【作者】 古静燕；

【导师】 崔承彬； 顾谦群；

【作者基本信息】 中国海洋大学 ， 生药学， 2004， 硕士

【摘要】 本文自采自胶东半岛沿海的24个海泥、海水样品中共分离、纯化出236株海洋放线菌，采用tsFT210小鼠乳腺癌温度敏感型细胞对这些菌株进行了体外抗肿瘤活性筛选，经过反复筛选共有9株放线菌活性稳定，阳性率为3.8％，其中3株为G₀／G₁周期抑制活性，5株为G₂／M期周期抑制活性，1株为细胞毒活性。A2002表现为强的细胞周期抑制活性，通过初发酵预实验，活性稳定，被确定为目的菌株，发酵7天其活性和菌丝体量达到最高。经中科院微生物所鉴定该菌为白浅灰链霉菌（Streptomyces albogriseolus）。 以tsFT210细胞系活性筛选模型为向导，采用跟踪活性部位进行分离的方式，确定了发酵物乙酸乙酯提取物的活性部位。利用硅胶柱层析、反相硅胶柱层析、Sephadex LH-20、HPLC等多种分离手段从A2002两次发酵产物乙酸乙酯提取物中分离纯化到5个化合物。根据理化性质和波谱数据鉴定了它们的结构，分为两类化合物，一类为三环缩醛内酯素类化合物，命名为5-羟基三环缩醛内酯素（1），三环缩醛内酯素（2）；一类为环二肽类化合物，命名为丙氨酸-脯氨酸环二肽（3），丙氨酸-缬氨酸环二肽（4），脯氨酸-酪氨酸环二肽（5）。以tsFT210小鼠乳腺癌细胞，采用SRB法测定化合物1和2的细胞增殖抑制活性，并结合流式细胞术测定及配以光学显微镜细胞形态学观察，系统地测试了化合物1-5的细胞周期抑制活性。结果表明，化合物1和化合物2具有细胞周期抑制活性、诱导细胞凋亡和细胞毒性作用，且其活性具有量效依赖关系。1，2在低浓度时为G₂／M期抑制活性，在中浓度为诱导细胞凋亡活性，而在高浓度为细胞毒活性，化合物1的活性强于化合物2，在此活性筛选模型下化合物3，4，5无明显抗肿瘤活性，其中化合物2为新化合物。更多还原

【Abstract】 In the present dissertation, 236 Actinomycete strains were isolated from 24 marine sedment samples collected from JIAODONG byland .They were all tested at the based of screening program for cell cycle inhibitors using tsFT210 cell line. After retested two times there were 9 strains with stable activity, the positive data arrived 3.9%.Among these 9 active strains 3 strains exihited Go/G1 phase inhibitory activity ,5 strains showed G2/M phase inhibitory activity and 1 strains showed cytotoxic activity. Strain A2002 with strong and stable G2/M phase inhibitory activity was selected as the target strain after a series of pre-experiments and the fermentation time was 7 days with the highest activity and largest mycelium amount. A2002 was identified as Streptomyces albogriseolus.Guided by cell cycle inhibitory activity in a mouse cdc2 mutant cell, tsFT210,the ethyl acetate extraction of mycelium was the active part. Successive purification of the active part by VLCX ODS, Sephadex LH-2CK HPLC ,led to the isolation of 5 compouds .By means of spectroscopic method according to their physicochemical properties,they were two kinds of compounds. One was tricyclacetalactonin and were named 5-hydroxyl-tricyclacetalactonin(l), tricyclacetalactonin (2) respectively,the other was cyclopeptides and named cyclo(-Pro-Ala)(3) - cyclo(-Ala-Val)(4) , cyclo(-Pro-Tyr)(5) ?We used the SRB on the tsFT210 cancer cell line to test the cell proliferation inhibitory activity of compound 1 and 2 .Also the bioactivities of compound 1 and 2 on ts FT210 cell line were evaluated with the aid of morphologic study and flow cytometry analysis. The experimented results exhibited that compound 1 and 2 had the cell cycle inhibitory activity , apoptosis inducing activity and cyctoxic activity.And their activites represented amount-effect relation.At low dose compound 1 and 2 were G2/M phase inhibition ,at middle dose apoptosis inducing and at hige dose cytotoxic.The bioactivity of compound 1 was stronger than compound 2.Compound 3,4,5 had no obvious antituomor activity against the tsFT210 cancer cell line, compoud 2 was identified as new compound.更多还原