基于骨组织差异蛋白质组的中医"肾主骨"理论的研究

As one of classical theories in Traditional Chinese Medicine(TCM), "Kidney governing bone" was used to describe the relationship between kidney and bone and guide the treatment and therapy of bone disease in clinical practice, however the pathogenesis were deficiency in essence. Based on the research of differential proteomics and corresponding study in early stages, we hypothesized that the theory of "Kidney governing bone" is the functional condition of Kidney in TCM which regulated the expression of differential proteomics and genome of bone tissues, the intrinsic relationship between Kidney and bone may be the connection between the functional status of Kidney in TCM and the expression of the special functional proteomics and genome of bone tissues. To demonstrate this hypothesis, this topic will show and compare the differenciation proteomics and genome of bone tissues(cortical bone and spongy bone)from human and rats under the status among deficiency of "Shenyin", "Shenyang" and sufficiency of "Shen" with the techniques and methods related to proteomics and molecular biology ,respectively. In order to explore the scientific connotation of the theory of "kidney governing bone" in TCM and the "deficiency of Kidney leading to bone disease", the aims are to conform the natrue relationship between the functional status of Kidney in TCM and the expression level of differential proteomics and genome of bone tissues, and finally to establish research basis for paying attention to Kidney for treating bone disease in clinical practice.

"肾主骨"是中医经典理论之一，是阐述肾与骨之间内在联系的重要理论，并一直指导着中医临床骨病的诊治，但目前对肾如何"主"骨及肾与骨之间内在联系的实质还不明确。依据蛋白质组学理论及前期相关研究，我们提出这样假说："肾主骨"可能是中医肾的功能状态对骨组织本身功能蛋白质组及其功能基因组表达调控的影响，肾与骨之间的内在联系可能就是中医肾的功能状态与骨组织特定功能蛋白质组及其基因组表达水平的内在关联性。为论证此假说，本课题利用蛋白质组学及分子生物学相关的技术与方法，分别展示人与大鼠的骨组织（皮质骨与松质骨）在肾阴虚、肾阳虚及无肾虚状态下差异的蛋白质组及其基因组，并进行互相比较验证。旨在从骨组织差异的功能蛋白质组及其基因组表达水平证实其与中医"肾"功能状态的内在关联性，从而深入阐释中医"肾主骨"、"肾虚骨病"理论的科学内涵及"肾"与骨联系的实质，为临床从肾为主论治骨病奠定研究基础。

课题采用蛋白质组学及分子生物学相关技术与方法，分别展示了肾阴虚、肾阳虚及正常状态下大鼠与人骨组织（皮质骨与松质骨）差异蛋白质组及其基因组，并通过“治疗反证”及多项技术证实差异表达的蛋白，为进一步阐释“肾主骨”理论的部分实质和提高“从肾为主论治骨病”的认识与实践提供了实验依据。. 首先，获取肾阴虚、肾阳虚大鼠及人骨组织标本。采用观察活动度、体温、体重等，检测骨密度、血清cAMP、cGMP及骨代谢等指标，分别获得成功且稳定的肾阴虚、肾阳虚证大鼠模型；制定了人肾阴虚、肾阳虚证临床评分标准；收集了大鼠与人骨组织研究标本。其次，展示并分析肾阴虚、肾阳虚及正常大鼠与人皮质骨、松质骨组织双向电泳凝胶图谱，结果显示大鼠（人）皮质骨中有12（15）个、松质骨14（18）个蛋白与肾阴虚有关，大鼠（人）皮质骨12（15）个、松质骨23（16）个蛋白与肾阳虚有关。第三，通过“治疗反证”，证实大鼠皮质骨中8（6）个、松质骨中10（7）个差异表达的蛋白经补肾阴（肾阳）治疗后原差异表达得到纠正。第四，随机选择经“治疗反证”明确的肾阴（肾阳）虚大鼠皮质骨6（6）个、松质骨7（7）个，经Western Blot检测证实，除肾阴虚松质骨1个蛋白表达差异不明显外，其它蛋白的差异表达都存在；另外，随机选择肾阴（肾阳）虚人皮质骨松质骨各3（3）个，经该技术检测证实它们的差异表达都存在。考虑肾阴虚、肾阳虚共有蛋白对于整体认识“肾主骨”更有意义，故选择经前面确认且两证共同差异表达的蛋白：大鼠皮质骨3个、松质骨2个及人皮质骨松质骨各3个蛋白，进行RT-PCR验证，结果证实这些差异表达蛋白的mRNA也存在差异。第五，选择经RT-PCR确认的大鼠5个、人6个蛋白，进行血清Elisa检测，证实大鼠Arhgdib，人血液中Hsp70、BMP-4、Arhgdib等3个蛋白与骨组织表达差异趋势一致，这些蛋白可以作为肾虚的血清潜在标志物开展进一步研究。. 由于时间关系，本研究只采取阶梯式对部分差异蛋白进行系列验证，另一些蛋白的验证研究有待后续开展。