阅读说明：本技术 B7-h4+单核细胞型mdsc在制备ins激素治疗疗效预测试剂中的应用 (B7-H4+Application of monocyte type MDSC in preparing INS hormone treatment efficacy prediction reagent ) 是由 李丽民 张明超 陈麒麟 曾科 于 2021-01-27 设计创作，主要内容包括：本发明公开了B7-H4～+单核细胞型MDSC在制备INS激素治疗疗效预测试剂中的应用。外周血中B7-H4～+单核细胞型MDSC作为检测靶点在制备预测激素治疗特发性肾病综合征疗效的试剂中的应用。检测外周血中B7-H4～+单核细胞型MDSC的试剂在制备预测激素治疗特发性肾病综合征疗效的试剂中的应用。B7-H4～+单核细胞型MDSC作为激素敏感型特发性肾病综合征分型标志物的应用。INS患者治疗早期出现B7-H4～+单核细胞型MDSC的升高与激素治疗敏感有关,早期观测INS患者激素治疗后B7-H4～+单核细胞型MDSC的变化可以有效预测激素疗效。(The invention discloses B7-H4 + Application of monocyte type MDSC in preparing INS hormone treatment efficacy prediction reagent. Peripheral blood B7-H4 + Application of the monocyte type MDSC as a detection target in preparing a reagent for predicting the curative effect of hormone therapy idiopathic nephrotic syndrome. Detection of B7-H4 in peripheral blood + Use of an agent for monocyte-type MDSC in the preparation of an agent for predicting the efficacy of hormone therapy for idiopathic nephrotic syndrome. B7-H4 + The application of the monocyte type MDSC as a hormone sensitive idiopathic nephrotic syndrome typing marker. Early appearance of INS patients treated with B7-H4 + The increase in monocyte-type MDSC is associated with hormone therapy sensitization, with early observation of post-hormone therapy B7-H4 in INS patients + Changes in monocyte-type MDSCs can be effective in predicting hormonal efficacy.)

1. Peripheral blood B7-H4⁺Application of the monocyte type MDSC as a detection target in preparing a reagent for predicting the curative effect of hormone therapy idiopathic nephrotic syndrome.

2. Detection of B7-H4 in peripheral blood⁺Use of an agent for monocyte-type MDSC in the preparation of an agent for predicting the efficacy of hormone therapy for idiopathic nephrotic syndrome.

3.B7-H4⁺The application of the monocyte type MDSC as a hormone sensitive idiopathic nephrotic syndrome typing marker.

4. Detection of B7-H4 in peripheral blood⁺Application of a reagent of monocyte type MDSC in preparing a hormone sensitive idiopathic nephrotic syndrome typing reagent.

5.B7-H4⁺Application of the monocyte type MDSC as a detection target in preparing a medicament for treating idiopathic nephrotic syndrome.

Technical Field

The invention belongs to the field of medical biology, and relates to B7-H4⁺Application of monocyte type MDSC in preparing INS hormone treatment efficacy prediction reagent.

Background

Idiopathic Nephrotic Syndrome (INS) is a type of glomerular disease with edema, hypoproteinemia and proteinuria as the main clinical manifestations, which can be classified into minimal-lesion nephropathy (MCD) and Focal Segmental Glomerulosclerosis (FSGS) according to the pathological manifestations. Immunosuppressive therapy with Glucocorticoids (GCs) as the main component is the first-line treatment option of the INS patients at present, and the treatment effect of hormone therapy is judged according to the condition of urine protein after sufficient GCs treatment for 8 to 16 weeks, so that the patients with hormone therapy sensitivity (SSNS) and the patients with hormone resistance (SRNS) are divided. With the extensive use of gene sequencing technology, the improvement of disease models, the expansion of clinical registration research systems and the depth of kidney transplantation research, people have the ability to molecularly type INS based on disease pathogenesis. Single gene mutation-related INS patients are generally insensitive to immunosuppressive therapy, progress to ESRD quickly and have a low probability of relapse after transplantation. Non-monogenic related INS patients can currently be divided into two subtypes: immunosuppressive therapy is sensitive, with little progress to immune-related INS of ESRD; immunosuppressive therapy is poorly effective and gradually progresses to ESRD, and circulating pathogenic factor-related INS with a high recurrence rate after transplantation. Descriptive typing of this through pathological manifestations and hormonal effects has limited the progress of INS diagnostic methods. Although some studies have attempted to treat INS with targeted molecular drugs, the broad disease typing makes it difficult for many interventional studies to demonstrate overall efficacy, leading to false negative results. The development of pathogenesis-based disease typing will effectively advance the recognition of such diseases and the possibility of developing new therapeutic drugs. The need to find the characteristic molecular changes of different subtypes of INS and to classify diseases accordingly will greatly drive the understanding and comprehension of INS.

The specific expansion of myeloid-derived immunosuppressive cells (MDSCs) was first found in peripheral blood of FSGS hormone-sensitive patients by this research team, suggesting that immunosuppressive cells may be a molecular marker of hormone efficacy in FSGS patients. Bone marrow-derived immunosuppressive cells play an important immunomodulatory role in tumors, autoimmune diseases, infectious diseases, obesity, and pregnancy, and it is currently believed that such cells consist of mononuclear-like cells and polymorphonuclear-like cells. The two types of cells have great difference in differentiation sources, functional pathways and the like, and meanwhile, the immunosuppressive capacity of the mononuclear-like cells is remarkably stronger than that of the polymorphonuclear-like cells. This suggests that mononuclear-like cells with significant anti-inflammatory function may play a more important role in INS patients.

B7-H4 is also named as B7x, B7S1 or VTCN1, is discovered approximately simultaneously in 2003 by three independent laboratories by bioinformatics methods, and plays a similar inhibitory regulatory role as CTLA-4 and PD-1 as a member of the B7 superfamily of proteins. The DNA encoding human B7-H4 is located on chromosome 1p12/13.1, has 6 exons and 5 introns, and has a total of 66kb, and the 6 th exon is alternatively spliced, and can generate two different transcripts. The B7-H4 protein consists of 282 amino acids and is divided into an extracellular segment, a hydrophobic transmembrane segment and an extremely short intracellular segment (consisting of only two amino acids). The extracellular domain is similar to other B7 families and has an antibody-like structure. The B7-H4 protein has the complete structure of a type I transmembrane protein with a signal peptide at the N end, the extracellular region is an IgV and IgC-like domain, and the protein has 4 conserved cysteine residues and 7N-like glycosylation sites. The B7-H4 protein is 25% homologous protein compared with other B7 family members, which shows that the B7-H4 is greatly different from other B7 family members, and the biological functions of the B7-H4 are probably greatly different. Rodent and human B7-H4 homology is approximately 87%, suggesting that B7-H4 are highly conserved evolutionarily.

Disclosure of Invention

The object of the present invention is to overcome the above-mentioned disadvantages of the prior art by providing B7-H4⁺Application of monocyte type MDSC in preparing INS hormone treatment efficacy prediction reagent.

Previous studies by the inventors have found that hormones can induce the expression of B7-H4 in monocyte type MDSCs. The kidney injury of B7-H4-/-mice is aggravated under inflammatory stimulation, and the administration of B7-H4-Ig can improve the kidney injury performance of model mice, which indicates that B7-H4 with immune suppression function plays a protective role in kidney injury, and suggests that B7-H4+ mononuclear cells have potential clinical value for predicting the curative effect of hormone therapy INS.

The purpose of the invention can be realized by the following technical scheme:

peripheral blood B7-H4⁺Application of the monocyte type MDSC as a detection target in preparing a reagent for predicting the curative effect of hormone therapy idiopathic nephrotic syndrome.

Detection of B7-H4 in peripheral blood⁺Use of an agent for monocyte-type MDSC in the preparation of an agent for predicting the efficacy of hormone therapy for idiopathic nephrotic syndrome.

B7-H4⁺The application of the monocyte type MDSC as a hormone sensitive idiopathic nephrotic syndrome typing marker.

Detection of B7-H4 in peripheral blood⁺Application of a reagent of monocyte type MDSC in preparing a hormone sensitive idiopathic nephrotic syndrome typing reagent.

B7-H4⁺Application of the monocyte type MDSC as a detection target in preparing a medicament for treating idiopathic nephrotic syndrome.

B7-H4⁺Monocyte type MDSC may also be described as CD11b⁺CD14⁺CD16⁺B7-H4⁺Monocyte, B7-H4⁺CD14⁺CD16⁺A cell.

Has the advantages that:

the invention utilizes prospective clinical observation of patients who are included in INS according to standards, and the observation of B7-H4 in peripheral blood of the patients on days 1, 3, 7 and 14 after hormone treatment⁺Monocytic MDSC (B7-H4)⁺CD14⁺CD16⁺) Finally, the curative effect of the hormone therapy of the patient is judged according to the proteinuria condition of 8 weeks after the hormone therapy. INS patients with complete remission on hormonal therapy B7-H4 in peripheral blood⁺Monocytic MDSC (B7-H4)⁺CD14⁺CD16⁺) Within one week of treatment, significant expansion occurred, and hormone treatment partially relieved the patient that the population of cells did not change significantly. Early appearance of INS patients treated with B7-H4⁺The increase in monocyte-type MDSC is associated with hormone therapy sensitization, with early observation of post-hormone therapy B7-H4 in INS patients⁺Changes in monocyte-type MDSCs can be effective in predicting hormonal efficacy. B7-H4⁺Monocyte type MDSC can be used as hormone sensitive INS patientThe candidate disease typing marker of (1).

Drawings

FIG. 1 is a schematic representation of the clinical patient study procedure for glucocorticoid therapy of idiopathic nephrotic syndrome targeted cell populations

FIG. 2 peripheral blood CD14 from FSGS patients before and after glucocorticoid treatment⁺CD16⁺And CD15⁺Analysis of B7-H4 expression in cells. A represents a flow analysis diagram; b is the statistical analysis of the expression ratios of B7-H4 in each group. Data are representative of three independent replicates. P<0.05,**P<0.01,***P<0.001。

FIG. 3 dexamethasone-induced T cell proliferation inhibition of peripheral blood B7-H4+ CD14+ CD16+ and B7-H4-CD14+ CD16+ cells; b-graph represents a statistical analysis of the inhibitory capacity of T-cells. Data are representative of three independent replicates. P <0.05, P <0.01, P < 0.001.

FIG. 4 significant expansion of B7-H4+ monocytic MDSCs in patients with complete remission of INS during early treatment

Detailed Description

Example 1

1. Experimental methods

1.1. Patient's health

Selecting patients with kidney biopsy pathological diagnosis INS from Shen medical hospital affiliated with Nanjing university medical college (Dongjing general war hospital) from 7 months to 11 months in 2018, and screening according to the following inclusion and exclusion criteria. All final patients in the cohort received sufficient prednisone treatment and signed an informed consent. At follow-up baseline (within 1 week before treatment), 2ml of peripheral blood was collected from patients on days 1, 3, 7, 14 and 28 after hormone treatment for analysis of B7-H4+ immunosuppressive monocyte dynamics and clinical markers at the corresponding time points. After 8 weeks of prednisone induction treatment, various clinical indexes of the patients are recorded, and the treatment effect is judged (as shown in figure 1). The study protocol was approved by the ethical committee of the jinling hospital affiliated with the medical college of Nanjing university.

1.1.1. Group entry criteria

1) Age <65 years old;

2) the clinical manifestations are consistent with nephrotic syndrome;

3) (ii) number of glomeruli under PAS staining > 7;

4) serum creatinine <3mg/d (265.2 umol/L);

5) glomerular filtration rate (eGFR, CKD-EPI formula) is more than or equal to 30ml/min.1.73m 2;

6) the immunosuppressive agents such as glucocorticoid, cyclosporine A, tacrolimus, mycophenolate mofetil, cyclophosphamide, leflunomide, CD20 monoclonal antibody and the like are not used for 1 month before the group is added.

1.1.2. Exclusion criteria

1) It is known that absolute contraindications for glucocorticoid therapy (allergy to adrenocortical hormone drugs) are defined;

2) patients with active viral hepatitis, hepatitis virus quantitative value exceeding normal value, or glutamic-pyruvic transaminase and/or glutamic-oxalacetic transaminase increase by 2 times of upper limit of normal value;

3) patients with active infection, tumor, metabolic disorder and other kidney disease complications;

4) patients with type 2 diabetes or obesity (BMI greater than 28kg/m2 before onset);

5) a pregnant patient;

6) patients who are diagnosed unequivocally as hereditary kidney disease or who have unequivocal genetic mutations, secondary to metal poisoning, drug damage, or other systemic diseases leading to nephrotic syndrome;

7) follow-up compliance was poor.

1.2. Clinical medication scheme

The study medication is prednisone acetate (prednisone), 5 mg/tablet, 1mg/kg per day, maximum dose of 60 mg/day, three times of postprandial administration, induction time of 8 weeks, or equal dose of methylprednisolone tablet. Other medications include those that allow the use of renin angiotensin aldosterone system inhibitors (RAAS inhibitors), diuretics, anticoagulants, and the like.

Immunosuppressive agents such as cyclosporin A, tacrolimus, mycophenolate mofetil, cyclophosphamide and tripterygium glycosides tablet are forbidden.

1.3. Clinical observation index

Age, sex, and renal biopsy baseline of the patient and 24 hour urine protein quantification (Upro), urine red blood cell count (U-RBC), renal tubular function index (NAG enzyme, RB protein, C3, alpha 2 macroglobulin), blood white cell count (WBC), blood neutrophil Proportion (PMN), blood monocyte proportion (MNC), serum creatinine (SCr), serum photoinhibitin C, serum Albumin (ALB), alanine/glutamic oxaloacetic transaminase, serum cholesterol (TC), serum Triglycerides (TG), peripheral blood CD3+, CD4+, CD8+ T cells, and peripheral blood B7-H4+ monocytes (B7-H4+ CD14+ CD16+ cells) during follow-up were collected.

1.4. Kidney pathological examination and observation index

The patient takes the prone position and adopts a percutaneous oblique needle insertion negative pressure suction method under the guidance of B ultrasonic to carry out renal biopsy puncture. Conventional light-transmitting lens, immunofluorescence and electron microscopy. Light mirrors include HE, PAS, PASM-Masson and Masson trichrome stains. Immunofluorescence staining of kidney tissues comprises direct staining of IgG, IgA, IgM, C3 and C1q and indirect staining of kappa and lambda light chains. The electron microscope tissue is fixed before and after being dyed and then is observed under a Hitachi 7500 transmission electron microscope. The following indices were evaluated by 2 renal pathologists, with pathology scoring performed on PAS sections: the number of glomeruli, the glomerular sclerosis ratio, the segment glomerular sclerosis ratio, acute tubulointerstitial injury (no; mild:nomore than 25%, moderate: 26-50%, severe: > 50%), tubular atrophic interstitial fibrosis (no; mild:nomore than 25%, moderate: 26-50%, severe: 50%), arterial transparence degeneration and arteriosclerosis, and the degree of podocyte foot process fusion is observed under an electron microscope.

1.5. Flow cytometry analysis of B7-H4+ monocytes

Peripheral blood from patients was collected with EDTA anticoagulant tubes before prednisone treatment (baseline level), on days 1, 3, 7, 14 and 28 after treatment, and immediately transferred by the specialist to the laboratory on ice (4 ℃). Ensure a transit time of less than 2 hours. The peripheral blood volume was adjusted according to the number of leukocytes in the peripheral blood, and the antibody was added to the peripheral blood containing 1X 106 leukocytes, and incubated at room temperature for 20 minutes. After the flow antibody incubation was completed, the erythrocyte lysate was added to human peripheral blood. After standing for 10 minutes in the dark at room temperature, washing was performed twice, and finally the sample was analyzed by a flow cytometer. A single flow antibody positive and negative control sample were incubated for flow cytometry analysis according to the same method. The proportion of B7-H4+ monocytes was calculated according to the following equation: B7-H4+ CD14+ + CD16+ absolute number of cells/CD 14+ + CD16+ absolute number of cells) × 100. Flow cytometry analysis was performed using the software Flow-jover 8.8software (Tree Star, Inc Ashland, OR).

1.6. Evaluation of therapeutic efficacy

Efficacy definition 12, Complete Remission (CR) according to the 2012 KDIGO guidelines recommendation: the quantity of urine protein is less than or equal to 0.4 g/day, the serum albumin is more than or equal to 35g/L, and the renal function is normal; partial Remission (PR): urinary protein quantification >0.4 g/day but <3.5 g/day, and decline over baseline 50%, renal function is stable (SCr normal or rise < 25%); treatment-ineffective (NR): the CR or PR criteria were not met.

1.7. Exit indication

Patients in the group had one or more of the following events during the follow-up visit, i.e. as withdrawal cases:

1) adequate hormone induction phase prednisone continuous withdrawal for more than 1 week for various reasons;

2) treatment protocol changes occurred during the 8 week follow-up visit;

3) the SCr value doubled and exceeded the normal range (2 weeks apart, two consecutive measurements);

4) severe complications or drug side effects result in intolerance of hormone-induced therapy patients;

5) patients voluntarily quit or are out of follow-up due to various reasons.

1.8. Statistical method

Data analysis was performed using SPSS 25.0 software. For continuous variable data, the fit to the positive-distribution metric is expressed as mean ± standard deviation (x ± s), and the comparisons between groups are performed using t-test of independent samples. The non-positive-distribution of the metrology data is expressed in terms of median (interquartile range) and the comparison between groups is performed using the ManWhitney U test. The data are counted in cases and percentages (%) and comparisons between groups are by chi-square test or exact probability.

Repeated measurements of data at different follow-up time points were analyzed using repeated measurements of variance. Wherein, the comparison among groups adopts a two-factor repeated measurement variance analysis method to analyze the main effect and the interaction effect, and judges the influence of the change of different intervention measures along with the time on the observation index. And (3) judging whether each group of data obeys normal distribution or not by analyzing the student residual error and carrying out Shapiro-Wilk test, and judging whether each group of data has no abnormal value or not by judging whether the student residual error exceeds 3 times of standard deviation or not. And judging whether the data conforms to the spherical hypothesis or not through Mauchly's spherical hypothesis test, wherein the variance covariance matrixes of the dependent variables are equal (P >0.05) for the interactive items. Then, carrying out independent effect analysis in sequence aiming at grouping and time factors, wherein the grouping factor independent effect analysis needs fixed time factors, and carrying out independent sample t test or t' test (when variance is not uniform) on each time point; the time factor independent effect analysis needs to fix grouping factors, and single factor repeated measurement variance analysis is adopted to compare different time points. All tests are bilateral tests, and the difference is shown to be P <0.05, and the difference is shown to be P <0.01, so that the statistical significance is obvious.

2. Results of the experiment

2.1. Effect of hormones on B7-H4 monocytes

Previous studies have demonstrated B7-H4⁺M-MDSC has obvious immunosuppressive function, and hormone can obviously amplify ADR mice B7-H4⁺M-MDSC, we hoped to further study hormone pairs in patients with B7-H4⁺Influence of M-MDSC. We selected INS patients before receiving hormone treatment as a control group and observed the effect of sufficient hormone treatment for 1 week on B7-H4 monocytes. Hormone treatment B7-H4 in peripheral blood of FSGS patients⁺CD14⁺CD16⁺The ratio is significantly higher than that before hormone treatment, while B7-H4⁺CD15⁺The ratio was not significantly different from the control group (fig. 2). In addition, we sorted B7-H4 by flow cytometry⁺CD14⁺CD16⁺And B7-H4^-CD14⁺CD16⁺T cell expansion experiments were performed. As a result, it was found that B7-H4⁺CD14⁺CD16⁺The immunosuppression capability of the cell subset is stronger than that of B7-H4^-CD14⁺CD16⁺. This result suggests that the hormone significantly amplified FSGS patient B7-H4⁺CD14⁺CD16⁺Cell subsets, B7-H4⁺CD14⁺CD16⁺Cell subsets exerted significant immunosuppressive functions (figure 3).

2.2 hormone treatment within 1 week B7-H4⁺Amplification of monocyte-type MDSC enables prediction of efficacy 8 weeks after hormone treatment

As shown in FIG. 1, to observe changes in hormone therapy and B7-H4+ monocytes in INS patients, we finally performed a prospective clinical observation of 13 INS patients according to the inclusion exclusion criteria. Patients were divided into two groups according to their urinary protein level at week 8 after hormone therapy (hormone effect results), with complete remission patients (CR) at 24 hours with urinary protein below 0.3g/d and partial remission Patients (PR) at 24 hours with urinary protein 0.3-3.5 g/d. Flow analysis of peripheral blood was performed on patients before hormone treatment, on days 1, 3, 7, 14, and 28 after hormone treatment, and changes in B7-H4+ monocyte-type MDSV were observed. Third day after hormone treatment, CR patients B7-H4⁺CD14⁺CD16⁺The cells were significantly higher than the levels in PR patients (fig. 4), indicating that expansion of B7-H4+ monocytes within 1 week of hormone treatment could predict hormone 8 week treatment efficacy.