本文選題：應(yīng)激性衰老　切入點(diǎn)：阿霉素腎病　出處：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過檢測(cè)STAT1、P53及P21在阿霉素腎病小鼠腎臟中的表達(dá),研究應(yīng)激性衰老在阿霉素小鼠腎病發(fā)生發(fā)展中的作用。進(jìn)一步干預(yù)STAT1-p53-p21通路表達(dá)是否可調(diào)控衰老機(jī)制,從而延緩了阿霉素腎病小鼠的疾病進(jìn)展。方法:將90只6周齡BALB/c雄性小鼠隨機(jī)分為3組:正常對(duì)照組、阿霉素腎病模型組,纈沙坦治療干預(yù)組。模型組與干預(yù)組于實(shí)驗(yàn)開始后分別從尾靜脈注射阿霉素10mg/kg誘發(fā)應(yīng)激衰老模型,對(duì)照組于實(shí)驗(yàn)開始后分別從尾靜脈注射等容積生理鹽水,放入代謝籠內(nèi)飼養(yǎng),自由攝食、飲水。存活的小鼠注射阿霉素后3d時(shí)檢測(cè)尿蛋白,應(yīng)用試紙法測(cè)定尿蛋白2+提示模型復(fù)制成功,計(jì)入實(shí)驗(yàn),治療組給予纈沙坦20mg/kg灌胃,1次/d;正常對(duì)照組及模型組給予等量生理鹽水灌胃,各組分別在0、2、4、6、8周隨機(jī)抽取6只小鼠收集24h尿液、血清及腎臟組織�？捡R斯亮藍(lán)法檢測(cè)24h尿蛋白定量,全自動(dòng)生化儀檢測(cè)生化指標(biāo):血清白蛋白(Albumin,ALB)、總膽固醇(Total cholesterin,TC)及肌酐(Creatinine,Cr)的含量。腎臟組織石蠟包埋后切片3μm,進(jìn)行HE、PAS染色觀察腎小球硬化程度及腎小管損害;免疫組織化學(xué)法檢測(cè)腎組織STAT1、P53、P21蛋白的表達(dá),新鮮腎臟組織提取RNA行RT-PCR檢測(cè)腎臟STAT1、P53、P21m RNA的表達(dá)。所有數(shù)據(jù)應(yīng)用SPSS 21.0統(tǒng)計(jì)分析軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理。計(jì)量資料數(shù)據(jù)進(jìn)行正態(tài)分布檢驗(yàn),符合正態(tài)分布的數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,兩組間的比較應(yīng)用方差齊性檢驗(yàn)后,采用t或t′檢驗(yàn),多組間的比較應(yīng)用單因素方差分析后,采用LSD檢驗(yàn)做各組間均數(shù)的兩兩比較,P0.05表示有統(tǒng)計(jì)學(xué)意義。結(jié)果:1造模后小鼠的一般情況模型組及藥物干預(yù)組小鼠于阿霉素注射后第1周均出現(xiàn)精神不振,毛質(zhì)粗糙及脫毛,活動(dòng)量減少,進(jìn)食差,有不同程度的腹瀉及體重減輕,小鼠注射阿霉素后,其平均體重迅速下降,第2周降至最低,然后逐漸回升,但與正常組相比仍有顯著差異。實(shí)驗(yàn)期間實(shí)驗(yàn)組4只小鼠因腹瀉及消瘦死亡;藥物干預(yù)組3只小鼠灌胃時(shí)出現(xiàn)消化道出血死亡;共死亡7只,死亡率為7.8%。2只復(fù)制模型失敗被剔除。對(duì)照組小鼠健康存活。2阿霉素腎病小鼠各期24小時(shí)尿蛋白及血生化指標(biāo)的變化模型組尿蛋白于第2周明顯升高且達(dá)到峰值,第4、6、8周與2周相比,尿蛋白稍有下降,與同期正常對(duì)照組相比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]差異有統(tǒng)計(jì)學(xué)意義(P0.05)。纈沙坦干預(yù)組尿蛋白顯著低于同期模型組:分別是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],差異有統(tǒng)計(jì)學(xué)差異(P0.05),但仍高于同期正常對(duì)照組,并且有顯著差異(P0.05)。對(duì)照組各周尿蛋白比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(見Table 1)模型組ALB隨著時(shí)間逐漸降低,第2周(19.77±1.50)g/L達(dá)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較第2周稍升高,但仍處于低水平狀態(tài),與對(duì)照組相比差異顯著有統(tǒng)計(jì)學(xué)意義(P0.05),干預(yù)組ALB較同期模型組值顯著升高,分別是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)],與模型組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),但仍低于同期正常組(P0.05)。(見Table 2)模型組CHOL隨時(shí)間逐漸升高,第2周(6.46±1.47)mmol/L達(dá)高峰,第4(4.60±0.64)mmol/L、6(3.90±0.80)mmol/L、8(3.56±0.43)mmol/L較第2周下降,但仍處于高水平狀態(tài),與同期對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。干預(yù)組CHOL較同期模型組顯著降低,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)],差異有統(tǒng)計(jì)學(xué)意義(P0.05),但仍高于同期正常組(P0.05)。(見Table 2)Scr在對(duì)照組、模型組及干預(yù)組中比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(見Table 2)3阿霉素腎病小鼠各期腎組織病理形態(tài)學(xué)改變:對(duì)照組各期腎小球及腎小管形態(tài)結(jié)構(gòu)無明顯變化。模型組2周末部分腎小球系膜基質(zhì)及系膜細(xì)胞增生,部分腎小管擴(kuò)張可見少量蛋白管型;4、6周末出現(xiàn)系膜基質(zhì)和系膜細(xì)胞輕、中度增生,伴足細(xì)胞損傷,血管袢和腎小球囊有輕度粘連。部分腎小球萎縮,腎小管蛋白管型增多、伴腎間質(zhì)炎性細(xì)胞浸潤(rùn);8周末:可見50%以上腎小球中出現(xiàn)節(jié)段性硬化,部分血管管腔縮小閉塞,血管袢坍塌,系膜細(xì)胞增生,系膜區(qū)擴(kuò)大,腎小管上皮細(xì)胞空泡樣改變,可見大量蛋白管型及局部間質(zhì)纖維化;纈沙坦干預(yù)組各時(shí)間段較同期模型組腎小球硬化和小管間質(zhì)損害等病變有明顯改善,其中炎癥較輕,蛋白管型少見。對(duì)照組各時(shí)間段腎小球硬化評(píng)分差異無統(tǒng)計(jì)學(xué)意義(P0.05),模型組隨時(shí)間進(jìn)展腎小球硬化程度逐漸加重,各時(shí)間段評(píng)分明顯高于同期對(duì)照組,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)],差異有統(tǒng)計(jì)學(xué)意義(P0.05);干預(yù)組腎小球硬化程度較模型組相對(duì)減輕,各時(shí)間段的評(píng)分均低于同期模型組,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)],差異有統(tǒng)計(jì)學(xué)意義(P0.05),但仍高于同期對(duì)照組(P0.05)。(見Table 3)對(duì)照組各時(shí)間段腎小管間質(zhì)損害評(píng)分,差異無統(tǒng)計(jì)學(xué)意義(P0.05),模型組隨時(shí)間進(jìn)展腎間質(zhì)損害程度逐漸加重,明顯高于同期對(duì)照組,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)],差異有統(tǒng)計(jì)學(xué)意義(P0.05);干預(yù)組腎小管間質(zhì)損害程度稍有緩解,低于同期模型組,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)],差異有統(tǒng)計(jì)學(xué)意義(P0.05),但仍高于同期對(duì)照組(P0.05)。(見Table 3)4阿霉素腎病小鼠腎組織STAT1、P53、P21蛋白的表達(dá)模型組STAT1、P21、P53蛋白均隨時(shí)間推移在腎小球中表達(dá)遞增,且第2周時(shí)較4、6、8周表達(dá)顯著增加,與同期正常組比較差異有顯著性,干預(yù)組STAT1、P53、P21表達(dá)量較同期模型組均明顯減少,STAT1表達(dá):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表達(dá):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表達(dá):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)。正常組和干預(yù)組之間表達(dá)量比較差異同樣有統(tǒng)計(jì)學(xué)意義(P0.05)。(見Fig.1、2、3,Table4)5阿霉素腎病小鼠腎組織STAT1、P53、P21m RNA的表達(dá)水平:模型組不同時(shí)間中三個(gè)指標(biāo)均隨著時(shí)間推移表達(dá)逐漸增加,且明顯高于同期對(duì)照組表達(dá),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。干預(yù)組與同期模型組相比表達(dá)量明顯減少,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),但仍明顯高于同期對(duì)照組(P0.05)。(見Table 5)結(jié)論:1衰老相關(guān)蛋白STAT1、P53、P21在阿霉素腎病模型中第2周開始表達(dá)顯著增加,提示應(yīng)激性衰老可能參與阿霉素腎病早期腎臟變化的發(fā)生發(fā)展。2應(yīng)用ARB可有效抑制了JAK-STAT信號(hào)通路與其下調(diào)基因P53、P21的表達(dá),干預(yù)STAT1-P53-P21通路可延緩阿霉素腎病的進(jìn)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.瀵圭収緇勫皬榧犲仴搴峰瓨?gòu)z，

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