【作者】 李成乾；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 目的研究高嘌呤饮食及罗格列酮干预对OLETF大鼠血尿酸水平及肾小管上皮细胞有机阴离子转运体1(organic anion transporter 1,OAT1)、尿酸盐转运子(uratetransporter,UAT)基因表达的影响。方法30只4周龄雄性OLETF大鼠普通饮食喂养至第25周龄时,随机分为对照组和高嘌呤饮食组,其中对照组10只、高嘌呤饮食组20只。高嘌呤饮食组给予10%高酵母饲料喂养,同时给予腺嘌呤溶液灌胃(50mg/kg/d),对照组给予普通饮食,同时每日给予同体积蒸馏水灌胃。同系4周龄雄性LETO大鼠30只,作为OLETF大鼠的实验对照组,其分组及处理方法同上。监测大鼠血尿酸(SUA)、甘油三酯(TG)、总胆固醇(TC)、肌酐(CRE)、尿素氮(BUN)、24小时尿量及尿尿酸(UUA),放免法测定血胰岛素水平,比较两种大鼠高尿酸血症的发病情况。待OLETF大鼠高嘌呤饮食组与对照组两组间血尿酸水平出现显著差异时,进一步将高嘌呤饮食组随机分为两组,其中一组继续给予高嘌呤饮食(即高嘌呤饮食组),另一组在高嘌呤饮食基础上进行罗格列酮干预(即罗格列酮干预组),血尿酸在各组间有显著统计学差异时,处死所有大鼠。取大鼠肾皮质抽提总RNA,应用Taq Man实时荧光定量逆转录-聚合酶链反应(RT-PCR)技术共扩增OAT1、UAT和内参照GAPDH目的片段,分析计算OAT1、UAT mRNA相对表达量。取大鼠肾组织制备石蜡切片,进行荧光免疫组织化学染色,Simple PCI图像分析软件分析OAT1、UAT的蛋白表达水平。结果①高嘌呤饮食后第11天,OLETF和LETO高嘌呤饮食组的血尿酸水平均较对照组显著升高(P=0.000),OLETF高嘌呤饮食组较LETO高嘌呤饮食组亦显著升高(P=0.008);②OLETF大鼠高嘌呤饮食组中,高尿酸血症的患病率为84.21%,LETO大鼠患病率为36.84%,两组发病率具有显著差异(P＜0.05);③高嘌呤饮食后,OLETF大鼠高嘌呤饮食组OAT1 mRNA的表达水平较对照组明显降低(t’=21.690,P=0.000),LETO大鼠高嘌呤饮食组与对照组相比无显著性差异(t’=-1.541,P=0.162)。OLETF大鼠高嘌呤饮食组OAT1 mRNA的表达水平与LETO大鼠高嘌呤饮食组相比亦显著降低(P=0.000);④高嘌呤饮食后,LETO大鼠高嘌呤饮食组UAT mRNA的表达水平较对照组明显降低(t’=8.241,P=0.000),OLETF大鼠高嘌呤饮食组UAT mRNA的表达水平较对照组亦明显降低(t’=23.090,P=0.000),且其降低程度较LETO鼠更甚(P＜0.05)。⑤罗格列酮干预后第21天,高嘌呤饮食组血尿酸水平显著高于对照组(P=0.002),而罗格列酮干预组显著低于高嘌呤饮食组(P=0.047),与对照组相比无显著差异(P=0.183);⑥实时荧光定量结果显示:OLETF大鼠高嘌呤饮食组及罗格列酮干预组OAT1、UAT mRNA的表达水平均显著低于对照组(P＜0.05),罗格列酮干预组OAT1、UAT mRNA的表达水平较高嘌呤饮食组显著升高(P＜0.05),但仍明显低于对照组(P＜0.05)。⑦荧光免疫组化结果显示:对照组、高嘌呤饮食组及罗格列酮干预组肾脏近端小管上皮细胞均有OAT1、UAT的表达。Simple PCI软件分析结果示:高嘌呤饮食组OAT1、UAT的吸光度值较对照组明显降低(P＜0.05),罗格列酮干预组OAT1、UAT的吸光度值较高嘌呤饮食组明显升高(P＜0.05)。结论高嘌呤饮食可导致代谢综合征大鼠(OLETF大鼠)血尿酸水平的显著升高,且其高尿酸血症的发病率显著高于同系正常大鼠(LETO大鼠),其机制可能与高嘌呤饮食后OLETF大鼠OAT1、UAT的表达较LETO大鼠显著降低有关;罗格列酮可以显著降低OLETF大鼠高嘌呤饮食后的血尿酸水平,其机制可能为罗格列酮显著改善胰岛素抵抗,降低胰岛素水平,间接上调OAT1、UAT基因的表达,进而显著增加肾脏尿尿酸的排泄,降低血尿酸水平。目的观察不同浓度脂蛋白对肾小管上皮细胞(HK-2细胞)人尿酸盐转运子(humanurate transporter hUAT)mRNA和人尿酸盐转运蛋白(human organic aniontransporter 1,hOAT1)mRNA表达的影响表达的影响。方法所有实验均在体外培养的HK-2细胞上进行。根据培养液中所含脂蛋白,将HK-2细胞分为3大组。①单纯采用DMEM培养基组(对照组);②极低密度脂蛋白(VLDL)组:V1组:DMEM/F-12+50ug/ml VLDLV2组:DMEM/F-12+400ug/ml VLDL。③高密度脂蛋白(HDL)组:H1组:DMEM/F-12+50ug/mlHDL H2组:DMEM/F-12+400ug/ml HDL。每种条件下,均培养6瓶细胞取均数,培养48小时,收集细胞,计数,抽提总RNA。取IugRNA逆转录为cDNA,荧光定量PCR共扩增hUAT、hOAT1和内参照GAPDH目的片段,分析计算hUAT和hOAT1 mRNA相对表达量。整个实验过程重复一次。统计学分析采用t检验和单因素方差分析,显著性标准为P＜0.05。结果①所有标本均能检测到hUAT和hOAT1 mRNA的表达。②随着VLDL浓度的增加,hUAT mRNA表达水平明显下降;对照组hUAT mRNA表达水平明显高于V1、V2组,差异有显著性(P=0.000),hUATmRNA表达水平V1组和V2组间差异有显著性(P＜0.001)。③随着VLDL浓度的增加,hOAT1mRNA表达水平明显下降;对照组hOAT1 mRNA表达水平明显高于V1、V2组,差异有显著性(P=0.000)。V1和V2组间差异无显著性(P=0.927)④随HDL浓度的增加,hUAT mRNA表达水平下降;对照组hUAT 1mRNA表达水平明显高于H1、H2组,差异有显著性(P=0.000),H1组和H2组间差异有显著性(P＜0.05)。⑤随HDL浓度的增加,hOAT1 mRNA表达水平增加;对照组hOAT1 mRNA表达水平明显低于H1、H2组,差异有显著性(P=0.000),H1组和H2组间差异无显著性(P＞0.05)。结论VLDL,HDL水平升高所导致的hUATmRNA表达水平下调,可能是脂代谢紊乱引起高尿酸血症的重要机制。HDL对hOAT1 mRNA表达水平上调作用,提示HDL可能具有一定的增加尿酸排泄作用。更多还原

【Abstract】 Objective:To observe the effect of high purine diet and the intervention of rosiglitazone on the blood uric acid and the expressions of gene OAT1 and UAT in the renal epithelial cells of OLETF rats.Methods:30 male OLETF rats of 4 weeks age were raised up to 25 weeks age, and were divided randomly into control group（n=10）and high purine diet group（n=20）.The high purine diet group was administered with adenine（50mg/kg/d） by gavage and was fed ground standard rat chow containing 10%yeast.30 male LETO rats of 4 weeks age were processed the same way with OLETF.TG,TC,UA,CRE,BUN and 24h urine volume and UUA were detected.Insulin were detected by radioimmunity.The incidence rate of hyperuricemia was compared between the OLETF and LETO rats.Then devide the OLETF high purine diet group into high purine diet group（n=10） and intervention group（n=10）.Both were given high purine diet,besides the intervention group were given rosiglitazone（3 mg/kg/d） by gavage daily.Keep on monitoring the SUA until there were significant difference.Then extracte total RNA from the all rats’cortex of kidney.OAT1,UAT and GAPDH mRNA were detected by real time fluorescent quantification RT-PCR.The expression of OAT1,UAT were detected by immunohistochemistry.Results:①On the 11^th day of high purine diet, blood uric acid level of both OLTEF and LETO high purine diet group were significantly higher than their corresponding control group（P=0.000）.Moreover,blood uric acid of OLTEF high purine diet group was much higher than that of LETO high purine diet group（P =0.008 ）;②The incidence rate ofhyeruricemia of OLTEF high purine diet group（84.21%） was significantly higher than that of LETO high purine diet group（36.84%）（P＜0.05）;③On the 11^th day of high purine diet,OAT1 expression level was significantly decreased in the OLTEF high purine diet group compared with the OLTEF control group（t’ =21.690,P =0.000）.There was no difference between the high purine diet group and the control group in LETO rats（t’=-1.541,P =0.162）.OAT1 expression level of the OLTEF high purine diet group was significantly decreased than that of LETO high purine diet group（P =0.000）;④On the 11th day of high purine diet,UAT expression level of both OLTEF and LETO high purine diet group were significantly decreased than their corresponding control group（P =0.000）,UAT expression level of the OLTEF high purine diet group was significantly decreased than that of LETO high purine diet group（P=0.035）;⑤On the 21^th day of intervention,blood uric acid level of the intervention group was significantly decreased than that of the high purine diet group（P=0.002）,and there was no difference between the intervention group and the control group;⑥Both OAT1 and UAT expression level of the intervention group were significantly higher than that of the high purine diet group（P＜0.05 ）,but still were significantly decreased than that of the control group（P＜0.05 ）;⑦The expression of OAT1 and UAT were detected in all slices.Absorbance values of both OAT1 and UAT of the intervention group were significantly higher than that of the high purine diet group（P＜0.05）,but still were significantly decreased than that of the control group（P＜0.05）;Conclusion:（1） The incidence rate of hyeruricemia led by high purine diet in OLTEF rats is significantly higher than of LETO rats.It is probably because that under the same dosage of purine loads,OAT1 and UAT expression level of OLTEF rats is significantly decreased than that of LETO rats.（2） The high blood uric acid level of OLTEF rats led by high purine diet can be significantly reduced by rosiglitazone.It is probably because that rosiglitazone can indirectly up regulate the expression of OAT1 and UAT by improving insulin resistance. PART TWO Role of Lipid Metabolic Disorder in the Expression of hUAT and hOAT1 Gene in HK-2 CellsObjective:To observe the role of different concentration of lipoprotein and free fat acid in the expression of hUAT and hOAT1 mRNA in HK-2 cells.Methods:All experiments were performed with HK-2 cells cultured in vitro.According to the concentration of lipoprotein in culture medium,growth-arrested cells were devided into 3 groups.And according to the concentration of VLDL,HDL,cells treated with lipoprotein were devided into 1,2,group.①Control group:DMEM/F-12②VLDL group: V1:DMEM/F-12+50ug/ml VLDL.V2:DMEM/F-12+400ug/ml VLDL..③HDL group:H1: DMEM/F-12+50ug/ml HDL.H2:DMEM/F-12+40Oug/ml HDL.In each culture medium, 6 bottle of cells were cultured for 48h to get the mean.Then all were collected,counted and total RNA was extracted.1 ug mRNA was reverse transcripted to eDNA and hUAT, hOAT1 and GAPDH were amplified by real-time fluorescent quantitative PCR.Then relative expression quantity of hUAT and hOAT1 mRNA was analyzed.Repeat the whole experiment.The data was analyzed by t-test and one-factor ANOVA and P＜0.05 was considered significant.Results:①hUAT and hOAT1 mRNA could be detected in all the samples.②hUAT mRNA expression decreased with the increase of VLDL concentration after treated for 48h. The hUAT mRNA of control groups was significantly higher than that of V1,V2 group（P=0.000） and there was significant difference between V1,V2 groups（P＜0.05）.③hOAT1 mRNA expression decreased with the increase of VLDL concentration after treated for 48h.The hOAT1 mRNA of control groups was significantly higher than that of V1,V2 group（P=0.000） and there was no significant difference between V1,V2 groups（P=0.927 ）.④With the increase of HDL concentration,hUAT mRNA expression decreased after treated for 48h.hUAT mRNA of control group was significantly higher than that of H1,H2 groups （P=0.000）.There was significant difference between H1,H2 groups（P＜0.05）.⑤With the increase of HDL concentration,hOAT1 mRNA expression increased after treated for 48h. hOAT1 mRNA of control group was significantly lower than that of H1,H2 groups （P=0.000）.There was significant difference between H1,H2 groups（P＞0.05）. Conclusions:Increased concentration of VLDL,HDL down-regulate the expression of hUAT mRNA,which may be an important pathogenesis in hyperuricemia induced by lipid metabolic disorder.HDL can increase the expression of hOAT1 mRNA,Which indicate that HDL maybe can increase the excretion of uric acid.更多还原