mannosidase, peptide-N-glycanase F or endoglycosidases) and in vitro non-enzymatic catalysis, all of which will be further used to ensure which kind of glycation modification could markedly influence HDL mediated cellular cholesterol efflux and the underlying mechanisms.These expected results may provide evidence for the intervention target of HDL glycation modification.

糖尿病可改变高密度脂蛋白（HDL）糖基化修饰进而影响其对心血管系统的保护作用。申请人前期研究发现：与健康人相比，无论是HDL-C水平较低的I型糖尿病患者还是HDL-C水平略有升高的II型糖尿病患者，他们的HDL对RAW264.7巨噬细胞[H3]-胆固醇外排皆有不同程度的影响；而体外非酶催化HDL所得晚期糖基化终产物（AGEs）则可显著抑制细胞胆固醇外排。申请人认为，HDL糖基化修饰影响细胞胆固醇外排并不局限于AGEs,其寡糖链的修饰特别是唾液酸的修饰对细胞胆固醇外排有重要影响。基于前期样本收集、载脂蛋白分离、寡糖链释放、单糖组成分析等工作，申请人拟采用液相串联质谱、气相质谱、甲基化分析、工具酶等研究手段阐释糖尿病患者与健康人HDL寡糖链差异，并利用特异性糖苷酶去糖基化和非酶促糖基化，探讨HDL何种糖基化修饰显著影响细胞胆固醇外排及其分子机制。期待为锁定HDL糖基化修饰的干预靶点提供依据。

糖尿病可改变高密度脂蛋白（HDL）成分，特别是糖基化修饰，进而影响其对心血管系统的保护作用。胆固醇逆向转运是HDL的主要生物学功能之一。目前，对于晚期糖基化终产物（AGEs）对HDL介导的胆固醇外排存在矛盾性的研究结果。大多数研究者认为AGEs可显著降低HDL介导的胆固醇外排；少数研究者认为AGEs对HDL胆固醇外排无影响，甚至升高。研究团队提出假说，除AGEs外，糖尿病患者HDL的成分改变，特别是正常的糖基化修饰是影响其介导的胆固醇外排的重要影响因素。. 该研究的重要发现之一是糖尿病患者HDL与正常人相比具有显著差异，特别是唾液酸化修饰；采用糖苷酶处理的结果显示：糖尿病患者唾液酸化修饰程度可显著影响其接纳胆固醇的能力，其可能的分子机制为唾液酸可增强胆固醇转运子ABCG1的表达。该研究的另一个重要发现是AGEs对HDL接纳胆固醇的能力影响不显著，而以往报道的糖尿病患者HDL接纳胆固醇能力的下降或可由于糖尿病患者HDL的成分改变所致（包括，蛋白成分改变，脂质陈分改变和糖基化修饰改变）；而某些报道糖基化修饰对HDL接纳能力无影响或源于糖尿病患者血浆唾液酸显著升高，而这种升高可上调疾病状态下功能失常的HDL的胆固醇接纳能力所致。外源性N-乙酰神经氨酸，可在一定程度上降低apoE-/-小鼠主动脉的脂质蓄积，特别是可显著降低血浆中的甘油三酯含量。人工合成的唾液酸化修饰的槲皮素，亦可发挥良好的心血管保护作用。.我们认为，糖基化修饰对HDL胆固醇外排有显著影响；唾液酸在糖尿病患者血浆中的显著增高是机体的一种补偿机制，其存在可在一定程度上减缓动脉粥样硬化的发生。其分子机制可能与以下几方面相关：①唾液酸携带的负电荷可减少脂蛋白及其他颗粒之间以及与血管内皮细胞的黏附；②唾液酸在HDL上的增多可上调疾病状态下功能失调HDL的接纳胆固醇的能力，从而促进胆固醇在机体内的逆向转运；③外源性补充唾液酸或亦可改善疾病状态下HDL功能，这种改善作用或部分源于其抗氧化功能。. 研究期间发表相关论文十余篇，其中SCI论文5篇，另有两篇在审。申请专利1项 (尚未授权)。