CN110244064B

CN110244064B - Application of Cystatin SN in predicting sensitivity of chronic nasosinusitis and nasal polyp patient to glucocorticoid

Info

Publication number: CN110244064B
Application number: CN201910521050.7A
Authority: CN
Inventors: 张罗; 王成硕; 闫冰
Original assignee: Beijing Institute Of Otorhinolaryngology; Beijing Tongren Hospital
Current assignee: Beijing Institute Of Otorhinolaryngology; Beijing Tongren Hospital
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2021-04-02
Anticipated expiration: 2039-06-17
Also published as: CN110244064A

Abstract

The invention relates to the field of molecular biology detection, in particular to application of Cystatin SN in predicting sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid. The prediction method has the advantages of safe and noninvasive sample collection method, good tolerance, simple and quick detection method, good prediction performance and good clinical application prospect.

Description

Application of Cystatin SN in predicting sensitivity of chronic nasosinusitis and nasal polyp patient to glucocorticoid

Technical Field

The invention relates to the field of molecular biology detection, in particular to application of Cystatin SN in predicting sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid.

Background

The chronic nasosinusitis is accompanied by nasal polyp, the mouth of a patient in China exceeds 8000 thousands, the nasal sinusitis is a common intractable disease of the nose, has correlation with various chronic diseases (such as heart disease, hypertension and the like) and even mental diseases, seriously harms the health of the patient, and brings heavy burden to national medical expenditure. But the therapeutic effect is still unsatisfactory. Under the medical background of 'prominent prevention, early intervention and comprehensive treatment', clinical practice is guided through scientific research achievements, accurate, effective, safe and economic medical service is provided for patients, and the development of future disciplines is inevitable.

Chronic sinusitis with nasal polyp is manifested by symptoms of nasal obstruction, watery nasal discharge, sneezing, nasal itching, etc., and the inherent cause is caused by the increase of inflammatory factors. The TH1/TH2/TH17 factor imbalance is generally considered to be the immunological cause. This cause is closely related to the degree of response of the disease to clinical drugs. In general, the inflammatory response of TH2 type is more responsive to glucocorticoids, whereas TH1/TH17 type factors are less responsive.

However, in terms of clinical practice, how to judge the type of immunity is still a difficult point of diagnosis and treatment. The traditional gold standard adopts a pathological detection mode, namely, disease tissues are obtained by biopsy, various inflammatory cell values are obtained by paraffin embedding, slicing, hematoxylin and eosin staining and counting, and then the inflammation type is judged after the percentage is calculated. However, biopsy of the harvested tissue increases the likelihood of bleeding and infection in the patient. Therefore, in clinical practice, glucocorticoid is generally administered to patients without glucocorticoid contraindication, and then whether effective or not is judged according to endoscopic results to guide subsequent medication. Although short-term or local glucocorticoid use hardly brings about systemic side effects of the hormone, it still brings about an increase in the diagnosis and treatment costs for patients who are not glucocorticoid sensitive.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to find a noninvasive and economic method for predicting the glucocorticoid curative effect, which can effectively save the expenditure of patients and medical care and guide doctors to reasonably take medicine, thereby establishing an accurate medical system. Through previous work based on proteomics technology, the inventor screens Cystatin SN (CST1) which is a member of a Cystatin family, and can predict the sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid.

Specifically, the invention relates to an application of a Cystatin SN detection agent in preparation of a kit for predicting the sensitivity of a patient suffering from chronic nasosinusitis and nasal polyp to glucocorticoid.

The invention firstly provides that Cystatin SN can be used for predicting the sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid, and the detection method is simple, convenient and quick, has good prediction performance and has good clinical application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a ROC plot of CST1 concentration in nasal secretions versus percentage of eosinophils in polyp tissue in an embodiment of the present invention;

FIG. 2 is a scatter plot of CST1 concentration in nasal secretions detected between hormone sensitive and non-sensitive groups in accordance with an embodiment of the present invention;

FIG. 3 is a scatter plot of percentage eosinophils in polyp tissue detected between hormone sensitive and non-sensitive groups in accordance with an embodiment of the present invention;

FIG. 4 is a graph showing the change in CST1 concentration before and after hormone administration in a sensitive group according to one embodiment of the present invention;

FIG. 5 is a graph of the change in CST1 concentration before and after hormone administration in a non-sensitive group in accordance with one embodiment of the present invention;

FIG. 6 is a graph of the correlation of CST1 concentration in nasal secretions with the percentage of eosinophils in polyp tissue, in accordance with an embodiment of the present invention.

Detailed Description

The invention relates to application of Cystatin SN detection agent in preparation of a kit for predicting sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid.

In some embodiments, the kit further comprises a sample pretreatment reagent.

In some embodiments, the sample is selected from nasal secretions and/or nasal shed cells.

The sample is selected from nasal secretion and/or nasal exfoliative cells, the sample collection is safe and noninvasive, the tolerance is good, and the detection method is simple, convenient and quick.

The sample pretreatment reagents may include sample extraction reagents (e.g., reagents used in nasal sampling or swelling sponge absorption), or protein concentration detection reagents (e.g., BCA), or protein diluents (e.g., PBS or water, etc.).

In some embodiments, the kit further comprises reagents for detecting the percentage of eosinophils.

In some embodiments, the dosage form of the glucocorticoid comprises any one of an oral dosage form (e.g., oral methylprednisolone or dexamethasone phosphate tablets, etc.), an injection (e.g., hydrocortisone injection), an ointment (e.g., 0.1% or other concentration betamethasone valerate ointment), a spray (e.g., glucocorticoid nasal spray), and an inhalant (e.g., budesonide aerosol);

in some embodiments, the glucocorticoid comprises one or more of hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, and betamethasone.

In some embodiments, the Cystatin SN detection agent comprises a quantitative detection agent for Cystatin SN protein;

in some embodiments, the Cystatin SN detection agent comprises one or more of an antibody or antibody fragment capable of specifically binding Cystatin SN protein, a lectin, and an aptamer.

The specific binding agent has at least 10 for its corresponding target molecule⁷Affinity of l/mol. The specific binding agent preferably has 10 to its target molecule⁸l/mol, or more preferably 10⁹Affinity of l/mol. The skilled person will understand that the term "specific" is used to indicate that other biomolecules present in the sample do not significantly bind to the specific binding agent of the Cystatin SN protein. Preferably, the level of binding to biomolecules other than the target molecule (Cystatin SN protein) results in a binding affinity that is at most only 10% or less, only 5% or less, only 2% or less, or only 1% or less, respectively, of the affinity to the target molecule. Preferred specific binding agents will meet the above minimum criteria for both affinity and specificity.

The Cystatin SN detection agent is preferably selected from an antibody or an antibody fragment, and the antibody or the antibody fragment can be packaged and exist in the forms of an ELISA detection reagent, an antibody chip and an immunoassay kit.

In some embodiments, the Cystatin SN detection agent is used to detect Cystatin SN mRNA.

The term "for detecting Cystatin SN mRNA" is not to be construed as merely a detection agent for Cystatin SN mRNA in the present invention, but includes the remaining detection agents known to those skilled in the art to reflect the expression level of Cystatin SN mRNA. For example, the expression level of Cystatin SN mRNA can be indirectly detected by quantitatively detecting cDNA obtained by reverse transcription of Cystatin SN mRNA or polypeptide fragments obtained by transcription of the cDNA.

In some embodiments, the Cystatin SN detection agent comprises a reagent suitable for use in at least one of the following methods:

fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescent quantitative PCR, sequencing or biomass spectrometry.

In some embodiments, the Cystatin SN detector is a probe or primer that is capable of specifically binding Cystatin SN mRNA or Cystatin SN cDNA.

In some embodiments, the probe or primer bears a detectable label.

In some embodiments, the Cystatin SN detection agent is a qRT-PCR primer for Cystatin SN mRNA, whose upstream primer is set forth in SEQ ID NO: 1, the downstream primer is shown as SEQ ID NO: 2, respectively.

In some embodiments, the kit further comprises an internal reference gene primer;

in some embodiments, the internal reference is GAPDH, tubulin, or actin;

in some embodiments, the upstream primer of the qRT-PCR primer for GAPDH is as set forth in SEQ ID NO: 3, the downstream primer is shown as SEQ ID NO: 4, respectively.

According to one aspect of the invention, the invention also relates to the use of Cystatin SN for predicting the sensitivity of patients with chronic rhinosinusitis and nasal polyps to glucocorticoids; specifically, the method comprises the following steps:

a method of predicting the sensitivity to glucocorticoids in a patient with chronic rhinosinusitis with nasal polyps, comprising:

(a) measuring the amount of Cystatin SN mRNA or protein expression in the sample, (b) using the measurement of step (a) to assess the sensitivity of a patient with chronic rhinosinusitis complicated by nasal polyps to glucocorticoids, wherein an increased amount of Cystatin SN mRNA or protein expression is (is) indicative of sensitivity.

In some embodiments, the method further comprises detecting the percentage of eosinophils, e.g., peripheral blood eosinophils.

An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood. As the skilled artisan will appreciate, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Determining whether a subject sample has sensitivity compared to the normal control sample can be performed using statistical methods well known in the art and confirmed using confidence intervals and/or p-values. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Examples

First, research method

1. Patient grouping and hormone sensitivity evaluation indexes:

patients with chronic sinusitis and nasal polyp were enrolled: clinical information (sex, age, concomitant diseases, symptom score, sinus CT Lund-Mackay score, polyp size score before hormone treatment, etc.) was collected from chronic sinusitis patients who met the criteria for diagnosis of EPOS 2012 and had no oral glucocorticoid contraindication.

Each patient was treated with oral hormone for 2 weeks (methylprednisolone 24mg, morning, drench), and polyp size scoring was performed again after the end of oral hormone treatment, and the change in polyp score before and after oral hormone treatment was classified into hormone-sensitive group (polyp size score decreased by more than 1 point) and hormone-insensitive group (polyp size score decreased by no more than 1 point). The scoring criteria were as follows:

TABLE 1 nasal polyp size Scale Table

Note: all patients in the group had a unilateral score of no less than 2 points before oral hormone treatment.

2. Taking and leaving specimen

Nasal secretion retention: nasal secretion of the patient was retained by sponge-expanding absorption before the start of oral hormone therapy (a 0.5cm x 3cm sponge-expanding sponge was gently placed between the inferior turbinate and septum of the patient, the upper end of the sponge-expanding sponge was directed to the free edge of the middle turbinate, and the sponge-expanding sponge was kept for 5min and then taken out for use).

Taking the nasal cast-off cells: middle nasal sampling was performed under intranasal endoscopic guidance. A sterile cotton swab was gently pressed on the middle nasal mucosa surface for 30 seconds and rotated 5 cycles under an intranasal scope before being placed into a sterile collection tube containing transport medium and immediately snapped off at the red line. Information was marked and the swabs were stored at-80 deg.C

Polyp tissue specimen retention: the sterile sinus-sieving forceps are used for taking a small nasal polyp tissue for standby.

3. Sample treatment and experimental methods:

nasal secretions: placing the expanded sponge absorbing nasal secretion into a 15mL centrifuge tube, adding 500 microliter of normal saline, standing at 4 ℃ for 2h, placing the expanded sponge into an injector without a needle and a piston, placing the injector into the original centrifuge tube, centrifuging at 4 ℃ and 1500rpm for 15min, and taking the supernatant as a nasal secretion specimen of a patient. Determining total protein content of nasal secretion sample by BCA method, and diluting to total protein content of 5 × 10^-3mg/mL, measured for CST1 content by ELISA (Cloud-Clone Corp. USA, detection range 0.2ng/mL-10ng/mL)

Nasal shedding of cells: total RNA is extracted by a conventional method, the RNA is reversely transcribed into cDNA, and the expression of CST1 and an internal reference gene (such as GAPDH) is detected by fluorescent quantitative PCR.

Extracting total RNA: the extraction method is described below by taking Trizol reagent as an example, and any method capable of extracting total RNA may be applied. (1) 1mL of Trizol was added to the test tube containing the exfoliated cells, followed by shaking, then adding 200. mu.L of chloroform (chloroform), shaking vigorously and mixing well, and standing at room temperature for 5 minutes. (2) Centrifuge at 12000 rpm, 4 ℃ for 15 min. (3) About 250. mu.L of the supernatant was taken, added to an EP tube, and an equal amount (about 250. mu.L) of isopropanol was added. After mixing, standing for 10 minutes at 12000 r, centrifuging for 15min at 4 ℃. The supernatant was discarded and the precipitate was retained. (4) The precipitate was washed with 75% ethanol in equal volume (about 250. mu.L) to isopropanol and mixed well. 7500rpm, 4 deg.C, 15 min. The supernatant was discarded and the precipitate was retained. (5) Uncovering, standing in a fume hood for 15min to volatilize ethanol. (6) The precipitate was dissolved with 20-100. mu.L Nase-free water. RNA concentration was measured spectrophotometrically.

Reverse transcription of RNA into cDNA: again, by way of example only, any reagent that can reverse transcribe total RNA to cDNA can be used in the reaction. Using the reverse transcription kit (cat No. RR036A) from Takara as an example, the reaction system was 2. mu.L of prime mix (kit-of-reagents), 500ng of total RNA, supplemented to 10. mu.L with RNase Free dH 2O. The cDNA was obtained at 37 ℃ for 15 min.

Fluorescence quantitative PCR detection of expression of CST1 and internal reference gene GAPDH: similarly, any primer capable of detecting the expression level of CST1 or the expression level of the internal reference gene GAPDH is suitable for use in the present method, and any reagent capable of performing fluorescent quantitative PCR is suitable for use in the present method, and the following takes the fluorescent quantitative PCR kit (cat. No. RR820A) from Takara as an example,

TABLE 2 Target Gene primers

CST1-forward	5’-TAGGATAATCCCGGGTGGCA--3’
		CST1-reverse	5’-GTCTGTTGCCTGGCTCTTAGT-3’
GAPDH-forward	5’-CTCCTCCTGTTCGACAGTCAGC-3’
		GAPDH-reverse	5’-CCCAATACGACCAAATCCGTT-3’

TABLE 3 reaction System

Reaction conditions for PCR amplification (with three-step and two-step method being optional)

The three-step method comprises the following steps: pre-denaturation at 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 1 min, 55 ℃ for 1 min, and 72 ℃ for 1 min, and final extension at 72 ℃ for 7 min.

The two-step method comprises the following steps: annealing and extension are carried out at 95 ℃ for 30 seconds, 95 ℃ for 5 seconds and 60 ℃ for 1 minute, and the total number of cycles is 40.

Interpretation of experimental results: ct value of the target gene CST 1-Ct value of an internal reference gene (e.g., GAPDH) is defined as Δ Ct, and the lower the Δ Ct value, the higher the expression level of CST1 in exfoliated cells

Nasal polyp tissue: after the tissue fixative was fixed, paraffin was embedded, and then cut into 5 μm sections by a Leica RM2235 cryostat (Leica Microsystems, Bannockburn, IL, USA) microtome, HE stained, and the percentage of inflammatory cells (eosinophils, neutrophils, lymphocytes, and plasma cells) was counted under 400 × microscopic field, 3 non-overlapping fields were counted per section, 5 sections were counted per polyp tissue, and the average of the 15 fields was taken as the infiltration of inflammatory cells in the polyp tissue.

Second, CST1 content in nasal secretion predicts hormone sensitivity of chronic sinusitis complicated with nasal polyp

Patients were enrolled: the 111 patients with chronic sinusitis and nasal polyp, who meet the diagnosis standard of EPOS 2012 and have no oral glucocorticoid contraindication, were grouped together, and clinical information (sex, age, concomitant diseases, symptom score, sinus CT Lund-Mackay score, polyp size score before hormone treatment, etc.) was collected according to the method described above. And subjected to 2 weeks of oral hormone therapy (methylprednisolone 24mg, morning start, drench).

Nasal secretions: the methods of obtaining, treating and detecting the specimen are the same as above

The experimental results are as follows:

1. clinical data and laboratory data of hormone sensitive and non-sensitive groups show that: indexes such as CST1 concentration, nasal polyp tissue eosinophil percentage, nasal polyp tissue neutrophil percentage, nasal polyp tissue lymphocyte percentage, nasal polyp tissue plasma cell percentage, CT ethmoid sinus score/maxillary sinus score before treatment and the like in nasal secretions of patients are different between hormone sensitive groups and non-sensitive groups. (Table 4)

TABLE 4 patient clinical information and laboratory results

2. Inclusion of all the differential variables in result 1 into logistic regression analysis showed that only the concentration of CST1 in nasal secretions and the percentage of eosinophils in polyp tissue predicted oral hormone treatment sensitivity, so a subject working curve (ROC curve) was generated (fig. 1), indicating that the prediction of CST1 concentration in nasal secretions was 94.8% accurate and tissue eosinophils was 98.7% accurate, both predicted to be equally potent (statistical test P > 0.05). (FIG. 2) CST1 concentration in nasal secretions predicted a sensitivity of 0.880, specificity of 0.972 and a cutoff of 2575.5ng/mL for oral hormone treatment sensitivity; (FIG. 3) the percentage of eosinophils in polyp tissue predicted a sensitivity of 0.920 for oral hormone treatment sensitivity, a specificity of 1, and a predicted cutoff of 29.5%.

3. Oral hormone treatment reduced the concentration of CST1 in nasal secretions of hormone-sensitive patients (fig. 4), but there was no statistical difference in the effect of oral hormone treatment on the concentration of CST1 in nasal secretions of hormone-insensitive patients (fig. 5).

Third, the content of CST1 in the nose exfoliative cells predicts the hormone sensitivity of chronic nasosinusitis

Patients were enrolled: 83 chronic nasosinusitis patients with rhinopolypus who meet the diagnosis standard of EPOS 2012 and have no oral glucocorticoid contraindication are combined, and clinical information (sex, age, concomitant diseases, symptom score, sinus CT Lund-Mackay score, polyp size score before hormone treatment and the like) is collected according to the method. And subjected to 2 weeks of oral hormone therapy (methylprednisolone 24mg, morning start, drench).

Nasal shedding of cells: the methods of obtaining, treating and detecting the specimen are the same as above

Results of the experiment

1. Clinical data and laboratory data of hormone sensitive and non-sensitive groups show that: indexes such as CST1 value (delta Ct) in nasal cast-off cells, percentage of eosinophils in nasal polyp tissue, percentage of neutrophils in nasal polyp tissue, percentage of lymphocytes in nasal polyp tissue, percentage of plasma cells in nasal polyp tissue, CT (computed tomography) sinus screening score/maxillary sinus score before treatment and the like of a patient are different between a hormone sensitive group and a non-sensitive group. (Table 5)

TABLE 5 patient clinical information and laboratory results

2. Incorporating all of the differential variables in 1 into logistic regression analysis showed that only the value of CST1 in nasal exfoliated cells (Δ Ct) and the percentage of eosinophils in polyp tissue predicted oral hormone treatment sensitivity, so a subject working curve (ROC curve) was made (fig. 6), and the area under the ROC curve indicated that the accuracy of the prediction of CST1 values (Δ Ct) in nasal exfoliated cells was 95.2%.

Area under the curve

Variable VAR00001 of inspection result

a. By nonparametric assumptions

b. The original assumption is that: true area of 0.5

The above experiments show that:

the concentration of CST1 in nasal secretions and nasal shed cells can predict the sensitivity of CRSwNP patients to oral hormone treatment. The sample collection method is safe and noninvasive, has good tolerance, simple, convenient and quick detection method and good prediction performance, and has good clinical application prospect.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Beijing Hospital, Beijing university of medicine, otorhinolaryngology institute of Beijing

Application of <120> Cystatin SN in prediction of glucocorticoid sensitivity of patient suffering from chronic nasosinusitis and nasal polyp

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Claims

The application of Cystatin SN detection agent in the preparation of a kit for predicting the sensitivity of patients suffering from chronic nasosinusitis and nasal polyp to glucocorticoid;

the Cystatin SN detection agent comprises a quantitative detection agent for detecting the concentration of Cystatin SN protein in nasal secretion, a probe or primer which can be specifically combined with Cystatin SN mRNA or Cystatin SN cDNA in nasal exfoliative cells, a sample pretreatment reagent and a reagent for detecting the percentage of eosinophilic granulocyte in nasal polyp tissues;

the sample is nasal secretion, nasal cast-off cells and nasal polyp tissues;

the dosage form of the glucocorticoid is an oral dosage form.
2. The use of claim 1, wherein the glucocorticoid comprises one or more of hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, and betamethasone.
3. The use according to claim 1, wherein the agent for the quantitative detection of the concentration of Cystatin SN protein comprises one or more of an antibody or antibody fragment capable of specifically binding Cystatin SN protein, a lectin and an aptamer.
4. The use of claim 1, wherein the Cystatin SN detection agent comprises a reagent suitable for use in at least one of the following methods: fluorescent dye method, digital PCR, resonance light scattering method, real-time fluorescent quantitative PCR, sequencing or biomass spectrometry.
5. The use of claim 1, wherein the probe or primer is detectably labeled.
6. The use of claim 5, wherein the probe or primer capable of specifically binding Cystatin SN mRNA or Cystatin SN cDNA in nasal shedding cells is a qRT-PCR primer of Cystatin SN mRNA, and the upstream primer is shown in SEQ ID NO: 1, the downstream primer is shown as SEQ ID NO: 2, respectively.
7. The use of any one of claims 1 to 6, wherein the kit further comprises an internal reference gene primer.
8. The use of claim 7, wherein the internal reference is GAPDH, tubulin or actin.
9. The use of claim 8, wherein the upstream primer of the qRT-PCR primer of GAPDH is as shown in SEQ ID NO: 3, the downstream primer is shown as SEQ ID NO: 4, respectively.