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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文库,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文库,高效电转化大肠杆菌进行文库扩增,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文库。文库挑选得到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消减文库为大批量筛选、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。
Suppression subtractive hybridization was used to construct a mouse model of irradiated blood deficiency before and after the induction of the Chinese herbal medicine Siwu Tang by subtracting cDNA library. The genes related to the efficacy of Siwu Tang were cloned and identified. mRNA was extracted from bone marrow cells before and after induction of irradiated mice Siwu Decoction and cDNA was synthesized and used as tester and driver for subtractive hybridization and inhibitory PCR. The PCR product was ligated with the vector to construct a cDNA library, and E. coli transformed into E. coli for library expansion. Increase, randomly pick the clones for enzyme digestion, sequencing analysis. The subtractive cDNA library before and after induction of radiation-induced mouse Siwu Decoction with high depletion efficiency was successfully constructed. 512 positive clones were selected from the library. Thirty inserts were randomly picked and sequenced. Bioinformatics analysis showed that most of them were closely related to red blood cell differentiation, bone marrow tissue repair, and structural reconstruction, of which 8 were new gene fragments. The cDNA subtractive library before and after induction of radiation-induced mouse Siwu Decoction constructed by suppression subtractive hybridization laid a foundation for large-scale screening and cloning of the specific genes related to efficacy of Siwu Tang. And at the molecular level, Siwu Decoction has been found to have a regulatory effect on genes that inhibit apoptosis, promote bone marrow tissue repair and structural remodeling, and induce red blood cell differentiation, and may be related to Siwu Tang’s blood-supplementing effect.