CN118957051A - Application of UBC (UBC) as target in diagnosis and treatment of non-obstruction azoospermia - Google Patents

Abstract

The invention discloses application of UBC as a target in diagnosis and treatment of non-obstruction azoospermia. The UBC has obviously reduced expression level of testis support cells of patients with non-obstruction azoospermia, can be used as a biomarker for diagnosing the non-obstruction azoospermia or evaluating the curative effect of treatment, can also be used as a drug, a drug target or a target gene in gene therapy, and provides a new strategy for diagnosing, treating and relieving the non-obstruction azoospermia.

Description

Application of UBC (UBC) as target in diagnosis and treatment of non-obstruction azoospermia

Technical Field

The invention relates to the field of biological medicine, in particular to application of UBC as a target spot in diagnosis and treatment of non-obstruction azoospermia.

Background

Male infertility is a type of complex disease occurring in the male reproductive system and is largely classified into obstructive azoospermia (Obstructive azoospermia, OA) and Non-obstructive azoospermia (Non-obstructive azoospermia, NOA) according to etiology. The cause of OA is clear, namely, the flowing out of the sperm is blocked, a series of germ cells including sperms in testes are not damaged, and the patient can realize the subsequent normal fertility process by treating and dredging the blockage or extracting sperms through operation. NOA is mainly due to the fact that intracesticular dysspermia results in almost no sperm production, which accounts for about 10-15% of male infertility. The final morphological feature of testis tissue in NOA patients is the absence or lack of germ cells, which are found only in 10% of NOA patients by surgery, excessive apoptosis or necrosis of endotestis spermatogonial stem cells and developmental microenvironment somatic cells, leading to testicular atrophy, leading to the most severe NOA disease, known as support-only syndrome (SCOS). However, no drug is currently approved for treatment of NOA, and there is an urgent need to enhance the study of the relevant mechanisms of NOA progression to study and screen for effective targets for clinical diagnosis and treatment.

Ubiquitin C (UBC), one of the key genes involved in the synthesis of ubiquitin, is an indispensable role in ubiquitination. Ubiquitin-conjugating is not only a highly fine post-translational modification, but also a core mechanism for regulating numerous vital activities of cells, involving proteasome-mediated proteolysis. Furthermore, ubiquitination also exhibits a broad, multifaceted non-degrading function, covering regulation from signal transduction to gene transcription, from fine coordination of endocytosis to precise transport of proteins, even including repair of DNA damage and maintenance of cell survival and proliferation.

In view of the urgent need of the current clinic for the medicament for ubiquitination of testicle supporting cells in the treatment of non-obstruction azoospermia, a brand new treatment target is needed to be provided, and a new thought and method are developed for the prevention and treatment of the non-obstruction azoospermia.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of UBC as a target point in diagnosing and treating non-obstruction azoospermia. The nucleotide sequence of the UBC is shown as SEQ ID NO. 3.

The invention provides application of ubiquitin C as a target in developing and/or screening and/or preparing a medicament for preventing and/or treating non-obstruction azoospermia.

The invention also provides application of the ubiquitin C detection substance in preparing and/or screening products for diagnosing or assisting in diagnosing non-obstruction azoospermia.

The invention also provides application of the ubiquitin C detection substance in preparing and/or screening products for evaluating or assisting in evaluating therapeutic effects of non-obstruction azoospermia.

In some embodiments, the substance that detects ubiquitin C comprises reagents required to detect ubiquitin C protein content or gene expression levels by RT-PCR method, RT-qPCR method, biochip detection method, southern blotting method, in situ hybridization method, immunoblotting method, immunohistochemical method, spatial transcriptome technique.

In some embodiments, the ubiquitin C detecting agent is an agent that detects ubiquitin C protein by immunohistochemical techniques.

In some embodiments, the substance that detects ubiquitin C is an agent that can detect the amount of nucleic acid expression associated with ubiquitin C protein or the amount of ubiquitin C protein.

In some embodiments, the product comprises any one of a reagent, a kit, a chip, a test strip, a membrane strip, or an assay platform.

The invention also provides the use of a ubiquitin C inhibitor in at least one of the following:

(a) Use in screening for drugs for preventing non-obstructive azoospermia;

(b) Use in the manufacture and/or screening of a medicament for the treatment or co-treatment of non-obstructive azoospermia;

(c) The application in preparing and/or screening medicines for inhibiting occurrence and/or development of non-obstruction azoospermia.

In some embodiments, the ubiquitin C inhibitor comprises a substance that reduces the amount of ubiquitin C protein expression, a substance that reduces the amount of ubiquitin C protein, a substance that reduces ubiquitin C protein activity, or a substance that inhibits UBC gene expression.

In some embodiments, the substance includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, an antibody, a polypeptide, a protein, a gene editing vector, a lentivirus, or an adeno-associated virus.

The ubiquitin C inhibitor provided by the invention has the effect of destroying or preventing ubiquitin C from participating in ubiquitin protein synthesis and UBC transcriptional expression.

In some embodiments, the ubiquitin C inhibitor is an antibody, polypeptide, or small molecule that binds to ubiquitin C protein; or (b)

The ubiquitin C inhibitor is a shRNA (short hairpin RNA) or siRNA (small interfering RNA) which reduces the expression of the gene encoding ubiquitin C protein.

In some embodiments, the UBC inhibitor is an siRNA having a sequence set forth in SEQ ID No.1 and SEQ ID No. 2.

In conclusion, compared with the prior art, the invention achieves the following technical effects:

1. UBC has obviously reduced expression level of testis support cells of patients with non-obstruction and azoospermia, can be used as a biomarker for diagnosing the non-obstruction and azoospermia or evaluating the curative effect of treatment, can also be used as a drug, a drug target or a target gene in gene therapy, and provides a new strategy for diagnosing, treating and relieving the non-obstruction and azoospermia.

2. The invention provides a UBC inhibitor siRNA capable of knocking down the expression of a supporting cell (TM 4), which can simulate the change of the supporting cell in-vitro non-obstruction azoospermia and is used for evaluating and diagnosing the influence of IBC on the non-obstruction azoospermia.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the differential expression of genes and functional enrichment analysis of various somatic cells between NOA and control according to example 1 of the present invention; wherein A is the differential expression gene between various somatic cells of NOA group and control group, red represents up-regulating gene, and blue represents down-regulating gene; B. c is the difference expression gene GO enrichment analysis and KEGG enrichment analysis of the testis somatic cells of the NOA group and the control group respectively;

FIG. 2 is an analysis of energy metabolism characteristics of NOA testis support cells according to example 2 of the present invention; wherein A is enrichment analysis of 7 mitochondria-related signal paths of testis support cells of NOA group and control group; b is enrichment analysis of 4 oxidative phosphorylation related signal paths of the testis support cells of the NOA group and the testis support cells of the control group; c is an up-regulating gene and a down-regulating gene related to mitochondrial function in testis supporting cells; d is immunofluorescence double-label analysis of UBC expression conditions in human testis tissue samples of the NOA group and the control group;

FIG. 3 is an immunofluorescence of the transfected TM4 cells with UBC siRNA according to example 3 of the present invention;

FIG. 4 shows the results of UBC expression down-regulation affecting TM4 cell proliferation and migration levels in accordance with example 3 of the present invention; wherein A is a graph of proliferation of TM4 cells under the condition that EdU is detected to inhibit UBC expression; b is a PI staining flow type detection result graph of TM4 cell cycle change of each group; c is a fusion rate result graph of scratch experiment observation TM4 cells;

FIG. 5 is a graph showing the effect of UBC expression down-regulation on TM4 cell energy metabolism in accordance with example 4 of the present invention; a, B is a result chart of JC-1 probe flow detection TM4 cell mitochondrial membrane potential change; c is a chart of the change result of detecting TM4 cell Reactive Oxygen Species (ROS) by probe flow; d is a mitochondrial fluorescence map in Mito-TRACKER RED CMXRos labeled tracer cells;

FIG. 6 is a graph showing the effect of down-regulated expression of UBC in TM4 cells on SSCs survival in example 5 of the present invention; wherein A is a spermatogonial stem cell image extracted from a seminiferous tubule of a male mouse testis, B is an alkaline phosphatase staining image of the spermatogonial stem cell, and C is an immunofluorescence result image; d is a flow type result graph; e is a mode diagram of co-culture of TM4 cells and spermatogonial stem cells SSCs; f is a content detection result diagram of the cell factor GDNF of the culture fluid of the co-culture system; g is a survival rate detection result graph of spermatogonial stem cells in a co-culture system; h is a graph of the proliferation detection result of spermatogonial stem cells in the co-culture system.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

Aiming at the urgent need of testicular support cell ubiquitination drugs in the non-obstruction azoospermia treatment in the current clinic, the invention discloses a potential treatment target. According to the invention, through single-cell sequencing of NOA patients, the expression level of UBC in testis tissues of non-obstructive azoospermia patients is found to be remarkably reduced. The UBC siRNA sequence is designed to explore the influence of UBC on the mitochondrial function and the cytobehavioral function of a supporting cell (TM 4), and a new thought and method are provided for simulating the pathological process of the non-obstruction azoospermia in vitro and clinically preventing and treating the non-obstruction azoospermia.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used, unless otherwise specified, are all commercially available.

Example 1 clinical sample collection and sequencing

Collecting 3 NOA and 3 testis tissue samples of a control group through medical ethical approval, and carrying out single-cell transcriptome sequencing; the patient is subjected to ethical approval and sampled by a testis puncture biopsy operation of a special person in a reproductive medicine center of a women and young health care department, and the specific method comprises the following steps: the patient takes a lying position, and is conventionally disinfected and spread with towels. One side of the testis was selected and secured with the middle, ring and thumb of the left hand, leaving the testis exposed to the relatively avascular region between the thumb and index finger. After local anesthesia, a syringe was used to penetrate the parenchyma and a small amount of testis tissue was obtained by rapid aspiration. The puncture part is bandaged after operation, and anti-inflammatory medicines are orally taken.

The tissue together with the preservation solution is sent to Shanghai ice Biotechnology Co., ltd in 4 hours from body to carry out tissue sample dissociation and single cell suspension preparation work. Single cells were then captured by 10 x Genomics technology, cellular RNA information was collected, reverse transcribed into cDNA, and random primer PCR amplification of the cDNA was performed to construct a library. The data volume was 100G/sample by NovaSeq on-board machine sequencing.

Sequencing data analysis: raw data of each sample is read through the R language platform Rstudio, integrated using Seurat package (v4.4.0), converted into Seurat objects, and the samples are grouped to obtain sample main information: ncount_rna (number of molecules measured per cell), nFeature _rna (number of genes measured per cell), percentage. Low quality cells were filtered according to nFeature _rna < 200, nFeature _rna > 7500, ncount_rna < 500, ncount_rna > 28000, and percentage.mt > 25 criteria and differential genes were screened by multi-sample integration and batch correction, clustering of primary cell types, identification of environmental free RNA contaminating primary cell types to predict and remove single cell transcriptomes, differential analysis between cell groups, enrichment analysis, genome scoring.

In order to explore potential factors of spermatogonial stem cell development disorder and germ cell apoptosis of NOA patients, differential expression genes and functional enrichment analysis between NOA group samples and control group samples are analyzed based on biological information of single cell sequencing detection technology.

As shown in a of fig. 1, testis support cells contained 265 expression up-regulated genes and 125 expression down-regulated genes; the mesenchymal cells and peritubular myoid cells contain 70 expression up-regulating genes and 138 expression down-regulating genes; the immune cells contain 85 expression up-regulating genes and 49 expression down-regulating genes; the endothelial cells contained 22 up-regulated genes and 100 down-regulated genes; vascular smooth muscle cells contain 115 expression up-regulating genes and 38 expression down-regulating genes (fold difference |log2 (fold change) |gtoreq.0.5, P < 0.01). The testis support cells expressed the most differentially expressed genes in NOA group compared to other somatic cells. The GO analysis results of fig. 1B show that differentially expressed genes of testis support cells are mainly enriched in functions such as "ubiquitin protein ligase binding", "proton transmembrane transport", and "oxidoreductase activity". The KEGG analysis results of fig. 1C show that the differentially expressed genes of testis support cells are enriched in the signal pathways of nervous system related diseases such as "parkinson disease", "alzheimer disease" and the like and the signal pathways of mitochondrial functions such as "active oxygen", "oxidative phosphorylation" and the like, which indicate that mitochondrial dysfunction exists in the defect of testis support cells in the NOA group.

Example 2 influence of UBC on the energy metabolism function of non-obstructive azoospermia testis support cells

Mitochondrial dysfunction or damage exists in the NOA group testis support cells, mainly comprises the differences of oxidative phosphorylation, proton transport, oxidoreductase activity and the like, and can have a mitochondrial damage mechanism similar to the nervous system diseases. In this example, a mitochondrial-related signal pathway gene set was obtained from the mitocarta3.0 database, and the main enrichment signal of mitochondrial dysfunction of NOA group testis support cells was analyzed.

The results of fig. 2a show that, among the 7 mitochondria-related major signaling pathways, oxidative phosphorylation activity was significantly reduced in NOA group testis support cells, a potential factor responsible for other mitochondrial function differences.

Oxidative phosphorylation was broken down into oxidative phosphorylation subunits (OXPHOS subunits), oxidative phosphorylation assembly factors (OXPHOS assembly factors), respiratory chain assembly (Respirasome assembly), and cytochromes C (Cytochrome C), and the difference between different functions in supporting cells was analyzed, as shown by the B results in fig. 2, the activity of mitochondrial respiratory chain assembly functions of testis supporting cells in NOA group was significantly increased. Further analysis of the differentially expressed genes revealed mitochondrial function-related genes, and testis support cells mitochondrial function-related up-down-regulated genes, as shown in the C results of fig. 2, UBC expression in NOA group testis support cells was significantly down-regulated in the differentially expressed genes related to mitochondrial function. Further localization of the NOA group testis support cells (SOX 9) by immunofluorescence, as shown in D of fig. 2, significantly lower UBC expression in the NOA group was observed.

Example 3 Effect of UBC expression Down-regulation on TM4 cell proliferation and migration

Mouse testis support cell TM4 culture: primary testis support cell TM4 is extracted from C57BJ/6 wild male mice and is immortalized, and is cultured by using DMEM-F12 culture solution, 2.5% fetal bovine serum and 5% horse serum are added into the culture solution, and the cells are passaged every 2 days. Ubiquitin C (UBC) siRNA was constructed with the following sequence:

The optimal transfection ratio of siRNA to lipo3000 was determined by transfection with fluorescent marker siRNA (FAM siRNA) and its inhibitory effect on the transcription and expression of UBC in TM4 cells was examined. The results in FIG. 3 show that when the confluence of TM4 cells is 60-70%, the siRNA (pmol): lipo3000 (μl) =25: 1 proportion transfection, the fluorescence intensity in the observation hole is 131.1+/-4.809 after 48 hours, and the transfection efficiency of siRNA is highest.

Use of EdU as an analogue of thymidine can replace thymine (T) in DNA replication and penetrate into the DNA molecule being synthesized. After staining, the nuclei in the proliferated state will appear red fluorescent. The effect of UBC transcriptional interference on TM4 cell proliferation was analyzed by counting three groups of cell proliferation efficiencies (i.e., the percentage of red-fluorescent EdU positive cells over the total blue-fluorescent cells). The results of FIG. 4A show that the proliferation rate of UBC siRNA group cells is significantly reduced compared to the control group, indicating that down-regulation of UBC expression can affect proliferation of TM4 cells.

Further, the fluorescence intensity of PI combined with DNA is detected by PI single-dye flow type cells, so that the content of the DNA in the cells can be directly reflected. The B results of FIG. 4 show that down-regulation of UBC expression results in a decrease in the percentage of TM4 cells in the G0/G1 phase and an increase in the percentage of TM4 cells in the S phase. In FIG. 4C, down-regulation of UBC expression is detrimental to TM4 cell migration, suggesting that the cycle regulation of TM4 cells is disturbed after down-regulation of UBC expression, and normal proliferation is not possible.

Example 4 Effect of UBC expression Down-regulation on the energy metabolism level of TM4 cells

In order to detect mitochondrial changes in TM4 cells caused by inhibition of UBC expression, the mitochondrial membrane potential of the cells is detected by JC-1 probes, and at higher mitochondrial membrane potentials JC-1 aggregates in the mitochondrial matrix to form a polymer (Jaggregates) that can produce red fluorescence; at low mitochondrial membrane potential, JC-1 cannot aggregate in the matrix of mitochondria, and JC-1 is a Monomer (Monomer) and can generate green fluorescence. The red/green ratio at the different treatments was examined by flow cytometry, from which mitochondrial membrane potential changes were analyzed.

The results of FIG. 5A show that, when UBC expression is inhibited, the mitochondrial membrane potential of the TM4 cells decreases and mitochondrial respiratory chain electron transport is blocked. Further, the level of Reactive Oxygen Species (ROS) in TM4 cells is detected by a DCFH-DA probe, electrons can leak during oxidative phosphorylation during the transfer of electrons on the respiratory chain, a harmful substance called Reactive Oxygen Species (ROS) is formed, and the detection of ROS content can reflect laterally whether the oxidative phosphorylation process of the cells is impaired. The B-C results of FIG. 5 show that upon inhibition of UBC expression, the ROS level of TM4 cells is elevated and oxidative phosphorylation is compromised. Furthermore, mito-TRACKER RED CMXRos labeling of mitochondria within the tracer cell was observed to decrease the number of mitochondria within the TM4 cell by down-regulation of UBC expression (D of FIG. 5).

Example 5 Effect of UBC expression Down-regulation on TM4 cell Spermatogonial Stem Cells (SSCs)

In order to explore the influence of testicular support cells with down-regulated UBC expression on spermatogenic stem cell development, spermatogenic stem cells are extracted from 6-8 d male mouse testicular seminiferous tubules, and related identification work is carried out, and the specific steps are as follows: extracting spermatogonial stem cells:

① P4-P5 generation feeder cells (MEFs), mitomycin C, were prepared half an hour in advance using 0.1% gelatin coated cell culture flasks and placed in a 5% CO ₂ cell incubator at 37 ℃. ② Taking 6-8 d C57 strain mice, performing intraperitoneal injection for killing by using 1% of pentobarbital sodium, soaking the whole body in 75% of ethanol, then taking out, cutting the abdomen, taking out bilateral testes, and placing in a PBS (double antibody-containing) dish for washing. The testis is stripped of its tunica albuginea, washed, then transferred to a new PBS dish, rinsed, and carefully discarded. ③ Pulling to scatter the tissue, adding 5mL of 1 xIV collagenase solution, and placing in an incubator for 7-8 min. ④ Digestion was terminated by adding 1mL FBS, transferred to a 15mL centrifuge tube, and centrifuged at 1600rpm/min for 5min. The supernatant was discarded, and 3mL of PBS was added to resuspend 1600rpm/min for centrifugation for 5min. ⑤ The supernatant was discarded, resuspended in 0.05% EDTA-containing pancreatin, transferred to a new petri dish and placed in an incubator for 15min. The cells were then filtered using a 70 μm filter by repeating step ④.⑥, transferred to a 15mL centrifuge tube, and centrifuged at 1600rpm/min for 5min to separate the cells from the PBS. The supernatant was discarded, resuspended in spermatogonial stem cell culture medium, transferred to a 0.1% gelatin coated flask and incubated in a 5% CO ₂ cell incubator at 37 ℃. ⑦ After the cells are grown overnight and most of the cells are attached, the cells are repeatedly blown directly by a 1mL pipette, the incompletely attached cells and the suspended cells are collected into a 15mL centrifuge tube, centrifuged at 1600rpm/min for 5min, and then transferred into a new cell culture flask. ⑧ Culturing for 1-2 days, and observing whether grape-shaped cell clusters are formed or not, wherein half liquid exchange is adopted. After cells grew into a grape-like cell mass, the medium was discarded, washed 1-2 times with PBS, and then cells were digested with 0.05% EDTA pancreatin. ⑨ Digestion was stopped by adding twice the volume of complete medium, collected in a 15mL centrifuge tube, centrifuged at 1600rpm/min for 5min, differential adherent separation of SSCs from adherent cells, followed by transfer of SSCs into mitomycin C treated feeder cells for long term culture.

Alkaline phosphatase staining:

① Cells were seeded into 24-well plates. And fixing for 30s-3min by using an ALP fixing solution precooled at the temperature of 2-8 ℃.

② Washing with PBS for 1-2 times, and draining off excessive water.

③ And (3) dripping ALP incubation liquid to cover cells, and placing the cells in a 37 ℃ incubator for incubation for 15-20 min, and washing with PBS for 1-2 times.

④ Counterstaining: and (3) dyeing the core solid red or methyl green counterstain liquid for 3-5 min.

⑤ Live cell imager imaging (bright field).

The results are shown in FIG. 6A. The spermatogonial stem cells were stained with alkaline phosphatase, and the staining was positive as shown in FIG. 6B. Expressing the spermatogonial stem cell marker genes such as GFR alpha-1, OCT4, PLZF, KIT and the like, and specifically comprises the following steps:

And (3) fluorescent double-label identification:

① Cell climbing tablets are manufactured according to different groups. Cells were fixed with 4% paraformaldehyde for 15min.

The PBS was rinsed 3 times for 5min each.

② 1ML of PBS containing 0.5% Triton X-100 was added to each well, and the cells were permeabilized at room temperature for 20min.

③ Closing: a5% BSA blocking solution was prepared and added dropwise thereto, and the mixture was blocked at room temperature for 30 minutes.

④ Incubation resistance: after removal of the blocking solution, primary antibody was added dropwise and incubated overnight at 4 ℃. The PBS was rinsed 3 times for 5min each.

⑤ Secondary antibody incubation: the secondary antibody (diluted in PBS) was incubated at room temperature for 2h in the dark. The PBS was rinsed 3 times for 5min each.

⑥ DAPI (PBS dilution, ratio 1:1000) counterstain for 10min. PBS was rinsed 3 times for 5min each

⑦ And (5) picking out the cell climbing sheet, placing the cell climbing sheet in a glass slide, and dripping the anti-fluorescence quenching sealing tablet sealing sheet.

⑧ Observing expression condition of spermatogonial stem cell markers under laser confocal microscope

2. Flow cytometry identification

① Cells were digested with 0.05% EDTA pancreatin according to different groupings, transferred to 1.5mL EP tube and centrifuged at 1600rpm/min for 5min.

② The supernatant was discarded, 70% ethanol was added, and the mixture was placed on a shaker at 4℃for 4h to fix the cells.

③ 1ML of PBS containing 0.5% Triton X-100 was added, and the cells were permeabilized at room temperature for 20min. PBS is rinsed 1-2 times and centrifuged at 1600rpm/min for 5min.

④ A5% BSA blocking solution was prepared and added dropwise thereto, and the mixture was blocked at room temperature for 30 minutes.

⑤ Incubation resistance: after removal of the blocking solution, primary antibody was added dropwise and incubated overnight at 4℃on a shaker. PBS is rinsed for 1-2 times, each time for 5min.

⑥ Secondary antibody incubation: the secondary antibody (diluted in PBS) was incubated at room temperature for 2h in the dark. PBS is rinsed for 1-2 times, each time for 5min.

⑦ The result of the flow-on detection is shown as C in fig. 6.

The flow results showed a PLZF positive cell fraction of 99.3% (D of fig. 6), indicating that the extracted primary cells were spermatogenic stem cells. The TM4 cells were then co-cultured with spermatogonial stem cells by Transwell chamber (fig. 6E) and the content of glial cell-derived neurotrophic factor (GDNF) in the culture broth was examined by ELISA experiments. GDNF is a factor important for maintaining self-renewal of spermatogenic stem cells and is normally secreted by testicular support cells within the seminiferous tubules. GDNF secreted by the mouse testis support cells can promote self-renewal of SSCs and prevent differentiation of SSCs. Whereas in the mouse testis, where GDNF expression is inhibited, apoptosis of undifferentiated SSCs is promoted, resulting in the presence of only supporting cells in the seminiferous tubules. The expression level of GDNF was detected by ELISA assay, while cell proliferation was detected by CCK-8 and EdU.

CCK-8 operation steps: cells were inoculated into 96-well plates after digestion and treated in groups. At the time point of detection, 1/10 volume of CCK-8 was added to each well, and the mixture was cultured in a 5% CO ₂ incubator at 37℃for 2 to 4 hours. The absorbance (OD value) was then measured at a wavelength of 450nm by a microplate reader to calculate the cell viability.

EdU experiment procedure:

① Cells were seeded into 24-well plates and subjected to grouping treatment.

② Cell culture medium was changed at the set test time point, and diluted EdU working solution (diluted 1:1000 with complete medium) was added. Incubation was continued for 4 hours (specific time depends on the growth rate of the cells) at 37℃in a 5% CO ₂ cell incubator.

③ After the EdU labeling was completed, the medium was aspirated, and the cells were washed 2 to 3 times with PBS, then 1mL of 4% paraformaldehyde was added, and the cells were fixed at room temperature for 15 minutes.

④ After removal of the fixative, the plates were washed 3 times for 5 minutes with PBS.

⑤ 1ML of PBS containing 0.5% Triton X-100 was added to each well, and the cells were permeabilized at room temperature for 20 minutes. The supernatant was discarded and the well plate was then washed 3 times for 5 minutes with PBS.

⑥ And preparing a Click reaction solution according to the specification under the light-shielding condition. 0.3mL of Click reaction solution was added. Incubate at room temperature for 60 min in the dark. The supernatant was discarded and the well plate was then washed 3 times for 5 minutes with PBS.

⑦ 0.5ML of a1 Xhoechst 33342 solution (diluted 1:1000 with PBS) was added and incubated for 10 minutes in the absence of light. The supernatant was discarded and the well plate was then washed 3 times for 5 minutes with PBS.

⑧ Shooting was performed using a living cell imager (Hoechst 33342 shows blue fluorescence, edU shows red fluorescence).

The results in FIGS. 6F-H show that inhibition of UBC expression inhibits the secretion of the cytokine GDNF in TM4 cells, affecting the development of SSCs.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (9)

1. The ubiquitin C as a target is applied to the development and/or screening and/or preparation of medicaments for preventing and/or treating non-obstruction azoospermia.

2. Use of a substance for detecting ubiquitin C for the preparation and/or screening of a product for the diagnosis or for the assisted diagnosis of non-obstructive azoospermia.

3. Use of a substance for detecting ubiquitin C for the preparation and/or screening of a product for assessing or aiding in the assessment of the therapeutic efficacy of a non-obstructive azoospermia.

4. The use according to claim 2 or 3, wherein the substance for detecting ubiquitin C comprises reagents required for detecting UBC protein content or gene expression level by RT-PCR method, RT-qPCR method, biochip detection method, southern blotting method, in situ hybridization method, immunoblotting method, immunohistochemical method, spatial transcriptome technique.

5. The use according to claim 2 or 3, wherein the product comprises any one of a reagent, a kit, a chip, a test paper, a membrane strip or a detection platform.

6. Use of an ubiquitin C inhibitor in at least one of the following:

(a) Use in screening for drugs for preventing non-obstructive azoospermia;

(b) Use in the manufacture and/or screening of a medicament for the treatment or co-treatment of non-obstructive azoospermia;

(c) The application in preparing and/or screening medicines for inhibiting occurrence and/or development of non-obstruction azoospermia.

7. The use according to claim 6, wherein the ubiquitin C inhibitor comprises a substance that reduces the amount of ubiquitin C protein expression, a substance that reduces the amount of ubiquitin C protein, a substance that reduces ubiquitin C protein activity or a substance that inhibits ubiquitin C gene expression.

8. The use according to claim 6, wherein the ubiquitin C inhibitor is an antibody, polypeptide or small molecule which binds to ubiquitin C protein; or (b)

The ubiquitin C inhibitor is shRNA or siRNA.

9. The use according to claim 6, wherein said ubiquitin C inhibitor is an siRNA having the sequence shown in SEQ ID NO.1 and SEQ ID NO. 2.