p53、p21在阿霉素肾病小鼠中表达和意义

发布时间：2018-06-22 18:17

本文选题：p53 + p21　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:通过检测p53、p21在阿霉素肾病小鼠中的表达,探讨细胞衰老机制是否参与阿霉素肾病的发生和发展及其可能的机制。方法:将60只6周龄、雄性BALB/c小鼠随机分为两组:正常组和阿霉素肾病模型组(模型组),每组30只。小鼠适应性饲养1周后,模型组尾静脉一次性注射阿霉素10mg/kg,正常组尾静脉一次性注射等量生理盐水。模型组于给药7天后留取24h尿蛋白进行定量,24h尿蛋白定量≥50mg/kg者纳入本实验。两组分别于给药后第0、2、4、6周末随机选取6只小鼠处死,并留取24小时尿液、血清及肾脏组织。检测24小时尿蛋白定量及血生化指标。肾组织石蜡切片行HE和PAS染色,观察各组肾小球硬化程度和肾小管损伤程度。免疫组织化学法检测各组肾脏p53蛋白、p21蛋白的表达水平。Real-time PCR技术检测肾脏p53、p21的mRNA表达。分析p53、p21蛋白及p53、p21mRNA表达与其它指标的相关性。结果:1阿霉素肾病模型组和对照组各期24h尿蛋白定量的变化对照组0、2、4、6周末尿蛋白比较差异均无统计学意义(P0.05)。模型组尿蛋白在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]显著高于同时期对照组[(2.02±0.47)g/24h]、[(2.25±0.55)g/24h]、[(2.09±0.56)g/24h],差异均有统计学意义(P0.05)。模型组尿蛋白从2周末显著增高,且2周末达高峰,4、6周末与2周末相比,尿蛋白稍有下降(见表2)。2阿霉素肾病模型组和对照组各期血清生化指标的变化对照组0、2、4、6周末血白蛋白、总胆固醇比较差异均无统计学意义(P0.05)。模型组血白蛋白在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]明显低于同时期对照组[(36.77±1.81)g/L]、[(37.04±1.79)g/L]、[(36.74±1.49)g/L],差异均有统计学意义(P0.05),模型组血白蛋白从2周末明显降低,且2周末达最低值,4、6周末与2周末相比,血白蛋白稍增高。模型组胆固醇在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]明显低于同时期对照组[(2.17±0.15)mmol/l]、[(2.16±0.24)mmol/l]、[(2.25±0.25)mmol/l],差异均有统计学意义(p0.05),模型组总胆固醇从2周末显著增高,且4周末达峰值,6周末与4周末相比,总胆固醇稍降低。两组0、2、4、6周末血肌酐水平比较差异无统计学意义(p0.05,见表3)。3阿霉素肾病模型组和对照组各期肾组织病理学改变光镜下,对照组0、2、4、6周末及模型组0周末肾小球及肾小管形态结构正常。模型组2周末肾小球可见少量球囊粘连,肾小管出现蛋白管型,肾间质可见炎性细胞浸润;4周末可见系膜细胞轻中度增生,部分肾小球硬化,肾小管节段性扩张,肾间质较多炎性细胞浸润;6周末出现系膜细胞中重度增生,肾小球局灶节段性硬化,肾小管萎缩,肾间质大量炎性细胞浸润。对照组0、2、4、6周末肾小球硬化及肾小管间质损伤比较差异均无统计学意义(p0.05)。模型组肾小球硬化及肾小管间质损伤程度在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)显著高于同时期对照组(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),差异均有统计学意义(p0.05),随时间病变程度逐渐加重(见图1)。4阿霉素肾病模型组和对照组各期肾组织p53、p21蛋白表达水平的变化对照组0、2、4、6周末p53、p21蛋白表达水平比较差异均无统计学意义(p0.05)。模型组p53、p21蛋白表达水平在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)显著高于同时期对照组(8.111±1.744,11.883±1.863)、(7.869±1.940,12.652±2.174)、(8.537±1.262,12.422±2.848),差异均有统计学意义(p0.05),随时间p53、p21蛋白表达逐渐增多(见图2、表4)。5阿霉素肾病模型组和对照组各期肾组织p53、p21mRNA表达水平的变化对照组0、2、4、6周末p53、p21mRNA表达水平比较差异均无统计学意义(P0.05)。模型组p53、p21mRNA表达水平在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)显著高于同时期对照组(0.96±0.10,1.04±0.09)、(0.97±0.08,1.04±0.08)、(0.97±0.11,1.05±0.08),差异均有统计学意义(P0.05),随时间p53、p21mRNA表达水平逐渐增高(见表5)。6阿霉素肾病模型肾组织p53、p21的表达与不同指标的相关性模型组中,p53蛋白表达与肾小球硬化、肾小管间质损伤、尿蛋白及总胆固醇呈正相关(r=0.877,r=0.830,r=0.456,r=0.576,P0.05),与白蛋白呈负相关(r=-0.343,P0.05),与血肌酐不相关(P0.05);p53mRNA表达与肾小球硬化、肾小管间质损伤、尿蛋白及总胆固醇正相关(r=0.940,r=0.926,r=0.473,r=0.524,P0.05),与白蛋白呈负相关(r=-0.379,P0.05),与血肌酐不相关(P0.05);p21蛋白表达与肾小球硬化、肾小管间质损伤、尿蛋白及总胆固醇呈正相关(r=0.838,r=0.804,r=0.410,r=0.525,P0.05),与白蛋白呈负相关(r=-0.396,P0.05),与血肌酐不相关(P0.05);p21mRNA表达与肾小球硬化、肾小管间质损伤、尿蛋白及总胆固醇呈正相关(r=0.948,r=0.940,r=0.520,r=0.559,P0.05),与白蛋白呈负相关(r=-0.377,P0.05),与血肌酐不相关(P0.05)(见表6)。结论:阿霉素肾病小鼠模型可以模拟人类微小病变型肾病及局灶节段性肾小球硬化症,表现为大量蛋白尿、低白蛋白血症、高胆固醇血症。衰老基因p53、p21在阿霉素肾病小鼠模型2周末即表达增加,且与阿霉素肾病病变程度相关,提示细胞衰老存在于阿霉素肾脏病早期,参与阿霉素肾病的发生和发展。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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