救必应黄酮类成分抗产ESBLs大肠杆菌作用机理研究

大肠杆菌耐药性较为严重，寻求新的有效而安全抗感染药物成为亟待解决的问题。救必应具有清热解毒、止血抗菌等功效，前期研究该药具有较强的抗菌活性。本课题拟通过基因芯片筛选出救必应黄酮类作用大肠杆菌前后差异基因表达,分析与药物作用相关的基因以及药物对细菌生长繁殖、新陈代谢等多方面的影响,提出抗菌机理假设；从碱性磷酸酶（AKP）、电导率、胞外蛋白含量和SDS-PAGE 电泳分析中药对细胞壁、细胞膜和蛋白质的影响；采用凝胶阻滞实验和外光谱、圆二色谱和荧光光谱观察分析救必应提取物对细菌DNA的结构的影响及其方式，采用流式细胞术观察中药提取物作用于菌后对其细胞膜结构的影响。用PCR技术获得细菌DNA合成相关基因（DNA复制起始子基因、DNA聚合酶Ⅲ基因、DNA促旋酶基因等）并通过凝胶阻滞实验验证中药提取物与大肠杆菌DNA合成相关基因的作用及抑制蛋白质的合成，阐明救必应对产ESBLs大肠杆菌耐药抑制机理。

The phenomenon of antibiotic resistance of Escherichia Coli has been serious, It has become urgent problems to be solved to seek safe and effective anti-infection drugs.Cortex Ilecis Rotundae has been a remedy for clearing heat and removing toxicity, hemostasis and antibacterial and the other effects. Prophase researches have proved that this drug had high antibacterial activity. The hypothesis of antibacterial mechanism was put forward in this study by screening out differential gene expression before and after the medicine effect of E.coli with gene chips,and analyzing the gene related to drug effects, the influence of upon bacterial growth, reproduction,and metabolism. It was analyzed that the traditional Chinese medicine influenced on the cell wall, cell membrane and protein through AKP, conductivity, extra-cellular protein content and SDS-PAGE electrophoresis; with the gel retardation assay, external perimysium,circular dichroism and fluorescence spectra, we could analyze the effect of extracts of Cortex Ilicis Rotundae on the structure of bacterial DNA and influence mode, to observe the influence of the extractss of the Chinese medicine on the structure of cell membrane after acting on the bacteria.Obtaining the bacterial DNA by PCR to synthesize the related gene(DNA replication initiators,DNA polymerase III gene,DNA gyrase gene,etc)and verify the combination of the extracts of Chinese medicine and the related genes synthesized by E.coli DNA could inhibit the protein synthesis,elucidate the mechanism of Cortex Ilecis Rotundae against ESBLs-producing E.coli.