The Extraction Of Nucleic Acids From Lactobacillus DM9811 Cultured Filtrate And Its Effects On HT-29 Cell Line And VSV

OBJECTIVES: Lactobacillus is one of the predominant micro- organisms in the host commensal bacteria. Researches have proved that it plays an important role in maintaining host intestinal homeostasis, stimulating host immune system maturity and promoting host health. We have found that there are lots of nucleic acids from cultured filtrate of lactobacillus DM9811 exponential growth phase, which are about 100 bp. It is worth to study the characteristics, source, nucleotide sequence and biological functions of the nucleic acids. Our study is to study the effects of the nucleic acids from cultured filtrate on the HT-29 human colonic epithelial cell line and its possible mechanism. We also want to explore the potential beneficial effects on inhibition of vesicular stomatitis virus (VSV) infection and the induction of interferon (IFN) by the nucleic acids from cultured filtrate and to explore the role of the nucleic acids in intestinal virus infection disease and its possible mechanisms. Then by our study, we want to establish a new foundation of interaction disciplines and unraveled the molecular basis for their cross talk between lactobacillus DM9811 and its host.METHODS: 1. The extraction, purification and preserve of the nucleic acid from cultured filtrate of lactobacillus DM9811 exponential growth phase. Measured lactobacillus DM9811 growth curve and calculated the generation time of lactobacillus DM9811. The cultured filtrate was gained from the culture medium of lactobacillus DM9811 exponential growth phase by G6 vacuum filtration. It was treated with chloroform/ dimethylcarbinol,then washed with 75% ethanol, redissovled in distilled water without RNase,observed the correlation between the optical density at 260nm and lactobacillus DM9811 colony forming units. The nucleic acid was quantitated by nucleic acid quantitative analyzer. The sample treated with RNase (Free DNase) was analyzed by agarose gel electrophoresis. As a control, the culture medium without fermented by lactobacillus DM9811 was extracted and analysed by the same way above. The nucleic acid was preserved in liquid nitrogen about one month and then observes the change of the nucleic acid. Then analyze the possible source of the nucleic acid from cultured filtrate of lactobacillus DM9811 exponential growth phase. 2. The effects on proliferation of HT-29 human colonic adenocarcinoma cell line by nucleic acids and its molecular mechanism in vitro. For the in vitro proliferation analysis, cell viability was determined by trypan blue exclusion, and 1×105/ml HT-29 cells were suspended in the RPMI-1640/F12 cell growth medium with 10% newborn calf serum (NCS), then the cell suspension was added into 96 well/0.1ml culture plate for 24hrs. The cells were rinsed by RPMI-1640/F12 cell culture medium without NCS for three times. Then the cells were treated with 0, 10, 20, 30, 50, 100, 150, 200μg/mL nucleic acids for 24h, 48h, 72h and 96h. The cell proliferation was detected by MTT colorimetry and calculated the inhibition rate of cell growth. The cell cycle of HT-29 cell was analyzed in the concentration of 200μg/mL of nucleic acid in different times by Flow Cytometry. Then detect the mRNA express level of cell cycle associated protein according to the results of the cell cycle analysis. 3. The inhibition effect of nucleic acid from cultured filtrate of lactobacillus DM9811 exponential growth phase to VSV and its possible mechanism. Virus passage was performed in HEp-2 monolayers and TCID₅₀ was detected in WI-38 monolayers. To perform biological assays, the WI-38 cells were separately seeded in 96 well plates at concentration 2.5×10⁵ viable cells/mL (2.5×10⁴ cells/well) and incubated for 24h at 37°C in atmosphere of 5% CO₂ to reach the monolayer. First, the cytotoxicitic effect on WI-38 cells was detected by different final concentrations from 0μg/mL to 200μg/mL nucleic acid from cultured filtrate. Then detect the competitive inhibition effects, the blockage of viral invasion effects and the inhibition of biosynthetic effects of nucleic acids from cultured filtrate to VSV. And observe the induction of IFN production effects of nucleic acids from cultured filtrate to WI-38 cells and to BALB/c mouse splenocytes. The antiviral effects were showed by cell survival in MTT or crystal violet. Immunofluorescence was performed to detect the possible molecular mechanism by nucleic acid from cultured filtrate of lactobacillus DM9811 exponential growth phase to VSV according to the results of biological assays.RESULTS: 1. Lactobacillus DM9811 exponential growth phase was 0-12 hours after incubation and its generation time is 1.95h. The nucleic acids from cultured filtrate of lactobacillus DM9811 exponential growth phase were RNA and its molecular size is approximately 100bp by agarose gel electrophoresis. And the relationship between the OD260 and lactobacillus DM9811 colony forming units is significant positive correlation. There is no any strap left after the RNase (Free DNase) treatment; it implies that there is no significant lysate in exponential phase of bacteria growth and it suggest that the nucleic acid may be secreted by the lactobacillus DM9811. And the nucleic acid was no significantly degradation after preserved in liquid nitrogen about one month by agarose gel electrophoresis. 2. The inhibition effects to HT-29 cell lines proliferation of nucleic acids. The nucleic acid from cultured filtrate showed significant inhibition on HT-29 cell line on the concentration from 0μg/mL to 200μg/mL and the inhibition rate showed significantly dose and time dependent. The inhibition rate can reach to 63.29±3.49% on the concentration of 200μg/mL in 96 hours. The result of nucleic acids from cultured filtrate to HT-29 cell cycle phase on the concentration of 200μg/mL in 24 hours by FCM is that the percentage of G0/G1 phase is 83.95±0.328%, S phase is 13.68±1.021% in test group, and correspondingly the percentage of G0/G1 phase is 66.33±0.471%, S phase is 27.94±0.210% in control group. The results showed that HT-29 cell cycle was arrested in G1/S phase, and the apoptotic rate increased greatly along with the interaction time, and the apoptotic rate can reach to14.5±1.35% in 72 hours. The cell cycle associated proteins were detected by RT-PCR. It showed that the mRNA level of Cyclin D1, Cyclin E was significantly lower than the control group, CDK2 and CDK4 was slightly low, CDK6 was slightly high, p27^Kip1, p53 was significantly higher than the control group, PCNA was significantly lower than the control group. 3. The inhibition effects to VSV infection by nucleic acids from cultured filtrate in vitro. The TCID50 of the VSV is 10^-5.56/0.1mL. There is no significant cytotoxicitic effects of nucleic acids from cultured filtrate to WI-38 cells at the concentration of 0μg/mL to 200μg/mL. In the competitive inhibition assay, there are significantly difference between the test group and the virus control group and the percentage of the cell survival shows significant dose-dependent, and the cell survival rate in the concentration of 200μg/mL of nucleic acids from cultured filtrate, 90.9±3.67%, is significantly higher than the virus control group. The blockage of viral invasion effects of nucleic acids to VSV is significant, and the cell survival rate is 96.6±1.47% in the concentration of 200μg/mL. The inhibition of biosynthetic effects of nucleic acids to VSV is not significant, and there is no statistically difference between test groups and the virus control group. There is no significantly antiviral effects by induction WI-38 cells which was incubated with nucleic acids from cultured filtrate of lactobacillus DM9811 exponential growth phase to product IFN. But we find that the nucleic acids from cultured filtrate can induce the splenocytes from BALB/c mouse to product IFN-α, and IFN-αis dose-dependence by ELISA. The cell survival in the test group is obviously higher than virus control group. The immunofluorescence showed that nucleic acids from cultured filtrate may compete with VSV for the cell surface receptor, and result in the infection rate decreased and the cell survival increased. The results showed that the nucleic acids from cultured filtrate may hinder the absorption and cell internalization of the VSV due to the competitive binding to the receptor of the cell surface.CONCLUSIONS: 1.There is a lot of nucleic acids which is approximately 100bp RNA from cultured filtrate of lactobacillus DM9811 exponential growth phase. The nucleic acids may be secreted by lactobacillus DM9811. 2. The nucleic acids from cultured filtrate of lactobacillus DM9811 exponential growth phase can inhibit the proliferation of HT-29 human colonic adenocarcinoma cell line, and the inhibition rate is dose-and-time dependence. The cell cycle analysis of HT-29 showed that the cell cycle was arrested in G1/S phase, and the mRNA level of the cell cycle associated protein showed correspondingly changes. 3. The nucleic acids from cultured filtrate of lactobacillus DM9811 exponential growth phase have no cytotoxicitic effects on WI-38 cells. The nucleic acids from cultured filtrate show competitive inhibition effects to VSV infection and block the cell internalization of the VSV. It can also induce the splenocytes of BALB/c mouse to product IFN-α, and the antiviral effects show dose dependence. The results of immunofluorescence show that the nucleic acids from cultured filtrate can competitive binding to the specific viral receptor of the cell surface to decrease the infection rate.