The Research On The Screening Model And Primary Screening The Potential Anti-tumor Active Strains

Tumor is a serious disease to human health and life, and there still no have effective drugs to cure it completely. However, the diversity of microbes and the metabolites of microbes provide us so many resources. Especially of extreme environmental microbial, its metabolites have more highly diversity in chemical structure and function mechanisms, and it will be an important way to find and research potential anti-fungal, anti-tumor substance.It’s particularly important to build a reasonable anti-tumor pesticide screening system according to new research target, new model and detection methods in natural anti-cancer drugs research. The new screening method of active material with microtubules system has caused wide public concern. Because of fungal cells and animal cells have common physiological basis of eukaryotic cells, and between anti-tumor effect and fungal form mutation there has good corresponding relations, so many anti-tumor drugs in the appropriate concentration can change the fungal form, inhibit fungal spores germinate or make mycelia distortion. So the development and utilization of fungi as target model strain with its shape variation index, screening for anti-tumor activity material, have very profound research value.Until now, fungal screening model which is wide used for many researchers is Pyricularia oryzae that is established by Iwasaki et al.The thousands of active substances for fungi or tumer have been selected with this model by both at home and abroad. Among them, Rhizoxin、Fusarielin A,nsamitocin P-3,and some others have been developed into clinical new drugs for curing tumer. However, some problems were found by researchers in practical utilization of P.oryzae model: 1)some active substances was not detected by this model; 2)culture for P.oryzae is difficult and long time for conidia production; 3) conidia is small and pale in color,difficult to observe and evaluate. Therefore, reserachers begin to explore and establish somne new fungal screening models.Wen et al and Bao et al established Alternaria solani model and Fusarium oxysporum T-6 model were much better than the P.oryzae model,especially for spores production is easier and observation is more convinient. During the process of selecting antimicroorganisms against potato late blight (Phytophtora infestans) in our laboraaatory, we found some antimicroorganisms could significantly inhibit the growth, and result in the protoplast concentration, or deformation of mycelia, sporangia of P.infestans. These phenomena are similar to that P.oryzae effected by other antibiotic organisms or active substances. Moreover, these effects observed in our experimental elicit us to explore the new screening model for antifugal or antitumer in comparison with these models.This research mainly screens active antifugal strains from the air dry soil sample of Yuncheng salt lake in Shanxi, and selects the strain as the pre-model one, and then treat the pre-model strain and P.oryzae with various known antitumer drugs in order to establish a new screening model. After that, using the this screening model to select some active organisms and their metaboltes, as well as investigate the characteristics and function. The main results were as follows:1. Isolation and selection of strians. From the air dry soil sample of Yuncheng salt lake in Shanxi, using two methods of the soil directly separation and the soil suspending liquid separation with 6 different salt concentrations : 0%, 2.5%, 5%, 7.5%, 10%, 12.5%, 43 strains were isolated and purificated with well growth and steadily genetics. Of YH-1 strain had some different characteristics: 1) life history is simple; 2) culture is easy; 3) conidia is obtained easily, growth period short, conidia production time short, with large number of conidia; 4) obseration is easy since the conidaphore and conidia are duck in color. Therefore, YH-1 strain could be as continueous strain in potential.2. Evaluation and establishment of screening model strain. P.oryzae and YH-1 spore were treated with 11 kinds of anti-tumor drugs by the method of 96-well analysis plate. The shape variation of fungal spores and mycelia were observed in different period(2h,4h,6h)and different anti-tumor drugs dosage. The characteristics of fungal spores and mycelia (suppression, curly, terminal expansion, etc) were used as a diplex index for antifungal/ anti-mitotic activity material screening. In these data we could find that YH–1 had more sensitivity and detection range than P.oryzae to anti-tumor drug. Through the microscope observation and comparing with P.oryzae, YH-1 strain had the characteristics with more sporulation yields, lager, dark black conidia and their distortion of body for easy observation. The most proper observation time of YH-1 was in 6 to 8 hours, while that of P.oryzae in 14 hours. So YH-1 was quite reasonable to be developed as target fungus in primary screening system of anticancer active drugs.According to the YH-1 morphological characteristics, culture characteristics and the physiological and biochemical feature, YH-1 was identified as Alternaria tenuissima.3. Primary application of screening model strain YH-1 and selection of active strains. Active substances were screened from 42 strains microorganisms using P.oryzae and YH-1 activity screening model. Most strains had significant inhibitory effect to P.oryzae, of them, inhibition rates of 16 strains were more than 60%, with Yhx-4 strains had the highest inhibition rate (90.81%). It was found that most strains inhibited P.oryzae also showed obvious inhibition to YH-1. Inhibition rates of 12 strains were also more than 60% to YH-1, in which Yhx-4 strains still had the most inhibition rate (89.02%). To further investigate the effect of fermented liquid from different strains, the results showed that YH-7 had the highest inhibition rate to both P.oryzae and YH-1, the inhibition rates reached 68.34% and 64.29%, respectively.4. Primary research of active substances from antibiotic strains. Effective antifungal substances in YH-7 strain fermentation liquid were preliminarily studied. Firstly, the best fermentation time was determined at 9 days in that fermented products had the strongest antifungal action. Secondly, main anti-fungal substance were determined as the proteins by ammonium sulfate fractionation, and then the best saturation of ammonium sulfate precipitation was determined as 85%. It was carried on a further verification to prove the anti-bacterial substances that was mainly protein, using protease K and denaturant phenol-chloroform method. By the method of 96-well analysis plate, the effective antimicrobial protein of YH-7 in the highest concentrations could inhibit the spores of P.oryzae and YH-1 growth, and also could make the hyphaes of YH-1 terminal expansion in the appropriate concentration. Therefore, the antimicrobial protein of YH- 7 was likely to be developed into antifungal, anti-tumor effective ingredients.