一种Parkin基因敲除小鼠肾虚证动物模型的建立

Establishment of an Animal Model of Parkin Gene Knockout Mice with Kidney Deficiency Syndrome

目的:构建Parkin基因敲除(knock out,KO)小鼠肾虚证动物模型。方法:本研究设置对照组、Parkin KO组、氢化可的松组,观察并记录小鼠一般情况,完善行为学(旷场实验、轮替实验和新臂探索实验)、检测血清指标(生长激素水平、促肾上腺皮质激素、皮质醇、TSH、T3、T4、雌二醇、睾酮),分析各组小鼠临床表现和相关血清指标与肾虚症是否相符。两组间检测均数的比较采用t检验,多组间比较采用单因素方差分析。结果:Parkin KO组、氢化可的松组表现为拱背蜷缩、反应迟钝、自主活动减少、倦怠、精神萎靡、饮食减少、畏寒喜暖等,与肾虚症候相符,KO组表现更加明显。KO组生长激素水平较对照组明显下降(P<0.01)。旷场实验中,KO组直立探索次数、总探索距离、运动速度明显下降(P<0.01),氢化可的松组直立探索次数明显下降(P<0.01);自发轮替实验中KO组总的进臂次数显著降低(P<0.01),氢化可的松组小鼠总的进臂次数也明显降低,KO组下降更加明显;新臂探索实验中,KO组新臂探索次数及总进臂次数明显降低(P<0.05),新臂停留时间显著降低(P<0.01);上述指标提示KO组、氢化可的松组小鼠对外界事物的自发探索能力、自发活动能力空间学习记忆能力、工作记忆能力显著降低,KO组下降更加明显。与对照组比较,KO组雌鼠雌二醇(E2)水平(P<0.05)、E2/T水平(P<0.01)、产仔量(P<0.01)均显著下降,氢化可的松组雌鼠产仔量明显下降(P<0.05),KO组雄鼠血清睾酮(T)水平明显下降(P<0.01),血清E2水平(P<0.05)、E2/T水平(P<0.01)显著升高,提示KO组、氢化可的松组性腺功能降低,生殖能力下降,且KO组下降更加明显。KO组促肾上腺皮质激素、TSH、T3、T4显著降低(P<0.01),皮质醇也明显降低(P<0.05),氢化可的松组促肾上腺皮质激素、皮质醇、TSH、T4均明显下降(P<0.05),KO组下降更明显。与对照组比较,KO组小鼠肾组织Parkin无表达,氢化可的松组Parkin表达呈下降趋势。结论:Parkin基因敲除鼠可作为一种理想的肾虚证动物模型。

Objective:To construct an animal model of Parkin gene knockout mice with kidney deficiency syndrome.Methods:In this study,a control group,a Parkin KO group,and a hydrocortisone group were set up to observe and record the general condition of mice,complete behavioral studies(open field test,Spontaneous alternatin test and novel arm exploration test),and detect serum indicators(growth hormone levels,Adrenocorticotropic hormone,cortisol,TSH,T3,T4,estradiol,testosterone),analyze whether the clinical manifestations and related serum indexes of mice in each group are consistent with kidney deficiency.T-test was used for the comparison of the detection mean between two groups,and one-way ANOVA was used for the comparison between multiple groups.Results:The Parkin KO group and the hydrocortisone group showed arch-back,curl up,slow response,reduced autonomic activity,burnout,listlessness,reduced diet,afraid of the chills and like warmth,etc.which was consistent with the kidney deficiency syndrome,and the KO group performance was more obvious.The growth hormone level in the KO group was significantly lower than that in the control group(P<0.01).In the open field test,the number of rearing,total exploration distance,and movement speed of the KO group decreased significantly(P<0.01),and the number of upright exploration of the hydrocortisone group decreased significantly(P<0.01);In the spontaneous rotation test,the total number of arm advances in the KO group was significantly reduced(P<0.01),the total number of arm advances in the hydrocortisone group was also significantly reduced,and the decrease in the KO group was more obvious;In the new arm exploration experiment,the number of new arm explorations and the total number of arm advances in the KO group were significantly reduced(P<0.05),and the residence time of the new arm was significantly reduced(P<0.01);the above indicators suggested that the spontaneous exploration ability,spontaneous activity ability,spatial learning and memory ability and working memory ability of external things of the mice in the KO group and hydrocortisone group were significantly reduced,and the KO group's decline was more obvious.Compared with the control group,the estradiol(E2)level(P<0.05),E2/T level(P<0.01),and the number of litter(P<0.01)of female rats in the KO group were significantly decreased.The the number of litter of the females in the hydrocortisone group decreased significantly(P<0.05).The serum testosterone(T)levels of male rats in the KO group decreased significantly(P<0.01),the serum E2 levels(P<0.05)and E2/T levels(P<0.01)increased significantly High,suggesting that the KO group and hydrocortisone group had reduced gonad function and decreased reproductive capacity,and the KO group had a more obvious decline.Corticotropin,TSH,T3,T4 were significantly decreased in the KO group(P<0.01),and cortisol was also significantly decreased(P<0.05).The corticotropin,cortisol,TSH,and T4 in the hydrocortisone group were significantly reduced(P<0.05),the decrease in KO group was more obvious.Compared with the control group,there was no Parkin expression in the kidney tissue of the KO group,and Parkin expression in the hydrocortisone group showed a downward trend.Conclusion:Parkin knockout mice can be used as an ideal animal model of kidney deficiency syndrome.