CN112266956A - Application of MAP3K8 detection reagent in preparation of pulpitis screening kit and inhibitor in preparation of medicine for treating pulpitis - Google Patents
Application of MAP3K8 detection reagent in preparation of pulpitis screening kit and inhibitor in preparation of medicine for treating pulpitis Download PDFInfo
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- CN112266956A CN112266956A CN202011265990.3A CN202011265990A CN112266956A CN 112266956 A CN112266956 A CN 112266956A CN 202011265990 A CN202011265990 A CN 202011265990A CN 112266956 A CN112266956 A CN 112266956A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/18—Dental and oral disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention provides application of a MAP3K8 expression level detection reagent in preparation of an pulpitis screening kit and an inhibitor in preparation of a medicine for treating pulpitis. The expression of MAP3K8 mRNA is obviously increased in human dental pulp inflammation tissues, and the risk of the population to be tested suffering from pulpitis can be screened by detecting the expression of MAP3K8 mRNA in human dental pulp tissues: if the expression of MAP3K8 mRNA is obviously increased, the risk of suffering from the pulpitis is high. Meanwhile, the inhibition of the expression of MAP3K8 can effectively inhibit the inflammatory reaction of dental pulp and reduce the tissue necrosis. Therefore, the kit for detecting the expression level of MAP3K8 can be used for auxiliary diagnosis of pulpitis and provides effective basis for patients to take relevant treatment measures or decisions; the MAP3K8 inhibitor can be used for preparing medicines for treating pulpitis. The detection kit and the MAP3K8 inhibitor have good application prospects.
Description
Technical Field
The invention relates to application of a MAP3K8 detection reagent in preparation of an pulpitis screening kit and application of an inhibitor in preparation of a medicine for treating pulpitis.
Background
Pulpitis refers to an inflammatory condition that occurs in the pulp tissue. The pulp tissue is a loose connective tissue rich in blood vessels, nerves and extracellular matrix, located within the pulp chamber surrounded by dentin. When a tooth is subjected to caries, trauma, etc., exposure of the pulp can result in the invasion of bacteria and toxins into the pulp through the dentinal tubules, causing irritation of the pulp tissue resulting in inflammation. The main symptoms of pulpitis are pain, such as spontaneous pain or pain occurring when eating or drinking, which seriously affects the quality of life of people. If the biomarkers related to the pulpitis can be found, the pulpitis can be accurately detected through the detection of the biomarkers, clinical medication can be guided, and the method is greatly helpful for patients.
Mitogen-activated protein kinase 8(MAP3K8, Gene SEQ ID NO: ENsembl: ENSG00000107968) is widely expressed in systemic tissues, and has a prominent role in the immune system, inflammatory response, and carcinogenesis. Meanwhile, the MAP3K8 deletion is found to have an effect on various inflammations, such as bronchitis, enteritis and the like. However, studies have shown that MAP3K8 depletion has different effects on the inflammatory response of different diseases. Such as: research shows that in the process of bronchitis reaction caused by mouse ovalbumin, the deletion of MAP3K8 causes the increase of IgE level and severe asthma reaction due to the differentiation of Th2 cells, namely the deletion of MAP3K8 causes the exacerbation of inflammatory reaction; in a Crohn's disease (IBD) mouse inflammation model, the MAP3K8 deletion leads to a decrease in lymphocyte number and alleviates the occurrence and exacerbation of the disease.
However, the prior art related to pulpitis is not seen in MAP3K8 at present.
Disclosure of Invention
The invention aims to provide application of a MAP3K8 detection reagent in preparation of an pulpitis screening kit and an inhibitor in preparation of a medicine for treating pulpitis.
The invention provides application of a reagent for detecting the expression level of MAP3K8 in preparing a pulpitis screening kit.
Further, the reagent for detecting the expression level of MAP3K8 is a reagent for sequencing;
and/or the reagent for detecting the expression level of the MAP3K8 is a PCR reagent; preferably, the reagent for detecting the expression level of MAP3K8 is qPCR reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a western blot reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for a protein chip detection method.
Further, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting MAP3K8 in human tissues;
preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting MAP3K8 in human dental pulp tissue;
more preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting the expression level of MAP3K8 mRNA.
The invention also provides a pulpitis screening kit, which comprises a reagent for detecting the expression level of MAP3K 8;
preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for sequencing;
and/or the reagent for detecting the expression level of the MAP3K8 is a PCR reagent; preferably, the reagent for detecting the expression level of MAP3K8 is qPCR reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a western blot reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for a protein chip detection method.
Further, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting MAP3K8 in human tissues;
preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting MAP3K8 in human dental pulp tissue;
more preferably, the detecting the expression level of MAP3K8 is detecting the level of MAP3K8 mRNA.
The invention also provides the application of the MAP3K8 inhibitor in preparing a medicament for treating pulpitis;
preferably, the medicament is a medicament for reducing necrosis of pulp tissue.
Further, the drug contains the target sequence GGCGCAGTCGAATACCTAA.
Furthermore, the medicine takes adenovirus, adeno-associated virus, lentivirus or cells as a vector.
The invention also provides a medicament for treating pulpitis, which takes a MAP3K8 inhibitor as an active ingredient;
preferably, the medicament is a medicament to reduce necrosis of pulp tissue;
more preferably, the drug contains target sequence GGCGCAGTCGAATACCTAA.
Furthermore, the medicine takes adenovirus, adeno-associated virus, lentivirus or cells as a vector.
The key point of the invention is that the mRNA expression level of MAP3K8 in human tooth pulp tissue is determined to be obviously related to the risk of suffering from pulpitis, so that the mRNA expression level of MAP3K8 in the tooth pulp tissue can be detected under the condition that tooth pulp can be obtained when a patient performs other dental operations, the risk of suffering from pulpitis of the patient is predicted, and prevention is well done in advance. As for the means for specifically detecting the mRNA expression level of MAP3K8 in human dental pulp tissue, various means disclosed in the prior art can be adopted, and not only the means in the present embodiment, but also any method capable of detecting the mRNA expression level of MAP3K8 can be used for pulpitis screening.
The research of the invention shows that the expression of MAP3K8 mRNA in human dental pulp inflammation tissue is obviously increased, and the risk of the population to be tested suffering from pulpitis can be screened by detecting the expression of MAP3K8 mRNA in human dental pulp tissue: if the expression of MAP3K8 mRNA is obviously increased, the risk of suffering from the pulpitis is high. Meanwhile, the inhibition of the expression of MAP3K8 can effectively inhibit the inflammatory reaction of dental pulp and reduce the tissue necrosis. The invention provides a novel pulpitis screening marker and a novel pulpitis screening kit, which can realize effective screening of pulpitis; and human tooth marrow tissue can be used as a detection sample, so that the harm to a patient is low. The invention also provides a medicine for treating pulpitis by taking the MAP3K8 inhibitor as an active ingredient. The detection kit and the MAP3K8 inhibitor have good application prospects.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the results of gene expression in normal dental pulp tissue (Control pulp tissue) and inflammatory dental pulp tissue (inflamed pulp tissue): a is a thermographic display of each gene in normal pulp tissue and inflammatory pulp tissue; b is the expression of MAP3K8 gene in normal pulp tissue and inflammatory pulp tissue.
Figure 2 is a result of the down-regulation of MAP3K8 expression in the virus to inhibit the inflammatory response of dental pulp tissue: a is the Sham group H & E results; b is AAV-Scramble group H & E result; c is AAV-MAP3K8-down group H & E result.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
Example 1, MAP3K8 expression was significantly elevated in inflammatory dental pulp tissue
1. Method of producing a composite material
Collected in the department of dental pulp, the doctor visits the oral cavity hospital of western Sichuan university in 10-2020 in 2019, and clinically diagnoses the dental pulp as irreversible pulpitis due to spontaneous pain and pain induced by cold and hot stimulation, and plans to carry out 3 cases of inflammation dental pulp tissues for root canal treatment. Meanwhile, the patient is diagnosed in maxillofacial surgery for 3 cases of normal pulp tissues of orthodontic teeth are required to be removed due to orthodontic requirements. Pulp tissues of each patient were collected, total RNA of the tissues was extracted using Trizol reagent (Invitrogen, CA, USA), whole genome transcriptome sequencing (RNA-seq) was performed according to a conventional method in the art, that is, total RNA quantity and purity were detected using Bioanalyzer 2100 and RNA 6000NanoLabChip Kit (Agilent, CA, USA), polyA RNA was extracted, RNA was cleaved and reverse-transcribed into cDNA, a cDNA library was constructed, and whole transcriptome sequence detection was performed using an illumina X10 high throughput sequencing platform. Genes with expression differences were screened and statistically analyzed according to the statistical t-test method (log2 fold difference > 1, or log2 fold difference < -1, and p value < 0.05).
2. Results
The results of the dental pulp tissue transcriptome analysis are shown in fig. 1. Fig. 1 shows that, when the quality Control is qualified, the mRNA expression of MAP3K8 in the inflammatory pulp tissue (inflamed pulp tissue) is significantly increased compared to the normal pulp tissue (Control pulp tissue), with a factor of 3.9 fold increase and significant statistical differences.
3. Conclusion
In human dental pulp tissue, mRNA expression of MAP3K8 was significantly elevated under inflammatory stimulation. That is, mRNA expression of MAP3K8 was significantly elevated in inflammatory pulp relative to normal pulp tissue. Therefore, by detecting the expression level of MAP3K8 mRNA in the dental pulp tissue, the purpose of screening pulpitis can be achieved.
Example 2 composition of pulpitis screening kit and method of use
Any reagent for detecting the expression level of MAP3K8 mRNA can be used for preparing the pulpitis screening kit.
Example 3 downregulation of MAP3K8 expression Virus inhibits inflammatory response in dental pulp tissue
1. Method of producing a composite material
1.1 plasmid construction and Virus embedding
The AAV Helper-Free System is used for producing recombinant AAV particles (namely MAP3K8 expression downregulation virus) which can downregulate the expression of MAP3K8 in dental pulp stem cells, and is a preferable MAP3K8 inhibitor of the invention, and the process is as follows:
1) a target fragment of rat-derived MAP3K8(rno-MAP3K8) is designed, and the target sequence is GGCGCAGTCGAATACCTAA (SEQ ID NO. 1). The above sequences were cloned into the viral vector GV479 to obtain an experimental group (AAV-MAP3K8-down group). The control group (AAV-Scramble group) had the inserted sequence TTCTCCGAACGTCTCACGT (SEQ ID NO. 2). The viral vector comprises an Inverted Terminal Repeat (ITR) sequence and a Multiple Cloning Site (MCS) fragment. The sequence of the elements is U6-MCS-CAG-EGFP.
2) The recombinant expression plasmids described above were co-transfected into AAV-293 cells (providing the trans-acting factors required for AAV replication and packaging) with pHelper (carrying the adenovirus-derived genes) and pAAV-RC (carrying the AAV replication and capsid genes).
3) The AAV viral particle supernatant was collected from the infected AAV-293 cells.
4) The supernatant was subjected to 2 CsCl density gradient centrifugation and 1 ultrafiltration to remove cellular proteins and residual CsCl ions in the supernatant, thereby purifying the virus.
5) The titer of the resulting virus was determined by quantitative PCR. The final virus titer obtained was 3.15X 1012v.g./mL. Subpackaging and storing the virus at-80 ℃.
The experimental group obtains MAP3K8 expression down-regulation virus; control group received control virus.
1.2 construction of rat pulpitis model
Injecting chloral hydrate into abdominal cavity of SD rat for anesthesia, randomly dividing into 3 groups, fixing in supine position, grinding teeth in upper jaw at two sides, opening marrow on occlusal surface with 1/4# ball drill, exposing complete marrow cavity, flushing dentin detritus in marrow cavity with physiological saline, placing small cotton ball of physiological saline in pulp section and marrow cavity, and exposing pulp tissue to oral environment for infection. 3 days later, the cotton ball is taken out under anesthesia, the pulp section is flushed by normal saline, the absorbable gel sponge is placed on the pulp section, 1 mu L of medicinal reagent is injected into the pulp section by a microinjector, and the cavity is filled with resin material. According to the difference of the injected medicinal agents, the injection is divided into the following groups: (1) untreated group (sham group), no open pith operation; (2) a control group (AAV-scramble group) injected with a control virus (virus carrying DNA having a sequence shown in SEQ ID NO. 2); (3) in the experimental group (AAV-MAP3K8-down group), MAP3K8 expression-down-regulated virus (virus carrying DNA having a sequence shown in SEQ ID NO.1) was injected. Rats were sacrificed 72 hours after treatment. And (3) whole body formalin perfusion, taking out the rectangular jawbone block at the periodontal of the experimental molar, placing the rectangular jawbone block in 4% paraformaldehyde for fixation for 24 hours, performing paraffin section immunostaining, and detecting the inflammation condition of dental pulp.
2. Results
H & E staining results show: the pulp section of the first molar tooth of the rat in which AAV-script drugs were placed was in a typical inflammatory state, the coronal pulp was in a necrotic and disintegrated state, the nucleus was broken and dissolved, and the inflammatory region was expanded to the root pulp (FIG. 2B). The inflammation state of the pulp of the rat placed with AAV-MAP3K8-down drug is obviously relieved, the state is changed from necrosis disintegration state to fibrosis repair state, the section of the pulp is in a fiber-like structure, the necrotic area is separated from the root pulp area by bundled fiber tissue, and angiogenesis is shown (figure 2C).
3. Conclusion
AAV-MAP3K8-down drug can effectively inhibit inflammatory reaction of dental pulp, inhibit inflammatory cell reaction, and reduce tissue necrosis. The MAP3K8 inhibitor can be used for preparing medicines for treating pulpitis.
In conclusion, the expression of MAP3K8 mRNA is obviously increased in human dental pulp inflammation tissues, and the risk of pulpitis of the people to be tested can be screened by detecting the expression of MAP3K8 mRNA in human dental pulp tissues: if the expression of MAP3K8 mRNA is obviously increased, the risk of suffering from the pulpitis is high. Meanwhile, the inhibition of the expression of MAP3K8 can effectively inhibit the inflammatory reaction of dental pulp and reduce the tissue necrosis. Therefore, the kit for detecting MAP3K8 can be used for auxiliary diagnosis of pulpitis and provides effective basis for patients to take relevant treatment measures or decisions; the MAP3K8 inhibitor can be used for preparing medicines for treating pulpitis. The detection kit and the MAP3K8 inhibitor have good application prospects.
Claims (10)
1. Use of a reagent for detecting expression level of MAP3K8 in preparation of a pulpitis screening kit.
2. Use according to claim 1, characterized in that: the reagent for detecting the expression level of the MAP3K8 is a reagent for sequencing;
and/or the reagent for detecting the expression level of the MAP3K8 is a PCR reagent; preferably, the reagent for detecting the expression level of MAP3K8 is qPCR reagent;
and/or, the reagent for enzyme-linked immunosorbent assay or enzyme-linked immunoassay reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a western blot reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for a protein chip detection method.
3. Use according to claim 1 or 2, characterized in that: the reagent for detecting the expression level of MAP3K8 is a reagent for detecting the expression level of MAP3K8 in human tissues;
preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting the expression level of MAP3K8 in human dental pulp tissue;
more preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for detecting the mRNA level of MAP3K 8.
4. A pulpitis screening kit is characterized in that: it comprises reagents for detecting the expression level of MAP3K 8;
preferably, the reagent for detecting the expression level of MAP3K8 is a reagent for sequencing;
and/or the reagent for detecting the expression level of the MAP3K8 is a PCR reagent; preferably, the reagent for detecting the expression level of MAP3K8 is qPCR reagent;
and/or the reagent for detecting the MAP3K8 is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a western blot reagent;
and/or the reagent for detecting the expression level of the MAP3K8 is a reagent for a protein chip detection method.
5. The kit of claim 4, wherein: the reagent for detecting MAP3K8 is a reagent for detecting the expression level of MAP3K8 in human tissues;
preferably, the reagent for detecting MAP3K8 is a reagent for detecting the expression level of MAP3K8 in human dental pulp tissues;
more preferably, the detecting the expression level of MAP3K8 is detecting the mRNA level of MAP3K 8.
Use of a MAP3K8 inhibitor in the manufacture of a medicament for the treatment of pulpitis;
preferably, the medicament is a medicament for reducing necrosis of pulp tissue.
7. Use according to claim 6, characterized in that: the drug contains the target sequence GGCGCAGTCGAATACCTAA.
8. Use according to claim 6 or 7, characterized in that: the medicine takes adenovirus, adeno-associated virus, lentivirus or cells as a carrier.
9. A medicine for treating pulpitis is characterized in that: the medicine takes a MAP3K8 inhibitor as an active ingredient;
preferably, the medicament is a medicament to reduce necrosis of pulp tissue;
more preferably, the drug contains target sequence GGCGCAGTCGAATACCTAA.
10. The medicament of claim 9, wherein: the medicine takes adenovirus, adeno-associated virus, lentivirus or cells as a carrier.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080247955A1 (en) * | 2007-01-16 | 2008-10-09 | Jun Kuai | Inflammation treatment, detection and monitoring via TREM-1 |
| US20170242015A1 (en) * | 2014-09-17 | 2017-08-24 | Institut Curie | Map3k8 as a marker for selecting a patient affected with an ovarian cancer for a treatment with a mek inhibitor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080247955A1 (en) * | 2007-01-16 | 2008-10-09 | Jun Kuai | Inflammation treatment, detection and monitoring via TREM-1 |
| US20170242015A1 (en) * | 2014-09-17 | 2017-08-24 | Institut Curie | Map3k8 as a marker for selecting a patient affected with an ovarian cancer for a treatment with a mek inhibitor |
Non-Patent Citations (3)
| Title |
|---|
| MCLACHLAN,J.L. ET AL: "Gene Expression profiling of pulpal tissue reveals the molecular complexity of dental caries", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| 余元勋 等: "《中国疾病信号通路与靶向治疗学》", 31 May 2013 * |
| 李敏 等: "p38MAPK信号通路在慢性牙髓炎疼痛大鼠中的表达及作用机制研究", 《中国临床药理学与治疗学》 * |
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