STAT1-P53-P21通路在阿霉素肾病小鼠应激性衰老作用机制的研究

发布时间：2018-04-07 17:14

  本文选题：应激性衰老　切入点：阿霉素肾病　出处：《河北医科大学》2017年硕士论文

【摘要】：目的:通过检测STAT1、P53及P21在阿霉素肾病小鼠肾脏中的表达,研究应激性衰老在阿霉素小鼠肾病发生发展中的作用。进一步干预STAT1-p53-p21通路表达是否可调控衰老机制,从而延缓了阿霉素肾病小鼠的疾病进展。方法:将90只6周龄BALB/c雄性小鼠随机分为3组:正常对照组、阿霉素肾病模型组,缬沙坦治疗干预组。模型组与干预组于实验开始后分别从尾静脉注射阿霉素10mg/kg诱发应激衰老模型,对照组于实验开始后分别从尾静脉注射等容积生理盐水,放入代谢笼内饲养,自由摄食、饮水。存活的小鼠注射阿霉素后3d时检测尿蛋白,应用试纸法测定尿蛋白2+提示模型复制成功,计入实验,治疗组给予缬沙坦20mg/kg灌胃,1次/d;正常对照组及模型组给予等量生理盐水灌胃,各组分别在0、2、4、6、8周随机抽取6只小鼠收集24h尿液、血清及肾脏组织。考马斯亮蓝法检测24h尿蛋白定量,全自动生化仪检测生化指标:血清白蛋白(Albumin,ALB)、总胆固醇(Total cholesterin,TC)及肌酐(Creatinine,Cr)的含量。肾脏组织石蜡包埋后切片3μm,进行HE、PAS染色观察肾小球硬化程度及肾小管损害;免疫组织化学法检测肾组织STAT1、P53、P21蛋白的表达,新鲜肾脏组织提取RNA行RT-PCR检测肾脏STAT1、P53、P21m RNA的表达。所有数据应用SPSS 21.0统计分析软件对实验数据进行统计学处理。计量资料数据进行正态分布检验,符合正态分布的数据采用均数±标准差(x±s)表示,两组间的比较应用方差齐性检验后,采用t或t′检验,多组间的比较应用单因素方差分析后,采用LSD检验做各组间均数的两两比较,P0.05表示有统计学意义。结果:1造模后小鼠的一般情况模型组及药物干预组小鼠于阿霉素注射后第1周均出现精神不振,毛质粗糙及脱毛,活动量减少,进食差,有不同程度的腹泻及体重减轻,小鼠注射阿霉素后,其平均体重迅速下降,第2周降至最低,然后逐渐回升,但与正常组相比仍有显著差异。实验期间实验组4只小鼠因腹泻及消瘦死亡;药物干预组3只小鼠灌胃时出现消化道出血死亡;共死亡7只,死亡率为7.8%。2只复制模型失败被剔除。对照组小鼠健康存活。2阿霉素肾病小鼠各期24小时尿蛋白及血生化指标的变化模型组尿蛋白于第2周明显升高且达到峰值,第4、6、8周与2周相比,尿蛋白稍有下降,与同期正常对照组相比2W[(16.78±1.35)vs(2.21±0.79)mg/d]、4 W[(13.88±0.89)vs(2.20±0.70)mg/d]、6 W[(12.67±1.47)vs(2.13±0.42)mg/d]和8 W[(10.35±0.92)vs(2.25±1.01)mg/d]差异有统计学意义(P0.05)。缬沙坦干预组尿蛋白显著低于同期模型组:分别是2W[(11.62±1.96)vs(16.78±1.35)mg/d]、4W[(9.96±1.31)vs(13.88±0.89)mg/d]、6W[(6.73±1.87)vs(12.67±1.47)mg/d]、8W[(4.10±0.87)vs(10.35±0.92)mg/d],差异有统计学差异(P0.05),但仍高于同期正常对照组,并且有显著差异(P0.05)。对照组各周尿蛋白比较差异无统计学意义(P0.05)。(见Table 1)模型组ALB随着时间逐渐降低,第2周(19.77±1.50)g/L达最低值,4周(21.73±3.43)g/L、6周(24.33±3.14)g/L、8周(28.05±3.22)g/L较第2周稍升高,但仍处于低水平状态,与对照组相比差异显著有统计学意义(P0.05),干预组ALB较同期模型组值显著升高,分别是2W[(23.8±2.73)vs(19.77±1.50)g/L]、4W[(25.90±1.71)vs(21.73±3.43)g/L]、6W[(27.23±1.06)vs(24.33±3.14)]以及8W[(32.93±3.73)vs(28.05±3.22)],与模型组比较差异有统计学意义(P0.05),但仍低于同期正常组(P0.05)。(见Table 2)模型组CHOL随时间逐渐升高,第2周(6.46±1.47)mmol/L达高峰,第4(4.60±0.64)mmol/L、6(3.90±0.80)mmol/L、8(3.56±0.43)mmol/L较第2周下降,但仍处于高水平状态,与同期对照组相比差异有统计学意义(P0.05)。干预组CHOL较同期模型组显著降低,2W[(8.34±0.89)vs(6.46±1.47)mmol/L]、4W[(6.21±0.48)vs(4.60±0.64)mmol/L]、6W[(5.38±0.57)vs(3.90±0.80)mmol/L]、8W[(4.87±0.59)vs(3.56±0.43)],差异有统计学意义(P0.05),但仍高于同期正常组(P0.05)。(见Table 2)Scr在对照组、模型组及干预组中比较差异无统计学意义(P0.05)。(见Table 2)3阿霉素肾病小鼠各期肾组织病理形态学改变:对照组各期肾小球及肾小管形态结构无明显变化。模型组2周末部分肾小球系膜基质及系膜细胞增生,部分肾小管扩张可见少量蛋白管型;4、6周末出现系膜基质和系膜细胞轻、中度增生,伴足细胞损伤,血管袢和肾小球囊有轻度粘连。部分肾小球萎缩,肾小管蛋白管型增多、伴肾间质炎性细胞浸润;8周末:可见50%以上肾小球中出现节段性硬化,部分血管管腔缩小闭塞,血管袢坍塌,系膜细胞增生,系膜区扩大,肾小管上皮细胞空泡样改变,可见大量蛋白管型及局部间质纤维化;缬沙坦干预组各时间段较同期模型组肾小球硬化和小管间质损害等病变有明显改善,其中炎症较轻,蛋白管型少见。对照组各时间段肾小球硬化评分差异无统计学意义(P0.05),模型组随时间进展肾小球硬化程度逐渐加重,各时间段评分明显高于同期对照组,2W[(0.42±0.03)vs(0.01±0.01)]、4W[(0.59±0.04)vs(0.01±0.03)]、6W[(0.78±0.05)vs(0.02±0.00)]8W[(1.19±0.63)vs(0.01±0.01)],差异有统计学意义(P0.05);干预组肾小球硬化程度较模型组相对减轻,各时间段的评分均低于同期模型组,2W[(0.17±0.00)vs(0.42±0.03)]、4W[(0.25±0.04)vs(0.59±0.04)]、6W[(0.45±0.04)vs(0.78±0.05)]8W[(0.52±0.06)vs(1.19±0.63)],差异有统计学意义(P0.05),但仍高于同期对照组(P0.05)。(见Table 3)对照组各时间段肾小管间质损害评分,差异无统计学意义(P0.05),模型组随时间进展肾间质损害程度逐渐加重,明显高于同期对照组,2W[(0.01±0.06)vs(1.08±0.10)]、4W[(1.35±0.05)vs(0.02±0.04)]、6W[(1.55±0.06)vs(0.01±0.01)]、8W[(1.93±0.12)vs(0.01±0.00)],差异有统计学意义(P0.05);干预组肾小管间质损害程度稍有缓解,低于同期模型组,2W[(0.24±0.05)vs(1.08±0.10)]、4W[(0.44±0.06)vs(1.35±0.05)]、6W[(0.64±0.05)vs(1.55±0.06)]、8W[(0.85±0.06)vs(1.93±0.12)],差异有统计学意义(P0.05),但仍高于同期对照组(P0.05)。(见Table 3)4阿霉素肾病小鼠肾组织STAT1、P53、P21蛋白的表达模型组STAT1、P21、P53蛋白均随时间推移在肾小球中表达递增,且第2周时较4、6、8周表达显著增加,与同期正常组比较差异有显著性,干预组STAT1、P53、P21表达量较同期模型组均明显减少,STAT1表达:2W[(75.40±9.65)vs(437.38±49.66)]、4W[(91.99±9.28)vs(534.06±45.81)]、6W[(136.31±12.03)vs(640.26±44.58)]、8W[(167.21±15.55)vs(777.11±59.19)](P均0.05);P53表达:2W[(24.24±2.60)vs(142.63±6.91)]、4W[(44.94±3.79)vs(172.08±8.12)]、6W[(55.00±3.38)vs(191.93±5.47)]、8W[(75.99±3.97)vs(216.26±9.15)](P均0.05);P21表达:2W[(50.16±4.13)vs(255.31±22.75)]、4W[(65.46±5.40)vs(294.61±22.13)]、6W[(76.97±5.50)vs(337.89±21.55)、8W(92.61±3.77)vs(380.96±21.43)](P均0.05)。正常组和干预组之间表达量比较差异同样有统计学意义(P0.05)。(见Fig.1、2、3,Table4)5阿霉素肾病小鼠肾组织STAT1、P53、P21m RNA的表达水平:模型组不同时间中三个指标均随着时间推移表达逐渐增加,且明显高于同期对照组表达,差异有统计学意义(P0.05)。干预组与同期模型组相比表达量明显减少,STAT1m RNA:2W[(9.2±0.81)vs(22.61±0.95)]、4W[(15.90±0.77)vs(64.22±1.31)]、6W[(45.05±1.16)vs(86.58±1.31)]、8W[(94.76±1.91)vs(125.21±3.63)](P均0.05);P53m RNA:2W[(2.83±0.23)vs(3.94±0.42)]、4W[(3.62±0.45)vs(6.46±0.26)]、6W[(5.27±1.07)vs(7.89±0.64)]、8W[(6.97±0.15)vs(9.75±0.73)](P均0.05);P21m RNA:2W[(3.89±0.23)vs(6.72±1.02)]、4W[(6.51±0.49)vs(10.94±0.61)]、6W[(5.82±0.29)vs(13.10±0.81)、8W(7.83±0.31)vs(15.21±0.48)](P均0.05),但仍明显高于同期对照组(P0.05)。(见Table 5)结论:1衰老相关蛋白STAT1、P53、P21在阿霉素肾病模型中第2周开始表达显著增加,提示应激性衰老可能参与阿霉素肾病早期肾脏变化的发生发展。2应用ARB可有效抑制了JAK-STAT信号通路与其下调基因P53、P21的表达,干预STAT1-P53-P21通路可延缓阿霉素肾病的进展。
[Abstract]:Objective: to detect the expression of STAT1, P53 and P21 in the kidney of adriamycin induced nephropathy in mice, study on stress aging in adriamycin nephropathy mice cancerdevelopment. Whether the expression of STAT1-p53-p21 pathway further intervention can regulate aging mechanism, thus delaying the progression of adriamycin nephropathy mice disease. Methods: 90 6 week old male BALB/c mice were randomly divided into 3 groups: normal control group, adriamycin nephropathy model group, valsartan treatment group. Model group and intervention group at the beginning of the experiment respectively from the intravenous injection of adriamycin induced by 10mg/kg stress aging model, at the beginning of the experiment were from tail vein injection of equal volume of normal saline control group, placed in metabolic cages. Free feeding, drinking water. The mice survived after the injection of adriamycin 3D urine protein, urine protein measurement indicated that the model was established successfully using 2+ test paper, included in the experiment, the treatment group was given Treated with valsartan 20mg/kg orally, 1 times /d; normal control group and model group were given normal saline groups respectively at 0,2,4,6,8 weeks, 6 mice were randomly selected to collect 24h urine, serum and kidney tissue. Detection of 24h urinary protein by Coomassie brilliant blue method, biochemical indicator detection automatic biochemical analyzer: serum albumin (Albumin total cholesterol (Total), ALB cholesterin, TC) and creatinine (Creatinine, Cr). The contents of kidney tissue paraffin sections after 3 m, HE, PAS staining was used to observe the degree of glomerular sclerosis and renal tubular damage; immunohistochemistry detection of renal tissue STAT1, P53, P21 protein expression, fresh kidney tissue RNA extraction for RT-PCR detection of renal STAT1, P53, expression of P21m RNA. All data used SPSS 21 statistical analysis software for statistical analysis of experimental data. The normal distribution test measurement data to conform to normal distribution number According to the standard deviation (x + s) said that the application of comparative test of homogeneity of variance between the two groups, using t or T '- test, analysis and comparison of application of single factor variance between groups, using LSD test between groups were 22, P0.05 said. The results were statistically significant 1: after modeling the general situation in mice model group and drug intervention group mice at first weeks after injection of adriamycin were Jingshenbuzhen, rough hair and hair removal, reduce the volume of activities, poor diet, diarrhea and weight reduction, mice after injection of adriamycin, the average body weight decreased rapidly, second weeks to the lowest and then gradually picked up, but there are still significant differences compared with the normal group. During the experiment, the experimental group of 4 mice died of diarrhea and emaciation; drug intervention group died of gastrointestinal bleeding 3 mice; total 7 rats died, the mortality rate was 7.8%.2 only copy the model of failure was 鍓旈櫎.瀵圭収缁勫皬榧犲仴搴峰瓨娲，

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