摘要
目的观察去甲肾上腺素（NE）、血管紧张素Ⅱ（AngⅡ）对培养乳鼠心肌细胞表达TNF-α的影响。方法取1～3天SD乳鼠100只进行心肌原代培养，分为7组，对照组、10～mol／L NE组（A组）、10^-6mol／LNE组（B组）、10^-7mol／LNE组（C组）、10^-7mol／L AngⅡ组（D组）、10^-8mol／L AngⅡ组（E组）和10^-9mol／L AngⅡ组（F组）。采用免疫组织化学法和RT—PCR测定上述不同浓度培养心肌细胞24、48h，评价不同浓度NE、AngⅡ对心肌细胞表达TNF-α的影响。用凝胶滞留实验验证NF-κB是否参与TNF-α的转录调控，筛选TNF-α可能启动子的位点。结果对照组心肌细胞TNF-α呈阴性，B、C、E、F组呈弱阳性，A组和D组呈阳性。对照组心肌细胞表达微量的TNF-α mRNA，NE和AngⅡ各组表达显著增高，并存在浓度和时间梯度。NE和AngⅡ各组的核蛋白与其上游含有潜在NF－κB结合位点的两段寡核苷酸的结合强于对照组。结论NE和AngⅡ能促使培养的心肌细胞表达TNF—α，NF-κB的核转移是激活TNF－α表达的共同通路。
Objective To observe the effects of norepinephrine（NE） or angiotensinⅡ （Ang Ⅱ ） on expression of TNF-α in cardiomyocytes. Methods Primarily cultured rat cardiomyocytes were incubated with 10^-5 mol/L（A Group）, 10^-6 mol/L （B Group） and 10^-7 mol/L （C Group） NE and 10^-7 mol/L （D Group）, 10^-8 mol/L （E Group） and 10^-9 mol/L （F Group） Ang Ⅱ for 24 or 48 hours. Immunohistochemistry,RT-PCR and electrophoretic motif shift assay（EMSA）were used to detect TNF-α expression and the possible nuclear factor NF-κB which could be translocated to the nucleus and to screen the probable promoter of TNF-α. Results Immunohistochemical study revealed that the cardiomyocytes of control group were TNF-α negative, while those of the B,C, E, and F groups were weakly positive, those of A and D groups were positive. TNF-α mRNA was detected in control cardiomyocytes, but was obviously increased in NE or Ang Ⅱ treated cardio myocytes and increased with increasing concentration and incubation time. The translocation of NF-κB to the nucleus was increased in cardiomyocytes treated with NE or Ang Ⅱ. The potential promoter may exist in two upstream oligonucleotides of TNF-α. Conclusion The mRNA level of TNF-α was very low in the control cultured new born rat cardiomyocytes,but it was obviously increased in cardiomyocytes treated with NE or Ang Ⅱ , with NF-κB activation and translocation to the nucleus, which may be the pathway leading to expression of TNF-α.
出处
《中华老年心脑血管病杂志》
CAS
北大核心
2008年第11期849-851,共3页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
国家科学自然基金（30600236）
