骨质疏松肾阴虚型小鼠病证结合模型建立的实验研究

摘要

目的 采用病、证模型复合方法建立骨质疏松肾阴虚型病证结合模型.方法 雌性昆明小鼠45只,随机分为对照组(A组)、骨质疏松症模型组(B组)、骨质疏松肾阴虚型模型组(C组),每组15只.骨质疏松模型采用去势法复制,骨质疏松肾阴虚模型采用去势结合甲状腺素灌服法诱导.通过观察小鼠的症状体征、下丘脑-垂体-靶腺轴(肾上腺,甲状腺)相关指标及环磷酸腺苷(cAMP)与环磷酸鸟苷(cGMP)等验证肾阴虚证；通过测定骨密度、骨组织形态学及护骨素(OPG)和细胞核因子κB受体激活物配基(RANKL)基因表达等评估骨质疏松症.结果 与A组小鼠相比,C组动物出现明显的毛发干枯,大便干结,体质量下降,肛温升高等类似肾阴虚表现,促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、促甲状腺激素(TSH)、游离三碘甲状腺原氨酸(FT3)含量升高,cAMP/cGMP升高,股骨及腰椎骨密度降低,骨组织HE染色及骨形态计量学示明显骨质疏松症征象,骨组织OPG表达下凋,RANKL表达上调.与B组相比,C组在骨密度、骨组织形态学、OPG及RANKL基因表达,差异均无统计学意义.结论 去势结合甲状腺素复制骨质疏松肾阴虚病证结合模型是可行的,该模型同时具有肾阴虚和骨质疏松症的特征改变,更接近临床实际情况,适合作为绝经后骨质疏松肾阴虚型病证结合模型进一步在中医药研究领域推广运用.%Objective:To establish the osteoporosis model with type of deficiency of Kidney-Yin.Methods:45 female Kunming mice were divided randomly into control group(group A),osteoporosis group(group B),osteoporosis integrated with syndrome group(group C).Osteoporosis model were replicated by castration.The model group C were established by excision bilateral ovaries and thyroxine.Through observing the signs and symptoms of mice,detecting related index of hypothalamus-pituitary-target gland axis (adrenal gland,thyroid gland),ring adenosine monophosphate(cAMP)and cyclic guanosine phosphate(cGMP)to verify the kidney Yin deficiency syndrome.By measuring osteoprotegerin(OPG)and nuclear factor kappa B receptors predominate activators ligands(RANKL)gene expression,bone density and bone tissue morphology to evaluate osteopomsis.Results:Compared with group A,mice in group C appeared obvious kidney-Yin deficiency symptoms,including dry hair,hard stool,frail and yellow urine,decreased weight etc.The level of CRH,ACTH,TSH,FT3(P ＜ 0.01,P＜ 0.01,P ＜0.05,P＜0.05),the ratio of cAMP/cGMP increased(P＜0.01),the bone density of lumbar vertebra and femur decreased significantly(P＜ 0.05);HE staining revealed osteoporosis.The level of OPG decreased(P＜ 0.01)and RANKL increased(P＜ 0.01).However there are no difference between group B and group C on bone density,morphological changes and the level of OPG and RANKL(P＞ 0.05).Conclusion:The model of osteoporosis with type of deficiency of Kidney-Yin could be established by excision bilateral ovaries and thyroxine.Since had characteristics of kidney Yin deficiency and osteoporosis,it may be a stable and reliable model for the studies of osteoporosis in TCM.