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扬州大学国际合作团队在柳叶刀杂志子刊《EBioMedicine》上发表研究成果
发布日期:2018-09-26浏览次数:字号:[ ]

 

  -Press ReleaseA new animal model for hypercholesterolemia research

    近日,扬州大学兽医学院和江苏省动物重要疫病与人兽共患病防控协同创新中心成勇教授、扬州大学转化医学研究院梁景岩教授、日本山梨大学医学院范江霖教授为通讯作者,扬州大学兽医学院和江苏省动物重要疫病与人兽共患病防控协同创新中心博士研究生陆睿、扬州大学转化医学研究院袁婷婷、王英歌为并列第一作者,联合在Lancet子刊EBioMedicine发表题为“Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7” 的研究长文(Research Article),为家族性高胆固醇血症的研究与治疗提供了崭新的疾病动物模型。

 

   

    受该杂志推荐,特发此新闻公告(Press release)。

    家族性高胆固醇血症(Familial hypercholesterolemia)是人类最常见的遗传性疾病之一。其特征是出生时低密度脂蛋白胆固醇(LDL-C)异常升高,如不及时治疗会导致过早的动脉粥样硬化、心血管疾病(CVD)发生,且致死年龄较低。多数情况下,这种疾病都是由位于常染体上的低密度脂蛋白受体(LDLR)基因突变引起的。

    动物模型在研究发病机理和治疗药物中起着极为重要的作用,目前临床前研究常用的高脂血症模型主要为低密度脂蛋白受体(LDLR)或载脂蛋白EApoE)基因敲除小鼠,然而小鼠与人类的脂代谢有较大差异且需要高脂饮食诱导,并不能较好的评价药物的疗效。家兔与人类脂代谢机制相似度较高,是合适的动物模型,但目前仅有一种自然突变产生的自发性高血脂兔(WHHL兔)可供研究者使用。由于缺乏胚胎干细胞,制备基因敲除兔的技术难题一直不能破解,新的基因编辑技术的出现使此难关已成为历史。

    该研究使用CRISPR/Cas9技术成功制备了低密度脂蛋白受体双等位基因敲除兔。研究者设计了识别LDLR基因第七外显子(对应LDLREGF-A区)的sgRNA,对成功获得的七只基因敲除兔分析结果表明,突变位点均位于预想区域。根据对转录和翻译结果的预测,可将突变分为两种类型,翻译提前终止与氨基酸插入/缺失/置换。这两种类型均可导致细胞表面功能性LDLR含量降低,进而使得血液内低密度脂蛋白大量积累,形成高脂血症。研究者对七只正常饮食下LDLR基因敲除兔血浆胆固醇分析的结果表明,血浆中总胆固醇(TC)、甘油三脂(TG)以及低密度脂蛋白胆固醇(LDL-C)均有不同程度的提高,而高密度脂蛋白胆固醇(HDL-C)均有明显的下降。对其中三只LDLR基因敲除兔的病理分析显示,兔主动脉和冠状动脉均产生严重的动脉粥样硬化病变,严重程度与血脂高低成正相关,且部分伴有严重的黄色瘤。

    世界范围内家族性高胆固醇血症发病率超过了1/500,已查明有超过1700种低密度脂蛋白受体突变,但我们因为研究工具的缺乏仍然对其所知甚少。本研究制备的LDLR基因敲除兔为今后深入研究家族性高胆固醇血症、动脉粥样硬化以及新一代降血脂药物的研发提供了合适的疾病动物模型,具有广泛的临床转化前景。

该研究成果受益于扬州大学推进国际科研合作与学科交叉等相关举措,并受国家重点研发计划国际创新合作重点专项、转基因重大专项、国家自然科学基金委疾病动物模型专项等项目资助。

 

 

 Press release

A new animal model for hypercholesterolemia research

(Rabbits with deficiency of LDLR)

Researchers from College of Veterinary Medicine and Medical College, Yangzhou University and Faculty of Medicine, University of Yamanashi established a new animal model for studying familial hypercholesterolemia (FH). The team established rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7 using Crispr/Cas9. The rabbits spontaneously developed severe hypercholesterolemia and atherosclerosis lesions on normal chow diet. The work has been published online in the international scientific journal EbioMedicine(2018.09.19).

About familial hypercholesterolemia

Familial hypercholesterolaemia is one of the most common inherited metabolic diseases in humans and can affect individuals from all ethnic groups. When left untreated, this genetic disorder of cholesterol metabolism can lead to premature atherosclerosis, cardiovascular disease (CVD) and often death at a very young age. The low-density lipoprotein receptor (LDLR) plays a key role in the regulation of cholesterol homeostasis. When the LDLR is defective, low-density lipoprotein (LDL) cannot enter cells by receptor-mediated endocytosis and the lipoprotein accumulates in plasma, eventually producing atherosclerosis.

Study method and findings

The team established rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7 using Crispr/Cas9. The genomic DNA sequences, theoretical amino acid sequences, and tertiary structure of the LDLR knockout alleles were analyzed. Mutations can be divided into two groups: frame-shift mutation and deletion/insertion, both of which resulted in reduced content of functional LDLR on the cell surface and lost the ability to eliminate LDL-C. As expected, on a NC diet, all the LDLR-KO rabbits showed hyperlipidemia and their plasma TC and LDL-C levels were significantly higher than those of wild-type rabbits. The team sacrificed three LDLR-KO founder rabbits maintained on a NC diet and all of them showed different degrees of aortic lesions (red areas stained with Sudan IV). Micrographs of aortic atherosclerosis lesions indicated typical fibrous plaques contained rich foam cells derived from macrophages, depositions of calcium and covered by thin fibrous caps. The team also checked coronary artery of the most severe rabbit and found advanced coronary artery atherosclerosis.

Research implications

The LDLR-KO rabbits with biallelic mutations via the CRISPR/Cas9 system anchored exon 7 spontaneously developed severe hypercholesterolemia and atherosclerosis on a normal chow (NC) diet. During the past two decades, LDLR-KO mice have been widely used for studying many facets of lipid metabolism and atherosclerosis. However, homozygous LDL receptor KO mice on a chow diet, TC levels have been found to be only mildly elevated (200–300 mg/dl) and developed no or only mild atherosclerosis.

There is currently only one strain of spontaneous endogenous hypercholesterolemia rabbit, the Watanabe heritable hyperlipidemic (WHHL) strain. However, more than 1700 human LDLR mutations have been reported worldwide. Limited hyperlipidemic rabbit models hamper the investigation of all these mutations, since the rabbits have features of lipoprotein metabolism more similar to those of humans than mouse. We can expect that the LDLR-KO rabbit model will provide new insights into the understanding of FH and atherosclerosis and expand the power of the rabbit model for translational research in cardiovascular disease.

Acknowledgements

    This work was supported by the National Key Research and Development Program of China, the National Natural Science Foundation of China, the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology, Japan.

Research Contact

Yangzhou University:

Yong Cheng (chengyong@yzu.edu.cn)

Jingyan Liang (jyliang@yzu.edu.cn);

University of Yamanashi:

Jianglin Fan (jianglin@yamanashi.ac.jp)

Journal article

Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7

Full article link:

https://doi.org/10.1016/j.ebiom.2018.09.020

 




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