【作者】 谢丹；

【导师】 陈友香； 夏振信；

【作者基本信息】 武汉大学 ， 内科学， 2005， 硕士

【摘要】 目的:采用链脲佐菌素（streptozotocin,STZ）腹腔注射所致糖尿病大鼠为动物模型,以苯那普利和当归补血汤作为阳性对照药物,探讨益气活血法及组方归芪方的疗效,观察其对糖尿病（diabetes mellitus,DM）大鼠肾脏转化生长因子β₁（transforming growth factor β₁,TGF-β₁）mRNA表达、血清及肾脏肝细胞生长因子（hepatocyte growth factor,HGF）的影响,为糖尿肾病（diabetic nephropathy,DN）的临床治疗提供新思路。 方法:127只Wistar大鼠,其中106只腹腔注射STZ60mg/kg,72h后取尾静脉血测空腹血糖,血糖≥16.7mmol/L几者为糖尿病大鼠,纳入实验:另外21只大鼠列为正常对照组,注射相当体积的柠檬酸缓冲液。糖尿病大鼠随机分为4组:糖尿病模型组、苯那普利组、当归补血汤组和归芪方组,分别给予自来水、苯那普利10mg/kg、当归补血汤3.6g/kg、归芪方4.5g/kg灌胃,每日一次。正常组给予相同体积的自来水灌胃。各组大鼠分别于实验第4、6、8周末分批处死,称取体重。处死前1～2d将大鼠置洗净的金属代谢笼收集24h尿,用放射免疫法测定尿微量白蛋白及β₂-微球蛋白（β₂-microglobin,β₂-MG）。腹主动脉取血,测血糖、血甘油三酯（triglyceride,TG）、胆固醇（cholesterol,CHO）、高密度脂蛋白（high density lipoprotein,HDL）、血肌酐（serum creatinine,Scr）、尿素氮（blood urea nitrogen,BUN）及内生肌酐清除率（endogenous creatininee clearance rate,Ccr）。处死后迅速剖腹取出双肾,剥离肾包膜,快速剔除结缔组织等,分离出肾皮质,生理盐水漂洗,微量电子天平称量肾重后,右肾固定后石蜡包埋,切成6μm厚的切片,行HE染色和PAS染色,采用医学图像分析系统测定平均肾小球面积（average glomerular area,GA）;左肾皮质部分取1mm×1mm×1mm大小用戊二醛固定,待行透射电镜检查并测定肾小球基底膜平均厚度（mean thickness of glomerular basement membrane,MGBM）;原位杂交法检测肾皮质TGF-β₁mRNA表达变化。酶联免疫吸附法（ELISA）检测血清及肾组织中HGF含量的变化。 统计学方法:用SPSS11.0统计软件进行分析。数据以均数±标准差（（?）±s）表示,多组资料间比较,方差齐者,采用单因素方差分析,LSD或SNK多重比更多还原

【Abstract】 Objective In the present study, the clinical effects of Chinese Medical therapy of YiQiHuoXue method （invigorating Qi-activating the blood） and Gui Qi Mixture （GQM） was investigated and compared with Benazepril and Danggui Buxue Decoction （DBD）in streptozotocin-induced diabetic rats mainly with respect to the effects on the expression of renal transforming growth factor- β₁（TGF- β₁） mRNA and levels of renal and serum hepatocyte growth factor（HGF）. It will provide us new insights and therapies for the clinical practices to diabetic nephropathy.Methods Diabetes was induced by the intraperitoneal administration of streptozotocin （STZ, 60mg/kg body weight）. Tail vein blood glucose levels were measured after 72h. Animals with blood glucose levels of more than 16. 7mmol/l were selected as diabetic rats and then randomly divided into four groups: （1） Benazepril-treated diabetic rats: received 10mg/kg Benazepril orally once a day by gastric tube; （2） Danggui Buxue Decoction-treated rats: received 3. 6g/kg DBD;（3） Gui Qi Mixture-treated rats: received 4. 5g/kg GQM; （4）diabetic control rats: received an equal volume of drinking water. At 4,6,8 weeks after STZ injection, the animals were housed in individual metabolic cages for 24h to obtain urine for the measurement of albumin and β₂-microglobin（β₂^MG） by radioimmunoassay. The urine creatine （Ucr） was also determined. Plasma was collected under 5% pentobarbital anesthesia （30mg/kg body weight ） and blood glucose, TG, CHO, HDL, Scr and BUN were measured. The two kidney of rats were rapidly removed, carefully decapsulated. The right kidney was weighed and immersed in methyl Carnoy’ s fixative, embedded in paraffin, amd sectioned for histology. The sections （6μm） were stained with hematoxylin and eosin （HE） and periodic acid Schiff （PAS）, thehistological morphology and average glomerular area were analyzed. A part of left kidney tissue （lmmXlmmXlmm） was immersion fixed in HistoChoice MB for the study of microstructure and mean thickness of glomerular basement membrane. In situ hybridization was adopted to assay the expression of TGF- P , mRNA in renal constitution of rats. Glomerular and tubular positive findings were assessed by medical pathological image analysis system and semi-quantitative method. Concentration of HGF in serum and kidney were determined by enzyme-linked immunosorbent assay （ELISA）.Statistical analysis: Results were expressed as mean±S （x±s）. Comparisions among 5 groups were analyzed by one-way analysis of variance, followed by either LSD or SNK multiple comparision test to evaluate statistical difference. Value of urinary protein excretion were log transformed before statistical analysis. P < 0.05 is regarded as significant.Results: Diabetic rats with plasma glucose levels higher than 16. 7mmol/l were polyuric and failed to gain weight in the experience. Their level of TG, CHO were significantly higher than those of control rats, and HDL level was decreased（P< 0.01）. Both DBD and GQM could decrease the the high plasma glucose level and improve lipid metabolism disorders （P<0.01, P<0.05）. Neither hyperglycemia or hyperlipidemia were affected by Benazepril treatment （P>0.05） .Ccr and urinary albumin and P2^MG excretion in diabetic rats were gradually and significantly increased compared with that in control rats at 4 weeks（P < 0. 01, respectively）, and increased further with time（P<0.01）. There were no significant difference between diabetic group and normal group in level of Scr and BUN at 4 and 6 week （P>0. 05）. This two index were increased at 8 week （P<0.01）. Daily administration of Benazepril, DBD or GQM improved the renal function and reduced urinary albumin and 0 2^MG toa similar extent（P > 0.05）. Kidney weight/body weight ratio, mean glomerular area and mean thickness of glomerular basement membrane were significantly increased in untreated diabetic rats than in control rats（P<0.05, P<0.01） and were reduced by the treatment of diabetic rats with Benazepril and DBD compared with the index in untreated diabetic rats（P<0. 01）. The index of hypertrophy of group GQM was lower than that of group Benazepril or group DBD. Compared with findings in control animals, the glomerular expression of TGF- P , genes in diabetic rats was significantly increased （P<0. 01）. And In situ hybridization showed that the positive position were major in glomerulus and tubular epithelial cells. Treatment with Benazepril or DBD significantly decreased the expression of TGF-3, mRNA in diabetic rats compared with findings in untreated diabetic rats （/’■■CO. 01） and there was no significant difference between this two groups （P>0. 05）. The expression of TGF-3 i mRNA in group GQM was lower than those in group Benazepril and DBD（/K0. 01）. Diabetic rats have increased serum HGF levels compared with controls （P<0. 01） and the HGF levels were not significantly different between the three stages （P>0. 05）. HGF level in the diabetic kidney transiently increased but then declined （P<0. 01）. The excretion of renal and serum HGF in GQM , DBD or Bnazepril treated group were significantly higher than those in the diabetic control group （P<0. 01） and there were no significant difference between these three groups（P>0. 05）. Conclusion:1. Diabetic mellitus and renal lesions occurred in four diabetic groups after STZ were used.2. GQM could improve lipid metabolism disorders , decrease the high plasma glucose level and the excretion rate of the 24h urine albumin andP2-MG ,and relieve the renal lesion.3. TGF-P! that produced in kidney may serve as major mediators ofthe renal changes observed in experimental diabetes. HGF level in the diabetic kidney transiently increased but then declined. Renal HGF but not serum HGF could reflect the progression of DN.4. GQM could up-regulate the expression of HGF in renal tissue and was superior to Benazepril and DBD in reduction the expression of renal TGF- 3 i mRNA and that could be the reasons for its renal-protecting effects in preventing nephropathy process.更多还原