| Porcine tr ansmiss ible gastroenter itis virus(TGEV) could infect swines of all ages and breeds. In particular piglets of less than two weeks ages, the mortality r ate reached up to 100%. The clinical symptoms were mainly vomiting, sever e diarrhea and dehydr ation. TGEV was the important pathogen of causing cdiarrhea dis eas e of piglets. As a result, it leaded to huge economic losses and s er ious ly thr eated to healthy development for the sw ine industry. Probiotics were able to improve animals growth perf ormance, enhance immunity and treat diarrhea diseases and so on. Theref ore, probiotics had been paid close extens ive attention from animal husbandry. In order to simulate the intest inal envir onment of swines, to explor e the inter active relationships between cells and probiotics, the maintenance of phys iological envir onment homeostas is, the inf luence for cells growth and prolifer ation. However, in consider ation of P K-15 cells which were susceptible cells of T GEV, this exper iment builded P K-15 cells and probiotics co-cultur e model in vitro by taking advantage of unders lung Transwell, and then, thr ough trypan blue staining to gr ope conditions that made the co-culture system into existenc e,such as, concentr ations of pr obiotics and co-culture times. After that, this test also us ed MTT color imetr ic method to measure antivir al activity when P K-15 cells and probiotics were co-cultured, so as to pr eliminary study the interactive relationships among probiotics, cells and virus. In addition, this exper iment applied real-time f luor escent quantitative PCR to detect m RNA express ion of anti-viral cytokines IFN- γ and IL-6 which secreted by P K-15 and to explor e the antiviral effector of cells under the effect of probiotics.In vitro w ith P K-15 cells and pr obiotics co-cultur e model, making us e of trypan blue staining to detect P K- 15 cells survival rates under the conditions of diff erent combinations of different probiotics concentr ations and diff erent co-cultur e times. The results showed that the survival rates of P K-15 cells were differ ent under the conditions of diff erent concentr ation of probiotics and differ ent co-culture times. When probiotics concentr ations were less than108 CF U/m L and co-cultur e times wer e less than 24 hours, the survival rates of P K- 15 cells were higher than 90%. When pr obiotics concentr ations were less than1012 CF U / m L and co-culture times wer e less than 3 hours, the survival rates of P K- 15 cells could be mor e than 80%. T hat was to say, P K- 15 cells and pr obiotics co-cultur e system was cons istent in the above mentioned two condtions.Us ing MTT color imetr ic method to measur e anti-TGEV activity of co-cultur e system of different concentr ations probiotics and P K-15 cells were co-cultur ed 3 hours as well as probiotics which the concentrations were 108 CF U/m L and P K- 15 cells were co-cultur ed at different times. The r esults showed that in the P K-15 cells and probiotics co-culture system, the inhibition rates for T GEV were higher when probiotics concentration wer e about 108 CF U/m L and co-culture time extended to about 12 hours. As a consequence, 108 CF U/m L was the optimal concentration of probiotic, 12 hours was the best time of P K-15 cells and probiotics were co-cultured. In the above mentioned two condtions, the inhibition rates for TGEV of Bacillus sub tili s co-culture gr oup wer e highest. Followed by Ent ero co ccu s f aeci um co-cultur e group. The inhibition rates for TGEV of Lactobacillus amylovorus gr oup wer e lowest.Af ter probiotics of concentration of 108 CF U/m L and P K-15 c ells were co-cultured 12, this co-culture system was infected by TGEV. And then, TGEV titers wer e detected at diff er ent time points. The changed curves of-lg T CID50 of TGEV were piotted. At first,- lg T CID50 of TGEV were r ise, and then- lg T CID5 0 of TGEV wer e decline. When the time of collecting TGEV was 48 hours, the- lg T CID50 of TGEV r eached up to maximum value 3.87 in the P K- 15 cells and Bacill u s sub tili s co-culture system. When the time of collecting T GEV was 24 hours, the-lg TCI D50 of TGEV reached up to maximum value 5.16 in the P K-15 cells and Enterococcus f aeciu m co-cultur e system. When the time of collecting T GEV was 48 hours, the- lg T CID50 of TGEV reached up to maximum value 4. 55 in the P K- 15 cells and Lactobacillus amylovorus co-culture system. In the co-cultur e system, the T GEV average titers of any tr e atment gr oup were less than normal TGEV group′s. Therefore, PK-15 cells and probiotics co-culture could reduce TGEV titers. The decreas ed magnitude of Bacillus subtilis co-cultur e group were biggest. Followed by Enterococcus faecium co-cultur e gr oup. Lactobacillus amylovorus group were lowest.Six holes cells cultur e plates cover ed w ith monolayer P K- 15 cells were dispos ed as follows: the control gr oup of normal cells( Control group), TGEV gr oup of inf ection(TGEV gr oup), the group w ith concentration of 108 CF U/m L of Bac illus subtilis co-cultur ed 12 hours( BS +Cell group) and the gr oup infected by TGEV which w ith concentr ation of 108 CF U/m L of Bac illus subtilis co-cultur ed 12 hours( BS+Cell+TGEV gr oup), and then collected P K-15 cells samples at 1 h, 3 h, 6 h, 12 h, 24 h. The m RNA expr ession level of IFN- γ and IL-6 s ecreted by P K- 15 cells in ever y gr oup at diff erent time points wer e detected by real-time f luor escent quantitative P CR. The results showed that P K-15 cells and Bacillus subtilis co-culture system which was inf ected by TGEV could increase IFN- γ and IL-6 m RNA express ion level, and the effect was rapid increas e at f irst, then gradually decr eases. BS +Cell+TGEV group could s ignif icantly increase IFN-γ m RNA expression at the points of 3 h and 6 h. BS+Cell+T GEV group also could signif icantly increase I L-6 m RNA express ion at the points of 1 h and 3 h. the m RNA expr ess ion of IFN- γ and IL-6 r eached up to maximum at 3 h in BS+Cell+TGEV group. TGEV and Bac illus subtilis also could increase the m RNA express ion leve l of IFN- γ and IL-6 of P K- 15 cells, and have the same tr end with BS+Cell+TGEV group. Wher ease T GEV gr oup and Bac illus subtilis group s ignif icantly increas ed the m RNA express ion level of IFN-γ only at 6 h. TGEV group signif icantly increased the m RNA express ion level of I L-6 only at 3 h. Bac illus subtilis gr oup signif icantly increas ed the m RNA expr ess ion level of I L- 6 only at 3 h. The r eached maximum value of above-mentioned two conditions were both lower than BS +Cell+TGEV group. |
PDF Full Text Request