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目的研究海洋微生物代谢产物萎蔫酸金属铜离子络合物与镉离子络合物体外抗结核活性,以及对Mtb H37Rv基因表达的影响。方法应用纸片扩散法(Kind-Bauer,K-B法)初步鉴定萎蔫酸铜离子络合物、萎蔫酸镉离子络合物的抗结核活性,重复实验3次;绝对浓度间接法测定萎蔫酸铜离子络合物、萎蔫酸镉离子络合物的最低抑菌浓度(minimum inhibitory concentration,MIC),重复实验确定更准确的MIC;Mtb cDNA芯片检测萎蔫酸铜离子络合物、萎蔫酸镉离子络合物处理Mtb组与溶剂对照处理Mtb组表达差异的基因,重复实验一次。结果萎蔫酸铜离子络合物、萎蔫酸镉离子络合物在卡介苗(BCG)平板抑菌圈直径分别为(30.4±0.6)mm、(30.0±0.8)mm。萎蔫酸铜离子络合物抗Mtb H37Rv的MIC为10μg/ml;对耐多药结核分枝杆菌,萎蔫酸镉离子络合物MIC为7.5μg/ml,低于S(MIC=25μg/ml)和RFP(MIC=25μg/ml);对Mtb临床分离株(0907961,耐S和EMB),萎蔫酸铜离子络合物的MIC(10μg/ml)低于S(S的MIC为20μg/ml),萎蔫酸镉离子络合物的MIC(5μg/ml)均低于S(MIC=20μg/ml)、EMB(MIC=6.4μg/ml)。Mtb cDNA芯片检测发现萎蔫酸铜离子络合物处理组与溶剂对照组比较,有差异表达的基因总数为23条,其中已知功能基因有10条;萎蔫酸镉离子络合物处理组与溶剂对照组比较,有差异表达的基因总数为28条,其中已知功能基因有11条。这些存在差异的基因功能涉及核苷酸、脂质、能量、辅酶、氨基酸代谢,DNA的复制、转录、翻译、翻译后修饰以及细胞膜的生物合成等。结论萎蔫酸铜离子络合物与萎蔫酸镉离子络合物具有较好的体外抗结核活性,尤其对部分耐药菌株的抑制作用甚至优于抗结核一线用药,可作为抗结核药物的前导化合物;运用基因芯片所探究出的差异表达的基因为进一步深入研究其抗菌作用机制提供了一定的实验依据。
Objective To study the antituberculous activity of metal ions of wilt acid metal complexes and cadmium ion complexes of marine microorganisms in vitro and their effects on the gene expression of Mtb H37Rv. Methods The anti-tubercular activity of copper wilt acid complex and cadicides wilt acid were preliminarily identified by Kind-Bauer method and repeated three times. The absolute concentration of copper wilt acid The minimum inhibitory concentration (MIC) of the complex and the wilt acid cadmium ion complex was determined by repeated experiments to determine the more accurate MIC. The Mtb cDNA chip was used to detect the wilting acid copper ion complex and the wilt acid cadmium ion complex The Mtb group was treated with the solvent control gene expression differences, and the experiment was repeated once. Results The inhibitory zone diameters of the wilting acid copper ion complex and the wilt acid cadmium ion complex in BCG plates were (30.4 ± 0.6) mm and (30.0 ± 0.8) mm, respectively. The MIC for the wilting acid copper ion complex against Mtb H37Rv was 10 μg / ml; for M. multidrug-resistant M. tuberculosis, the MIC for the wilt acid cadmium ion complex was 7.5 μg / ml below S (MIC = 25 μg / ml) (MIC = 25 μg / ml); MIC (10 μg / ml) of Mtb clinical isolates (0907961, S and EMB resistant) (5μg / ml) were lower than S (MIC = 20μg / ml) and EMB (MIC = 6.4μg / ml). Compared with the solvent control group, the total number of differentially expressed genes in the wilt acid copper ion complex treatment group was 23, of which 10 were known to be functional genes; Mtb cDNA complexes detected with Mtb cDNA microarray; Compared with the control group, the total number of differentially expressed genes was 28, of which 11 were known as functional genes. These differences in gene function involve nucleotides, lipids, energy, coenzymes, amino acid metabolism, DNA replication, transcription, translation, post-translational modification and cell membrane biosynthesis. Conclusion The wilt acid copper ion complex and wilt acid cadmium ion complex have good antituberculosis activity in vitro, especially for some drug-resistant strains even superior to the first-line anti-TB drug, which can be used as the leading compound of anti-TB drug The genes differentially expressed using the gene chip provided some experimental evidence for further study on the antimicrobial mechanism.