Application of Trem2 in the preparation of reproductive prognosis products for detecting and evaluating cryptorchidism

Abstract

The present invention provides the use of Trem2 in the preparation of a reproductive prognosis product for detecting and evaluating cryptorchidism, and relates to the field of biomedical technology. In this application, the role and mechanism of testicular macrophages in the occurrence of cryptorchidism are deeply explored from multiple levels such as molecules, cells, animals, and clinical. A new male reproductive gene, Trem2, is identified by using a variety of technologies including gene knockout mice and fetal testis in vitro culture. In the testicular tissue of the normal group, Trem2 is highly expressed in the seminiferous tubules and interstitium; while in the testicular tissue of cryptorchidism, Trem2 expression is decreased. Dap12, the upstream molecule of Syk, is highly expressed in the testicular tissue of wild-type mice, and in Trem2 ‑ / ‑ Low expression in mouse testis, Trem2 ‑ / ‑ The expression of Insl3 in mice was lower than that in the wild-type group. The study in this application suggested that Trem2 may be involved in the Dap12 / Syk signaling pathway, affecting the testicular descent and sperm development process.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of Trem2 in preparing a reproductive prognosis product for detecting and evaluating cryptorchidism. Background Art

[0002] Cryptorchidism is a common malformation of the urinary and reproductive system in children and one of the risk factors for male infertility and testicular tumors. It refers to a congenital disease in which the testicles cannot descend to the scrotum and remain in the retroperitoneum, inguinal canal or at the entrance of the scrotum. In boys of normal weight (≥2.5kg), the incidence of cryptorchidism is about 1.1%-8.4%; in boys who are premature or weigh less than 2.5kg, this proportion rises sharply, ranging from 1.1% to 45.3%. When cryptorchidism occurs, the testicles of the child will undergo vacuolar degeneration, and the size, weight, anogenital distance (AGD), number, motility and morphology of the testicles will change. Therefore, undescended testicles are one of the pathogenesis of infertility and testicular cancer. The occurrence of cryptorchidism is closely related to multiple factors, among which genetic changes play an important role in the occurrence of cryptorchidism. A large number of studies have confirmed that the formation of cryptorchidism is related to differential gene expression.

[0003] Therefore, we need to explore new cryptorchid differentially expressed genes at the transcriptome level, study the susceptibility genes of cryptorchid children, further explore the relationship between such susceptibility genes and the occurrence and development of cryptorchidism, and finally explore the pathogenesis of cryptorchidism, so as to provide new guidance and direction for the research of cryptorchidism. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that the key factors for the occurrence of cryptorchidism and their specific regulatory mechanisms have not yet been clarified in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of Trem2 gene as a target in the preparation of prenatal diagnostic products for detecting and evaluating cryptorchidism.

[0007] Preferably, the diagnostic product is a detection reagent or a detection kit targeting the Trem2 gene.

[0008] The present application also provides the use of Trem2 gene as a target in the preparation of a product for the treatment of cryptorchidism.

[0009] The present application also provides a detection kit, which uses the Trem2 gene as a target and is used for prenatal diagnosis of cryptorchidism.

[0010] The present application also provides a method for constructing a cryptorchidism model of Trem2 knockout mice, which is characterized by comprising the following steps:

[0011] S1: Preparation and observation of cryptorchidism model of Trem2 knockout mice;

[0012] S2: Total protein extraction and Western blot analysis of mouse testicular tissue;

[0013] S3: Western blot experiments were used to detect the expression levels of key proteins in the signaling pathway (Insl3, Dap12 and Syk).

[0014] The application of Trem2 provided in this application in the preparation of a product for detecting and evaluating the reproductive prognosis of cryptorchidism has been conducted by deeply exploring the role and mechanism of testicular macrophages in the occurrence of cryptorchidism from multiple levels, including molecular, cellular, animal, and clinical. A new male reproductive gene, Trem2, has been identified using a variety of techniques, including gene knockout mice and fetal testis in vitro culture. In normal testicular tissue, Trem2 is highly expressed in seminiferous tubules and interstitium; whereas in cryptorchid testicular tissue, Trem2 expression is decreased. Dap12, an upstream molecule of Syk, is highly expressed in the testicular tissue of wild-type mice, and in Trem2 - / - Low expression in mouse testis, Trem2 - / - The expression of Insl3 in mice was lower than that in wild-type mice. The research in this application suggests that Trem2 is involved in the Dap12 / Syk signaling pathway, affecting the testicular descent and sperm development process. The research in this application clarifies the relationship between Trem2 and cryptorchidism, providing pioneering results for the occurrence of cryptorchidism and the diagnosis and treatment of male infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of differentially expressed genes and Trem2 expression in cryptorchid children in Example 1 of the present invention;

[0016] Among them, A. Hierarchical clustering heat map shows 26 genes significantly downregulated and 23 genes significantly upregulated in cryptorchidism; B. Volcano plot shows the gene expression profile of cryptorchidism and normal control testes; C. Bar graph shows the three most significant differentially expressed genes in genes related to macrophage function; D. IHC method was used to detect the expression of Trem2 in testicular tissue. In the testicular tissue of the normal group, Trem2 was highly expressed in the seminiferous tubules and interstitium; while in the testicular tissue of cryptorchidism, Trem2 expression was decreased. CON (control): normal control; CRY (cryptorchidism): cryptorchidism.

[0017] Figure 2 This is a schematic diagram of the construction and verification of the Trem2 gene knockout mouse model in Example 2 of the present invention;

[0018] A: Trem2 gene knockout strategy; B: PCR genotype identification. M: DNA marker; WT1 and WT2: two wild-type mice. KO1-4: four Trem2 - / - Mouse. N: ddH2O as negative template DNA. The primers in rows 1, 3, 4, 6, 8, 10, and 12 are Trem2-5S-in-tF1 and Trem2-5S-in-tR1; PCR positive products indicate that the mouse is wild type. The primers in rows 2, 5, 7, 9, 11, 13, and 14 are Trem2-5S-in-tF1 and Trem2-3S-in-tR1. PCR positive products indicate that the mouse is Trem2 - / - Mouse.

[0019] Figure 3 This is a schematic diagram for verifying that Trem2 deficiency leads to cryptorchidism in Example 2 of the present invention;

[0020] Among them, A, the location of mouse bladder, penis and testicles. The red circle is the bladder, and the yellow circle is the testicle; B, the statistical results of the incidence of cryptorchidism in mice, Unilateral cryptorchidism: unilateral cryptorchidism, Bilateral cryptorchidism: bilateral cryptorchidism; C, the comparison of the testicular size of mice; D, the statistical graph of the testicular weight of mice. CON (control): normal control; CRY (cryptorchidism): cryptorchidism; *: P < 0.05.

[0021] Figure 4 Trem2 in Example 2 of the present invention - / - Changes in mouse testis and sperm morphology;

[0022] Among them, A, mouse wild type / Trem2 - / - Testis HE staining. The seminiferous tubules of normal mouse testis are composed of supporting cells and 5-8 layers of spermatogenic cells. From the base of the seminiferous tubules to the luminal surface, there are spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and sperm. The seminiferous tubules of cryptorchid mouse testis show obvious vacuolar changes (green arrows) and occlusion (blue arrows); CON (control):

[0023] Normal control; CRY (cryptorchidism): cryptorchidism; B, Trem2 - / - Representative images of mouse sperm morphological abnormalities. (a) Normal sperm; head abnormalities (be) include banana-shaped (b), double-headed (c), obvious hollow (d), and hookless (e); tail abnormalities (fg) include tail folding (f), sheath defect (g), and bundled sperm clumps (h). Black arrows indicate morphological abnormalities.

[0024] Figure 5 This is a schematic diagram for verifying the effect of Trem2 deficiency on the expression of Dap12 and Syk in Example 3 of the present invention;

[0025] Among them, A, wild type, Trem2 - / - Normal type and Trem2 - / - Expression of Trem2 / Dap12 / Syk in cryptorchid mice; B, Western blot statistical analysis, *: P < 0.05; C, detection of Dap12 expression in mouse testis; D, detection of Syk expression in mouse testis; E. IHC statistical analysis, *:

[0026] P<0.05, CRY (cryptorchidism): cryptorchidism.

[0027] Figure 6 This is a schematic diagram for verifying the activation of the Dap12 / Syk pathway by Trem2 in Example 3 of the present invention;

[0028] Among them, A. The expression of Trem2 / Dap12 / Syk in macrophages was detected by Western blot. After adding ketoconazole, the expression of related proteins decreased, *: P < 0.05; B. The expression of Trem2 / Dap12 / Syk in macrophages was detected by Western blot. After adding Trem2 agonist HSP60, the expression of related proteins increased, *: P < 0.05; C. The co-localization of Dap12 and Syk in the testicular interstitium of wild-type mice was detected by tissue immunofluorescence.

[0029] Figure 7 This is a schematic diagram for verifying the effect of Trem2 on the proliferation of Leydig cells of the present invention;

[0030] Among them, A, tissue immunofluorescence detection of direct contact between macrophages and Leydig cells; HSD3B1 and F4 / 80 are marker proteins of testicular Leydig cells and testicular macrophages, respectively; BC, flow cytometry detection of Leydig cell proliferation ability, compared with the control group, ketoconazole stimulation weakened the Leydig cell proliferation ability, and HSP60 enhanced the Leydig cell proliferation ability. *: P < 0.05; D, tissue immunofluorescence detection of Leydig cell number changes, compared with the wild type, Trem2- / - male mice reduced the number of Leydig cells.

[0031] Figure 8 This is a schematic diagram for verifying that ketoconazole of the present invention inhibits the expression of macrophage Trem2 and stromal cell Insl3 hormone proteins.

[0032] Among them, A, experimental flow chart of co-culture of macrophage culture supernatant and interstitial cells; B, expression of Insl3 in indirect co-culture of macrophage supernatant and interstitial cells, *: P < 0.05; C, experimental flow chart of indirect co-culture of macrophages and interstitial cells using transwell chamber; D, Western blot detection of Insl3 expression in interstitial cells in indirect co-culture of transwell, *: P < 0.05; E, Western blot detection of Trem2 expression in macrophages in indirect co-culture of transwell, *: P < 0.05.

[0033] Fig. 9 This is a schematic diagram for verifying the effect of ketoconazole on Trem2 and Insl3 in the in vitro culture of early pregnancy human embryonic testis of the present invention;

[0034] A, experimental flow chart of fetal testis in vitro culture; B, before and after 14 days of in vitro culture of fetal testis, IHC was used to detect BrdU expression and observe cell proliferation; C, in vitro culture, after two weeks of KET treatment, IHC was used to analyze the expression of Trem2 and Insl markers in fetal testis samples; D, IHC statistical analysis chart, *: P < 0.05; E, correlation analysis of Insl3 and Trem2; F, Western blot detection of Trem2 expression and statistical analysis chart, *: P < 0.05. DETAILED DESCRIPTION

[0035] The present application provides the use of the Trem2 gene as a target in the preparation of a prenatal diagnostic product for detecting and evaluating cryptorchidism.

[0036] In one embodiment, the diagnostic product is a detection reagent or a detection kit targeting the Trem2 gene.

[0037] Application of Trem2 gene as a target in the preparation of products for the treatment of cryptorchidism.

[0038] The present application also provides a detection kit, which uses the Trem2 gene as a target and is used for prenatal diagnosis of cryptorchidism.

[0039] The present application also provides a method for constructing a cryptorchidism model of Trem2 knockout mice, which is characterized by comprising the following steps:

[0040] S1: Preparation and observation of cryptorchidism model of Trem2 knockout mice;

[0041] S2: Total protein extraction and Western blot analysis of mouse testicular tissue;

[0042] S3: Western blot experiments were used to detect the expression levels of key proteins in the signaling pathway (Insl3, Dap12 and Syk).

[0043] The following is an explanation of the above content in combination with specific verification experiments:

[0044] Example 1: Analysis of the correlation between Trem2 and cryptorchidism

[0045] Patients were recruited at the Affiliated Hospital of Nantong University between January 2016 and June 2018. Neonates diagnosed with cryptorchidism were re-examined at 3 months of age. Preterm or low birth weight infants and patients with a family history of cryptorchidism or systemic disease were excluded from the study.

[0046] This application was conducted in accordance with the basic principles of the Declaration of Helsinki and approved by the Ethics Committee of the Affiliated Hospital of Nantong University (2017-L102). All patients were of Han ethnicity living in Jiangsu Province, eastern China, and their parents gave written informed consent to participate. Six patients with cryptorchidism (mean age 1.7 years, 14 months to 3.3 years) (isolated cryptorchidism cases only) were included in this application, and the diagnosis of cryptorchidism was reconfirmed before orchiopexy. The six patients with cryptorchidism included five unilateral cases and one bilateral case. Three control patients were included in this application, including two patients with testicular trauma (7 and 10 years old) and one patient with incarcerated inguinal hernia (4 months old). Testicular biopsy of the lower pole of the testicular parenchyma was performed in the six patients with cryptorchidism. The necrotic part of the testis in the three normal control groups underwent debridement of the testicular necrotic tissue. During the operation, the parenchymal necrotic tissue was trimmed, and a very small sample of normal testicular tissue was collected at the edge of the necrotic tissue.

[0047] According to the high-throughput sequencing results of the samples in the previous application, bioinformatics analysis was used to analyze the differential genes between the control group and the experimental group. IHC was used to analyze the difference in Trem2 protein expression between cryptorchid tissue and testicular tissue of the control group.

[0048] Peripheral venous blood samples were collected from children with cryptorchidism (≥150 cases) and the control group (≥100 cases). The serum concentrations of sTrem2 and Insl3 were detected by ELISA, and the differential expression of sTrem2 in the serum of cryptorchidism patients and the healthy control group was analyzed.

[0049] Differential gene expression analysis and IHC validation of testicular tissues between cryptorchidism patients and healthy controls. In order to explore the mechanism of cryptorchidism, this application collected 6 human testicular tissues from boys with cryptorchidism and 3 normal testicular tissues from healthy controls for RNA sequencing analysis.

[0050] See also Figure 1The results in Table 1 show that compared with the control group, this application identified more than 5000 DEGs (differentially expressed genes, p-adj < 0.01, log2foldchange > 1 or < -1) in the patient cohort ( Figure 1 -A). The results showed that the dominant genes associated with cryptorchidism were screened out by hierarchical clustering. The figure only shows 26 genes that were significantly downregulated and 23 genes that were significantly upregulated in cryptorchidism ( Figure 1 -B). Since this application mainly focuses on testicular descent abnormalities caused by disorders in the testicular immune microenvironment, the screening of genes focuses on genes related to macrophage function. Referring to the immune cell-related dataset published by PornpimolCharoentong et al. in Cell reports in 2017, there are 33 genes related to macrophage function.

[0051] The present application counted the expression of these 33 genes in the sequencing results, of which 28 were differentially expressed genes (Table 1), and thus the three genes with the most significant differential expression were screened out: FPR1, Trem2, and VNN1 ( Figure 1 -C).

[0052] Table 1. Differentially expressed genes related to macrophage function in testicular tissues of normal and cryptorchid children

[0053]

[0054] In 2022, Luz Garcia-Alonso et al. used single-cell sequencing technology to find that Trem2 + ftMs plays an active role as a dominant gene in the development and function of fetal testis. Among these three genes, only Trem2 currently has definite literature confirming that it is related to testicular development. Therefore, this application chooses to explore the correlation between Trem2 and cryptorchidism. This application found that Trem2 specifically expressed in macrophages in the testis of cryptorchid patients was downregulated by 3.70174 times. In order to further verify the correlation between Trem2 and cryptorchidism, the IHC method was used to verify the expression of Trem2 in cryptorchidism patients and healthy controls. The results showed that compared with the healthy control group, Trem2 was significantly downregulated in the testis of cryptorchidism patients ( Figure 1 -D).

[0055] Example 2: Observation of mouse testicular morphology and detection of reproductive function

[0056] (1) Construction of Trem2 - / - Mouse and pregnant mouse model of macrophage clearance

[0057] 1) Trem2 - / -The mouse model has been successfully constructed by Jicui Yaokang Biotechnology Co., Ltd., and all animal experiments were approved by the Animal Committee of Nantong University (No. P20230302-013).

[0058] 2) Construction of a macrophage-clearing pregnant mouse model: On E1.5 and E4.5 days, 200 μl and 100 μl of clodronate liposomes were intraperitoneally injected into wild-type pregnant mice to clear macrophages in the pregnant mice and construct a macrophage-clearing pregnant mouse model. At the same time, combined with sTrem2 stimulation experiments, it was confirmed that Trem2 acts on macrophages to maintain the testicular immune microenvironment.

[0059] (2) Observe the morphology and structure of the testicles

[0060] C57BL / 6, Trem2 - / - The size, weight, and AGD of the testes of mice and their offspring were measured and photographed. HE staining was used to analyze C57BL / 6 and Trem2 - / -Testicular morphological changes in mice and their offspring after macrophage-depleted pregnant mice: Testes were fixed in 10% neutral buffered formalin at room temperature and embedded in paraffin. At least 6 testes of mice were sectioned and stained with hematoxylin and eosin (H&E) to observe changes in testicular morphology.

[0061] (3) Testing reproductive function

[0062] 1) Sperm motility and count: Semen analysis was performed by the casa method. Sperm volume, sperm concentration and sperm motility were determined according to the WHO laboratory manual. Items for evaluating sperm motility and count included: rapid progressive parameter motility (MOT, %), progressive motility (PROG, %), linear velocity (VSL, μm / s), curvilinear velocity (VCL, μm / s), average path velocity (VAP, μm / s), lateral head displacement amplitude (ALH, μm / s), linearity (LIN, %), straightness (STR, %) and wobble (WOB, %). Mouse epididymal cauda sperm was collected and the sperm concentration was ensured to be 106 / ml. One side of the epididymal cauda was placed in 1 ml of modified benchtop solution. The epididymal cauda was cut into 3 pieces and placed in a 37°C water bath for 15 min to release the sperm. 20 μl of sperm suspension was taken for evaluating sperm motility and count by the casa method.

[0063] 2) Detect sperm morphology: Detect sperm morphology according to the instructions of the Diff-quick staining kit. Spread the sperm sample on a slide and dry it, then fix it for 20 seconds. Slice with Diff-quickI staining for 5-10 seconds, stain the sperm midsection and tail red, then use Diff-quickII staining for 10-20 seconds, stain the sperm acrosome blue, and gently wash the slide three times with PBS. According to the WHO Laboratory Manual for Semen Morphology, sperm morphology was observed by Diff-quick staining. At least 200 sperm were analyzed each time to determine the percentage of sperm with abnormal morphology.

[0064] See also Figure 2 , Figure 3 , Figure 4 and Table 2. In this application, the Trem2 gene knockout mouse model has been successfully constructed using CRISPR / Cas9 technology. The strategy is shown in Figure 2 -A, completed by Jicui Yaokang Biotechnology Co., Ltd. After the mouse tail was cut, DNA was extracted for PCR amplification. When Trem2-5S-in-tF1 and Trem2-5S-in-tR1 were used as upstream and downstream primers, the PCR positive product indicated that the mouse was wild type. When Trem2-5S-in-tF1 and Trem2-3S-in-tR1 were used as upstream and downstream primers, the PCR positive product indicated that the mouse was Trem2 - / - Therefore, gel electrophoresis confirmed that Trem2 - / - (homozygous) mouse model was successfully constructed ( Figure 2 -B). Studies have shown that the morphology, size, weight, AGD, sperm count, motility and movement parameters, and sperm morphology of cryptorchid testes are altered. In order to explore the relationship between Trem2 and cryptorchidism in animal models, this experiment used WT and Trem2 - / - The experiment was conducted on mice in the two groups, and HE staining, Diff-quick and Casa methods were used to detect the location and reproductive function of the mouse testicles. The results showed that compared with the control group, Trem2 - / - Cryptorchidism occurs in mice Figure 3 -A), the cryptorchidism rate was 66.67%, of which the bilateral cryptorchidism rate was 46.67% and the unilateral cryptorchidism rate was 20% ( Figure 3 -B). At the same time, compared with the control group, the size of the testicles in the cryptorchid group ( Figure 3 -C), weight( Figure 3 -D), AGD, sperm count, motility and motility parameters were abnormal (Table 2).

[0065] Table 2. Wildtype and Trem2 - / - Tissue characteristics and exercise parameters of mice

[0066]

[0067] (*: P < 0.01, compared with Wildtype)

[0068] HE staining results showed that the seminiferous tubules became thinner; the seminiferous epithelium was reduced, including spermatogenic cells and supporting cells; the seminiferous tubules atrophied, and the testicular interstitial tissue thickened, showing varying degrees of fibrosis ( Figure 4 -A). Diff-quick method shows Trem2 - / - Abnormal sperm morphogenesis in cryptorchid mice Figure 4 -B).

[0069] Example 3: Detection of the effect of Trem2 on the secretion of inflammatory factors by macrophages and the signaling pathways involved

[0070] Lentivirus was used to infect cells to overexpress Trem2. Combined with Trem2 activators, the effects on the secretion of inflammatory factors by macrophages and the expression of signaling pathway proteins were observed. Co-IP and immunofluorescence confocal microscopy were used to verify the interaction of proteins.

[0071] (1) Detection of the levels of proinflammatory cytokines TNF-α and IL-1β

[0072] Collect WT, Trem2 - / - The serum of WT and Trem2 mice and their offspring were collected and the expression levels of pro-inflammatory cytokines TNF-α and IL-1β were detected by ELISA. - / - The expression levels of TNF-α and IL-1β were detected by ELISA in the testicular tissues of mice and their offspring after macrophage depletion in pregnant mice.

[0073] (2) Effects of Trem2 on signaling pathways

[0074] 1) Animal level: Western blot and IHC were used to detect wild type and Trem2 - / - Expression of DAP12 / Syk in mouse testis tissue.

[0075] 2) Cell level: Trem2 overexpression lentivirus and its control were used to infect macrophages to observe the expression of DAP12 / Syk. At the same time, macrophages were treated with Trem2 agonist HSP60 to observe the expression changes of related proteins.

[0076] 3) Gene level: RT-qPCR was used to detect changes in Trem2 mRNA expression levels after ketoconazole stimulation of macrophages; by constructing recombinant plasmids containing the full-length Trem2 promoter and a series of truncated fragments, the active region of the Trem2 promoter was identified by luciferase reporter gene assay. The candidate transcription factors located in the active region of the Trem2 promoter were predicted and analyzed by websites such as Jasper (https: / / jaspar.genereg.net / ), and a recombinant plasmid of the Trem2 promoter containing binding site mutations was constructed to identify the transcription factors that can bind to the Trem2 promoter.

[0077] 4) Protein level: RNA sequencing technology was used to analyze and detect differentially expressed genes in normal and Trem2 lentivirus-overexpressed macrophages, and functional enrichment analysis and KEGG analysis were performed on the differentially expressed proteins. The top 10 candidate target proteins with the largest fold changes and significant differences were verified by Western blot. The selected candidate proteins were verified for their interaction with Trem2 by co-immunoprecipitation (Co-IP) and GST-Pull down. Immunofluorescence confocal microscopy was used to detect whether they co-localized with Trem2. Western blot and immunofluorescence confocal microscopy were used to detect whether the expression and localization of the corresponding proteins in each mutant cell were affected by the mutation, so as to further clarify the function of Trem2 and explore new signaling pathways that may be involved.

[0078] Experimental results:

[0079] See also Figure 5 and Figure 6 To further verify the signaling pathway downstream of Trem2, Western blot and IHC were used to detect wild-type / Trem2 - / - The expression of Dap12 / Syk in mouse testis. The results showed that compared with wild type, Trem2, Dap12 and Syk were expressed in Trem2 - / - In normal type, both remain unchanged, Trem2 - / - Cryptorchidism type decreased ( Figure 5 -A, B, C, D and E).

[0080] The present application uses Western blot to detect the expression of Dap12 / Syk in macrophages before and after the application of ketoconazole. The expression of Trem2 / Dap12 / Syk was detected by ketoconazole-stimulated macrophages. The results showed that compared with the control group, the expression of Trem2, Dap12, and Syk in the ketoconazole-treated group decreased ( Figure 6-A). The Trem2 agonist HSP60 was used to detect the expression of downstream pathways after Trem2 activation. The results showed that Dap12 / Syk increased after HSP60 activation ( Figure 6 -B). At the same time, in order to verify the interaction between Dap12 and Syk, the experiment used tissue immunofluorescence to detect the co-localization of Syk and Dap12 in the testicular interstitium of wild-type male mice. The results showed that Dap12 and Syk co-localized ( Figure 6 -C), suggesting that Dap12 and Syk can interact.

[0081] Example 4: Detection of the effect of Trem2 deficiency on the number and function of mesenchymal cells

[0082] (1) Effect of Trem2 deficiency on the number of interstitial cells

[0083] Isolation of primary Leydig cells and establishment of culture system: Digest the testicular parenchyma with 0.5 g / L type I collagenase at 34°C and filter after shaking. Prepare percoll separation solution at concentrations of 60%, 37%, 26%, and 21%. Take 21%-26% of Leydig cells and establish in vitro culture at 34°C and 5% CO2. Co-culture primary Leydig cells or Leydig cell line (TM3) with macrophages stimulated with ketoconazole / Trem2 activator using transwell system to establish in vitro culture system.

[0084] 1) Interstitial cell proliferation assay

[0085] Macrophages stimulated with ketoconazole / Trem2 activator were co-cultured with stromal cells pre-stained with CFSE (FITC channel) using a transwell system. After 12 hours of co-culture, the cells were resuspended in PBS containing 1% fetal bovine serum, and the mean fluorescence intensity of stromal cells was observed by flow cytometry to determine the proliferation capacity of stromal cells.

[0086] 2) Interstitial cell ferroptosis and pyroptosis experiments

[0087] ① Detection of interstitial cell ferroptosis: Isolation and purification of wild-type and Trem2 - / - In primary testicular Leydig cells of mice, RT-qPCR was used to detect the mRNA levels of SLC3A2, VDAC3, TFRC and SLC40A1. Tissue immunofluorescence was used to perform Perls staining to detect iron ion localization. Tissue iron changes were detected using a kit. Serum hepcidin levels were detected using ELISA. Ferropoptosis of Leydig cell lines was detected using a transwell system. This showed that Trem2 deficiency caused a decrease in the number of Leydig cells by affecting ferroptosis.

[0088] ② Detection of interstitial cell pyroptosis: Isolation and purification of wild-type and Trem2 - / - Mouse primary testicular Leydig cells were analyzed by RT-qPCR, Western blot and IHC to determine the expression of wild-type and Trem2 - / - The expression levels of inflammatory response-rich pyroptosis factors (NLRP3, Caspase-1p20, GSDMD, IL-1β, and IL-18) in mice were different; the pyroptosis of mesenchymal cell lines was detected using a transwell system; it was confirmed that Trem2 deficiency could also lead to a decrease in the number of mesenchymal cells by affecting their pyroptosis.

[0089] (2) Effects of Trem2 deficiency on interstitial cell function

[0090] 1) Effect of Trem2 on hormone secretion of interstitial cells

[0091] ① Detection of hormone levels in fetal rat girdle tissue

[0092] Three-day-old wild-type and Trem2 - / - The mice were euthanized by CO2 asphyxiation, and the girdle tissue was immediately collected using a surgical magnifier. IHC and Western blot were used to detect the expression of Insl3 in the girdle tissue.

[0093] ② Detection of hormone changes by co-culture of macrophages and interstitial cells

[0094] Conditioned medium from macrophage culture was collected and added to interstitial cells as a source of macrophage inflammatory stimulators. The protein expression of Insl3 in interstitial cells was detected by Western blot. In addition, macrophages and interstitial cells were co-cultured using a transwell system. Macrophages were inoculated in transwell chambers and 20 μM ketoconazole was added. The next day, the chambers were replaced with medium and slowly placed in six-well plates for culture for 24 hours. Proteins were extracted from macrophages and interstitial cells, respectively, and the protein expression of Insl3 in interstitial cells and Trem2 in macrophages was detected by Western blot.

[0095] 2) Interstitial cell autophagy experiment

[0096] Isolation and purification of wild type and Trem2 - / -Mouse primary testicular Leydig cells were cultured and the autophagy of Leydig cells was detected by Western blot using the ratio of LC3B-II / LC3B-I and P62. The expression of PINK1 and PARKIN was detected by Western blot. The formation of autophagosomes was observed by transmission electron microscopy. The effects of blocking autophagy with autophagy inhibitors and activating autophagy with agonists on the autophagy of Leydig cells were detected. The expression of inflammatory pathway proteins such as NLRP3 / CASPASE-1 / ASC / IL-1β was detected by Western blot. The effect of Trem2 on the autophagy of Leydig cell lines was detected using a transwell system to determine the changes in autophagy of Leydig cells.

[0097] See also Figure 7 and Figure 8 , it has been well-documented that macrophages and interstitial cells have functional crosstalk. HSD3B1 and F4 / 80 are marker proteins for testicular interstitial cells and testicular macrophages, respectively, and are used to mark these two types of cells. In order to verify whether there is direct contact between them, tissue immunofluorescence was used to detect the co-localization of HSD3B1 and F4 / 80, and the results showed that macrophages and interstitial cells co-localized ( Figure 7 -A). Under the direct influence of macrophages, the proliferation function of interstitial cells will change. + / + The effect of macrophages on the proliferation of interstitial cells. This experiment used flow cytometry to indirectly co-culture interstitial cells with macrophages using a transwell chamber, and pre-stained interstitial cells with CFSE to detect the mean fluorescence intensity of interstitial cells. This application found that compared with the macrophage group, after ketoconazole stimulated macrophages, the mean fluorescence intensity of interstitial cells increased, indicating that the proliferation ability of interstitial cells decreased ( Figure 7 -B).

[0098] In order to further clarify the effect of Trem2 agonist on the number of interstitial cells, Trem2 agonist HSP60 was added to the transwell co-culture system. The flow cytometry results showed that Trem2 agonist HSP60 induced a significant decrease in the mean fluorescence intensity of interstitial cells, suggesting that Trem2 agonist HSP60 can promote the proliferation of interstitial cells ( Figure 7 -C). Tissue immunofluorescence was used to observe the effect of Trem2 gene knockout on the number of interstitial cells. Figure 7 -D shows that compared with wild type, Trem2 - / - The number of Leydig cells in the testes of mice is reduced. Both cell and animal experimental data show that the loss of Trem2 leads to a decrease in the number of Leydig cells.

[0099] Ketoconazole is an aromatase inhibitor that can inhibit the number and function of macrophages. Therefore, ketoconazole further aggravates the secretion disorder of steroid hormones by affecting the functional crosstalk between macrophages and interstitial cells. In this experiment, macrophages were treated with 20 μM ketoconazole for 24 hours, and the supernatant was used to treat interstitial cells for 24 hours ( Figure 8 -A) or co-cultured with mesenchymal cells using transwell chambers for 24 hours ( Figure 8 -C). The results showed that macrophage supernatant stimulation reduced the secretion of macrophage Insl3 ( Figure 8 -B), using the transwell co-culture system to co-culture macrophages and stromal cells in a non-contact manner. Co-culture of the two cells resulted in downregulation of Insl3 on stromal cells ( Figure 8 -D), Trem2 downregulation in macrophages ( Figure 8 -E). This suggests that when Trem2 expression is reduced on macrophages, it inhibits the secretion of steroid hormones.

[0100] Example 5: Establishing a fetal testis in vitro culture model to verify the relationship between Trem2 and cryptorchidism

[0101] (1) Collection of fetal testicular specimens

[0102] Human fetal testes were isolated from material obtained after elective termination of pregnancy in the Department of Gynecology, Affiliated Hospital of Nantong University in early pregnancy (8-13 weeks). The project was approved by the regional ethics committee (2024-L060). Written informed consent was given by women in accordance with national guidelines. No termination of pregnancy was associated with fetal abnormality, and all fetuses were morphologically normal. This application refers to the 2018 A, Macdonald J, Nielsen JE, etc. published a method in Cell reports. They collected 6 male fetuses, placed the fetuses in pre-cooled PBS to remove testicular tissue, placed it in Liebowitz L-15 culture medium and immediately transported it to the laboratory for in vitro culture. Before and after culture, a portion of testicular tissue was taken for BrdU detection to verify the activity of testicular tissue cultured in vitro. The testicles of 3 male fetuses were immersed in Trizol for RNA sequencing. The testicular tissues of the 3 cases were quickly frozen for the study of Trem2 and other signaling pathway proteins.

[0103] (2) Testis in vitro culture

[0104] Within 2 hours of surgical removal of the testes, the testicular tissue was cut into 1 mm3 pieces (average seminiferous tubule diameter is 150 μm). Fetal testicular tissue was cultured in vitro in hanging drops. 40 μl of culture medium was dripped on the lid of the culture dish, and a single tissue piece was placed in each drop of culture medium. The culture dish was then carefully inverted to keep the culture medium drop and the suspended tissue intact. 10 ml of DPBS was added to the bottom of the culture medium to prevent dehydration. The culture medium composition was: MEMα medium supplemented with 13 non-essential amino acids, sodium pyruvate (2 mM), L-glutamine (2 mM), 1× insulin-transferrin and selenium (ITS) supplement, 10% FBS and 1% penicillin-streptomycin. Testicular tissue was cultured under 37%, 5% CO2 conditions, and the culture medium was changed every 48 hours.

[0105] The testicular tissues of the experimental groups were cultured in culture medium containing 10-6M ketoconazole and 1IU / ml LH, respectively. The use of ketoconazole can reduce the production of androgens, and the use of 1IU / ml LH can stimulate the production of late hormones. During the 14 days of culture, a total of 6 stimulations were performed. At the end of the in vitro tissue culture, the testicular tissue fragments were fixed with 10% neutral buffered formalin for IHC detection. At the same time, single-cell sequencing was performed on in vitro cultured human testicular tissue to map the testicular cell landscape of fetal testes after ketoconazole and LH stimulation in vitro, and to infer the heterogeneity and dynamic change trajectories of various testicular cell subpopulations, especially testicular immune cells, to explore key proteins with potential, and to conduct subsequent biological experimental verification and mechanism research.

[0106] See also Fig. 9 In order to verify the correlation between Trem2 and Insl3, this application constructed an in vitro culture model of embryonic testis, simulated cryptorchidism by administering ketoconazole (reducing steroid hormone secretion), and administered LH as a positive control. Previous studies have shown that in vitro culture can maintain the hormone production activity of human fetal testicular tissue, and can induce hormone production through drugs and induction. In order to study the effect of testicular macrophages on steroid hormone production, this experiment used ketoconazole (10 -6 M) and luteinizing hormone (LH) (1 IU / ml) were used to treat aborted male fetuses for 14 days, and the culture medium was changed every two days ( Fig. 9 -A).

[0107] IHC was used to detect BrdU and observe the activity of testicular tissue before and after culture; the expression of macrophage marker protein Trem2 and testicular interstitial cell marker hormone protein Insl3 was detected. By comparing the expression of BrdU in testicular tissue before and after culture, the present application found that BrdU was highly expressed before and after culture. The results of BrdU histochemistry suggest that the testicular tissue of the ex vivo male fetus survived before culture, and after 14 days of in vitro culture, the testicular tissue was still alive ( Fig. 9 -B).

[0108] These results confirmed that the human embryonic testis in vitro culture system was successfully constructed. Compared with the control group, ketoconazole caused a decrease in Trem2 expression in macrophages, while at the same time, Insl3 hormone protein decreased in interstitial cells; LH induced an increase in Trem2 expression and Insl3. At the same time, the three groups of data were subjected to correlation analysis, and the results showed that Trem2 and Insl3 were positively correlated ( Fig. 9 -C, D, E, F). In summary, ketoconazole can inhibit the decrease of Trem2 and Insl3 expression during fetal testicular descent.

[0109] In summary, this application collected 6 boys with cryptorchidism and 3 normal testicular tissues as a control group, and performed RNA sequencing analysis on the testicular tissues.

[0110] The present application found that among the genes specifically expressed in macrophages, the differentially expressed genes FPR1, Trem2 and VNN1 were most significantly down-regulated. However, only Trem2 has literature that clearly points out its important role in testicular development.

[0111] In order to verify the correlation between Trem2 and cryptorchidism, the present application uses immunohistochemistry (IHC) to confirm that the expression of Trem2 is decreased in the testicular tissue of cryptorchidism patients (see Example 1 for details).

[0112] To further explore the role of Trem2 in cryptorchidism, CRISPER / CAS9 technology was used to construct the Trem2 - / - WT and Trem2 mice were observed by hematoxylin and eosin (HE) staining, Diff-quick and computer-aided sperm analysis (casa) methods. - / - The location of the testicles of mice in the Trem2 group was compared with the control group. - / - The cryptorchidism rate of mice was 66.67%, of which 46.67% were bilateral and 20% were unilateral. - / - The size, weight, AGD, number, motility and movement parameters of the testicles and sperm morphology of the cryptorchid group of mice were abnormal (see Example 2 for details).

[0113] In order to explore the mechanism by which Trem2 affects macrophages, this application detected the expression of the Trem2 / Dap12 / Syk pathway. - / -The results of Western blot and IHC of mouse testicular tissue showed that the expression of Dap12 and Syk proteins decreased; after ketoconazole stimulation of macrophages, the expression of Dap12 and Syk proteins decreased, and after Trem2 agonist stimulation, the expression increased (see Example 3 for details).

[0114] In order to explore the effect of Trem2 on the number of interstitial cells, tissue immunofluorescence, flow cytometry, and Western blot were used to study the proliferation function of interstitial cells. The results of tissue immunofluorescence suggested that Trem2 - / - In mouse testis, interstitial cells and macrophages are in direct contact; ketoconazole stimulation of macrophages can reduce the proliferation of interstitial cells, which is restored after stimulation with Trem2 agonists. In order to study the effect of Trem2 on hormone secretion by interstitial cells, testicular macrophages and interstitial cells were indirectly co-cultured using transwell chambers to detect the expression of Trem2 and Insl3. Western blot results showed that Trem2 expression was downregulated after ketoconazole stimulation of macrophages, accompanied by downregulation of Insl3 expression in interstitial cells in the lower chamber (see Example 4 for details).

[0115] Finally, the present application collected testicles of aborted fetuses of 8-13 weeks for in vitro culture, detected BrdU before and after culture to ensure tissue survival, and simultaneously used ketoconazole and luteinizing hormone (LH) to stimulate testicular tissue, respectively, to simulate hormonal changes in vitro. The results showed that Trem2 was downregulated under ketoconazole stimulation, while Trem2 was upregulated under LH induction (see Example 5 for details).

[0116] Therefore, the present application has concluded that Trem2 / Dap12 / Syk is downregulated in cryptorchidism from the cellular and animal levels. The present application will establish Trem2 as a new target for regulating the function of cryptorchid macrophages, and Dap12 / Syk as a new signaling pathway. Based on the results of previous studies, it has been proven that Trem2 is a new target for the occurrence of cryptorchidism, and that the lack of Trem2 in testicular macrophages promotes the secretion of inflammatory factors through signaling pathways such as Trem2 / Dap12 / Syk, interferes with the crosstalk between macrophages and interstitial cells, and then leads to a decrease in Insl3, which ultimately participates in the formation of cryptorchidism and affects sperm production.

Claims

1. Application of Trem2 gene as a target in the preparation of a prenatal diagnostic product for detecting and evaluating cryptorchidism: the diagnostic product is a detection reagent or a detection kit using Trem2 gene as a target.

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