CN116162699A - Biomarker for identifying active phase and inactive phase of ulcerative colitis and application thereof - Google Patents
Biomarker for identifying active phase and inactive phase of ulcerative colitis and application thereof Download PDFInfo
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Abstract
The invention discloses a biomarker for identifying active phase and inactive phase of ulcerative colitis and application thereof, wherein the biomarker is sulfate reducing bacteria. The invention expounds the important difference of intestinal microbiota in the active phase and the inactive phase of ulcerative colitis diseases, and finds that bacteria belonging to sulfate reducing bacteria can be effectively used as detection markers and treatment targets of the active phase and the inactive phase of ulcerative colitis, and the active phase and the inactive phase of ulcerative colitis can be easily and reliably detected through the markers.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker for identifying active phase and inactive phase of ulcerative colitis and application thereof.
Background
Inflammatory bowel disease is a chronic inflammatory disease of the intestinal mucosa, and is currently largely divided into two types, ulcerative Colitis (UC) and Crohn's Disease (CD). Ulcerative colitis is limited to the colon and is a superficial mucosal inflammation, mostly continuous, which can lead to ulcers, severe bleeding, toxic megacolon and fulminant colitis, severely affecting the quality of life of the patient and also leading to an increased risk of colorectal cancer. The prevalence of ulcerative colitis has increased year by year in recent decades and has become a global economic burden for disease. The etiology is not clear at present, but the result of the combined actions of genetic, environmental, microbial and immune-mediated factors is well accepted. Recent findings underscores the major role of intestinal microorganisms, the reduction in the total number, diversity and abundance of microbial species and the close association of microbial produced metabolites with ulcerative colitis.
Ulcerative colitis is caused by the combined action of genetic factors, environmental factors, intestinal microorganisms, immune responses and other factors. Intestinal microbial dysbiosis has been shown to be associated with the development of a variety of diseases, and sulfate-reducing bacteria are also involved in the development of a number of diseases. When the patient is exposed to the corresponding environment, the intestinal microorganisms of the patient with ulcerative colitis are disturbed, the bacteria producing short chain fatty acids are reduced, and the Proteus is increased. The current clinical treatment for ulcerative colitis is mainly to use large doses of drugs, including antibiotics, non-steroidal anti-inflammatory drugs, biological agents, immunomodulators, etc., to reduce the generation of inflammation. Patients with active ulcerative colitis are often treated with maintenance medications to prevent disease recurrence. However, the pharmaceutical biologicals commonly used at present, such as Infliximab (IFX), are expensive and bring great economic burden to patients. Therefore, the stage of the disease of the patient is monitored, and the use of medicines can be effectively reduced, so that the economic burden of the patient is lightened, and the life quality of the patient is improved.
Disclosure of Invention
To solve the problems, the invention utilizes the collected stool samples from 10 healthy volunteers, 18 non-active ulcerative colitis patients and 24 active ulcerative colitis patients in Jiang Nada affiliated hospitals to study intestinal flora changes in the active and non-active stool samples of the ulcerative colitis patients. The result shows that the intestinal microorganism composition in the feces of patients with ulcerative colitis in active phase and inactive phase is different, and particularly the bacterial content of the sulfate reducing bacteria is obviously different, so the discovery can be used as a novel biomarker for ulcerative colitis in active phase.
It is a first object of the present invention to provide a biomarker for identifying active and inactive phases of ulcerative colitis, the biomarker being a sulfate-reducing bacterium.
Further, the sulfate-reducing bacteria include the following species: desulfovibrio, desulfotomaculum, desulfobulbus, desulfomonas, desulfobacter, desulfomicrobium, desulfacinum.
Further, sulfate-reducing bacteria levels are elevated in patients with active ulcerative colitis.
A second object of the invention is to provide the use of the above biomarker for the preparation of a reagent for the detection of ulcerative colitis active and inactive phases.
The third object of the invention is to provide the application of the biomarker in preparing a medicament for treating ulcerative colitis in active period, wherein the medicament is designed by taking sulfate reducing bacteria as targets.
A fourth object of the invention is to provide the use of the above-mentioned biomarker in the screening of drugs for the treatment of active ulcerative colitis.
Further, the drugs to be screened are evaluated according to the content difference of sulfate-reducing bacteria after different drugs are administered.
It is a fifth object of the present invention to provide a method for evaluating the therapeutic effect of a drug for ulcerative colitis in active phase, comprising the step of detecting the content of sulfate-reducing bacteria.
Further, the disease stage of the patient is judged according to the detection condition of the biomarker.
A sixth object of the invention is to provide a kit for detecting active and inactive phases of ulcerative colitis, the kit comprising reagents for detecting sulfate-reducing bacteria.
Further, the sample to be tested is feces.
The invention has the beneficial effects that:
the invention explains that intestinal microbiota plays a key role in the development of ulcerative colitis, and finds that bacteria belonging to the genus sulfate reducing bacteria can be effectively used as a detection marker and a treatment target of the ulcerative colitis, and the active period and the inactive period of the ulcerative colitis can be easily and reliably distinguished through the marker.
Drawings
FIG. 1 is a graph of intestinal microbiota of healthy volunteers, non-active ulcerative colitis and active ulcerative colitis patients compared at the phylum and genus taxonomic level;
FIG. 2 is a comparison of the ratio of the individual flora (A-C) to the differential flora (D) in SRB in the stool of healthy volunteers, patients with non-active ulcerative colitis and patients with active ulcerative colitis;
FIG. 3 is a comparison of mainly differential sulfate-reducing bacteria from intestinal microorganisms in healthy volunteers, non-active ulcerative colitis and active ulcerative colitis patients (A), with higher sulfate-reducing bacteria content in active ulcerative colitis patients, and with a major metabolite H of the sulfate-reducing bacteria 2 The S content is also obviously higher than that of the inactive period patient (B), ns, and no obvious difference exists; * P, P<0.05，**，P<0.01。
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Term interpretation:
sulfate-reducing bacteria (sulfate-reducing bacteria, SRB): sulfate-reducing bacteria are a group of bacteria that are capable of reducing sulfate to hydrogen sulfide and are found in the gastrointestinal tract of humans and animals. The five most common sulfate-reducing bacteria in the human intestinal tract include: desulfovibrio, desulfobacterium, desulfomonas, desulfobulbus and Desulfotomaculum. Other sulfate-reducing flora also includes: desulfomicrobium; desulfacinum; lawsonia and Bilophila.
As described in the background art, in order to reduce the medication of a patient and reduce the economic burden of the patient, the prior art cannot accurately distinguish whether the disease stage of the patient is active or inactive, so the present invention proposes to distinguish the disease stage of the patient by using the great difference between the compositions of the intestinal microorganisms of the patient, particularly the composition of the sulfate-reducing flora, including:
use as a biomarker for distinguishing between active and inactive phases of ulcerative colitis; or,
use in the identification of the effect of a drug for ulcerative colitis active phase.
Preferably, the sulfate-reducing bacteria genus comprises: desulfovibrio, desulfobacterium; desulfomonas; desulfobulbus; desulfomamacum; desulfomicrobium; desulfacinum.
Wherein the detection kit for distinguishing the active period from the inactive period of ulcerative colitis is used for measuring the composition difference of intestinal microorganisms, particularly sulfate reducing bacteria, by collecting a patient excrement sample in vitro.
In a preferred embodiment of the invention, there is also provided the use of an agent which inhibits the breakdown of bacteria belonging to the genus sulfate-reducing bacteria to produce hydrogen sulfide in the identification of a pharmaceutical effect for the treatment of ulcerative colitis.
In particular, there is provided the use of an agent which inhibits the increase in IL-6, IL-1 beta and/or TNF-alpha concentration caused by bacteria belonging to the genus sulfate-reducing bacteria in the manufacture of a medicament for the treatment of ulcerative colitis.
In a preferred embodiment of the invention, there is also provided a method of identifying an agent or drug for the treatment of ulcerative colitis active phase, the method comprising the step of detecting a change in the level of bacteria belonging to the genus sulfate-reducing bacteria in faeces.
The specific steps are exemplified as follows:
(1) Detecting the content level of bacteria belonging to the genus sulfate reducing bacteria in the feces of healthy volunteers, inactive ulcerative colitis and active ulcerative colitis patients;
(2) Detecting the bacterial content level of the sulfate reducing bacteria in the feces of the patient in the ulcerative colitis active period before and after the treatment by using the medicine, and comparing the change of the bacterial content level of the sulfate reducing bacteria before and after the treatment by using the medicine.
Example 1
1. Patient sample collection
Stool samples were from 52 volunteers [10 healthy volunteers, 24 ulcerative colitis active patients and 18 ulcerative colitis inactive patients ] in the Jiang Nada subject hospital. All volunteers had obtained informed consent prior to inclusion in the study. The fecal samples were kept in a-80 ℃ freezer for subsequent analytical study. All studies were passed by the ethical committee of university of south of the Yangtze river (Jiangsu Wuxi).
2.16S sequencing
(1) Extraction of fecal genomic DNA
(1) Patient feces (about the size of rice grains) were taken into 1.5mL sterile centrifuge tubes, 500. Mu. L tail digestion buffer and 2.5. Mu.L proteinase K (20 mg/mL) were added to each tube, mixed well, placed in a 55℃metal bath and shaken at 1500rpm for 3h.
(2) 500. Mu.L of a phenol/chloroform isoamyl alcohol mixture (1:1; chloroform: isoamyl alcohol=24:1) was added to each tube, and after thorough mixing, centrifuged at 12000rpm for 10min
(3) Adding 600 μl of absolute ethanol (pre-cooled in advance at 4deg.C) into 300 μl to 1.5mL centrifuge tube, mixing, centrifuging at 12000rpm for 5min, and discarding supernatant
(4) 300. Mu.L of sterile ddH was added 2 O, adding 700 mu L absolute ethyl alcohol (pre-cooled in advance by a refrigerator at 4 ℃), mixing uniformly upside down, centrifuging at 12000rpm for 5min, and discarding the supernatant; after brief centrifugation, the remaining supernatant was aspirated away
(5) Air-drying at room temperature. 40 mu LRNase-free H was added 2 O,37 ℃ metal is dissolved in an assisted mode until the adherence transparent solid disappears, and the concentration of DNA is measured by using Nanodrop; preserving at-20 ℃.
(2) Amplicon production
The primer is 16S V3-V4338F-806R,
338F：ACTCCTACGGGAGGCAGCAG，
806R：GGACTACHVGGGTWTCTAAT
the extracted DNA was diluted to 25 ng/. Mu.L and the PCR reaction was as follows:
the reaction procedure: 95 ℃,3min,1 cycle; 95 ℃,30s,55 ℃,30s,72 ℃,30s,30 cycles; 72℃for 5min.
(3) The PCR products were detected by electrophoresis using 1.5% agarose gel, and the PCR amplified products were purified by Agencourt AMPure XP and dissolved in an elision Buffer, and labeled to complete the library construction. Fragment ranges and concentrations of the library were detected using an Agilent 2100 Bioanalyzer. The library that was qualified was sequenced by selecting the HiSeg platform based on insert size.
(4) Information analysis flow
Filtering the off-machine data to obtain high-quality effective data (Clean ready); splicing reads into Tags through an overlap relation between the reads; and clustering the spliced Tags with the similarity of more than 97% into OTU (Operational Taxonomic Unit) by using software USEARCH (v7.0.1090), and comparing the clustered Tags with a database to carry out species annotation.
3. Metagenomic sequencing
Illumina library construction
(1) DNA disruption
(1) Transferring a 60 mu LDNA sample into a covarias disruption tube;
(2) vortex oscillation is carried out for 5s and evenly mixed;
(3) randomly breaking the DNA into fragments of about 350bp by using a covarias M220 breaker;
(4) vortex oscillation is carried out for 5s after the breaking is finished, and the mixture is uniformly mixed;
(5) transfer to EP tube and wait for product purification.
(2) Purification of the product
(3) End repair, namely physical damage can occur at the two ends of the broken DNA fragment, and the end repair can be influenced by the fact that the DNA short fragment is not a blunt end when a joint is added. And A is added at the 3' end.
(4) And (3) adding a joint at two ends, namely connecting the Barcode joint with the DNA fragment in a mode of TA semi-sticky ends. Even if the library of different samples is mixed for sequencing, the data can be split according to the number of Barcode after the sequencing is completed.
(5) Library quality inspection
1% agarose gel electrophoresis is used for checking whether the product length reaches the standard, and whether the product has a mixed band, a dimer byproduct and other genome pollution or not; and (3) detecting the quality and concentration of the library by using a Qubit quantitative instrument, and then carrying out concentration normalization treatment.
(6) IlluminaNovaSeq high throughput sequencing
Metagenomic sequencing was performed using an Illumina sequencing platform, novaSeg sequencer.
(7) Performing off-the-shelf data analysis, including: data filtering, metagenomic assembly, species analysis, gene prediction, functional annotation, and the like.
4. H2S in fecal samples was determined using a human H2S ELISA kit (Luyuan Bode biotechnology co.ltd., beijing, china) according to the manufacturer' S instructions.
Rna extraction and real-time PCR
RNA extraction
Taking 2mL EP tubes, adding 1mL Trizol into each tube, placing into sample tissue, adding about 10 ceramic beads into each tube, shaking by precooled MP shaking instrument until the tissue is broken completely, adding chloroform (200 μL/mL), shaking by vortex for 20s (or shaking by hand) at room temperature for 5-10 min to separate layers (colorless upper layer, white middle layer, lower layer)Red), centrifuging (4 ℃,1,2000 Xg, 15 min), carefully sucking the upper colorless aqueous phase into another EP tube (about 400-500 mu L), adding an equal volume of isopropanol (about 400-500 mu L), gently inverting and mixing, standing at room temperature for 5-10 min, centrifuging (4 ℃,12000 Xg, 10 min), discarding the supernatant, adding 70-75% ethanol (4 ℃, vortex and shake centrifuging (4 ℃,7500 Xg, 5 min) to the RNA equal volume (1 mL), discarding the supernatant (quick centrifuging, discarding again, sucking cleanly with a gun head), airing at room temperature for 5-10 min, adding 10-40 mu L of RNA free H 2 Placing in O ice water mixture for 30min, metal bath (65deg.C, 5 min), immediately ice-bath for 5min to measure OD value, detecting RNA purity and concentration, and performing nucleic acid electrophoresis (180V, 10-15 min), and further detecting RNA extraction condition
Reverse transcription RT-cDNA preparation
Reverse transcription reaction system: 50 mu L system
The reaction procedure: 25 ℃ for 10min;48 ℃ for 40min;95 ℃ for 5min;4 ℃ and infinity
Real-time fluorescence quantitative PCR (qRT-PCR)
Primers used for Vibrio are as follows:
DSV-691-F primer(5’-CCGTAGATATCTGGAGGAACATCAG-3’)、
DSV-826-R primer(5’-ACATCTAGCATCCATCGTTTACAGC-3’)
fluorescent quantitative PCR reaction system: 17 mu L system
The reaction procedure: 50 ℃,2min,95 ℃,10min,1cycle,95 ℃,15s,60 ℃,1min,40cycles.
6. Results
In fig. 1 a-L are microbiota composition analyses of healthy volunteers (C), non-active ulcerative colitis patients (N) and active ulcerative colitis patients (a) using high throughput sequencing of the 16S rRNA amplicon, including bacteroides (a), proteasomes (B), firmicutes (C), clostridia (D), actinomycetes (E) (at the phylum level) and Desulfovibrio (F), desulfotomaculum (G) Desulfobulbus (H), desulfomonas (I), desulfobacter (J), desulfomicrobium (K), desulfacinum (L) (at the genus level), with varying summaries of genus. ns, no significant difference; * P <0.05, P <0.01. Compositional analysis by high throughput sequencing of the 16S rRNA amplicon indicated that the entire dataset was divided into 16 phylum, 37 class, 71 mesh, 127 family and 145 genus. The most typical of these are Bacteroides (FIG. 1A), proteus (FIG. 1B), and Thick-walled (FIG. 1C). At the genus level (FIGS. 1F-L), desulfovibrio among the genus sulfate-reducing bacteria of patients with active ulcerative colitis; desulfomamacum; desulfobulbus; desulfomonas; desulfobacteria; desulfomicrobium; desulfacinum levels were significantly higher than those of non-active ulcerative colitis.
Functional characterization of intestinal microorganisms associated with ulcerative colitis: by studying fecal microorganisms and genes involved at the level of active ulcerative colitis species, patients with active ulcerative colitis were found to have higher amounts of Vibrio desulphurisation and hydrogen sulphide. The difference in the content of Vibrio desulphatus between 24 patients with active ulcerative colitis and 18 patients with inactive ulcerative colitis was identified by real-time PCR (FIG. 2D). The sulfate reducing bacteria are the main flora capable of reducing sulfate into hydrogen sulfide in human body, compared with the patients with non-active ulcerative colitis, the proportion of sulfate reducing bacteria in intestinal flora of the patients with active ulcerative colitis is obviously increased (figure 3A), and one of metabolites of the sulfate reducing bacteria is also obviously higher than that of the patients with non-active ulcerative colitis (figure 3B).
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A biomarker for identifying active and inactive phases of ulcerative colitis, characterized by: the biomarker is sulfate reducing bacteria.
2. The biomarker of claim 1, wherein: the sulfate reducing bacteria include one or more of Desulfovibrio, desulfobacter, desulfomonas, desulfobulbus, desulfotomaculum, desulfomicrobium, desulfacinum.
3. The biomarker of claim 1, wherein: in patients with active ulcerative colitis, sulfate-reducing bacteria levels are elevated.
4. Use of the biomarker of claim 1 in the preparation of a reagent for detecting active and inactive phases of ulcerative colitis.
5. The use of the biomarker of claim 1 in the manufacture of a medicament for the treatment of active ulcerative colitis.
6. Use of the biomarker of claim 1 in screening for active ulcerative colitis treatment drugs.
7. The use according to claim 6, characterized in that: and evaluating the medicines to be screened according to the content difference of sulfate reducing bacteria after different medicines are applied.
8. A method for evaluating the therapeutic effect of a drug for treating active ulcerative colitis, comprising the steps of: comprising the step of detecting the sulfate-reducing bacteria content.
9. A kit for detecting active and inactive phases of ulcerative colitis, characterized in that: the kit comprises a reagent for detecting sulfate reducing bacteria.
10. The kit of claim 9, wherein: the sample to be tested is feces.
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