發(fā)布時間：2018-06-22 18:17

  本文選題：p53 + p21　； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過檢測p53、p21在阿霉素腎病小鼠中的表達,探討細(xì)胞衰老機制是否參與阿霉素腎病的發(fā)生和發(fā)展及其可能的機制。方法:將60只6周齡、雄性BALB/c小鼠隨機分為兩組:正常組和阿霉素腎病模型組(模型組),每組30只。小鼠適應(yīng)性飼養(yǎng)1周后,模型組尾靜脈一次性注射阿霉素10mg/kg,正常組尾靜脈一次性注射等量生理鹽水。模型組于給藥7天后留取24h尿蛋白進行定量,24h尿蛋白定量≥50mg/kg者納入本實驗。兩組分別于給藥后第0、2、4、6周末隨機選取6只小鼠處死,并留取24小時尿液、血清及腎臟組織。檢測24小時尿蛋白定量及血生化指標(biāo)。腎組織石蠟切片行HE和PAS染色,觀察各組腎小球硬化程度和腎小管損傷程度。免疫組織化學(xué)法檢測各組腎臟p53蛋白、p21蛋白的表達水平。Real-time PCR技術(shù)檢測腎臟p53、p21的mRNA表達。分析p53、p21蛋白及p53、p21mRNA表達與其它指標(biāo)的相關(guān)性。結(jié)果:1阿霉素腎病模型組和對照組各期24h尿蛋白定量的變化對照組0、2、4、6周末尿蛋白比較差異均無統(tǒng)計學(xué)意義(P0.05)。模型組尿蛋白在2周末[(16.19±1.13)g/24h]、4周末[(14.50±1.46)g/24h]、6周末[(12.52±1.2)g/24h]顯著高于同時期對照組[(2.02±0.47)g/24h]、[(2.25±0.55)g/24h]、[(2.09±0.56)g/24h],差異均有統(tǒng)計學(xué)意義(P0.05)。模型組尿蛋白從2周末顯著增高,且2周末達高峰,4、6周末與2周末相比,尿蛋白稍有下降(見表2)。2阿霉素腎病模型組和對照組各期血清生化指標(biāo)的變化對照組0、2、4、6周末血白蛋白、總膽固醇比較差異均無統(tǒng)計學(xué)意義(P0.05)。模型組血白蛋白在2周末[(19.63±1.28)g/L]、4周末[(23.09±1.91)g/L]、6周末[(26.51±1.34)g/L]明顯低于同時期對照組[(36.77±1.81)g/L]、[(37.04±1.79)g/L]、[(36.74±1.49)g/L],差異均有統(tǒng)計學(xué)意義(P0.05),模型組血白蛋白從2周末明顯降低,且2周末達最低值,4、6周末與2周末相比,血白蛋白稍增高。模型組膽固醇在2周末[(7.56±1.10)mmol/l]、4周末[(9.07±1.02)mmol/l]、6周末[(7.18±0.62)mmol/l]明顯低于同時期對照組[(2.17±0.15)mmol/l]、[(2.16±0.24)mmol/l]、[(2.25±0.25)mmol/l],差異均有統(tǒng)計學(xué)意義(p0.05),模型組總膽固醇從2周末顯著增高,且4周末達峰值,6周末與4周末相比,總膽固醇稍降低。兩組0、2、4、6周末血肌酐水平比較差異無統(tǒng)計學(xué)意義(p0.05,見表3)。3阿霉素腎病模型組和對照組各期腎組織病理學(xué)改變光鏡下,對照組0、2、4、6周末及模型組0周末腎小球及腎小管形態(tài)結(jié)構(gòu)正常。模型組2周末腎小球可見少量球囊粘連,腎小管出現(xiàn)蛋白管型,腎間質(zhì)可見炎性細(xì)胞浸潤;4周末可見系膜細(xì)胞輕中度增生,部分腎小球硬化,腎小管節(jié)段性擴張,腎間質(zhì)較多炎性細(xì)胞浸潤;6周末出現(xiàn)系膜細(xì)胞中重度增生,腎小球局灶節(jié)段性硬化,腎小管萎縮,腎間質(zhì)大量炎性細(xì)胞浸潤。對照組0、2、4、6周末腎小球硬化及腎小管間質(zhì)損傷比較差異均無統(tǒng)計學(xué)意義(p0.05)。模型組腎小球硬化及腎小管間質(zhì)損傷程度在2周末(0.535±0.116,1.133±0.136)、4周末(0.738±0.112,1.417±0.172)、6周末(0.908±0.113,1.633±0.136)顯著高于同時期對照組(0.020±0.020,0.067±0.061)、(0.022±0.017,0.083±0.075)、6周末(0.018±0.015,0.067±0.052),差異均有統(tǒng)計學(xué)意義(p0.05),隨時間病變程度逐漸加重(見圖1)。4阿霉素腎病模型組和對照組各期腎組織p53、p21蛋白表達水平的變化對照組0、2、4、6周末p53、p21蛋白表達水平比較差異均無統(tǒng)計學(xué)意義(p0.05)。模型組p53、p21蛋白表達水平在2周末(51.944±15.081,59.674±15.234)、4周末(164.902±12.852,202.428±24.098)、6周末(254.673±24.703,348.115±25.180)顯著高于同時期對照組(8.111±1.744,11.883±1.863)、(7.869±1.940,12.652±2.174)、(8.537±1.262,12.422±2.848),差異均有統(tǒng)計學(xué)意義(p0.05),隨時間p53、p21蛋白表達逐漸增多(見圖2、表4)。5阿霉素腎病模型組和對照組各期腎組織p53、p21mRNA表達水平的變化對照組0、2、4、6周末p53、p21mRNA表達水平比較差異均無統(tǒng)計學(xué)意義(P0.05)。模型組p53、p21mRNA表達水平在2周末(4.52±0.44,6.99±0.80)、4周末(6.43±0.68,9.36±0.69)、6周末(8.55±0.84,11.96±0.79)顯著高于同時期對照組(0.96±0.10,1.04±0.09)、(0.97±0.08,1.04±0.08)、(0.97±0.11,1.05±0.08),差異均有統(tǒng)計學(xué)意義(P0.05),隨時間p53、p21mRNA表達水平逐漸增高(見表5)。6阿霉素腎病模型腎組織p53、p21的表達與不同指標(biāo)的相關(guān)性模型組中,p53蛋白表達與腎小球硬化、腎小管間質(zhì)損傷、尿蛋白及總膽固醇呈正相關(guān)(r=0.877,r=0.830,r=0.456,r=0.576,P0.05),與白蛋白呈負(fù)相關(guān)(r=-0.343,P0.05),與血肌酐不相關(guān)(P0.05);p53mRNA表達與腎小球硬化、腎小管間質(zhì)損傷、尿蛋白及總膽固醇正相關(guān)(r=0.940,r=0.926,r=0.473,r=0.524,P0.05),與白蛋白呈負(fù)相關(guān)(r=-0.379,P0.05),與血肌酐不相關(guān)(P0.05);p21蛋白表達與腎小球硬化、腎小管間質(zhì)損傷、尿蛋白及總膽固醇呈正相關(guān)(r=0.838,r=0.804,r=0.410,r=0.525,P0.05),與白蛋白呈負(fù)相關(guān)(r=-0.396,P0.05),與血肌酐不相關(guān)(P0.05);p21mRNA表達與腎小球硬化、腎小管間質(zhì)損傷、尿蛋白及總膽固醇呈正相關(guān)(r=0.948,r=0.940,r=0.520,r=0.559,P0.05),與白蛋白呈負(fù)相關(guān)(r=-0.377,P0.05),與血肌酐不相關(guān)(P0.05)(見表6)。結(jié)論:阿霉素腎病小鼠模型可以模擬人類微小病變型腎病及局灶節(jié)段性腎小球硬化癥,表現(xiàn)為大量蛋白尿、低白蛋白血癥、高膽固醇血癥。衰老基因p53、p21在阿霉素腎病小鼠模型2周末即表達增加,且與阿霉素腎病病變程度相關(guān),提示細(xì)胞衰老存在于阿霉素腎臟病早期,參與阿霉素腎病的發(fā)生和發(fā)展。
[Abstract]:Objective: To investigate the expression of p53 and p21 in adriamycin nephropathy mice, and to explore whether the mechanism of cell aging is involved in the occurrence and development of adriamycin nephropathy and its possible mechanism. Methods: 60 6 weeks old male BALB/c mice were randomly divided into two groups: normal group and adriamycin nephropathy model group (model group), 30 mice in each group for 1 weeks. Then, the tail vein of the model group was injected with adriamycin 10mg/kg in one time, and the normal group was injected with the same amount of normal saline in the tail vein. The model group was given 24h urine protein for 7 days after administration, and the urine protein of 24h was more than 50mg/kg in the experiment. The two groups were killed at random in 6 mice at the end of 0,2,4,6 after the administration and left for 24 hours of urine. The quantitative and biochemical indexes of urine protein were measured for 24 hours. The renal tissue paraffin sections were stained with HE and PAS. The degree of glomerular sclerosis and renal tubule injury were observed. Immunohistochemistry was used to detect the p53 protein of kidney in each group and the expression level of p21 protein by.Real-time PCR technique to detect the mRNA expression of p53 and p21 in kidney. Analysis of the correlation between p53, p21 protein and p53, p21mRNA expression with other indicators. Results: there was no significant difference in the comparison of 0,2,4,6 weekend urine protein between the 1 adriamycin nephropathy model group and the control group at the 0,2,4,6 weekend (P0.05). The urine protein in the model group was at the 2 weekend [(16.19 + 1.13) g/24h], 4 weekend [(14.50 + 1.46) g/24h], 6 weeks. The end [(12.52 + 1.2) g/24h]] was significantly higher than that of the control group [(2.02 + 0.47) g/24h], [(2.25 + 0.55)) g/24h], [(2.09 + 0.56)) g/24h], the difference was statistically significant (P0.05). The urine protein in the model group increased significantly from the 2 weekend and reached the peak at the end of 2, and the urine protein was slightly lower than the end 2 at the end of the week (see Table 2).2 adriamycin nephropathy model group and the control group. There was no significant difference in serum albumin and total cholesterol in the control group at the weekend of 0,2,4,6 (P0.05). The serum albumin in the model group was 2 weekend [(19.63 + 1.28) g/L], 4 weekend (23.09 + 1.91) g/L], and 6 weekend [(26.51 + 1.34) g/L] (36.77 + 1.81) g/L], [37.04 + 1.79) g/L], [37.04 + 1.79) g/L], [(36.7)] 4 + 1.49) g/L], the difference was statistically significant (P0.05). The serum albumin in the model group decreased significantly at the end of the 2 week and reached the lowest at the 2 weekend. The 4,6 weekend was slightly higher than the 2 weekend. The cholesterol in the model group was 2 weekend [(7.56 + 1.10) mmol/l], 4 weekend [(9.07 + 1.02) mmol/l], 6 weekend [7.18 +] mmol/l] was significantly lower than that of the simultaneous control group [(7.18 +]). 2.17 + 0.15) mmol/l], [2.16 + 0.24) mmol/l], [(2.25 + 0.25)) mmol/l], the difference was statistically significant (P0.05). The total cholesterol in the model group increased significantly from the 2 weekend and reached the peak at the 4 weekend. The total cholesterol decreased slightly at the 6 weekend and 4 weekend. There was no significant difference in the level of serum creatinine at the weekend of the two group 0,2,4,6 (P0.05, see Table 3).3 adriamycin kidney The glomerular and renal tubules in the control group 0,2,4,6 weekend and the model group were normal in the control group and the control group. The glomerular and renal tubules were normal in the control group at the weekend of 0,2,4,6 and in the model group. In the model group, the glomeruli showed a small amount of conglutination at the end of the 2 week, the renal tubule appeared protein tube type, the renal interstitium showed inflammatory cell infiltration, and the 4 weekend showed mesangial cells mild to moderate hyperplasia. Partial glomerulosclerosis, segmental dilatation of renal tubules, more inflammatory cell infiltration of renal interstitium, severe mesangial cell hyperplasia, glomerular focal segmental sclerosis, renal tubule atrophy, and massive inflammatory cell infiltration in the renal interstitium at the weekend of 6, there was no significant difference in glomerular sclerosis and tubulointerstitial damage between the 0,2,4,6 and the renal tubules at the weekend of the control group (P 0.05). The degree of glomerular sclerosis and tubulointerstitial damage in the model group was 2 weekend (0.535 + 0.116,1.133 + 0.136), 4 weekend (0.738 + 0.112,1.417 + 0.172), and 6 weekend (0.908 + 0.113,1.633 + 0.136) significantly higher than that in the simultaneous control group (0.020 + 0.020,0.067 + 0.061), (0.022 + 0.017,0.083 + 0.535), and the difference was all There were statistical significance (P0.05), the degree of pathological changes gradually increased with time (see Figure 1).4 adriamycin nephropathy model group and control group p53, p21 protein expression level changes in the control group 0,2,4,6 weekend p53, p21 protein expression level difference was not statistically significant (P0.05). The model group p53, the level of p21 protein at the 2 weekend (51.944 + 15.) 081,59.674 + 15.234), the 4 weekend (164.902 + 12.852202.428 + 24.098), 6 weekend (254.673 + 24.703348.115 + 25.180) was significantly higher than that of the control group (8.111 + 1.744,11.883 + 1.863), (7.869 + 1.940,12.652 + 2.174), (8.537 + 1.262,12.422 + 2.848), and the difference was statistically significant (P0.05). As time p53, the expression of p21 protein increased gradually. Figures 2, table 4).5 adriamycin nephropathy model group and control group p53, p21mRNA expression level changes in the control group, the 0,2,4,6 weekend p53, p21mRNA expression level difference was not statistically significant (P0.05). The model group p53, the p21mRNA expression level at the 2 weekend (4.52 + 0.44,6.99 + 0.80), the 4 weekend (6.43 +. 0.69), 6 weekend (8.55 + 0.84,11) .96 + 0.79) was significantly higher than that in the control group (0.96 + 0.10,1.04 + 0.09), (0.97 + 0.08,1.04 + 0.08), (0.97 + 0.11,1.05 + 0.08), and the difference was statistically significant (P0.05). With time p53, the expression level of p21mRNA increased gradually (see Table 5).6 adriamycin nephropathy model renal tissue p53, p21 expression and the correlation model group, p53 protein The expression was positively correlated with glomerular sclerosis, tubulointerstitial damage, urinary protein and total cholesterol (r=0.877, r=0.830, r=0.456, r=0.576, P0.05), negative correlation with albumin (r=-0.343, P0.05), and blood creatinine unrelated (P0.05); p53mRNA expression was associated with glomerular hardening, renal tubulointerstitial damage, urinary protein and total cholesterol (r=0.940, r=0.926, r=) 0.473, r=0.524, P0.05), negative correlation with albumin (r=-0.379, P0.05), not related to serum creatinine (P0.05); p21 protein expression is positively correlated with glomerulosclerosis, tubulointerstitial damage, urinary protein and total cholesterol (r=0.838, r=0.804, r=0.410, r=0.525, P0.05), and negative correlation with albumin (r=-0.396,), and blood creatinine; A expression was positively correlated with glomerular sclerosis, tubulointerstitial damage, urinary protein and total cholesterol (r=0.948, r=0.940, r=0.520, r=0.559, P0.05), negative correlation with albumin (r=-0.377, P0.05), and blood creatinine (P0.05) (see Table 6). Conclusion: adriamycin nephropathy mice model can simulate human minitiform nephropathy and focal segmental kidney Glomerulosclerosis, characterized by a large number of albuminuria, hypoalbuminemia, hypercholesterolemia, and hypercholesterolemia. The expression of aging gene p53, p21 increased at the end of the 2 week of adriamycin nephropathy, which is associated with the degree of adriamycin nephropathy, suggesting that cell senescence exists in the early stage of adriamycin kidney disease and participates in the occurrence and development of adriamycin nephrosis.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692

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