CN116042875A - Microbial marker for early warning of type II diabetes mellitus and application thereof - Google Patents

Microbial marker for early warning of type II diabetes mellitus and application thereof Download PDF

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CN116042875A
CN116042875A CN202211611736.3A CN202211611736A CN116042875A CN 116042875 A CN116042875 A CN 116042875A CN 202211611736 A CN202211611736 A CN 202211611736A CN 116042875 A CN116042875 A CN 116042875A
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ruminococcus
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张学丽
邵华
吴鑫
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Southeast University
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Abstract

The invention discloses a microbial marker for early warning of occurrence of type II diabetes and application thereof, belongs to the technical field of biological medicines, and comprises a type II diabetes detection kit, wherein the kit is used for detecting a combination of microbial markers bacteroides faecalis (bacteroides faecalis) and ruminococcus (Ruminococcus gnavus). The increase of the abundance of the microorganism combination marker has high correlation degree with the genetic risk of the type II diabetes mellitus, can be used for singly pre-warning the risk of the type II diabetes mellitus, and is beneficial to assisting in gene screening to reduce the risk of individuals suffering from the type II diabetes mellitus.

Description

Microbial marker for early warning of type II diabetes mellitus and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a microbial marker for early warning of type II diabetes mellitus and application thereof.
Background
Diabetes is a result of co-participation and interaction of factors considered genetic, environmental (e.g., diet, intestinal flora), psychological, etc., and more than 95% of type ii diabetics have a significant family genetic history. To date, whole genome association analysis (genome wide association study, GWAS) has found 250 or more loci closely associated with type 2 diabetes, with potential value in driving its clinical typing and risk control. The genetic variation of the Gpr35 gene was found to be closely related to type 2 diabetes. GPR35 is an orphan receptor belonging to the G protein-coupled receptor family and is expressed mainly in the gastrointestinal tract and immune cells. The Gpr35 coding region is highly polymorphic, with UCSNP-51, -52, -38 and-40 showing a significant association with type 2 diabetes. However, because the mechanism of action of genetic variation in metabolic disorder complex pathological networks is not well understood, the current precise clinical diagnosis and treatment transformation research on the GWAS risk genes is still limited.
The intestinal tract of human body contains over 100 trillion microbial cells, and plays a vital role in metabolic regulation. Intestinal flora structure and function are susceptible to host intrinsic (genetic, psychological) and extrinsic (dietary, pharmaceutical) factors, and have high plasticity. Recent researches show that intestinal flora participates in the occurrence and development of metabolic diseases such as obesity, type II diabetes and the like induced by dietary factors, and meanwhile, the change of specific intestinal flora is closely related to the progress of the metabolic diseases such as diabetes and the generation of partial drug curative effects, so that the structural representation of the flora has potential value in diagnosis and treatment of the diabetes. The current diagnostic methods for type II diabetes mainly include: random (or occasional) plasma glucose test, fasting plasma glucose test, oral glucose tolerance test, A1c (glycosylated hemoglobin). However, for pre-diabetic patients, particularly those with reduced glucose tolerance, it is difficult for conventional detection methods to accurately pre-warn of the risk of developing diabetes. In addition, specific flora markers which are not developed at present are not used as auxiliary evaluation aiming at early warning of diabetes related to genetic risk factors.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a microbial marker for early warning of type II diabetes mellitus and application thereof.
The aim of the invention can be achieved by the following technical scheme:
the application of the reagent in preparing the type II diabetes detection kit can detect the expression level of the bacteroides faecalis and the ruminococcus.
A type II diabetes detection kit comprises a reagent capable of detecting the expression level of bacteroides faecalis and ruminococcus.
Further, the reagent includes a microbial probe or primer.
Further, the microbial probes or primers can be combined with bacteroides faecalis and ruminococcus by molecular hybridization to generate hybridization signals, and amplified by PCR technology.
Further, the reagent can detect the expression level of bacteroides faecalis and ruminococcus by 16SrRNA sequencing.
Further, the 16SrRNA sequencing includes quantitative PCR, gene chip, second generation high throughput sequencing, and Panomics or Nanostring techniques.
Further, the bacteroides faecalis and the ruminococcus are from the faecal genome.
The application of the kit in the evaluation and early warning of type II diabetes mellitus.
The application of bacteroides faecalis and ruminococcus in constructing early warning gene and model of risk of type II diabetes caused by environmental factors.
The invention has the beneficial effects that:
the invention creatively develops a method for early warning the genetic risk factor related type II diabetes mellitus by taking the special flora marker as an index, namely, the bacteroides faecalis and the gastric coccus form a microbial marker, the obvious increase of the abundance of the microbiome in faeces is taken as an early warning index, and compared with the traditional blood drawing inspection method, the detection of the faecal flora can be widely used for screening the diabetes mellitus of normal people, including patients with blood drawing contraindications. The method is simple and noninvasive, breaks through the limit of specific experimental equipment and experimental skills, and provides a new reference value for the type II diabetes, thereby being beneficial to the establishment of accurate treatment and early intervention measures of the type II diabetes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to those skilled in the art that other drawings can be obtained according to these drawings without inventive effort.
FIG. 1 is a graph showing weight gain and glucose metabolism index of Wild Type (WT) and Gpr35 Knockout (KO) mice of the present invention, FIG. 1A is a graph showing percentage of weight gain of each group of mice, FIG. 1B is a graph showing the appearance of mice at 10 weeks, FIG. 1C is weights of epididymal white fat (eWAT), subcutaneous white fat (sWAT) and perirenal white fat (pWAT) of mice, FIG. 1D is a graph showing blood glucose concentration-time change curve of each group of mice in a glucose tolerance test, and FIG. 1E is an insulin resistance index (HOMA-1R) value;
FIG. 2 shows the difference between Wild Type (WT) and Gpr35 Knockout (KO) mice in intestinal flora, the left graph shows the relative abundance of representative species, the middle graph shows the relative abundance values after Log2 conversion, and the right graph shows the p-value and FDR-value;
FIG. 3 is a graph showing the effect of Bacteroides (Bactoidescoca) of the present invention on metabolic index of normal and high fat diet mice, FIG. 3A is a graph showing the percentage of weight gain of each group of mice, FIG. 3B is a graph showing the change of blood glucose concentration versus time of each group of mice in a glucose tolerance experiment, FIG. 3C is daily intake of mice, FIG. 3D is inguinal white fat (iWAT) weight, and FIG. 3E is liver weight;
FIG. 4 is a graph showing the effect of ruminococcus (Ruminococcus gnavus) on the metabolic index of normal and high fat diet mice according to the present invention, wherein FIG. 4A is a graph showing the percentage of weight gain of each group of mice, FIG. 4B is a graph showing the blood glucose concentration-time curve and the area under the curve of each group of mice in a glucose tolerance test, FIG. 4C is the feeding amount, FIG. 4D is the epididymal white fat (eWAT) weight, and FIG. 4E is the liver weight.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: comparison of Wild Type (WT) and Gpr35 knockout (Gpr 35 KO) mouse glycolipid metabolism with intestinal flora composition
(1) Experimental animal
WT and Gpr35KO mice (C57 BL/6 genetic background, male, 8 week old, purchased from shanghai yao biotechnology limited) were kept in an SPF environment, were free to drink water, and after 7 days of adaptation the experiment was started. All mice were randomly divided into 4 groups: normal feed (chowdiet, CD,10% kcalfat) WT group (n=7), high fat feed (highfasday, HFD,60% kcalfat, n=9) WT group (n=8), normal feed Gpr35KO group (n=7), high fat feed Gpr35KO group (n=8). In the experimental process, body weight and food intake are measured, and the energy intake is obtained by converting the total food intake energy into the energy corresponding to each gram of body weight per day; mice were sacrificed after insulin sensitivity and glucose tolerance testing at week 9, livers and adipose tissues were weighed, and samples such as liver, adipose tissues, ileocecal valve contents, serum and the like were collected and stored for testing.
(2) Abdominal glucose tolerance test
After 9 weeks of HFD feeding, all mice were fasted overnight and D-glucose (2 g/kg body weight) was given by intraperitoneal injection, and blood glucose levels were determined at the mouse tail tip veins at 0, 15, 30, 60 and 120 minutes after administration, respectively
(3) Flora 16S rRNA sequencing
Bacterial DNA from the mouse ileocecal valve content was extracted using the fecal genomic DNA extraction kit (solebao), followed by detection of DNA concentration and purity using Onedrop instrument and agarose gel electrophoresis. 2.5ng of diluted genomic DNA was used as a template, and PCR amplification was performed using a 16SV4 universal primer (515F-806R) with Barcode, using Hot Start Colorless Master Mix high-efficiency high-fidelity enzyme. The PCR products were subjected to DNA concentration measurement, then subjected to isoconcentration mixing according to the concentration of the PCR products, and purified and recovered by using PCR Purification Kit kit after being sufficiently mixed. And (3) carrying out second round of amplification on the recovered product, detecting the amplified product, and carrying out on-machine sequencing on the Illumina Miseq after the result is qualified. Because the machine-down Data (Raw Data) obtained by adopting the Illumina MiSeq sequencing platform has certain low-quality Data, the analysis result is interfered, and therefore, the Raw Data is preprocessed before further analysis, and the specific operation steps are as follows: 1) For the original data of high-throughput sequencing, each sample data is split independently according to the information of Barcode, so that a primer sequence is taken out for quality control of the sequencing sequence; 2) Sequences passing quality inspection were aligned using a Silva database using Ribosomal Database Project (RDP) Classifier 2.3 to determine classification ranking (kingdom, class, phylum, order, family, genus, species) for each sequence. 3) And finally, classifying operation units (operational taxonomic units, OTUs) by using Mothur, classifying the sequences by taking 97% of sequence similarity as a standard, and generating OUTs redundancy spectrum according to the number of the sequences.
(4) Data analysis method
Experimental data are expressed in mean±sem, and the results were analyzed using GraphPad Prism 8 software, and data were analyzed using t-test, where p <0.05, p < 0.01, p < 0.001, p < 0.0001, and p <0.05 indicates that the data difference is statistically significant.
Example 2: effect of Bacteroides (Bacteroides caccae) on metabolism index of normal and high-fat diet mice
2.1 bacterial culture
Re-dissolving Bacteroides (B.caccae, ATCC 43182) powder with sterile water, adding into high-pressure sterilized ATCC medium 1490 liquid culture medium, and culturing in three-gas incubator (10% CO 2 ，10％H 2 ,80％N 2 The seed was cultured at 37℃for 48 hours. Washing the culture with pre-reduced sterile PBS under anaerobic condition, centrifuging 10ml of bacterial liquid at 3000rpm/min for 3 min, discarding supernatant, and diluting with pre-reduced sterile PBS to 5×10 under anaerobic condition 8 CFU/mL。
2.2 field planting of bacteroides faecalis
Male C57BL/6J mice (8 weeks old) were kept under the same SPF conditions. The adaptive environment is randomly divided into four groups: the wt+cd group (n=6), wt+cd administration group (wt+cd (+) (n=6), wt+hfd group (n=8) and wt+hfd administration group (wt+hfd (+)) (n=8), the administration groups were perfused with freshly prepared bacteroides faecalis suspension, 200 μl each time, once every day for two weeks. After 14 days, a sample of the mouse feces was collected for PCR to confirm successful colonization.
2.3 analysis of glycolipid metabolism index
The method for analyzing the glycolipid metabolism index is the same as in example 1, and will not be described in detail here.
Example 3: influence of ruminococcus (Ruminococcus gnavus) on the metabolic index of normal and high-fat diet mice
3.1 bacterial culture
The ruminococcus (r.gnavus, ATCC 29149) powder was reconstituted with sterile water and grown on autoclaved columbia blood plates (jx 601, shandong tuo pu bioengineering limited) and incubated in a three-gas incubator (10%CO2, 10%H2,80%N2, 37 ℃) for 48 hours. Washing the culture medium with sterile water under anaerobic condition, centrifuging 10ml of bacterial liquid at 4000rpm/min for 10 min, discarding supernatant, and diluting with sterile physiological saline under anaerobic condition to 5×10 8 CFU/min。
3.2 ruminococcus colonization
Male C57BL/6J mice (8 weeks old) were kept under the same SPF conditions. The adaptive environment is randomly divided into four groups: the wt+cd group (n=8), wt+cd administration group (wt+cd (+) (n=8), wt+hfd group (n=8) and wt+hfd administration group (wt+hfd (+)) (n=8), ampicillin 0.2g, metronidazole 0.2g, neomycin sulfate 0.2g and vancomycin hydrochloride 0.1g were freshly prepared and dissolved in 400mL of water to prepare a tetraantibiotic mixed solution (ABX) drinking water, which was drunk for 10 days (once every 3 days) to deplete the original intestinal flora of the mice. The mice faeces were collected for PCR to confirm the depletion of intestinal flora. Freshly prepared ruminococcus suspension was gavaged for the panel, 200uL per day, once daily for two weeks. After 14 days, a sample of the mouse feces was collected for PCR to confirm successful colonization.
3.3 analysis of glycolipid metabolism index
The method for analyzing the glycolipid metabolism index is the same as in example 1, and will not be described in detail here.
Flora 16SrRNA sequencing includes quantitative PCR, gene chip, second generation high throughput sequencing, panomics or Nanostring techniques.
As shown in FIGS. 1-4, the Gpr35 gene polymorphism is a risk site for type II diabetes mellitus, and in the present invention, gpr35 gene knockout can aggravate highFat diet induces weight gain and glucose metabolism disorder in obese mice, but has no significant effect on low-fat diet mice, suggesting that diet and genetic factors together affect the development and progression of type ii diabetes. As a result of 16SrRNA gene sequencing, gpr35 was found -/- There is a wide variation in intestinal flora composition between mice and wild-type mice, with the most significant difference between bacteroides faecalis and ruminococci. Subsequently, a wild type mouse single bacterial colonization experiment is adopted, and the microorganism combination can increase the sensitivity of the mice to obesity and glucose metabolism disorder induced by high-fat diet, and has similar effect to Gpr35 deletion, and the correlation of gene deficiency and intestinal flora disorder is suggested, so that the microorganism combination can be used as a pre-warning or diagnosis factor of genetic risk related type II diabetes. Compared with the traditional blood drawing inspection method, the invention uses the microbial abundance in the feces as an early warning index, and the fecal flora detection can be widely used for the diabetes screening of normal people, including patients with blood drawing contraindications. The method is simple and noninvasive, breaks through the limit of specific experimental equipment and experimental skills, and provides a new reference value for diagnosis of the type II diabetes, thereby being beneficial to the establishment of accurate treatment and early intervention measures of the type II diabetes.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Claims (9)

1. The application of the reagent in preparing the type II diabetes detection kit is characterized in that the reagent can detect the expression level of bacteroides faecalis and ruminococcus.
2. A type II diabetes detection kit is characterized by comprising a reagent capable of detecting the expression level of bacteroides faecalis and ruminococcus.
3. The type ii diabetes detection kit of claim 2, wherein the reagent comprises a microbial probe or primer.
4. A type ii diabetes mellitus detection kit according to claim 3, wherein said microbial probes or primers are capable of generating hybridization signals by molecular hybridization in combination with bacteroides faecalis and ruminococcus and are amplified by PCR technique.
5. The use of a reagent according to claim 1 for preparing a type II diabetes mellitus detection kit, wherein the reagent can detect the expression level of bacteroides faecalis and ruminococcus by 16SrRNA sequencing.
6. The use of a reagent according to claim 1 for preparing a type ii diabetes detection kit, wherein the 16SrRNA sequencing comprises quantitative PCR, gene chip, second generation high throughput sequencing, panomics or Nanostring techniques.
7. The use of a reagent according to claim 1 for the preparation of a type ii diabetes test kit, wherein the bacteroides faecalis and the ruminococcus are from the faecal genome.
8. The use of the type ii diabetes test of claim 2 for the assessment and pre-warning of type ii diabetes.
9. The use of bacteroides faecalis and ruminococcus in the construction of a model of risk of developing type ii diabetes caused by early warning genes and environmental factors as claimed in claim 1.
CN202211611736.3A 2022-12-14 2022-12-14 Microbial marker for early warning of type II diabetes mellitus and application thereof Pending CN116042875A (en)

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