CN116144764A

CN116144764A - Product for detecting glaucoma

Info

Publication number: CN116144764A
Application number: CN202310359430.1A
Authority: CN
Inventors: 于晓光; 吴文辉; 李小玲
Original assignee: Wenzhou Puxi Gene Technology Co ltd
Current assignee: Wenzhou Puxi Gene Technology Co ltd
Priority date: 2023-03-31
Filing date: 2023-03-31
Publication date: 2023-05-23

Abstract

The invention provides application of a reagent for detecting a biomarker in a sample in preparation of a kit for detecting glaucoma, wherein the biomarker is selected from PTGS1 and/or EDN1, and belongs to the technical field of biology. Also disclosed in the present invention is a product for detecting glaucoma, which comprises a reagent for detecting the expression level of PTGS1 gene or EDN1 gene or PTGS1 protein or EDN1 protein. According to the invention, by detecting the expression level of PTGS1 and/or EDN 1in the sample and comparing the expression level with normal people, whether the subject suffers from glaucoma can be judged, and a theoretical basis can be provided for detecting glaucoma.

Description

Product for detecting glaucoma

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a product for detecting glaucoma.

Background

Glaucoma is a generic term for a progressive optic neuropathy caused by pathological increases in ocular tension, the major pathological change being loss of optic ganglion cells and their axons, thinning of the nerve fiber layer, leading to glaucomatous optic papillary depressions and corresponding visual field defects. Glaucoma is the leading cause of second-to-world blindness next to cataracts, and is also the leading cause of irreversible blindness. Glaucoma progresses from retinal ganglion cell damage, degeneration to apoptosis, with corresponding atrophy and visual field defects eventually occurring.

Glaucoma is classified into primary open-angle glaucoma and primary closed-angle glaucoma. Risk factors for glaucoma include age, intraocular pressure, race, family history, myopia, hypertension, hypotension, diabetes, vasospasm, smoking, and the like. Glaucoma manifests itself mainly as a sudden decrease in vision, eye pain, eye distension, elevated intraocular pressure, advanced progression to irreversible optic nerve and visual field damage. The underlying purpose of glaucoma treatment is to preserve the visual function of the patient, stop or halt the continued progression or worsening of the condition. Traditional treatment methods of glaucoma include drug treatment and surgical treatment, and the methods have a certain recurrent property and have more adverse reactions to patients.

The use of the SP8 gene as a biomarker for diagnosing and treating glaucoma is disclosed in chinese patent CN202010383914.6, in which it is demonstrated that the SP8 gene expression of glaucoma patients is significantly down-regulated, glaucoma and normal persons can be distinguished according to the SP8 gene expression difference, in which the number of down-regulated SP8 genes in control populations and glaucoma patients is counted, and the results show that: 7 cases of SP8 gene expression down-regulation in control population, 43 cases of SP8 gene expression down-regulation in glaucoma patients, sensitivity of 86% and specificity of 84%, show that the SP8 gene can be used as a molecular marker for diagnosing glaucoma. However, the specificity and sensitivity of diagnosing glaucoma using the SP8 gene as a marker are relatively poor.

Chinese patent CN202211074096.7 discloses a primer for detecting serum lncRNA after glaucoma patient operation and an application method thereof, wherein the method mainly comprises the steps of extracting total RNA from clinical tissues by using TRIzol reagent, marking a sample, performing hybridization experiments, washing a chip, scanning the washed chip, collecting chip probe signal values by using software, performing chip data homogenization treatment and difference analysis by using software, and screening all differentially expressed lncRNAs and mRNAs by folding multiplying power, wherein the screening standard is that the multiplying power change is more than 1.5, and P <0.05. The kit disclosed by the invention can be used for rapidly, accurately and quantitatively determining the expression level of the lncRNANR_003923, and effectively eliminating the defects of low repeatability and low accuracy of conventional fluorescent quantitative PCR. But the detection method is relatively complex.

Chinese patent CN202010814367.2 discloses a COL5A3 mutant gene, wherein the COL5A3 mutant gene is formed by inserting a g.[49881insACCG ] mutation into a wild type COL5A3 gene, and the COL5A3 mutant gene carrying the g.[49881insACCG ] mutation causes primary glaucoma, and the patient often accompanies joint pain symptoms. The invention provides 4 specific pathogenic mutant forms of a primary glaucoma pathogenic gene COL5A3, a pathogenic gene detection kit based on PCR amplification and Sanger sequencing and a corresponding detection method, which can provide basis for detection of the pathogenic gene of the primary glaucoma and lay basis for analysis of pathogenic mechanism, development of therapeutic drugs, targeted prevention and treatment and the like.

The prior art proves that genetic factors play an important role in detecting glaucoma, and the discovery of relevant pathogenic genes or sites of glaucoma can further define the pathogenesis of glaucoma and has important significance in diagnosing or screening or prognosis evaluation of glaucoma.

Disclosure of Invention

Term interpretation:

as used herein, the singular forms "a," "an," and "the" include the singular and plural referents unless the context clearly dictates otherwise. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that corresponding range, and the endpoints recited.

The term "primer" refers to a short nucleic acid sequence having a short free 3 hydroxyl group capable of forming base pairs with a complementary template that serves as an origin for replication of the template strand.

Aiming at the problems in the prior art, the invention researches the biomarker related to occurrence and development of glaucoma and provides a product for detecting the glaucoma, thereby providing a new direction for detecting the glaucoma.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in one aspect, the invention provides the use of a reagent for detecting a biomarker selected from PTGS1 and/or EDN 1in a sample in the preparation of a kit for detecting glaucoma.

Preferably, the reagent detects the expression level of the PTGS1 gene or the EDN1 gene or the PTGS1 protein or the EDN1 protein.

Further preferably, when the expression level of PTGS1 and/or EDN1 is up-regulated in a sample of the subject compared to a normal person, then the subject suffers from glaucoma or the risk of the subject suffering from glaucoma is high.

Preferably, the sample is selected from one or more of tissue, plasma, serum, lymph, synovial fluid, aqueous humor, and tears.

Further preferably, the sample is tissue.

Still further preferably, the sample is trabecular tissue.

Preferably, the reagent comprises:

primers specifically amplifying the PTGS1 or EDN1 gene;

probes specifically recognizing the PTGS1 or EDN1 gene; or (b)

Binding agents that specifically bind to a protein encoded by PTGS1 or EDN1.

Preferably, the primer sequence for specifically amplifying the PTGS1 gene is shown as SEQ ID NO. 1-2; the probe for specifically recognizing the PTGS1 gene is shown as SEQ ID NO. 3; the primer sequence of the specific amplification EDN1 gene is shown as SEQ ID NO. 4-5; the specific EDN1 gene recognition probe is shown as SEQ ID NO. 6.

Preferably, the probe is marked with a fluorescent reporter gene at the 5 'end and a fluorescent quenching gene at the 3' end.

Further preferably, the fluorescent reporter group is selected from at least one of FAM, ROX, HEX, CY, VIC, TET, JOE, cy3, cy7, RED610, texas Red, RED670, NED, AMCA, pacific Blue, atto 425, BODIPY FL, alexa Fluor 488, yakima Yellow, quasar 570, aqua Phuor 593, atto 590, cy5.5.

Further preferably, the fluorescent reporter group is selected from at least one of VIC, ROX, FAM.

Still further preferably, the 5' end of the probe PTGS1-probe is marked with a FAM fluorescent reporter group;

still further preferably, the 5' end of the probe EDN1-probe is marked with a VIC fluorescent reporter group;

preferably, the fluorescence quenching group is at least one selected from 6-TAMRA, BHQ-1, BHQ-2, BHQ-3 and Dabcyl, eclipse, MGB, QYS-7.

Further preferably, the fluorescence quenching group is selected from one or more of BHQ-1 and BHQ-2.

Still further preferably, the 3' -end of the probe PTGS1-probe is marked with a BHQ1 fluorescence quenching group;

still further preferably, the 3' -end of the probe EDN1-probe is labeled with a BHQ1 fluorescence quenching group.

In yet another aspect, the invention provides a product for detecting glaucoma, said product comprising an agent for detecting the expression level of the PTGS1 gene or the EDN1 gene or the PTGS1 protein or the EDN1 protein.

Preferably, the method for detecting the mRNA expression level of the product is selected from one or more of polymerase chain reaction, real-time fluorescence quantitative reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection analysis, in situ hybridization method, nucleic acid microarray, RNA blot and DNA chip.

Preferably, the method for detecting the protein expression level of the product is selected from one or more of immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunoelectrophoresis, tissue immunostaining and immunoprecipitation analysis.

Preferably, the product is a nucleic acid membrane strip, a chip or a kit.

Further preferably, the chip comprises a gene chip comprising a primer or an oligonucleotide probe directed against PTGS1 or EDN1, a protein chip comprising a binding agent that specifically binds to PTGS1 or EDN1 protein.

Specifically, the specific binding agent is selected from one or more of a receptor of the protein PTGS1 or EDN1, lectin binding to the protein PTGS1 or EDN1, antibody against the protein PTGS1 or EDN1, and peptide antibody against the protein PTGS1 or EDN1.

Further specifically, the specific binding agent is selected from one or more of a peptide, a peptidomimetic, an ankyrin repeat protein, an antibody, a single domain antibody, and a single domain antibody fragment.

Still further specifically, the specific binding agent is a PTGS1 or EDN1 specific antibody.

Preferably, the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises primers and probes for specifically amplifying PTGS1 genes or EDN1 genes; the protein detection kit comprises a binding agent which specifically binds to PTGS1 or EDN1 protein.

Further preferably, the primer sequence for specifically amplifying the PTGS1 gene is shown as SEQ ID NO. 1-2; the probe for specifically recognizing the PTGS1 gene is shown as SEQ ID NO. 3; the primer sequence of the specific amplification EDN1 gene is shown as SEQ ID NO. 4-5; the specific EDN1 gene recognition probe is shown as SEQ ID NO. 6.

Preferably, the kit further comprises other reagents required for the PCR reaction.

Preferably, the reagent is selected from at least one of DNA polymerase, dNTPs, buffer, and metal ions that promote enzymatic reactions.

Preferably, the kit further comprises a primer for detecting the reference gene.

Further preferably, the reference gene is GAPDH.

Still further preferably, the primer sequence for specifically amplifying the GAPDH gene is shown in SEQ ID NO. 7-8; the specific GAPDH gene recognition probe is shown as SEQ ID NO. 9.

Preferably, the 5' end of the probe GAPDH-probe is marked with a ROX fluorescent reporter group;

preferably, the 3' -end of the probe GAPDH-probe is labeled with a BHQ2 fluorescence quenching group.

Preferably, the invention provides a use method of the kit, which comprises the following steps:

(1) Extracting RNA in a sample;

(2) Detecting PTGS1 mRNA and EDN1 mRNA expression levels in the RNA;

(3) When the expression level of PTGS1 mRNA and/or EDN1 mRNA is significantly increased compared to normal humans, then the subject is at high risk of suffering from glaucoma or suffering from glaucoma.

In yet another aspect, the invention provides the use of an inhibitor of PTGS1 and/or EDN 1in the manufacture of a medicament for the treatment of glaucoma.

Preferably, the inhibitor reduces expression of PTGS1 and/or EDN1 at the transcriptional or translational level.

Preferably, the medicament comprises a pharmaceutically acceptable carrier;

further preferably, the pharmaceutically acceptable carrier is selected from one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants.

Preferably, the dosage forms of the medicament include, but are not limited to, injections, drops, tablets, capsules, oral liquid dosage forms, granules, ointments, suspensions, powders, emulsions, solutions, drop pills, suppositories, aerosols.

Compared with the prior art, the invention has the following beneficial effects:

(1) According to the invention, by detecting the expression level of PTGS1 and/or EDN 1in the sample and comparing the expression level with normal people, whether the subject suffers from glaucoma can be judged, and a theoretical basis can be provided for detecting or diagnosing or treating glaucoma.

(2) The invention improves the sensitivity of detecting glaucoma and improves the detection efficiency and accuracy.

Drawings

Fig. 1 is a graph showing the expression levels of PTGS1 mRNA in the experimental and control groups in aqueous humor samples, where p < 0.001.

Fig. 2 is a graph of EDN1 mRNA expression levels in aqueous samples of experimental and control groups, where p < 0.001.

Fig. 3 is a graph showing expression levels of PTGS1 mRNA in the trabecular tissue in the experimental and control groups, where p < 0.001.

Fig. 4 is a graph showing EDN1 mRNA expression levels in trabecular tissue for the experimental and control groups, where p < 0.01.

Detailed Description

The following description of the present invention is provided by way of specific examples to facilitate understanding and grasping of the technical solution of the present invention, but the present invention is not limited thereto, and the described examples are only some, but not all, examples of the present application. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

Example 1

1. Sample collection

Patients diagnosed with glaucoma were 20 (experimental group) and eye-state normal subjects were 15 (control group).

Experimental group inclusion criteria:

(1) Meets the diagnostic criteria for glaucoma;

(2) Age between 18-65 years old;

(3) Eyes were not dosed one month prior to group entry;

(4) And signing an informed consent form.

Experimental group exclusion criteria:

(1) Eliminating diabetes and hypertension;

(2) Other ocular diseases besides glaucoma;

(3) Patients with systemic diseases;

(4) Malignant tumor.

Control group inclusion criteria:

age, sex and experimental group patients were matched.

2. Extracting RNA in a sample to be tested:

(1) The tissue (aqueous tissue or trabecular tissue) was ground with nitrogen, 1mL of Trizol lysate and 200 μl of chloroform (Trizol 1 lysate: chloroform=5:1) were added, mixed by shaking for 15s, incubated at 37℃for 2min, and centrifuged at 12000rpm for 15min at 4 ℃.

(2) The upper colorless liquid was aspirated into a clean centrifuge tube, and an equal volume of isopropanol was added and mixed well and incubated for 10min at 37 ℃.

(3) Centrifuging at 12000rpm at 4deg.C for 10min, discarding supernatant, washing RNA precipitate with 75% ethanol, drying at room temperature for 5-10min, and dissolving RNA with RNase-free sterile water.

(4) Concentration detection: mu.l of RNA sample was taken and absorbance was measured on a nucleic acid protein detector. The OD260/280 ratio is all in the range of 1.8-2.2.

3. Reverse transcription experiment:

reverse transcription uses Prime Script ^TM RT reagent Kit, sample addition was performed according to Table 1 below.

Table 1.

Reagent(s)	Sample addition amount
		5×PrimeScript ^TM Buffer	4μl
PrimeScript RT Enzyme Mix I	1μl
		0ligo dT Prime(50μM)	1μl
Random 6mers(100μM)	4μl
		Total RNA	2μl
RNase-free ddH ₂ O	8μl

The above system was placed in an EP tube and the cDNA was inverted according to the following procedure: the obtained cDNA was stored at-20℃for 15min at 37℃and 5s at 85 ℃.

4. Real-time fluorescent quantitative PCR

(1) Primer design

Primers were designed based on the gene sequences of PTGS1 and EDN1, and specific primer sequences are shown in Table 2 below;

table 2.

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(2) Real-time fluorescent quantitative PCR amplification test

By TaqMan ^TM The PCR reaction system was prepared from the multiplex premix, and PCR amplification was performed on an amplification apparatus, and the amplification system (Table 3) and the reaction conditions were as follows:

TABLE 3 real-time fluorescent quantitative PCR amplification System

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The PCR conditions were as follows: 95 ℃ for 20s; (95 ℃,5s;60 ℃,40s;40 cycles).

5. Data analysis

SPSS19.0 version of statistical software is adopted for data processing, and the P value less than 0.05 is statistically significant for the difference.

6. Experimental results

As shown in fig. 1-4, in aqueous humor samples, PTGS1 and EDN1 were significantly up-regulated in glaucoma patients compared with normal persons, and the difference was statistically significant, and the expression amounts of PTGS1 and EDN 1in glaucoma patients were 4.83 times and 4.70 times the expression amounts of the control group. In trabecular tissue, the expression levels of PTGS1 and EDN1 were statistically different from those of normal human samples, and the expression levels thereof in glaucoma patients were 3.48 times and 2.83 times the expression levels of the control group. The above results indicate that PTGS1 and EDN1 can be applied to glaucoma detection.

7. Validity analysis:

ROC analysis is performed on the expression levels of PTGS1 and EDN1 by using R language, and the application of PTGS1 and EDN1 to glaucoma diagnosis is found to have higher accuracy, sensitivity and specificity. In aqueous samples, PTGS1 had an AUC value of 0.880, a sensitivity of 0.988 and a specificity of 0.991 for detecting glaucoma; the AUC value of EDN1 was 0.892, its sensitivity for detecting glaucoma was 0.992, and the specificity was 0.995. In trabecular tissue, PTGS1 has an AUC value of 0.874, a sensitivity of 0.976 for detecting glaucoma, and a specificity of 0.989; the AUC value of EDN1 was 0.881, the sensitivity for detecting glaucoma was 0.988, and the specificity was 0.986. As shown in tables 4-5 below, table 4 shows the AUC values of the 2 genes in aqueous humor samples, and Table 5 shows the AUC values of the 2 genes in trabecular tissue.

TABLE 4 Performance of PTGS1 and EDN1 mRNA in aqueous humor samples for glaucoma detection

	PTGS1	EDN1
			AUC	0.880	0.892
Sensitivity of	0.988	0.992
			Specificity (specificity)	0.991	0.995

TABLE 5 Performance of PTGS1 and EDN1 mRNA in trabecular tissue for glaucoma detection

	PTGS1	EDN1
			AUC	0.874	0.881
Sensitivity of	0.976	0.988
			Specificity (specificity)	0.989	0.986

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting a biomarker in a sample for the preparation of a kit for detecting glaucoma, wherein the biomarker is selected from the group consisting of PTGS1 and EDN1.

2. The use according to claim 1, wherein the agent detects the expression level of the PTGS1 gene or EDN1 gene or PTGS1 protein or EDN1 protein.

3. The use according to claim 2, wherein when the expression level of PTGS1 and/or EDN 1in the sample of the subject is up-regulated compared to normal humans, then the subject suffers from glaucoma or the risk of the subject suffering from glaucoma is high.

4. The use according to claim 3, wherein the sample is selected from one or more of tissue, plasma, serum, lymph, synovial fluid, aqueous humor, tears.

5. The use according to any one of claims 1 to 4, wherein the agent comprises:

primers specifically amplifying the PTGS1 or EDN1 gene;

probes specifically recognizing the PTGS1 or EDN1 gene; or (b)

Binding agents that specifically bind to a protein encoded by PTGS1 or EDN1.

6. The use according to claim 5, wherein the primer sequence for specifically amplifying the PTGS1 gene is shown in SEQ ID NO. 1-2; the probe for specifically recognizing the PTGS1 gene is shown as SEQ ID NO. 3; the primer sequence of the specific amplification EDN1 gene is shown as SEQ ID NO. 4-5; the specific EDN1 gene recognition probe is shown as SEQ ID NO. 6.

7. A product for detecting glaucoma, said product comprising an agent for detecting the expression level of a PTGS1 gene or EDN1 gene or PTGS1 protein or EDN1 protein.

8. The product of claim 7, wherein the product is a nucleic acid membrane strip, a chip or a kit.

9. The product according to claim 8, wherein the kit comprises a gene detection kit and a protein detection kit, and the gene detection kit comprises primers and probes for specifically amplifying the PTGS1 gene or the EDN1 gene; the protein detection kit comprises a binding agent which specifically binds to PTGS1 or EDN1 protein.

10. The product according to claim 9, wherein the primer sequence for specifically amplifying the PTGS1 gene is shown in SEQ ID NO. 1-2; the probe for specifically recognizing the PTGS1 gene is shown as SEQ ID NO. 3; the primer sequence of the specific amplification EDN1 gene is shown as SEQ ID NO. 4-5; the specific EDN1 gene recognition probe is shown as SEQ ID NO. 6.

Use of an inhibitor of ptgs1 and/or EDN 1in the preparation of a medicament for the treatment of glaucoma.