CN112553358A - Application of microbial flora in cerebral apoplexy - Google Patents
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Abstract
The invention discloses application of a microbial flora in cerebral apoplexy, particularly relates to a microorganism of Streptophyta, and firstly discovers that the abundance of Streptophyta in a sample after the cerebral apoplexy is treated by statins is reduced, so that the detection of the level of the Streptophyta can be used as a basis for assisting in judging the treatment effect of the statins.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to application of microbial flora in stroke.
Background
Stroke, also known as cerebral infarction or cerebral infarction, is a leading cause of disability and death, and poses a heavy social and economic burden worldwide. In clinical practice, intravenous thrombolysis and intravascular intervention have proven to be effective therapies. However, due to strict therapeutic indications and time windows, only about 7% of stroke patients are eligible for thrombolytic therapy. The therapy for treating the core symptoms of stroke, particularly acute brain injury following stroke, remains limited. In recent years, more and more researches show that the imbalance of intestinal flora is closely related to risk factors of stroke, such as obesity, hypertension, hyperlipidemia, diabetes and the like, and can influence the occurrence and prognosis of stroke through various action mechanisms. There is a very close and complex relationship between the intestinal tract and the central nervous system. The intestinal flora plays an important role in the development and normal physiological functions of the central nervous system. It was found that germ free mice were unable to develop a normal immune system and blood brain barrier. After the use of antibiotics to inhibit the intestinal flora, the structure and function of microglia in the brain tissue of mice are destroyed. The intestinal microorganisms can participate in development and physiological functions of the central nervous system through mechanisms such as nerves, immunity and endocrine, and meanwhile, the central nervous system can influence the intestinal microenvironment through regulating intestinal motility and an intestinal endocrine system and can also regulate intestinal mucosal immune response through the intestinal nervous system. In summary, the intestinal tract and the central nervous system interact and interact with each other through a variety of complex mechanisms.
The function of intestinal flora imbalance in cardiovascular and cerebrovascular diseases is increasingly emphasized, and a new idea is provided for the essential research of stroke on the level of intestinal flora for treating stroke; meanwhile, a theoretical basis is provided for treating the cerebral apoplexy by statins.
Disclosure of Invention
The invention aims to research intestinal flora related to the effect of statins on treating stroke, thereby providing a new idea and means for treating stroke.
The invention provides an intestinal flora marker related to statin drug treatment, which is characterized in that the intestinal flora marker is Streptophyta.
Further, the statin is atorvastatin.
The invention provides application of a reagent for detecting Streptophyta in preparation of a product for diagnosing cerebral apoplexy or evaluating an auxiliary treatment effect.
Further, the reagent is a reagent for detecting the abundance of Streptophyta.
Further, the agent includes a primer, probe, antisense oligonucleotide, aptamer, or antibody specific for Streptophyta.
The invention provides a product for diagnosing cerebral stroke or evaluating auxiliary treatment effect, which comprises a reagent for detecting the abundance of Streptophyta.
Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer or an antibody for detecting the specificity of the intestinal flora marker.
Furthermore, the product also comprises a reagent for extracting microbial genome DNA, microbial protein or thallus components.
The invention provides application of Streptophyta in preparing a medicament for treating cerebral apoplexy.
Further, the medicament comprises an inhibitor of Streptophyta.
The invention has the advantages and beneficial effects that:
according to the invention, by comparing the abundance of the intestinal flora in the samples after stroke and medication, the correlation between the intestinal flora Streptomyces and the stroke treated by statins is found for the first time, and a means is provided for the auxiliary diagnosis of the treatment effect of statins and the treatment of the stroke.
Drawings
FIG. 1 is a graph of the abundance of Streptophyta in different samples;
FIG. 2 is a ROC plot of Streptophyta as the sensed variable.
FIG. 3 is a graph showing the results of FMT transplantation experiments; wherein panel a is an mNSS score plot; FIG. B is an experimental run wheel; fig. C is a contact time detection chart in the sticker removal experiment; fig. D is a contact time detection chart in the sticker removal experiment.
Detailed Description
According to the invention, the microbial sequencing is carried out on the samples of the stroke group and the statin treatment group, so that the relevance of Streptophyta and statin treatment of stroke is found for the first time, and the fecal transplantation technology proves that the intestinal flora can improve neuroethology and reduce the volume of cerebral infarction, thereby playing a role in treating stroke.
Based on the application, the invention provides the application of the reagent for detecting the Streptophyta in preparing a product for diagnosing cerebral apoplexy or evaluating the auxiliary treatment effect; the reagent may be any conventional reagent in the art as long as the abundance of Streptophyta can be detected. As alternative embodiments, the agent includes, but is not limited to, a primer, probe, antisense oligonucleotide, aptamer, or antibody specific for Streptophyta.
The term "primer" is a 7-50 nucleic acid sequence capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.
As one embodiment, the invention can use primers to detect the abundance of Streptophyta. In the present invention, primers can be used to amplify the sequence of 16S rRNA retained in the microorganism Streptophyta, and after amplification of the sequence, the presence of the microorganism can be detected or the level of the microorganism can be determined by the presence or absence of the production of the desired product. The term "16S rRNA" refers to rRNA constituting a 30S small subunit of a prokaryotic ribosome, which has a large part of a base sequence highly retained on the one hand and a partial region showing high base sequence diversity on the other hand. In particular, since there is little diversity between species and there is diversity between species, it is possible to efficiently identify prokaryotes by comparing the sequences of 16S rRNA.
The sequence amplification method using the primer may use various methods known in the art. For example, Polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot start PCR, nested PCR, boost PCR, real-time PCR, differential PCR (differential display PCR: DD-PCR), rapid amplification of cDNA ends (rapid amplification of cDNA ends: RACE), reverse (inversion) polymerase chain reaction, vector-mediated PCR, thermal asymmetric cross-PCR (TAIL-PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-sustained sequence replication (selected-sustained sequence replication), selective amplification of a target base sequence may be used, but the scope of the present invention is not limited thereto.
As one embodiment, the present invention may use a probe as a reagent for detecting Streptophyta. The probes of the invention may be used with molecules that bind to specific sequences or subsequences or other portions of the Streptophyta. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to a target nucleotide by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
In the present invention, "complementary" or "complementarity" is used to refer to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing principle. For example, the sequence "5 '-A-G-T-3'" is complementary to the sequence "3 '-T-C-A-5'". Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Alternatively, "complete" or "total" complementarity may also exist between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.
As one embodiment, the present invention can detect the abundance of Streptophyta using an antibody. By using immunological methods based on antigen-antibody reactions, the corresponding microorganisms can be detected or the level of microorganisms can be determined. Examples of the Assay method used for this purpose include western blotting, enzyme linked immunosorbent Assay (ELISA), Radioimmunoassay (RIA), radioimmunodification (radioimmunodification), Ouchenkia (Ouchterlony) immunodiffusion, rocket (rocket) immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay (Immunoprecipitation Assay), Complement Fixation Assay (complementary hybridization Assay), Fluorescence Activated Cell Sorter (FACS), and protein chip (protein chip).
In the present invention, the term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.
The term "sample" or "test sample" as used herein refers to any liquid or solid material containing nucleic acids. In suitable embodiments, the test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture, or is a tissue sample from an animal, and most preferably from a human. In an exemplary embodiment, the sample is stool.
The methods and compositions of the present invention can be used to detect nucleic acids associated with various bacteria using a biological sample obtained from an individual. The nucleic acid (DNA or RNA) may be isolated from the sample according to any method known to those skilled in the art. The biological sample may be obtained by standard procedures and may be used immediately or may be stored for later use under conditions appropriate for that type of biological sample.
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1 biomarkers associated with cerebral infarction
1. Laboratory animal
Specific pathogen-free grade adult male C57BL/6 mice (body weight 23.0 to 26.0g, 8 to 12 weeks old) were purchased from beijing vintonia. All C57BL/6 mice were housed under specific pathogen-free conditions with relatively constant humidity (60% + -5%) and temperature (22 deg.C + -3 deg.C) for 12 h/dark for 12h, and all animals were free to eat and drink. A mouse focal cortical cerebral infarction model is established by an electrocoagulation method, and mice are randomly divided into two groups, namely a control group (pMCAO, 25) and a medicine group (25).
2. And (3) medicine intervention: atorvastatin (pfeiy, usa) was dissolved in 0.9% saline at a concentration of 2 mg/ml. The drug was administered by gavage at 20 mg/kg/day. The cerebral infarction group was treated with an equal volume of physiological saline. Collecting a fecal sample of the mice 1 day after administration; meanwhile, the behavioral characteristics and the cerebral infarction volume of the mice are detected.
3.16S rRNA Gene sequencing
Bacterial DNA was extracted from the samples using a DNA extraction kit and the procedure was as described.
After the DNA sample is received, detecting the sample; and (3) detecting qualified samples to construct a library: recovering the target Amplicon fragment, repairing the broken sticky end into a flat end by using T4 DNA Polymerase, Klenow DNA Polymerase and T4 PNK, and adding a base A at the 3 'end to ensure that the DNA fragment can be connected with a special adaptor with a base T at the 3' end; or designing and synthesizing a double-Index fusion primer containing a sequencing joint, carrying out fusion primer PCR by taking genome DNA as a template, screening a target Amplicon fragment by using magnetic beads, and finally carrying out cluster preparation and sequencing by using a qualified library.
4. Data analysis
And carrying out corresponding biological information analysis by using data obtained by off-line. The data of the off-line data is filtered to remove low-quality reads, and the remaining high-quality Clean data can be used for later analysis; splicing reads into Tags through an Overlap relation between the reads; gathering Tags into OTU under a given similarity, and then performing species annotation on the OTU by comparing the OTU with a database; sample species complexity analysis and inter-group species difference analysis were performed based on OTU and species annotation results.
4.1 data processing
When Data processing and filtering are carried out, the original sequencing Data is processed by using an internal written program as follows to obtain clear Data, and the specific steps are as follows:
1) the method for removing the low quality according to the window is adopted, and the specific operation is as follows: setting 30bp as the window length, if the average quality value of the window is lower than 20, cutting the end sequence of the read from the window, and removing reads with the final read length lower than 75% of the original read length;
2) removing the linker-contaminated reads (the default adapter sequence and the read sequence have 15bp overlap, the overlap is set as 15bp, and the number of allowed mismatches is 3);
3) removing the reads containing N;
4) low complexity reads are removed (the length of continuous appearance of a certain base in default reads is more than or equal to 10, and 10bp is set).
If the sample is merged and built by the barcode, and clear Data is obtained, the sample is split by an internal written program by utilizing the barcode sequence. The number of mismatches allowed for alignment of the barcode sequence with sequencing reads was 0 bp.
4.2 sequence splicing
The sequence splicing uses software FLASH (Fast Length Adjustment of Short reads, v1.2.11), and pairs of reads obtained by double-end sequencing are assembled into a sequence by utilizing an overlapping relation, so as to obtain the Tags of the hypervariable region. The splicing conditions were as follows:
1) the minimum matching length is 15 bp;
2) the overlap region allows a mismatch ratio of 0.1. Reads without overlap relationships are removed. The Paired End Reads are spliced into Tags through overlap relation between Reads.
4.3 species Classification and abundance analysis
And performing OTU clustering on the treated Clean Tags, and then completing OTU species classification by annotating the OTU.
And clustering the spliced Tags into OTUs by using software USEARCH (v7.0.1090). The method mainly comprises the following steps:
1) clustering by using UPARSE under 97% similarity to obtain a representative sequence of the OTU;
2) removing chimeras generated by PCR amplification from the OTU representative sequences using UCHIME (v4.2.40); 16S and ITS adopt a method of comparing with the existing chimera database to remove chimera. 18S adopts a Denovo method to remove the chimera 16S chimera database: gold database (v20110519) ITS chimera database: UNITE (v20140703), divided into ITS full length, ITS1 and ITS2, selected by sequencing region.
3) All Tags were aligned back to OTU representative sequences using the usearch _ global method, resulting in a statistical table of the abundance of each sample at each OTU. After obtaining the OTU representative sequence, the OTU representative sequence is aligned with the database by RDP classifier (v2.2) software for species annotation, and the confidence threshold is set to 1. Wherein, the annotation result is filtered as follows:
removing OTUs with no annotated results;
the removed annotation result does not belong to the species in the analysis project. For example, the sample is bacterial 16S, which is removed if OTU is annotated with archaea. The remaining OTU side is available for later analysis.
Comparing the databases: 16S (including bacteria and archaea): greenene (default): v201305; RDP (remote desktop protocol): release 9201203
4) Species difference analysis
Species differential analysis is analyzed by bioinformatics analysis methods to detect differences in abundance exhibited by different groups (or samples) of microbial communities based on the obtained community abundance data. The content of species differential analysis includes: and (3) carrying out difference significance test between groups and Lefse multi-level species difference discriminant analysis. This project used the significance test of differences between groups to screen for different species.
Significance test of differences between groups species exhibiting abundance differences among different groups (samples) of microbial communities can be detected using rigorous statistical methods based on the obtained community abundance data, and a hypothesis test is performed to assess the significance of the observed differences. The analysis can select different classification levels of domains, kingdoms, phyla, classes, orders, families, genera, species, OTU, etc.
1) The Wilcox rank-sum test, also known as the Mann-Whitney U test, is a method of nonparametric testing of two independent sets of samples. The original assumption is that two populations of independent samples have no significant difference in distribution, and the average ranks of the two populations of samples are researched to judge whether the two populations of samples have difference in distribution, so that the analysis can be used for performing significant difference analysis on the species of the two populations of samples and correcting the P value by various methods.
2) The multiple test correction, i.e. the multiple test correction method for P value is "fdr".
3) And a two-tailed test for specifying the type of confidence interval to be evaluated, and selecting the two-tailed test (confidence interval).
4) A CI calculation method, i.e., a method of calculating a confidence interval, the method being DP: welch's confidence updated. Selecting confidence: 0.95.
calculating the influence size (effect size) by using a DP method, namely mean1-mean 2; confidence intervals were calculated using the Welch T test and the screening criteria FDR < 0.05.
5) And (3) according to the abundance of the differential flora, drawing a receiver operating characteristic curve (ROC), calculating two accurate confidence spaces, and analyzing the diagnostic efficiency of the differential flora.
5. Results
1) The results of the behavioral tests showed that the mNSS score of the mice after the administration was decreased, the Rotarod running time was increased, and the contact time and the removal time in the adherent removal experiment were decreased, compared to the control group; while the volume of cerebral infarction decreases.
2) Bioinformatic analysis results showed that Streptophyta exhibited significant differences in the drug administration groups compared to the model group (fig. 1), with an average abundance of Streptophyta in the model group of 0.02212432 and an abundance of 0.00063512 in the drug administration group.
3) The diagnostic efficacy results showed that the area under the curve of Streptophyta as an indicator of detection was 0.869, the cutoff value was 0.003, the sensitivity at this point was 0.960, and the specificity was 0.760 (FIG. 2), which is highly sensitive and specific.
Example 2 stool transplant experiment (FMT)
Fecal material from atorvastatin treated mice was collected under sterile conditions and donor mice feces (0.1g) were mixed and suspended in 1ml sterile PBS. The mixture was mixed using a bench top Vortex (Vortex-Genie 2, Science Industries, usa) for 15s, centrifuged at 800g for 3min at room temperature, and 500 μ L of the supernatant was gavaged orally to pmcaco mice (n ═ 12). Fresh stool preparation was done on the day of stool transplantation within 15min before gavage. Neuro-behavior of FMT mice was determined and compared to the pMCAO group (n-12).
Results as shown in fig. 3, following fecal transplantation, neuro-behavioral of mice was improved, mNSS score was decreased (fig. 3A), Rotarod race time was increased (fig. 3B), contact time and removal time were decreased in the adherent removal experiment (fig. 3C) and removal time (fig. 3D).
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
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