抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库，并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA，分别作为tester和driver进行消减杂交及抑制性PCR，将PCR产物与载体连接构建cDNA库，高效电转化大肠杆菌进行库扩增，随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序，生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系，其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用，可能和四物汤补血作用有关。

The isolation of differentially expressed cDNAs from Siwutang-induced mouses with SSH

Differentially expressed genes Siwutang induced were identified by Suppression Subtractive Hybridization(SSH),and the function of Siwutang was studied.SSH has been used to compare mouse bone marrow cells of radia-tion destroy model treated with Siwutang with untreated cells,and cDNA library was made.518clones was selected in cDNA library made by SSH.The mRNAs for at least8novel gene fragments were elevated by such treatment,including a cDNA clone which shared high homology with human proto oncogene MDM2,mouse alpha-globin,mouse neuronal apoptosis inhibitory protein6,mouse WNT-2,mouse neural precursor,mouse Bmp7,et al.Siwutang will be well elucidated.Siwutang can inhibit apoptosis,induce marrow repair and RBC differentiation.SSH technology can be successfully applied to identify differentially expressed genes.This approach allowed the identification of differ-entially expressed genes without the need to obtain previously cloned cDNAs.The molecular mechanism of traditional Chinese medicine could be studied.