经典气血阴阳虚证模型小鼠在“肾藏象”层面的证候物质基础研究

Study on material basis of model mice with classic deficiency syndrome of qi,blood,yin and yang in viscera state of kidney

目的研究气血阴阳虚证模型小鼠在"肾藏象"层面的证候物质基础。方法以雄性ICR小鼠为对象,以控食法复制气虚证模型,以乙酰苯肼复制血虚证模型,以甲状腺素复制阴虚证模型,以氢化可的松复制阳虚证模型。动态观察小鼠体质量变化,检测各脏器变化;取肾脏、肾上腺和骨髓组织抽提RNA,以实时荧光定量PCR技术检测相关基因表达;以ELISA方法检测血清皮质酮含量。结果 (1)与正常对照组比较,控食组小鼠体质量下降最显著(P<0.01),乙酰苯肼和氢化可的松组小鼠体质量也显著下降(P<0.05);控食组和氢化可的松组小鼠脾脏与胸腺明显萎缩(P<0.01),乙酰苯肼组小鼠脾脏显著肿大、而胸腺严重萎缩(P<0.01)。(2)与正常对照组比较,控食组和乙酰苯肼组血清皮质酮水平显著升高(P<0.05),而氢化可的松组显著下降(P<0.001)。(3)控食组小鼠肾上腺合成皮质激素的重要酶Star、Cyp21a1、Cyp11b1表达显著升高(P<0.05);肾脏Epo表达显著升高(P<0.05),Csf2显著下降(P<0.05);骨髓Tpo、IL-4表达显著上调(P<0.05)。(4)乙酰苯肼组小鼠肾上腺Cyp11a1、Cyp21a1、Cyp11b1表达显著升高(P<0.05);肾脏Epo、Csf2、Csf3表达显著升高(P<0.05);骨髓Csf3、IL-4、IL-5、IL-6表达显著升高,而IL-1b和IL-7显著下降(P<0.05)。(5)甲状腺素组小鼠肾上腺Cyp11b1表达显著升高(P<0.05);肾脏Epo表达显著升高,Csf2显著下降(P<0.05);骨髓Csf3、IL-2和IL-5表达显著下降(P<0.05)。(6)氢化可的松组小鼠Star、Cyp21a1、Cyp11b1、Cyp11b2表达显著下降(P<0.05),肾脏Epo表达显著升高,而Csf1和Csf2显著下降(P<0.05),骨髓Csf3、IL-1b、IL-5和IL-7表达显著下降,IL-4却显著升高(P<0.05)。结论通过控制摄食量,给予乙酰苯肼、甲状腺素及氢化可的松,可以复制出相关的中医虚证模型,且在肾上腺、肾脏及骨髓等"肾藏象"层面存在相应的物质功能变化。

Objective To study the material basis of model mice with deficiency syndrome of qi,blood,yin and yang in viscera state of kidney. Methods Taken the male ICR mice as objects,the model of syndrome of qi deficiency was replicated by dietary restriction method,the model of syndrome of blood deficiency was replicated by acetylphenylhydrazine,the model of syndrome of yin deficiency was replicated by thyroxine,the model of syndrome of yang deficiency was replicated by hydrocortisone. The body weight of mice was observed everyday,and the changes on different organs were compared. RNA was extracted in the kidney,adrenal and bone marrow tissue. The related gene expressions were detected by real time fluorescence quantitative PCR. The serum content of corticosterone was detected by ELISA. Results(1)Compared with the normal control group,the reduction on body weight of mice was most significant in the dietary restriction group( P<0.01),the body weight of mice in the acetylphenylhydrazine group and hydrocortisone group were also significantly decreased( P<0.05).The obvious atrophy in the spleen and thymus was observed in the dietary restriction group and hydrocortisone group( P<0.01),the significant swelling in the spleen and severe atrophy in the thymus were found in the acetylphenylhydrazine group( P<0.01).(2)Compared with the normal control group,the serum level of corticosterone was significantly increased in the dietary restriction group and acetylphenylhydrazine group( P < 0.05),while the serum level of corticosterone was significantly decreased in the hydrocortisone group( P<0.001).(3)In the dietary restriction group,the m RNA expressions of Star,Cyp21 a1 and Cyp11 b1 in the adrenal gland were significantly increased( P <0.05),and the Epo m RNA expression in the kidney was significantly increased( P<0.05) and the Csf2 m RNA expression in the kidney significantly decreased( P<0.05),the m RNA expressions of Tpo and IL-4 in the bone marrow were significantly up-regulated( P < 0.05).(4) In the acetylphenylhydrazine group,the m RNA expressions of Cyp11 a1,Cyp21 a1 and Cyp11 b1 in the adrenal gland were significantly increased( P < 0.05),the m RNA expressions of Epo,Csf2 and Csf3 in the kidney were significantly increased( P<0.05),the m RNA expressions of Csf3,IL-4,IL-5 and IL-6 in the bone marrow were significantly increased,but the m RNA expressions of IL-1 b and IL-7 in the bone marrow were significantly decreased( P<0.05).(5)In the thyroxine group,the Cyp11 b1 m RNA expression in the adrenal gland was significantly increased( P<0.05),the Epo m RNA expression was significantly increased and Csf2 m RNA expression was significantly decreased in the kidney( P<0.05),the m RNA expressions of Csf3,IL-2 and IL-5 in the bone marrow were significantly decreased( P < 0.05).(6)In the hydrocortisone group,the m RNA expressions of Star,Cyp21 a1,Cyp11 b1 and Cyp11 b2 in the adrenal gland were significantly decreased( P<0.05),the Epo m RNA expression was significantly increased and m RNA expressions of Csf1 and Csf2 were significantly decreased in the kidney( P<0.05),the m RNA expressions of Csf3,IL-1 b,IL-5 and IL-7 were significantly decreased and IL-4 m RNA expression was significantly increased in the bone marrow( P<0.05). Conclusion The related model of Chinese medical deficiency syndrome can be replicated by dietary restriction,administration of acetylphenylhydrazine,thyroxine and hydrocortisone. And there are corresponding changes on the material function in viscera state of kidney such as adrenal gland,kidney and bone marrow.