JP6836756B2 - How to predict the composition ratio in the oral bacterial flora - Google Patents

Description

The present invention relates to a method for easily predicting the compositional plexus ratio of the oral bacterial flora.

The major constituents of the oral flora are Bacteroidetes, Proteobacteria and Firmicutes. It is said that abnormal composition of the oral bacterial flora is involved not only in oral diseases such as periodontal disease but also in serious systemic diseases such as inflammatory bowel disease (Non-Patent Document 1). It has been reported that Bacteria phylum bacteria in the oral flora increase and Proteobacteria phylum bacteria decrease (Non-Patent Document 2), and similar reports have been made in cancer (Non-Patent Document 3). Furthermore, the inventors have found that the content of Bacteroidetes and Proteobacteria is involved in the therapeutic effect of sublingual immunotherapy, which is a method for treating allergic rhinitis (Fig. 1).

On the other hand, meta 16sRNA analysis and metagenomic analysis are currently the mainstream methods for comprehensively analyzing the flora structure of the oral cavity, etc., but these methods can detect detailed flora, but the time required for analysis. And there is a problem in terms of cost. That is, meta 16sRNA analysis per sample requires a high cost, and metagenome analysis is even more expensive. In addition, since it is difficult to perform analysis within a medical institution, saliva is collected at the medical institution, sent to a specialized laboratory, and the oral bacterial flora (flora) analysis is requested until the analysis result is obtained. It may take more than two months.

Wade WG. The oral microbiome in health and disease. Pharmacological Research 69: 137-143, 2013 Said HS, et al. Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA Research 21: 15-25, 2014. Le Bars P, et al. The oral cavity microbiota: between health, oral disease, and cancers of the aerodigestive tract. Canadian Journal of Microbiology 63: 475-492, 2017.

From these facts, there is a demand for an inexpensive method for analyzing oral bacterial flora in a short period of time, which can be easily analyzed at a facility in a medical institution.

The present inventors added saliva collected from a subject to the culture solution of THP-1 cells, further cultured the cells, and then measured the amount of interleukin-10 (IL-10) in the culture solution to measure the amount of Bacteroidetes phylum bacteria. We found that it is possible to predict the abundance ratio of Proteobacteria to Proteobacteria, the content ratio of Bacteroidetes phylum bacteria in the oral flora (hereinafter, also referred to simply as the ratio), and the content ratio of Proteobacteria in the oral flora. The present invention has been completed.

That is, the present invention has the following features.
[1] A method for predicting the composition ratio in the oral bacterial flora, which comprises a step of adding saliva to a monocyte cell culture medium, further culturing the culture solution, and measuring the amount of interleukin-10 in the culture medium.
[2] The method according to [1], wherein the monocyte cell is a human monocyte cell.
[3] The method according to [2], wherein the human monocyte cell is a THP-1 cell.
[4] The method according to [1], wherein the composition plexus ratio is the content ratio of Bacteroidetes phylum bacteria in the oral plexus.
[5] The method according to [1], wherein the constituent plexus ratio is the content ratio of Proteobacteria phylum bacteria in the oral bacterial flora.
[6] The method according to [1], wherein the compositional flora ratio is an amount ratio of the content ratio of Bacteroidetes phylum bacteria to the content ratio of Proteobacteria phylum bacteria, and [7] for predicting the compositional flora ratio in the oral bacterial flora. Interleukin-10 quantity measurement kit.

According to the present invention, the oral bacterial flora can be easily predicted.

The relationship between the effectiveness of sublingual immunotherapy and the composition ratio of the oral bacterial flora is shown. That is, patients who responded significantly to sublingual immunotherapy (visual analog scale (VAS) score 0) had a higher proportion of Bacteroidetes phylum bacteria than patients who did not respond (VAS score 95 or higher), and the Proteobacteria phylum Indicates a low proportion of bacteria. In the figure, P indicates a significant probability (Mann-Whitney test). The correlation between the IL-10 concentration in the culture medium and the Bacteroidetes phylum bacteria content ratio in the oral flora is shown. In the figure, R indicates the correlation coefficient, and P indicates the significance probability (Mann-Whitney test). The correlation between the IL-10 concentration in the culture medium and the Proteobacteria content ratio in the oral flora is shown. In the figure, R indicates the correlation coefficient, and P indicates the significance probability (Mann-Whitney test). The IL-10 concentration in the culture solution and the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria in the oral flora (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria) are shown. In the figure, R indicates the correlation coefficient, and P indicates the significance probability (Mann-Whitney test).

The monocyte cell used in the present invention can be appropriately used as long as it is a monocyte cell, but human monocyte cell is preferable, and THP-1 cell is more preferable. THP-1 cells are commercially available, for example from Cosmo Bio Co., Ltd.

Culturing of THP-1 cells can be appropriately used as long as it is a method generally used as a method for culturing THP-1 cells, but a medium such as RPMI1640 is used as 10% fetal bovine serum (FCS). In the presence, it is preferable to culture in an environment of _{5% CO 2 (carbon dioxide) and 37 ° C.} During culturing, it is preferable to change the medium once every 2 to 3 days, ^{maintain the number of cells at 2 to 8x10 5} cells / mL, and subculture if necessary.

Monocyte cells are cultured under the above conditions, and saliva collected from the subject is added to the culture medium. The method of collecting saliva can be appropriately selected, but Saliva collection aid (Salimetrics) or a similar device can be used. Saliva can be used without being diluted, but it can also be used after being appropriately diluted. Dilution can be carried out by appropriately using a commonly used buffer solution, but it is preferable to use PBS (phosphate buffered saline). In addition, saliva can be used after collection and refrigerated, but it is preferable to use it immediately after collection.

Saliva can be added from 0.05 μL to 50 μL (without dilution) per 0.5 mL of THP-1 cell culture, preferably 5 μL.

After adding saliva, monocyte cells (eg, THP-1 cells) are further cultured. Monocyte cells (for example, THP-1 cells) can be subjected to the same culture method as described above. The culturing time is preferably 12 to 48 hours, more preferably 12 to 36 hours, and even more preferably 24 hours.

After completion of the culture, the amount of IL-10 contained in the culture supernatant is measured. The measurement of the amount of IL-10 can be performed by a method known per se, and is not particularly limited. Western blotting, RIA method, ELISA method and the like can be used, but ELISA method is preferable. The ELISA method may be any as long as it can measure the amount of IL-10, but a culture solution containing IL-10 is added to a plate coated with an anti-human IL-10 monoclonal antibody (capture antibody), and further anti-IL- A sandwich ELISA method in which 10 monoclonal antibodies (detection antibodies) are added and the amount of the detected antibodies is measured is preferable. The detection antibody can be labeled, and when biotinylated labeling is selected as the labeling method, the amount of IL-10 is measured by measuring the enzyme activity using an enzyme bound to avidin (such as horseradish peroxidase). be able to.

Examples of the interleukin-10 amount measurement kit for predicting the composition ratio in the oral bacterial flora include those for measuring the IL-10 amount by the sandwich ELISA method, and the kit includes, for example, anti-human IL-10. As a capture antibody such as a monoclonal antibody, a detection antibody such as a biotinylated anti-IL-10 monoclonal antibody, an enzyme bound to avidin (such as horseradish peroxydase), a reagent for measuring enzyme activity, and a standard for preparing a calibration line. Includes human IL-10 for use, various buffers, 96-well plates, instruction manuals and the like.

When the measured value of IL-10 in the culture solution is, for example, 30 pg / mL or more, it can be predicted that the content ratio of Bacteroidetes phylum bacteria in the oral flora is 30% or more (sensitivity 89%, specificity 83%). ). When the measured value of IL-10 in the culture medium is, for example, 30 pg / mL or more, it can be predicted that the Proteobacteria content ratio in the oral flora is less than 20% (sensitivity 83%, specificity 89%). ). Further, when the measured value of IL-10 in the culture medium is, for example, 85 pg / mL or more, the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria in the oral flora (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria). ) Can be predicted to be 1.7 or more (sensitivity 100%, specificity 88%).

The higher the IL-10 concentration in the culture solution, the higher the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria in the oral flora (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria), and the risk of developing inflammatory bowel disease. It can be predicted to be high (or affected).

The higher the IL-10 concentration in the culture medium, the greater the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria in the oral flora (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria), and the effectiveness of sublingual immunotherapy is high. It can be predicted to be high.

Meta 16s RNA analysis was performed on Archaea predominate among ammonia-oxidizing prokaryotes in soils. Leininger, S. et al. Nature. 2006 442, 806-809., Pyrosequencing enumerates and contrasts soil microbial diversity. Roesch, LF et al. ISME J. 2007 1, 283-290., A core gut microbiome in obese and lean twins. Turnbaugh, PJ et al. Nature. 2009 457, 480-484., Human gut microbiota in obesity and after gastric bypass. Zhang, H. et al. This can be done by referring to documents such as Proc. Natl. Acad. Sci. USA 2009 106, 2365-2370.

[Example 1]
Saliva was collected from 15 subjects, stored refrigerated, and diluted with PBS to 1% saliva. The cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FCS) at 37 ° C. and 5% CO _{2 so} that the concentration in the culture medium was 0.8 × 10 ⁶ cells / mL. To 500 μL of THP-1 cell culture medium, 5 μL of saliva was added. Further, after culturing at 37 ° C. and 5% CO ₂ for 24 hours, the culture supernatant was collected, and the amount of IL-10 in the supernatant was measured by the ELISA method. ELISA was performed using Human IL-10 Elisa Set (catalog number: 555157) from BD Biosciences, as described in the attached Technical Data Sheet.

The amount ratio of Bacteroidetes phylum, Proteobacteria phylum and Firmicutes phylum bacteria contained in the same saliva sample collected from the subject was calculated by meta 16sRNA analysis (Macrogen Japan Co., Ltd.).

Table 1 shows the IL-10 concentration and the meta 16sRNA analysis results (ratio of Bacteroidetes phylum, Proteobacteria phylum and Firmicutes phylum bacteria) for each subject.

The IL-10 concentration in the culture medium and the ratio of Bacteroidetes phylum bacteria showed a positive correlation (R = 0.814) (Fig. 2), and the IL-10 concentration in the culture medium and the Proteobacteria phylum bacteria had a negative correlation (R = 0.814). −0.873) was shown (Fig. 3). Similarly, the IL-10 concentration in the culture solution and the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria) have a positive correlation (R = 0.896). It became clear to show (Fig. 4). As the correlation coefficient, Spearman's rank correlation coefficient was calculated using GraphPad Prism.

From the above, it was clarified that the composition ratio of the oral bacterial flora can be predicted by measuring the amount of IL-10.

According to the present invention, the composition of oral bacteria (major bacterial flora) can be predicted inexpensively and in a short time. For example, if the superiority of Bacteroidetes phylum bacteria (the ratio of Bacteroidetes phylum bacteria / Proteobacteria phylum bacteria is large) can be confirmed by measuring the amount of IL-10 by this test method, the risk of inflammatory bowel disease can be shown. .. This suggests that the present invention may be useful for early detection and treatment of diseases associated with the oral flora, prediction of therapeutic effect of sublingual immunotherapy, and the like.

Claims (5)

By adding saliva to the monocyte cell culture medium, further culturing, and measuring the amount of interleukin-10 in the culture medium.
・ Content ratio of Bacteroidetes phylum bacteria in the oral flora,
-Content ratio of Proteobacteria phylum bacteria in the oral flora, or
-Amount ratio of the content ratio of Bacteroidetes phylum bacteria to the content ratio of Proteobacteria phylum bacteria
A method for predicting the compositional plexus ratio in any of the oral bacterial flora. The method according to claim 1, wherein the monocyte cell is a human monocyte cell. The method according to claim 2, wherein the human monocyte cell is a THP-1 cell. ・ Content ratio of Bacteroidetes phylum bacteria in the oral flora,
-Content ratio of Proteobacteria phylum bacteria in the oral flora, or
-Amount ratio of the content ratio of Bacteroidetes phylum bacteria to the content ratio of Proteobacteria phylum bacteria
An interleukin-10 amount measurement kit containing an antibody against interleukin-10 for predicting the composition ratio in any of the oral bacterial flora. The composition ratio in the oral bacterial plexus is one of the following:
-Information that when the measured value of IL-10 in the culture solution is 30 pg / mL or more, the content ratio of Bacteroidetes phylum bacteria in the oral flora is predicted to be 30% or more;
-Information that when the measured value of IL-10 in the culture solution is 30 pg / mL or more, the content ratio of Proteobacteria phylum bacteria in the oral flora is predicted to be less than 20%;
-When the measured value of IL-10 in the culture medium is 85 pg / mL or more, the amount ratio of Bacteroidetes phylum bacteria to Proteobacteria phylum bacteria in the oral flora (ratio of Bacteroidetes phylum bacteria / ratio of Proteobacteria phylum bacteria) is 1.7. Information that is predicted to be above;
The interleukin-10 quantity measurement kit according to claim 4, which is predicted based on the above.

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