应用抑制消减杂交研究惊恐致肾虚及金匮肾气丸治疗后小鼠的差异表达基因(英文)

Using suppression subtractive hybridization to research the effects of jinguishenqi pills on the gene expression of the panic-induced kidney deficiency model mice

背景:中药金匮肾气丸能改善肾虚症状,但其分子机制还不清楚。目的:用抑制消减杂交技术观察惊恐致肾虚及中药金匮肾气丸治疗后小鼠的差异表达基因。设计:随机对照动物实验。单位:成都中医药大学中医遗传学研究室,四川大学生命科学学院分子生物学与生物技术四川省重点实验室。材料:实验在成都中医药大学实验动物中心进行。取BALB/c雄性成年小鼠30只,单纯随机分为模型组、药物治疗组和对照组3组,每组10只小鼠。方法:模型组和药物治疗组小鼠应用“恐伤肾”法和悬吊应激法制备肾虚鼠模型。药物治疗组小鼠每天灌服金匮肾气丸(北京同仁堂制药厂生产,由桂枝、附子、熟地、山药、山茱萸、泽泻、茯苓、丹皮等组成)1次,给药剂量按成人用药量的20倍。造模及给药时间总共为14d。对照组小鼠自然生长,不施加任何外界刺激。最后一次给药后24h,3组小鼠均取血100μL,应用SMART技术反转录并扩增以获取各组小鼠血液的全长总cDNA;运用正向和反向抑制消减杂交技术获得正反两个方向、3种状态下(肾虚、治疗后以及正常生理状态)的6组差异表达cDNA片段。主要观察指标:金匮肾气丸对肾虚小鼠基因表达的影响。结果:①各组均有差异表达的cDNA片段;中药治疗后,肾虚小鼠的差异基因表达谱有向正常生理状态下接近的趋势;用同一种cDNA作为抑制消减杂交的tester和driver,同源片段全部被消减掉,消减效率较高。②获得了6个cDNA文库。结论:惊恐所致肾虚与一些基因的差异表达相关,而中药金匮肾气丸能影响这种改变,使其差异表达基因谱趋近于正常生理状态。

BACKGROUND： Jinguishenqi pills （JP） can ameliorate the symptoms of kidney deficiency, but the molecular mechanism of the effect is unclear. OBJECTIVE： To observe the differentially expressed genes of panic-induced kidney deficiency model mice treated with JP using suppression subtractive hybridization （SSH）. DESIGN： Randomized control animal experiment. SETTING： Institute of Traditional Chinese Medicine Genetics, Chengdu University of Traditional Chinese Medicine; Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Science, Sichuan University. MATERIALS： This experiment was carried out at the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine. Totally 30 BALB/c male adult mice were used in the experiment, and they were randomly divided into model group, JP-treated group and control group, with 10 in each. METHODS： Mice models of kidney deficiency were created in the mice of model group and JP-treated group with the methods of “terrifying mouse with cat” and “suspending mouse over water”. Mice of the JP-treated group were intragastrically administrated of JP （produced in Tongrentang Pharmacy Co., Beijing, Z11020147, composed of Guizhi, Fuzi, Shudi, Shanyao, Shanzhuyu, Zexie, Fuling, Danpi, etc） 20-fold as human taken according to the bodyweight once per day. Modeling and administration were conducted in 14 days. Mice of normal control group were fed normally without any extra stimulation. Twenty-four hours after the last administration, 100 μL blood was collected respectively from mice of each group. Switch Mechanism at 5＇ end of RNA Template （SMART） technique was used in reverse transcription and amplification to obtain the whole length of total cDNA of mice in each group; Forward and reverse SSHs were used to acquire 6 groups of differential expression cDNA segments which were under 3 states （at kidney deficiency, after treatment and at normal physiological status）. MAIN OUTCOME MEASURES： Effect of JP on gene expression of kidney deficiency mice RESULTS： ① Differentially expressed cDNA segments were found in each group; The differentially expressed cDNA profile of model mice had restored to that of the normal physiological state by JP treatment, cDNA of the same kind was used as the tester and driver of SSH, and almost all the homologous fragments were subtracted completely, which showed that the subtraction efficiency was very high. ② 6 cDNA libraries were acquired. CONCLUSION： Panic-induced kidney deficiency is related to differential expression of some genes. The gene expression of the kindey deficiency model mice could be affected by JP treatment,making it trend to normal physiological status.