运用蛋白芯片联合质谱检测技术研究阴虚阳虚体质方药干预的机制

Under the guidance of the theory of "formula matched constitution".In accordance with the previous research on yin-deficiency and yang-deficiency constitution and the neuro- endocrine immune system factors, genomics, metabolomics result,we put forward the hypothesis of "Different constitution has different adaptability and response to certain formula.This is based on the human constitution type and state.And the molecular mechanism of adjustment of yin-deficiency and yang-deficiency constitution by corresponding formula is to regulate the imbalance of energy metabolism and neuro- endocrine system foctors "Thus,in this study,we use the technique of SELDI-TOF-MS to detect the differences of energy metabolism and neuro- endocrine system foctors between yin-deficiency and yang-deficiency constitution. Then, we plan to give Liuweidihuang and Guifudihuang pills to the corresponding constitution, and to observe the effect of them on regulating the constitutions.Through the above research, the connotation of yin-deficiency and yang-deficiency constitutions will be revealed. The theory of "formula matched constitution"and "constitution can be adjusted" will be proved. And the thought of "individual diagnosis and treatment" will be manifested. It has great significance for the promotion of "treatment based on constitution" and its application in the individual medicine and health medicine.

基于辨体用方思想，以中医阴虚和阳虚体质人群及其对应干预方剂为切入点，根据前期阴虚、阳虚体质与神经-内分泌-免疫学、基因组学和代谢组学研究结果，提出"不同体质对所应用方剂有不同的适应性与反应表达，方-体相应是以人的体质类型与状态为依据，使之'方为人所宜'；阴虚和阳虚体质方药干预的分子机制可能与调控能量代谢和神经-内分泌表达失调有关"的假说。本课题运用蛋白芯片联合质谱（SELDI）技术，检测阴虚和阳虚体质人群的能量代谢和神经-内分泌相关分子的差异表达及其应用六味地黄丸和桂附地黄丸对应干预的调控效应及其机制。本课题对于揭示中医阳虚、阴虚体质的现代内涵，论证 "方-体相应"假说、"体质可调"的科学原理，彰显中医个体化诊疗思想，推动辨体论治在个体医学、健康医学的临床应用，具有重要的理论和实践意义。

主要研究内容：.阳虚和阴虚体质人群蛋白差异表达的比较：依照中华中医药学会《中医体质分类与判定》标准，在一般人群中筛选阳虚、阴虚体质人群，并设平和体质对照组。运用RayBio Label-based (L-Series)Human Antibody Array 1000蛋白芯片联合质谱技术检测并分析比较各组人群外周血单个核细胞（PBMC）和血清中能量代谢和神经-内分泌相关分子的差异表达。检测结果经生物信息学分析，得出各组有差异表达的蛋白分子及通路，Western blot法对蛋白芯片检测得出差异的分子进行验证。.阳虚、阴虚体质人群差异蛋白的方药干预：前后比较分别以桂附地黄丸干预阳虚体质人群，以六味地黄丸干预阴虚体质人群，疗程为3个月。干预前和干预结束后抽取空腹静脉血，检测血清差异蛋白的表达，生物信息学分析后对差异表达分子进行验证。.重要结果：.经蛋白芯片技术检测，检测到阴虚体质、阳虚体质分别与平和体质在能量代谢、神经内分泌、免疫功能等方面存在蛋白表达的差异，其差异蛋白表达均为同向、；经质谱技术检测，检测到阴虚体质、阳虚体质分别与平和体质在能量代谢、神经内分泌、免疫功能等方面存在蛋白表达的差异，发现富集到同一GO功能的蛋白在二者之间存在相反表达，并用westernblot对其进行进一步验证；对阴虚体质、阳虚体质分别在六味地黄丸、桂附地黄丸干预前后的蛋白表达差异进行检测，结果显示阴虚体质、阳虚体质与平和体质相比分别存在神经内分泌及免疫功能紊乱，六味地黄丸与桂附地黄丸分别能对其表达紊乱进行调节。.科学意义：.本课题研究发现阴虚体质与阳虚体质在蛋白层面能量代谢和神经-内分泌存在异常，与基因组学、代谢组学已有研究结果相印证，进一步推进完善了阴虚体质与阳虚体质的微观机制研究。通过研究初步筛选到阴虚体质和阳虚体质潜在的微观生物标记物，有助于九体医学对体质模块的辨识；与对每个个体分别进行基因测序的精准医学相比，九体医学通过模块化思想将人群分类，彰显中医个体化诊疗思想，推动辨体论治在个体医学、健康医学的临床应用