体外合成的ABC转运蛋白2 mRNA在小鼠体内表达促进尿酸排泄

The ABCG2 mRNA Synthesized in vitro Promoted Uric Acid Excretion in Mice

高尿酸血症是指嘌呤代谢紊乱尿酸在体内堆积所导致的慢性代谢疾病,近年来其发病率增高且发病年龄呈现年轻化趋势,寻找有效的治疗靶点以及治疗方法是当前研究的热点。尿酸盐转运蛋白ABC转运蛋白2(ATP-binding cassette subfamily G member 2,ABCG2)主要表达于肾,促进尿酸排泄。本研究通过体外合成ABCG2 mRNA,将其转染至高尿酸血症模型小鼠体内,观察对小鼠尿酸的影响及其相关作用。化学合成ABCG2 mRNA的DNA模板,体外转录mRNA修饰后转染到小鼠肾小管上皮细胞TCMK-1,Western印迹检测细胞中的蛋白质表达。结果显示,TCMK-1细胞中的蛋白质表达量与mRNA转染量呈正相关(P<0.01),提示转染成功。动物实验,将24只SPF级小鼠随机分为正常组、高尿酸血症模型组、苯溴马隆治疗组[20 mg/(kg·d)]和mRNA治疗组[2 mg/(kg·3d)],共4组(n=6)。边造模边治疗持续28 d,治疗结束后检测其血尿酸、尿液中尿酸、血肌酐、血尿素氮和肝中黄嘌呤氧化酶指标。结果显示,与模型组小鼠相比,mRNA治疗能显著降低小鼠血尿酸(100.38±10.94)及血尿素氮(6.30±1.10)、肌酐(30.86±5.78)水平(P<0.05或P<0.01),升高小鼠尿液尿酸(617.48±50.34)水平(P<0.05),促进尿酸排泄,对肝中黄嘌呤氧化酶活性(26.19±2.58)无显著影响。HE染色观察小鼠肾病理结构改变。结果显示,与模型组小鼠相比,mRNA治疗组小鼠肾内肾小管细胞水肿、炎细胞浸润等病理损伤得到明显改善。qRT-PCR检测小鼠肾组织mRNA相对表达量,免疫组化、Western印迹检测小鼠肾ABCG2蛋白质表达水平。结果显示,mRNA组小鼠肾组织中ABCG2 mRNA及其蛋白质相对表达量明显上调(P<0.01)。综上所述,体外修饰合成的ABCG2 mRNA可以在高尿酸小鼠模型体内成功表达,并促进尿酸及其它有机离子排泄,同时改善小鼠肾损伤。该结果为临床利用ABCG2基因作为治疗高尿酸血症相关疾病的靶点提供了实验依据。

Hyperuricemia is a chronic metabolic disease caused by the accumulation of uric acid in the body caused by purine metabolism disorder.In recent years,the incidence of hyperuricemia has increased and the age of onset is showing a younger trend.Finding effective therapeutic targets and treatment methods is a hot spot of current research.The urate transporter ATP-binding cassette subfamily G member 2(ABCG2)is mainly expressed in the kidney and promotes uric acid excretion.In this study,ABCG2 mRNA was synthesized in vitro and transfected into hyperuricemia model mice to observe its effect on mouse uric acid levels.Firstly,the DNA template of ABCG2 mRNA was chemically synthesized,and then transcribed into mRNA in vitro,followed by modification and transfection into mouse TCMK-1 renal tubular epithelial cells.Finally,the protein expression in the cells was detected by Western blot.The results showed that the amount of protein expression in TCMK-1 cells was positively correlated with the amount of transfected mRNA(P<0.01),indicating a successful transfection.In animal experiments,twenty-four SPF mice were randomly divided into four groups(n=6):control group,hyperuricemia model group,benzbromarone group[20 mg/(kg·d)]and mRNA group[2 mg/(kg·3 d)].The mice have been modeled and treated for 28 days.During this period,the body weight and growth status of the mice were monitored daily.After the treatment,the levels of serum uric acid,urine uric acid,serum creatinine,blood urea nitrogen and liver xanthine oxidase were analyzed.The results showed that compared with the model group of mice,mRNA treatment can significantly reduce the levels of serum uric acid(100.38±10.94),blood urea nitrogen(6.30±1.10),and serum creatinine(30.86±5.78,P<0.05 or P<0.01).It can also increase the level of urine uric acid(617.48±50.34,P<0.05)in mice and promote the excretion of uric acid.But it has no significant effect on the activity of xanthine oxidase(26.19±2.58)in the liver.The pathological changes of mice kidney were observed by HE staining.The results showed that compared with mice in the model group,pathological damages such as renal tubular cell edema and inflammatory cell infiltration in the mRNA treatment group were significantly improved.The relative expression of mRNA in mice kidney was detected by qRT-PCR,and the protein expression of ABCG2 in mice kidney was detected by immunohistochemistry and Western blot.The results showed that the relative expression of ABCG2 mRNA and its protein were significantly up-regulated in the kidney tissues of mice in the mRNA group(P<0.01),indicating that the transfection was successful in vivo.In conclusion,ABCG2 mRNA synthetized and modified in vitro can be successfully expressed in hyperuricemia mice and promote excretion of uric acid and other organic ions,as well as improvement of renal injury in mice.These results provide experimental basis for the clinical application of ABCG2 as a target for the treatment of hyperuricemia related diseases.