上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (11): 1455-.doi: 10.3969/j.issn.1674-8115.2012.11.013

• 论著（基础研究） • 上一篇    下一篇

阿霉素肾病大鼠凋亡相关基因FADD和APAF1启动子DNA甲基化的变化

刘 剑, 仲 芳, 徐丽梨, 周 桥, 卢 颖, 郝 旭, 王伟铭, 陈 楠   

  1. 上海交通大学 医学院附属瑞金医院肾脏科, 上海 200025
  • 出版日期:2012-11-28 发布日期:2012-11-30
  • 通讯作者: 王伟铭, 电子信箱: wweiming@medmail.com.cn。
  • 作者简介:刘 剑（1987—）, 男, 硕士生；电子信箱: jim_liu@yeah.net。
  • 基金资助:

    国家自然科学基金（81270782）；国家重点基础研究发展计划（“九七三”计划）（2012CB517700）；上海市科委科研计划项目（08dz1900502）

DNA methylation of promoter of apoptosis-related FADD and APAF1 genes in rats with adriamycin nephropathy

LIU Jian, ZHONG Fang, XU Li-li, ZHOU Qiao, LU Ying, HAO Xu, WANG Wei-ming, CHEN Nan   

  1. Department of Nephrology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2012-11-28 Published:2012-11-30
  • Supported by:

    National Natural Science Foundation of China, 81270782；National Basic Research Program of China, “973” Program, 2012CB517700；Shanghai Science and Technology Committee Foundation, 08dz1900502

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摘要：

目的 探讨阿霉素肾病大鼠肾脏组织中凋亡相关基因DNA甲基化的变化与疾病进展的关系。方法 12只SD雄性大鼠随机分为阿霉素肾病组和正常对照组，每组6只。阿霉素肾病组大鼠分别于首次和1周后阴茎静脉注射5 mg/kg和2.5 mg/kg阿霉素造模。于造模后8周经腹主动脉采血，处死大鼠并留取肾脏组织。对两组大鼠血肌酐(Crea)、血尿素氮(BUN)、血清白蛋白(ALB)及尿白蛋白/肌酐比值(UACR)等生化指标进行检测和比较；Masson染色观察肾脏组织病理学改变；TUNEL染色观察大鼠肾小管和肾小球细胞凋亡情况；采用Real-Time PCR技术经高分辨率熔解曲线 (HRM)分析法对两组大鼠肾皮质Fas相关死亡区（FADD）和凋亡蛋白酶活化因子1（APAF1）启动子DNA甲基化进行检测，Real-Time PCR检测FADD mRNA表达。结果 与正常对照组比较，阿霉素肾病组大鼠Crea、BUN和UACR显著升高（P<0.05或P<0.01），ALB明显降低（P<0.01）。病理组织学检查发现：阿霉素肾病组大鼠肾小球系膜细胞增生明显，毛细血管受压，球囊粘连；肾小管上皮细胞肿胀，间质增生、炎症细胞浸润。TUNEL染色细胞凋亡检测发现，与正常对照组比较，阿霉素肾病组大鼠肾小管和肾小球凋亡细胞数量明显增加(P<0.01)。HRM分析法和Real-Time PCR检测结果显示：与正常对照组比较，阿霉素肾病组FADD启动子DNA呈较高程度的甲基化，FADD mRNA表达明显上调（P<0.05）。结论 在阿霉素肾病大鼠中，FADD基因启动子检测序列DNA甲基化程度增高，但其可能并非为影响FADD mRNA表达的主要因素。

关键词: 凋亡, DNA甲基化, 高分辨率熔解曲线, 肾脏

Abstract:

Objective To investigate the relationship between DNA methylation of apoptosis-related genes in renal tissues and disease progression in rats with adriamycin nephropathy. Methods Twelve male SD rats were randomly divided into adriamycin nephropathy group and normal control group, with 6 rats in each group. Adriamycin nephropathy model was established in adriamycin nephropathy group by injection of 5 mg/kg adriamycin on the first day and 2.5 mg/kg adriamycin one week later through penis vein. Blood samples were collected via abdominal aorta 8 weeks after model establishment, then rats were sacrificed, and renal tissues were harvested. The biochemical parameters such as serum creatinine (Crea), blood urea nitrogen (BUN), serum albumin (ALB) and urinary albumin to creatinine ratio (UACR) were measured. The pathological changes of renal tissues were observed with Masson staining. The apoptosis of renal tubular cells and glomerular cells was detected by TUNEL staining. The DNA methylation in the promoter of Fas-association protein with death (FADD) and apoptotic protease activating factor-1 (APAF1) genes was determined by high-resolution melting (HRM) analysis, and the expression of FADD mRNA was detected by Real-Time PCR. Results Crea, BUN and UACR in adriamycin nephropathy group were significantly higher than those in normal control group (P<0.05 or P<0.01), while ALB in adriamycin nephropathy group was significantly lower than that in normal control group (P<0.01). Pathological examination revealed that compared with normal control group, there were increased glomerular mesangial cell proliferation, swelling of renal tubular epithelial cells and inflammatory cell infiltration in adriamycin nephropathy group. TUNEL assay indicated that the number of apoptotic tubular cells and glomerular cells in adriamycin nephropathy group was significantly higher than that in normal control group (P<0.01). HRM analysis and Real-Time PCR demonstrated that compared with normal control group, the promoter of FADD was relatively hypermethylated, and the expression of FADD mRNA was significantly up-regulated in adriamycin nephropathy group (P<0.05). Conclusion In rats with adriamycin nephropathy, the DNA in the promoter of FADD is hypermethylated, but it may not be the major factor affecting the expression of FADD mRNA.

Key words: apoptosis, DNA methylation, high-resolution melting curve, kidney