壳寡糖降血脂作用及与RAP的表达相关性研究

Research on Chitosan Oligosaccharide Effect on Blood Lipid Level in Hypercholesterotemic and Its Correla-tionship with of Expression of Receptor-associated Protion

目的:研究壳寡糖降脂作用和受体相关蛋白RAP表达与壳寡糖在降血脂作用中的关系。方法:采用RT-PCR方法克隆RAP基因片段,插入表达载体PGEX获得了重组基因PGEX-RAP,转化入大肠杆菌DH5a,通过IPTG诱导表达了GST- RAP重组融合蛋白。运用单抗制备技术制备抗RAP单克隆抗体McAb-rap。用新西兰家兔制备高脂血症动物模型。分组分别观察和比较服用壳寡糖和辛伐他汀的降脂作用,TC、LDL-C含量的动态变化;观察实验各组RAP的表达量变化并进行分析。结果:体外成功诱导表达并纯化了GST-RAP融合蛋白,获得了单克隆抗体McAb-rap.其能识别天然膜抗原RAP。壳寡糖、辛伐他汀均能降低血清中TC、LDL-C浓度;壳寡糖降脂效果不如辛伐他汀显著(P<0.01);实验各组均表达RAP,但表达量各有差异。在高脂血症动物模型血管壁、心肌中RAP的表达量明显高于正常对照组。口服壳寡糖后高脂模型家兔血管壁、心肌中RAP表达量高于口服辛伐他汀组。在实验模型各组血管壁出现一条分子量约80 kD左右蛋白特异带,在正常家兔血管壁中未见。结论:壳寡糖具有一定的降血脂作用;血脂紊乱可引起血管壁、心肌RAP的表达增加;RAP的表达量与血脂水平的调控、壳寡糖在高脂血症中的降脂作用机制密切相关。80 kD左右特异表达带的蛋白性质及在血脂调节中的作用有待进一步研究。

Objective：To study the effects of Chitosan oligosaccharide on reducing the blood lipid in hypercholesterotemic in rabbits and Expression of Receptor-associated Protion（RAP） is related with regulated blood lipid. Methods： One, fragment of gene （RAP） was gotten by RT-PCR and the recombinant plasmid （PGEX-RAP） was constructed by inserting the RAP-cDNA into the PGEX. The the recombinant plasmid was transfected into DH5α and expressed the GST-RAP fusion protein by IPTG inducing. After the fusion protion purificated, the single-clone antibody against RAP was obtained by single clone technique. The hypereholesterotemic model was established in rabbits. Chitosan oligosaccharide and simvastatin Tablets was taken respectively into the model groups, mensurating level of TC and LDL-C level in models. The level of expression of RAP was analysed with Western-bolt technique. Results： GST-RAP fusion protein was obtained and purificated. Obtaining the single-clone antibody （McAb-rap） can recognize natural RAP. Chitosan oligosaccharide can play clown TC and LDL-C level in hypercholesterotemic. Compared with control rabbits, the expression of RAP in cardiac muscle,vascular wall were increased in rabbits of hypercholesterotemic group. The level expression of RAP in cardiac muscle, vascular wall in Chitosan oligosaccharide group were more than in simvastation Tablets group. In addition, it＇s discoving a exceptional protein band about 80KD In hypercholesterotemic groups. That was＇t shown in the controls. Conclusion：Chitosan oligosaccharide play a certainal effect in reducing TC and LDL-C level in hypercholesterolemie. Turbulence of lipid were increased expression of RAP in cardiac muscle,vascular wall in rabbits. Tbe expression were correlatived with the role of Chitosan oligosaccharide reducing lipid or its mechanism. The effect of a discovering exceptional protein band the in 80KD is＇t clear now.