尿酸酶基因敲除模型大鼠的研制

Urate oxidase (Uox, uricase)- deficient (Uox-/-) animals are ideal model animals for studying hyperuricemia and gout and evaluating uric-acid-lowering drugs. So far, there are no model rats with Uox-deficiency though a model mouse of C57BL/6J was generated. However, the model mice should be housed in a sterile environment with a low rate of survival (40%) for 63 weeks, which make it difficult for scientists to use the animal widely. Therefore, the present project will create a Uox-deficient rat to overcome the shortcomings. Uox gene of Sprague-Dawley rat will be precisely knocked out with CRISPR/Cas9 technology, and when Uox+/- rats were generated, the male and female will mate each other to produce Uox-/- offsprings. The genetically stable Uox-/- rats will be generated by making Uox-/- rats mate themselves for three or more generations. As for the stable Uox-/- rats, their genetic characteristic, quality of life, blood biochemistry, and renal microscopy will be evaluated. In order to ensure their high rate of survival, the strategies associated with decreasing uric acid synthesis, promoting uric acid degradation via intestinal tract, increasing uric acid solubility and excretion via renal tubules will be introduced. Eventually, the project will obtain genetically stable Uox-/- rats, and more than 80% will survive for one year in a clean environment rather than in a sterile one. This research will provide the rats which are highly similar to that of human in uric acid metabolic pathway with low expenditure for studying hyperuricemia and gout . This research will also provide reliable experimental basis for prevention and treatment of hyperuricemia and gout.

尿酸酶缺失模型动物是研究高尿酸血症和痛风、评价降尿酸药物的理想实验动物，但目前尚无尿酸酶缺失（Uox-/-）大鼠模型动物。最近虽成功建立了尿酸酶缺失的小鼠模型动物，但需无菌环境饲养且存活率低，很难推广使用。本项目以Sprague-Dawley大鼠为研究对象，采用CRISPR/Cas9技术精确敲除Uox基因，获得Uox+/-大鼠，通过反复交配获得Uox-/-大鼠，将Uox-/-大鼠再反复交配获得遗传性状稳定的尿酸酶缺失模型大鼠。采用分子生物学等技术对尿酸酶缺失大鼠从遗传、生命状况、血生化、肾功能等方面进行评价。为确保模型大鼠在清洁环境中1年成活率达80%以上，本研究必要时将对Uox-/-大鼠采用抑制尿酸生成、促进尿酸从肠道降解、增加肾小管尿酸溶解和排泄的新措施。本项目为高尿酸血症和痛风的研究提供与人尿酸代谢最接近且饲养管理成本较低的模型大鼠，为防治高尿酸血症和痛风提供确实可靠的实验基础。

尿酸酶缺失模型动物是研究高尿酸血症和痛风、评价降尿酸药物的理想实验动物，但目前尚无尿酸酶缺失（Uox-/-）的大鼠模型动物。最近虽成功建立了尿酸酶缺失的小鼠模型动物，但需无菌环境饲养，且存活率低，很难推广使用。本项目以Sprague-Dawley大鼠为研究对象，采用CRISPR/Cas9技术精确敲除Uox基因，获得尿酸酶基因敲除模型大鼠，并对其进行表型鉴定、分子鉴定及生物学评价。重要结果：1. 所研制的尿酸酶基因缺失模型大鼠已经繁育到第5代，在出生数、成活率、活力、饮食情况、性别比、寿命等方面与野生型SD大鼠无差异，6周时体重与野生型SD大鼠相比偏轻。尿酸酶基因缺失模型大鼠血清尿酸值明显高于野生型SD大鼠。其回肠、胰腺、肺、胃、肾上腺中的尿酸值比野生型SD大鼠明显升高，其他组织尿酸值无明显差异。尿酸酶缺失模型大鼠体内甘油三酯含量低于野生型SD大鼠、总胆固醇含量高于野生型SD大鼠、高、低密度脂蛋白含量与野生型SD大鼠体内含量无明显差异。尿酸酶缺失模型大鼠肾脏存在轻度损伤；2. 尿酸酶基因缺失模型大鼠体内无尿酸酶活性；外显子测序显示尿酸酶基因缺失模型大鼠体内尿酸酶基因3号外显子已被敲除；基因测序显示尿酸酶基因缺失模型大鼠体内尿酸酶基因2-4号外显子已被敲除；Western Blot实验结果表明尿酸酶基因缺失模型大鼠体内尿酸酶表达量降低。本研究研制的尿酸酶缺失模型大鼠为高尿酸血症和痛风研究及其新药研发提供一种稳定且经济的模型动物，为防治高尿酸血症和痛风研究提供坚实可靠的实验动物基础。