摘要
目的建立一种稳定、灵敏的人白介素-10(IL-10)荧光定量PCR的检测方法,比较白介素10在糖尿病肾病组和对照组中的表达差异。方法取糖尿病肾病Ⅳ期患者的外周静脉血淋巴细胞RNA标本,主要从模板浓度、引物筛选两方面进行探索,建立稳定、灵敏的实验方法;将建立的实验方法用于比较33例糖尿病肾病患者和35例正常健康组中血液白介素10的表达差异。结果①将475ng的总RNA逆转录成cDNA模板进行稀释,当模板浓度稀释范围在10~3倍以内,扩增引物为F:5’-GTGATGCCCCAAGCTGAGA-3’,R:5’-CACGGCCTTGCTCTTGTTTT-3’时可以获得白介素10荧光定量PCR检测的最佳条件。②与对照组相比,糖尿病肾病组患者外周血白介素10的表达水平明显下调(糖尿病肾病组16.21±1.84 vs对照组14.39±1.14),差异有统计学意义(t=7.049,P<0.01)。结论该研究建立了检测白介素10的稳定的荧光定量PCR方法;应用该检测方法发现,白介素10在糖尿病肾病组中的表达明显下降,为白介素10在糖尿病肾病中的功能研究和临床检测提供参考和借鉴。
Objective A stable and sensitive method for the detection of human interleukin-10(IL-10) real-time fluorescence quantitative PCR was established to compare the expression differences of IL-10 in diabetic nephropathy group and control group.Methods The peripheral venous blood lymphocyte RNA samples were obtained from patients with stage IV diabetic nephropathy.The expression level of IL-10 was detected by real-time quantitative PCR.This experiment mainly explored both template concentration and primer screening to establish a stable and sensitive experimental method for comparing the expression levels of IL-10 in 33 patients with diabetic nephropathy and 35 normal healthy groups.Results ①After reversing transcription of 475 ng of total RNA into cDNA,diluting the template,and when the template concentration dilution range was within 10~3 times,and the amplification primers were F:5’-GTGATGCCCCAAGCTGAGA-3’,R:5’-CACGGCCTTGCTCTTGT TTT-3’,it could obtain the optimal conditions for the detection of IL-10 fluorescence quantitative PCR.②Compared with the control group,the expression level of IL-10 in peripheral blood of patients with diabetic nephropathy was significantly down-regulated(diabetic nephropathy group 16.21±1.84 vs control group 14.39±1.14),and the difference was statistically significant(t=7.049,P<0.01).Conclusion The study established a stable real-time quantitative PCR method for the detection of IL-10.By this method,it was found that the expression of IL-10 was significantly decreased in the diabetic nephropathy group.It will provide reference for the functional research and clinical detection of IL-10 in diabetic nephropathy.
作者
岳芳芳
倪文娟
冷小敏
刘云
师晶晶
曹东东
郭明好
马东红
YUE Fang-fang;NI Wen-juan;LENG Xiao-min;LIU Yu;SHI Jing-jing;CAO Dong-dong;GUO Ming-hao;MA Dong-hong(Institute of Nephrologyand Immunology,the Hospital of Ngphrology,Henan Weihui 453100,China;Life Science Resfarch Center,the First Affiliated Hospital of Xinxiang Medical University,Henan Weihui 453100,China)
出处
《现代检验医学杂志》
CAS
2019年第4期11-14,共4页
Journal of Modern Laboratory Medicine
基金
河南省科技厅重点研发与推广项目(182102310585)
河南省高等学校重点科研项目(17A320026)
河南省卫生厅科技攻关省部共建备选项目(2018010013)
