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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: Traditional Chinese medicine Jinkui Shenqi Pill can improve the symptoms of kidney deficiency, but its molecular mechanism remains unclear. OBJECTIVE: To observe differentially expressed genes in mice treated with panic-induced kidney deficiency and traditional Chinese medicine Jinkui Shenqi Pills by suppression subtractive hybridization. Design: Randomized controlled animal experiments. Unit: Department of TCM Genetics, Chengdu University of Traditional Chinese Medicine, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University. Materials: Experiments were performed at the Experimental Animal Center of Chengdu University of Traditional Chinese Medicine. Thirty male BALB/c male mice were randomly divided into 3 groups: model group, drug treatment group and control group, with 10 mice in each group. METHODS: The mice of model group and drug treatment group were treated with “fear-inducing kidney” method and suspension stress method to prepare the kidney-rat model. The mice in the drug treatment group were fed with Jinkui Shenqi Pill every day (produced by Beijing Tongrentang Pharmaceutical Factory, consisting of Guizhi, Fuzi, Rehmannia, yam, hawthorn, Alisma, wolfberry, and paeonol, etc.), and the dosage was administered as adults. 20 times the dose. A total of 14 days were used for modeling and administration. The mice in the control group grew naturally and did not exert any external stimuli. At 24 h after the last administration, 100 μL of blood was collected from all three groups of mice, and SMART technology was used to reverse transcribe and expand to obtain the full-length total cDNA of each group of mice blood; positive and negative suppression subtractive hybridization techniques were used to obtain positive results. Six sets of differentially expressed cDNA fragments were inverted in two directions and under three conditions (kidney deficiency, after treatment, and normal physiological state). MAIN OUTCOME MEASURES: Effect of Jinkui Shenqi Pill on gene expression in kidney deficiency mice. RESULTS: 1The cDNA fragments were differentially expressed in each group; after treatment with traditional Chinese medicine, the differential gene expression profiles of kidney-deficient mice were close to normal physiological conditions; the same cDNA was used as the tester and driver for suppression subtractive hybridization, Fragments are all subtracted and the efficiency of elimination is high. 2 Six cDNA libraries were obtained. Conclusion: The kidney deficiency caused by panic attacks is related to the differential expression of some genes, but the traditional Chinese medicine Jinkui Shenqi Pill can influence this change, making the differentially expressed gene spectrum approach normal physiological state.