肾阴虚证的血浆标志蛋白研究

Kidney-Yin deficiency, the common syndrome of Traditional Chinese Medical in many clinical diseases, still lack of objective marker in clinical diagnosis. Our previous proteomics researches in kideny yin deficiency showed that it exist identical differential protein in blood plasma among different morbid state accompanied with kideny yin deficiency. In this study, we plan to apply the combination of syndrome and symptom, and enlarge our research subjects in which patients with Kidney-Yin deficiency are selected in different disease. SELDI-TOP-MS and other proteomics technology are used to analyze differential blood plasma protein in patients with Kidney-Yin deficiency and screen these differential expressed protein in Kidney-Yin deficiency. According to the theory of "Syndrome evaluation by Drug", blood plasm protein changes in Kidney-Yin deficiency will be compared after interfered with Liuweidihuang pills.Then we presume their signal effects in Kidney-Yin deficiency to get its marker protein in blood plasma, which could be synthesized in protein clips for the clinical diagnosis and prognosis of Kidney-Yin deficiency，in order to determine the plasma marker- protein of Kidney-Yin deficiency These will not only provide objective index in the diagnosis standardisation of Kidney-Yin deficiency, but also cinfirm the existence of common material basis in "the same syndrome in different diseases" which offer scientific evidence by the theory of "the same treatment for different diseases".

肾阴虚证是临床多种疾病的常见证侯之一，但迄今为止尚无公认的客观化诊断指标。课题组前期应用蛋白组学研究发现：不同疾病肾阴虚证血浆中存在相同的差异蛋白质，因而我们认为肾阴虚证血浆中可能存在标志性的蛋白质。本项目拟采用"病证结合"，扩大样本，首先在前期2－DE基础上引入SELDI-TOP-MS等蛋白组学技术对肾阴虚证常见疾病进行血浆蛋白质差异分析，筛选肾阴虚证的差异表达蛋白质；其次应用"以药测证"方法，对肾阴虚证患者进行六味地黄丸临床干预，分析干预前后血浆蛋白质的变化，进一步探讨上述筛选出的差异蛋白质对肾阴虚证的标志性意义；最后将上述获得的肾阴虚证差异蛋白质合成探针制成蛋白质芯片，扩大样本，与新收的临床肾阴虚证患者血浆进行杂交，进一步剔除假阳性差异蛋白，最终确定肾阴虚证的血浆标志蛋白。这将为肾阴虚证的临床诊断和预后评估提供客观化指标，同时也将为"异病同治"理论提供科学依据。

肾阴虚证是中医学的基本证候之一，历代医家对肾阴虚证的理论与防治研究都颇为重视，但对于肾阴虚证的客观化研究较为局限，其临床诊断方法仍然依靠主观辨证，迄今为止尚无公认的客观化诊断指标。本项目在前期基础上，采用“病证结合”，首先应用SELDI-TOP-MS技术，筛选出肾阴虚证血浆中有19个蛋白质峰差异有统计学意义(P<0.05)，并以3个蛋白标志物(1847.8、5906.3、14814.8 Da)建立的筛选模型检测灵敏度为87.5%，特异性为75.0%。其次，应用RayBiotech 蛋白芯片技术，筛选出肾阴虚证患者血清中有 16个差异表达的细胞因子，其中 4个表达上调、12个表达下调(P＜0.05)；而肾阳虚证患者血清中有 19个差异表达的细胞因子，其中18个表达上调、1个表达下调(P＜0.05)。最后，应用“方证相应”方法，开展“六味地黄丸—肾阴虚证”的临床试验和动物实验，分析干预前后肾阴虚证血浆蛋白质的变化，进一步剔除假阳性差异蛋白，最终获得肾阴虚证血浆中有6个差异表达的蛋白质（Angiostatin、Chordin Like 2、FGF-23、Inhibin B、M-CSF R、OSM等）。总之，本项目初步明确了肾阴虚证的血浆标志蛋白，为肾阴虚证的临床诊断和预后客观评价奠定了基础，同时也为“异病同治”理论提供实验证据。