应用基因芯片研究针刺涌泉延缓衰老的差异表达基因谱

【作者】 张卫；

【导师】 罗永芬； 王米渠；

【作者基本信息】 成都中医药大学 ， 针灸学， 2002， 博士

【摘要】 目的：应用基因芯片筛选出正常大鼠与D-半乳糖衰老造模大鼠及针刺涌泉穴后与D-半乳糖衰老造模大鼠的差异表达基因谱，研究针刺延缓衰老的差异表达基因谱，初步探讨针刺延缓衰老的基因机理。方法：(1)将成年健康雄性大鼠随机分为正常成年对照组(简称正常组)、D—半乳糖亚急性衰老模型组(简称模型组)和D—半乳糖亚急性衰老模型+针刺治疗组(简称针刺组)。模型组每日皮下注射10％D-半乳糖0.5ml，正常对照组注射相同体积的生理盐水，针刺组在注射D—半乳糖造模的同时予以每周针刺涌泉穴6天，进行延缓衰老治疗。7周后同时处死动物，摘取肝脏提取mRNA，并逆转录为cDNA以备基因芯片检测及应用p53与bc1-2两条基因检验此模型的成功性。随机选用2305条鼠cDNA制备成表达谱芯片。分别用Cy3和Cy5两种荧光物质分别标记正常组与模型组鼠及针刺组与模型组肝组织的cDNA，并制备成探针与表达谱芯片进行杂交，通过计算机扫描后进行数据分析。结果：模型组与正常组相比较有168条基因的表达量发生了明显的变化，针刺组与模型组相比较有318条基因的表达量有显著差异。其中有37条基因在两次杂交中是共有的，可以认为这些是本次针刺涌泉穴延缓衰老实验得到的与针刺延缓衰老直接相关的差异表达基因，同时针刺可以明显改变衰老组织中p53的表达量。结论：针刺涌泉穴可以在改善衰老动物整体状态及各项生理生化指标的同时影响许多基因的表达量，这种影响是一种广泛的整体效应并不仅仅局限于某一范围之内。利用基因芯片技术结合实验动物模型能大规模、高通量、高效率地研究有关针刺治疗作用的基因表达谱的变化，对进一步从微观整体角度研究针刺治疗的基因机理，判定针刺治疗的基因疗效以及衰老与基因的关系有十分重要的作用。更多还原

【Abstract】 Objective: To study the Differentially expressed genes of the effect of acupuncture Yongquan point on aging rat model by gene microarrays. Methed: ffe depart adult male rats as normal adult group , aging model group and acupuncture group at radom, aging model group was made by subcutaneous injection of 10% D-galactose 0.5ml one time everyday, while normal adult group was made by the injection of the same volume physiological salt water, at same time acupuncture group treated by acupuncture Yongquan point every day for 6 times a week. After 7 weeks, the rat were sacrified, We harvested their livers to extract mRNA to reverse transcription for cDNA microarray analyses. For the microarray analyses and verify whether the model is correctly with p53 and bcl-2, 2305 cDNA were selected to made microarray. cDNA extracted from model rats were labeled with cy5~dUTP and cy3-dUTP, while cDNA extracted from ilk1 nuriiiiil ^roiip weIX1 hilx’led with cy3-dl’TP and cy5~dLTP for to\v hybridized. These probes were hybridized with the cDNA microarrays, scanned, and analyzed. Result: There are 168 genes expression were clearly changed between adult group and aging model group and 318 differentially expressed in ageing rats treated by acupuncture. In these differentially expressed genes, 37 genes are same. While through acupuncture we could change p53’ s expression. Conclusion: Through acupuncture Yongquan point we can not only ameliorate ageing rats’ physiological and biochemical change, but also affect their gene expression. This effect is on whole. Using gene microarray techniquecombining animal model experiments can study the differentially expressed genes about aging in large scale , high throughout and high efficiency. Gene microarray will act as an important role in study the molecular mechanism of acupuncture and the relationships between aging and gene.更多还原