Testis Leydig cell senescence marker and application thereof

Abstract

The invention discloses a testis Leydig cell senescence marker and application thereof, the senescence marker is CCN5 protein, the senescence condition of the Leydig cell can be specifically reflected by detecting the CCN5 level of testis tissue, the senescence marker can be used as an index for evaluating Leydig cell senescence and testis senescence, and meanwhile, the senescence marker is an effective supplement for a diagnostic scheme of late onset hypogonadism (LOH). Furthermore, the invention also provides application of a preparation for inhibiting CCN5 protein expression in preparation of a product for improving testis Leydig cell senescence or treating diseases caused by testis Leydig cell senescence, the testis Leydig cell senescence can be improved and the LOH symptom can be treated by reducing CCN5 in a targeted manner, and treatment can be performed aiming at the core phenotypes of'lipid droplet loss' and'fibrosis' of testis Leydig cell senescence. The method is a treatment scheme different from exogenous testosterone supplement, stem cell injection, Senlytics drugs and exosome therapy.

Description

Technical Field

[0001] The present invention belongs to the field of medical and health technology, and specifically relates to a testicular Leydig cell aging marker and application thereof. Background Art

[0002] Testicular Leydig cells (LC cells) produce more than 90% of the testosterone in men. Their aging will lead to male reproductive endocrine disorders, accompanied by symptoms such as osteoporosis, muscle atrophy, decreased energy, memory loss, sleep disorders and sexual dysfunction, which is called late-onset hypogonadism (LOH), which seriously affects the physical and mental health and family happiness of elderly men. At present, there is no clear diagnostic standard for LOH. According to a report in the New England Journal of Medicine in 2010, it is recommended to use the presence of three sexual symptoms (reduced morning erection frequency, decreased libido and erectile dysfunction, etc.) related to total testosterone levels below 11nmol per liter (3.2ng / mL) and free testosterone levels below 220pmol per liter (64pg / mL) as a reference.

[0003] Normal testicular Leydig cells are rich in lipid droplets, the main component of which is cholesterol ester, which is the raw material for synthesizing testosterone. The loss of lipid droplets in aged LC cells leads to a lack of raw materials for testosterone synthesis. Coupled with the decreased expression of testosterone synthesis-related enzymes and the deterioration of responsiveness to luteinizing hormone (LH), these factors together lead to a decrease in testosterone synthesis and secretion. Fibrosis in the area surrounding testicular Leydig cells is another aging phenotype associated with Leydig cells. Generally speaking, this change is often associated with aging-related inflammatory responses and leads to the degeneration of protocell function. There is currently a lack of specific aging markers for the two most important aging characteristics of testicular Leydig cells, namely "lipid droplet loss" and "fibrosis". Serum testosterone and LH levels are used clinically to indirectly indicate testicular Leydig cell function, but this method has certain shortcomings, because testosterone levels do not absolutely reflect testicular function, and some elderly men with low testosterone do not have symptoms related to hypogonadism, and sperm quality can be maintained at normal levels; secondly, testicular Leydig cells naturally have high β-gal activity, and the accuracy of this method in diagnosing testicular Leydig cells is general; finally, the detection of "lipid droplet loss" and "fibrosis", the aging characteristics of testicular Leydig cells, mainly relies on different pathological staining, and there is a lack of a single detection indicator that can take both into account at the same time.

[0004] In terms of treatment, testosterone replacement therapy is currently the first-line clinical treatment for late-onset hypogonadism, but exogenous androgen supplementation can easily lead to side effects such as testicular atrophy, sexual dysfunction and cardiovascular risks, and is a "symptomatic treatment" that does not address the root cause. Some new anti-testicular Leydig aging methods are almost all derived from some non-specific anti-aging programs, such as stem cell injections, Senolytics drugs and exosome therapy. These therapies have multiple targets, and there is no treatment specifically targeting the two core phenotypes of testicular Leydig cell aging, "lipid droplet loss" and "fibrosis". Therefore, providing a treatment program that specifically targets the two core phenotypes of testicular Leydig cell aging, "lipid droplet loss" and "fibrosis", is of great significance for improving testicular Leydig cell aging and testicular function, and expanding the treatment options for late-onset hypogonadism. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a testicular Leydig cell aging marker.

[0006] The second object of the present invention is to provide the application of the testicular Leydig cell aging marker.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention first provides the use of a preparation for detecting the expression level of a CCN5 protein with an amino acid sequence such as that shown in SEQ ID No. 1 in the preparation of a product for evaluating the aging of testicular Leydig cells.

[0009] The present invention provides the use of a preparation for detecting the expression level of a CCN5 protein with an amino acid sequence such as that shown in SEQ ID No. 1 in preparing a product for assisting in the diagnosis of diseases caused by testicular Leydig cell aging.

[0010] The present invention combines sequencing data, experimental animals and clinical samples to screen and verify differential secretory proteins that are highly expressed in Leydig cells and increase with age. It is the first time to find that serum testosterone in elderly men is negatively correlated with the level of testicular CCN5 protein. The characteristic phenomenon of CCN5 deposition in the testicular interstitium and around the seminiferous tubules can be seen in LOH patients. In addition to the correlation, through cell and mechanism studies, it is proved that CCN5 mediates Leydig cell fibrosis through the β-catenin / SMADs signaling pathway, and mediates lipid droplet loss by changing the expression of cholesterol transporter and lipid droplet stabilizing protein RNF213. In summary, CCN5 is the direct cause of the two aging phenotypes of Leydig cells, "fibrosis" and "lipid droplet loss". Therefore, the present invention proposes CCN5 protein as a biomarker related to Leydig cell aging. Further, it can also be used as a potential warning molecule for evaluating testicular aging, expanding the diagnostic index of LOH, which is of great significance to the field of LOH diagnosis.

[0011] Furthermore, the product for evaluating testicular Leydig cell aging can also be used to evaluate male testicular aging, functional decline, and male reproductive aging.

[0012] Furthermore, the diseases caused by Leydig cell aging include but are not limited to late-onset hypogonadism.

[0013] The present invention provides use of a preparation for inhibiting the expression of a CCN5 protein having an amino acid sequence such as that shown in SEQ ID No. 1 in preparing a product for improving testicular Leydig cell aging and / or testicular reproductive endocrine function.

[0014] The present invention also provides the use of a preparation for inhibiting the expression of a CCN5 protein having an amino acid sequence such as that shown in SEQ ID No. 1 in the preparation of a product for treating diseases caused by testicular Leydig cell aging.

[0015] The present invention reduces the expression level of CCN5 protein in testicular Leydig cells through tools such as AAV9, which can effectively improve Leydig cell SA-β-gal activity (a universal cell aging marker), cell cycle arrest, testosterone synthesis and other aging characteristics, and also specifically restores the content of lipid droplets in Leydig cells and reduces the expression level of cell fibrosis-related genes. At the individual level, targeting CCN5 improves the physical strength and endurance, sexual desire and sexual function of elderly mice. These results show that CCN5 is an effective target for improving Leydig cell aging and testicular reproductive endocrine function, and is a potential target for treating LOH symptoms.

[0016] Furthermore, the preparation for inhibiting the expression of the amino acid sequence CCN5 protein can also be used to prepare a product for improving the reproductive endocrine function of aged testicles.

[0017] Furthermore, the diseases caused by Leydig cell aging include but are not limited to late-onset hypogonadism.

[0018] Furthermore, the preparation includes but is not limited to RNAi molecules and vectors thereof that target and inhibit CCN5 expression, or neutralizing antibodies that recognize and bind to CCN5 protein, etc.

[0019] Furthermore, the RNAi molecule is shRNA.

[0020] Preferably, the target sequence of the shRNA is shown as SEQ ID No.2.

[0021] Preferably, the primer sequences of the shRNA are shown in SEQ ID No.3 to SEQ ID No.4.

[0022] Furthermore, the vector is selected from adeno-associated virus, lentivirus or liposome.

[0023] Furthermore, the vector is selected from adeno-associated virus.

[0024] Preferably, the adeno-associated virus is AAV9 (adeno-associated virus type 9).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention first provides a testicular Leydig cell aging marker, wherein the testicular Leydig cell aging marker is CCN5 protein. By detecting the CCN5 level in testicular tissue, the Leydig cell aging can be specifically reflected. Compared with the detection of sex hormones such as testosterone and LH, this scheme is more targeted at the two most important aging characteristics of "fat droplet loss" and "fibrosis", and can be used as an indicator for evaluating Leydig cell aging and male testicular aging, and is also an effective supplement to the current LOH diagnostic scheme. Furthermore, the present invention also provides the use of a preparation that inhibits the expression of CCN5 protein in the preparation of a product for improving testicular Leydig cell aging or treating diseases caused by it. By reducing the CCN5 content in testicular Leydig cells, Leydig cell aging and testicular function can be improved, and LOH symptoms can be further treated. Targeting CCN5 to improve testicular Leydig cell aging and treat LOH symptoms can specifically treat the two core phenotypes of testicular Leydig cell aging, "lipid droplet loss" and "fibrosis". It is a new treatment option different from exogenous testosterone supplementation, stem cell injection, Senolytics drugs and exosome therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1is the expression level of CCN5 protein mRNA in different testicular cells; among them, SPG is spermatogonia, SPC is spermatocyte, SPT is spermatid, SC is supporting cell, LC is Leydig cell (interstitial cell), PTM is peritubular myoid cell, EC is endothelial cell, VSM is vascular endothelial cell, MAC is macrophage, T is T cell, MC is mast cell, and RBC is red blood cell.

[0028] Figure 2 The results of immunohistochemical staining of CCN5 protein in the testis of men of different age groups (left figure) and the correlation diagram between serum testosterone and testicular CCN5 protein levels in elderly men (right figure).

[0029] Figure 3 Comparison of immunohistochemical staining results of CCN5 protein in the testis of normal young people and LOH patients.

[0030] Figure 4 The effect of overexpression of CCN5 in Leydig cells on their β-gal activity (left) and cell proliferation (right).

[0031] Figure 5 The effect of overexpression of CCN5 in the testes of young mice on testicular seminiferous tubules (left) and testosterone levels (right).

[0032] Figure 6 To investigate the effect of overexpression of CCN5 in the testis of young mice on the content of lipid droplets in the testicular interstitium.

[0033] Figure 7 To investigate the effects of overexpression of CCN5 in the testes of young mice on the physical strength and sexual desire of mice. Figure 7 The left picture shows the results of the rotation experiment, the middle picture shows the number and duration of the male mouse sniffing the female mouse in the same cage, and the right picture shows the number and duration of the male mouse climbing and riding on the female mouse in the same cage.

[0034] Figure 8 This is the effect of incubation with recombinant CCN5 protein on fibrosis-related transcription factors in Leydig cells.

[0035] Fig. 9 The results are the results of the study on the mechanism of CCN5-mediated loss of lipid droplets in Leydig cells; Fig. 9 A is a schematic diagram of the channels involved in cholesterol influx and efflux in Leydig cells, B is the effect of CCN5 overexpression on cholesterol efflux channel proteins ABCA1, ABCG1 and cholesterol influx channel protein LDLR, C is the result of co-immunoprecipitation, D is the result of immunohistochemical staining of testicular tissue, and E is the effect of overexpression of CCN5 in Leydig cells on RNF213 protein.

[0036] Fig.10Changes in lipid droplet content in Leydig cells after knockdown of RNF213.

[0037] Fig.11 The figure shows the result of restriction enzyme digestion.

[0038] Fig.12 To investigate the effect of knocking down CCN5 in the testis of aged mice on the content of lipid droplets in the testicular interstitium.

[0039] Fig.13 To investigate the effects of knocking down CCN5 in the testis of aged mice on seminiferous tubules and testosterone levels.

[0040] Fig.14 To investigate the effects of knocking down CCN5 in the testes of aged mice on the physical strength and sexual desire of mice. Fig.14 The middle left picture shows the results of the wheel experiment, the middle picture shows the number of times and duration the male mouse sniffed the female mouse in the same cage, and the right picture shows the number of times and duration the male mouse climbed and mounted the female mouse in the same cage. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] Example 1

[0044] 1. The expression level of CCN5 mRNA in different testicular cells and testicular Leydig cells at different stages was analyzed by single-cell sequencing. The different testicular cells included spermatogonia SPG, spermatocytes SPC, spermatids SPT, supporting cells SC, Leydig cells LC, peritubular cells PTM, endothelial cells EC, vascular smooth muscle cells VSM, macrophages MAC, T cells T, mast cells MC, and red blood cells RBC. The testicular Leydig cells at different stages included testicular Leydig cells in prepubertal, puberty, adulthood, and old age.

[0045] The amino acid sequence of the CCN5 protein is shown in SEQ ID No. 1:

[0046] MRGTPKTHLLAFSLLCLLSKVRTQLCPTPCTCPWPPPRCPLGVPLVLDGCGCCRVCARRLGEPCDQLHVCDASQGLVCQPGAGPGGRGALCLLAEDDSSCEVNGRLYREGETFQPHCSIRCRCED GGFTCVPLCSEDVRLPSWDCPHPRRVEVLGKCCPEWVCGQGGGLGTQPLPAQGPQFSGLVSSLPPGVPCPEWSTAWGPCSTTCGLGMATRVSNQNRFCRLETQRRLCLSRPCPPSRGRSPQNSAF.

[0047] 2. The testicles of young, middle-aged, elderly people and LOH patients were used as test samples. Testicular puncture was performed to obtain tissues, and immunohistochemistry was used to detect the expression of CCN5 in testicular tissue samples.

[0048] Single-cell sequencing results Figure 1 As shown, the expression level of CCN5 mRNA in different testicular cells was analyzed based on single-cell sequencing data, indicating that CCN5 is highly expressed in testicular Leydig cells, and the expression level in aged testicular Leydig cells is significantly increased.

[0049] Immunohistochemistry test results Figure 2 As shown in the figure, immunohistochemical staining showed that in the aged testis, CCN5 was mainly concentrated in the interstitial region and around the seminiferous tubules, and the protein level increased with age; at the same time, the expression level of CCN5 in the testis was negatively correlated with serum testosterone. Immunohistochemistry showed that CCN5 was expressed in the testicular tissue of young men with normal spermatogenesis and LOH patients. Under normal circumstances, the CCN5 signal in the testis was extremely weak. In LOH patients, the characteristic phenomenon of CCN5 deposition in the interstitial region and around the seminiferous tubules can be seen ( Figure 3 ).

[0050] Example 2

[0051] 1. Overexpress CCN5 in testicular Leydig cells and measure the β-gal activity and cell proliferation ability of testicular Leydig cells:

[0052] Determination of β-gal enzyme activity: Use the Solebol (Cat. No. G1580) kit for determination:

[0053] Adherent cell staining: For cells cultured in 6-well plates, remove the cell culture medium, wash once with PBS, add 1mL β-Gal fixative, and fix at room temperature for 15 minutes. For other types of culture plates, refer to this ratio for the amount of fixative and subsequent solutions. Remove the cell fixative and wash the cells 3 times with 1×PBS, 3 minutes each time. Prepare the staining working solution according to the ratio. Remove the β-Gal washing solution and add 1mL staining working solution to each well. Incubate at 37℃ overnight for 12 to 24 hours. The 6-well plate can be sealed with a sealing film or plastic wrap to prevent evaporation. Observe under an ordinary optical microscope. If it is not possible to observe and count in time, remove the staining working solution, add 2mL PBS, and store it at 2 to 8℃ for several days; or add an aqueous gelatin sealing agent to seal the plate, which can be stored for a longer time.

[0054] Tissue section staining: For frozen sections treated with enzyme protection, proceed directly as follows. Add an appropriate volume of β-Gal fixative to fully cover the tissue, and fix at room temperature for at least 15 minutes. Soak and wash the tissue 3 times with PBS, each time for at least 5 minutes. Prepare the staining working solution according to the proportion. Add an appropriate amount of staining working solution. Incubate at 37℃ overnight for 12 to 24 hours. It is recommended to use a wet box to prevent evaporation or soak the entire section in the staining working solution. Observe under an ordinary optical microscope. If it cannot be observed in time, it can be sealed with an aqueous gelatin sealing agent for preservation.

[0055] Determination of cell proliferation ability: CCK-8 kit (Biosharp, catalog number cck8-BS350C-25*100T) was used to evaluate cell viability. Cells were seeded in a 96-well plate with a suspension of 100 μL / well, with each well containing approximately 2,000 cells. 10 microliters of CCK-8 solution was added to each well and then incubated in a cell culture incubator for 1 hour. The absorbance was then measured at 450 nm using a microplate reader.

[0056] 2. Overexpress CCN5 in the testes of young mice to observe its effects on the physical strength and sexual desire of young mice, and determine its effects on the testes, testosterone, and testicular Leydig cells of young mice:

[0057] Behavioral experiments to determine the effects on physical strength and sexual desire in young mice: Female mice were given estradiol 48 hours before sexual behavior testing, with each mouse receiving an intraperitoneal injection of 20 μg dissolved in 0.1 mL corn oil. Four hours before sexual behavior testing, each mouse was given an intraperitoneal injection of 500 μg of progesterone, also dissolved in 0.1 mL corn oil. Vaginal smears were used to confirm the estrus of female mice. Male mice were placed in experimental cages before observing mating behavior. Under dim red light, estrus females were gently introduced into the cages and continuous video recording was started for 30 min.

[0058] The male mouse initiated sniffing behavior of the female mouse when the male began to approach the female's tail and eventually began to sniff around the female's genital or perianal area. Sniffing behavior was considered to have ended when the male mouse moved its head away from the female, moved in a direction inconsistent with the female, stopped moving, or began other non-sniffing behaviors. The number of sniffing events and the total duration of sniffing within the first 5 min after cohabitation were recorded and statistically analyzed.

[0059] Mounting behavior was defined as the male climbing on top of the female and initiating rapid, shallow pelvic thrusts. Mounting behavior was considered to have ended when the male was no longer on top of the female. The number of sniffing and mounting events and the total duration of the mount within 30 min were recorded and statistically analyzed.

[0060] ELISA experiment for determining testosterone levels in mouse testicular homogenate: To measure testosterone in the testes, testicular tissue (20-50 mg) was accurately weighed and homogenized using a tissue grinder until no obvious tissue pieces remained in 500 μL buffer (0.5% BSA, weight / volume, 5 mM EDTA in PBS, pH 7.4). The tissue was further disrupted using a Q800R ultrasonic system (QSONICA) with the following parameters: time, 1 min; pulse, 15 s on / 30 s off; amplitude of 30%. The homogenate was then centrifuged at 3000 rpm for 10 min at 4 ° C to separate insoluble debris. The supernatant was collected for testosterone measurement. Testosterone levels were measured using a total testosterone ELISA kit (abclonal, RK00724). Each sample was measured in three wells and the average was taken after measurement.

[0061] Effects of behavioral determination on physical strength of young mice: Endurance experiment: All mice received 7 adaptation trainings before the start of the rotating rod endurance test. Before each formal experiment, the mice were placed on the rotating rod (at a constant speed of 5rpm) for 60s to adapt, after which the speed was increased to 20rpm. The time the mice stayed on the rotating rod at a speed of 20rpm for 5min was recorded. Each test was repeated three times, with a rest of 15min between each test. The longest duration of the three tests was used for analysis.

[0062] 3. Incubate Leydig cells with recombinant CCN5 protein. Purchase recombinant human CCN5 protein (Cat. No. ab50040), prepare it in cell culture medium at a concentration of 100 ng / mL, add it to the culture medium and incubate the cells. Then explore the mechanism of action of CCN5 protein in mediating Leydig cell fibrosis and lipid droplet loss.

[0063] The results are as follows:

[0064] like Figure 4 As shown, Figure 4The left figure shows that overexpression of CCN5 leads to an increase in the positive rate of β-galactosidase staining associated with Leydig cell senescence. The right figure shows that overexpression of CCN5 leads to a decrease in the proliferation ability of Leydig cells, indicating that overexpression of CCN5 in Leydig cells leads to increased β-gal activity and decreased cell proliferation. Overexpression of CCN5 in the testis of young mice leads to atrophy of the testicular seminiferous tubules and decreased testosterone synthesis ( Figure 5 ), Figure 6 It was shown that overexpression of CCN5 in mouse testis resulted in a decrease in the content of lipid droplets in the testicular interstitium, indicating that overexpression of CCN5 in the testis of young mice can lead to the loss of lipid droplets in Leydig cells; Figure 7 The results showed that after overexpression of CCN5 in mouse testis, the wheel test indicated that the mice's physical endurance decreased ( Figure 7 Left middle image), the number and duration of male mice sniffing the same female mice decreased ( Figure 7 Middle picture), the number and duration of male mice climbing and riding on female mice in the same cage decreased, indicating that the mice's sexual desire decreased ( Figure 7 Middle right panel), showing that overexpression of CCN5 in the testes of young mice leads to decreased physical strength (rotarod persistence time) and sexual desire (pre-mating behavior: sniffing and climbing female mice). Figure 8 In vitro experiments showed that incubation of Leydig cells with recombinant CCN5 protein led to activation of the fibrosis pathway, activation of the fibrosis-related transcription factor SMAD2 / 3 in Leydig cells, and upregulation of the expression level of the fibrosis-related protein α-SMA, indicating that CCN5 mediated Leydig cell fibrosis through the β-catenin / SMADs signaling pathway; further, Fig. 9 As shown, by directly incubating with CCN5 protein (Rec-CCN5), the extracellular pathway of CCN5 was simulated. By constructing a CCN5 overexpression plasmid with a truncated signal peptide (CCN5(ΔSP)), the intracellular pathway of CCN5 was simulated. The full-length CCN5 overexpression plasmid CCN5 (wt) was constructed to simulate the effects of the coexisting intracellular and extracellular pathways. Fig. 9 Middle B shows that only the two groups of CCN5(ΔSP) and CCN5(wt) with intracellular pathways showed upregulation of cholesterol efflux channel proteins ABCA1 and ABCG1, and downregulation of cholesterol influx channel protein LDLR, indicating that CCN5 mediates the depletion of lipid droplets in Leydig cells through the intracellular pathway; Fig. 9 Middle C shows that immunoprecipitation experiments have proven that CCN5 binds to two proteins, DOCK7 and RNF213. RNF213 is a protein on intracellular lipid droplets that stabilizes the outer membrane of lipid droplets. The loss of RNF213 causes lipid droplets to be bound and engulfed by lysosomes, which is also one of the reasons why CCN5 intracellular pathways mediate lipid droplet depletion. Fig. 9Middle D shows that in aged testis, CCN5 is upregulated and RNF213 is downregulated; Fig. 9 E in the middle indicates that WB experiments have proved that in the groups overexpressing CCN5 (including two groups of intracellular pathways), CCN5(ΔSP) and CCN5(wt), RNF213 protein showed a down-regulation trend, and the two were negatively correlated, further indicating that this protein interaction is one of the reasons for the intracellular pathway-mediated lipid droplet depletion in Leydig cells, indicating that CCN5 mediates lipid droplet loss by changing the expression of cholesterol transporter and lipid droplet stabilizing protein RNF213. Fig.10 It was shown that knocking down RNF213 led to the loss of lipid droplets in Leydig cells. After knocking down RNF213, the lipid droplets of Leydig cells were depleted. At the same time, it was seen at the tissue level that overexpression of CCN5 led to the depletion of lipid droplets in Leydig cells, proving that after knocking down RNF213, which interacts with CCN5 that is highly expressed in Leydig cells, the stability of lipid droplets will be destroyed, and enzymes that digest lipid droplets will more easily bind to the surface of lipid droplets, causing the lipid droplets to be depleted.

[0065] Example 3

[0066] CCN5 was knocked down in the testes of aged mice, and its effects on the physical strength and sexual desire of young mice were observed. Its effects on the testes, testosterone, and testicular Leydig cells of young mice were also measured.

[0067] The experimental steps for knocking down CCN5 in the testes of aged mice are as follows:

[0068] (1) Construction of RNAi recombinant viral vector:

[0069] CCN5 knockdown, using AAV9 (adeno-associated virus type 9) as shRNA delivery vector, preferred shRNA oligo sequence: target is GCACACCGAAGACCCACCTCC (SEQ ID No. 2); primer design:

[0070] Primer-T1 (SEQ ID No. 3):

[0071] 5-GATCCGCACACCGAAGACCCACCTCCCTCGAGGGAGGTGGGTCTTC GGTGTGCTTTTTT-3;

[0072] Primer-B1 (SEQ ID No.4):

[0073] 5-AATTAAAAAAGCACACCGAAGACCCACCTCCCTCGAGGGAGGTGG GTCTTCGGTGTGCG-3.

[0074] Anneal the oligo single-stranded DNA into double strands, insert the double-stranded shRNA oligo into the shRNA vector, construct the shRNA recombinant virus vector, and transform it into the competent cell Stbl3. The specific steps are as follows:

[0075] 1) Annealing of shRNA:

[0076] Dilute the primers with sterile TE buffer to a final concentration of 100 μM. Pipette 10 μL of the upstream and downstream primers, mix them, pipette evenly, and place them in a PCR tube for annealing. The procedure is shown in Table 1:

[0077] Table 1 Annealing procedure of shRNA

[0078]

[0079] After completion, place on ice for a few minutes and connect directly or store in a freezer at -20°C.

[0080] 2) Enzyme digestion and recovery of shRNA vector (the vector is cut in advance from the same batch):

[0081] The enzyme digestion system is shown in Table 2:

[0082] Table 2 Enzyme digestion system

[0083]

[0084]

[0085] Digest at 37℃ for about 30min. During this period, 0.8% agarose gel can be prepared, and nucleic acid electrophoresis can be performed after the digestion is completed. After the electrophoresis is completed, cut the gel strip containing the target fragment. Weigh the total weight with a balance and subtract the weight of the empty tube to calculate the weight of the gel. Calculate the volume of the gel based on 100mg being approximately 100μL, and add 1 times the volume of the gel to the BindingSolution and place it in a 65℃ water bath to completely melt the gel. Shake the EP tube appropriately during this period to accelerate the dissolution of the gel. Transfer all of the above liquid to the filter column and centrifuge at 13000 rpm for 30s (can be repeated once). Then discard the liquid in the tube, add 500μL of Wash Solution to the column, and centrifuge at 13000 rpm for 30s. Discard the liquid in the tube, add 500μL of WashSolution to the column, and centrifuge at 13000 rpm for 30s (can be repeated once). Then leave it empty for 3min. Place the filter column in a new 1.5mL EP tube and dry it at room temperature. Finally, add 35 μL of ddH2O to the column, let it stand for 5 minutes, and centrifuge at 13,000 rpm for 1.5 minutes. In order to improve the recovery rate, the dissolved DNA can be added to the column again and centrifuged for one minute. Discard the column to obtain the recovered vector fragment and measure its concentration. Fig.11 shown.

[0086] 3) Connection between shRNA vector and primer:

[0087] The connection system is shown in Table 3:

[0088] Table 3 Connection system

[0089]

[0090] Incubate in a 25°C water bath for 30 min.

[0091] 4) Conversion:

[0092] Place competent cells Stbl3 on ice and wait for them to thaw naturally. Add all the ligation products to competent cells Stbl3, place on ice for 20 minutes, and then heat shock in a 42℃ water bath for 90 seconds. Then quickly place on ice for 2-3 minutes. Add 1000μL LB culture medium without antibiotics and culture at 37℃, 150rpm shaking for 45 minutes. Centrifuge at 3000rpm for 2 minutes, discard about 850μL of supernatant, blow and disperse the bacterial liquid at the bottom of the tube, add it to the culture dish containing the corresponding resistance, spread it evenly with a sterilized applicator, and invert it in a 37℃ constant temperature incubator for overnight culture.

[0093] 5) Preparation of shRNA recombinant viral vector:

[0094] Pick several single colonies from step 4) and perform small-scale shaking culture.

[0095] 6) Sequencing to identify positive clones.

[0096] 7) Preparation of AVV9 virus solution:

[0097] a. Adeno-associated virus packaging:

[0098] AAV Pro-293T cell splitting: One day before transfection, subculture the cells in good condition into 10 cm culture dishes at an appropriate ratio, and prepare for transfection when the cell density reaches 80% to 90%.

[0099] Change medium before transfection: Replace cells with fresh culture medium 1 to 2 hours before transfection.

[0100] Transfection: Take a sterile 15mL centrifuge tube, and the transfection system / plate is as follows: DMEM 850μL, expression plasmid (i.e., constructed overexpression recombinant lentiviral vector) 10μg, serum type plasmid (aav vector plasmid, aav9 serum type) 10μg, auxiliary plasmid (pHelper vector) 20μg, HG transgene reagent 120μg. After mixing, place at room temperature for 15min to 20min, and then evenly drop into the culture dish that has been replaced with liquid in advance, and then place it in a CO2 incubator for culture.

[0101] Change medium: 6-8 hours after transfection, carefully remove the cell culture medium and discard it in a waste liquid cup containing disinfectant, and replace the medium after AAVPro-293T Cell Line transfection. Add Enhancing buffer: After replacing the medium after AAV Pro-293T CellLine transfection, evenly add 100×Enhancing buffer to promote transfection, 100μL / dish.

[0102] Virus collection: 72 hours after transfection, cells and cell supernatant were collected for subsequent concentration and purification.

[0103] b. Adeno-associated virus purification:

[0104] Transfer the cell suspension repeatedly between a dry ice foam box containing ethanol and a 37°C water bath, and freeze and thaw three times. Centrifuge at 4000rpm for 10min. Transfer the supernatant after centrifugation to an ultracentrifuge tube, centrifuge for 2h at 10WG. Add Benzonase at a final concentration of 50U / mL and MgCl2 at a final concentration of 0.002M, 37°C for 20min. Filter with a 0.22μm filter and transfer the supernatant to a horizontal ultracentrifuge tube. Prepare 60%, 40%, 25%, and 15% iodixanol, and lay a gradient. The gradient order is: 60% iodixanol, 40% iodixanol, 25% iodixanol, 15% iodixanol, and balance. 27.5WG, 4°C, centrifuge for 4h. Transfer to a 50KD ultrafiltration tube, centrifuge at 4500rpm to the required amount of virus, and discard the waste liquid at the bottom. After the virus liquid is divided, store it in a -80°C refrigerator.

[0105] (2) Testicular injection of recombinant AAV9 virus solution:

[0106] Mice were anesthetized, and a longitudinal incision was made in the lower abdomen to free the testicles. 0.4% trypan blue and recombinant AAV virus solution were prepared in a ratio of 1:9 (working titer was 1×10 12 VG / mL), the injection dose for each testis was about 10-20 μL, and the injection endpoint was when the entire testicle was filled with blue.

[0107] The results are as follows:

[0108] Fig.12 Knockdown of CCN5 in the testis of aged mice promoted the restoration of lipid droplets in the interstitial region. Fig.13 The results showed that knocking down CCN5 in the testes of aged mice reduced seminiferous tubule atrophy and testosterone decline, indicating that knocking down CCN5 in the testes of aged mice can restore the lipid droplet content in Leydig cells and improve seminiferous tubule atrophy and testosterone decline. Fig.14 The results showed that after knocking down CCN5 in the testis of aged mice, the wheel test showed that the mice's physical endurance increased ( Fig.14 Middle left picture), the male rats sniffed the female rats in the same cage more often and for longer periods of time ( Fig.14 Middle figure), the number and duration of male mice climbing and riding on female mice in the same cage increased ( Fig.14 Middle right figure), indicating improved sexual desire in mice, indicating that knocking down CCN5 in the testes of aged mice restored the mice's physical strength (rotarod persistence time) and sexual desire (pre-mating behavior: sniffing and climbing female mice).

[0109] In summary, the present invention provides a novel Leydig cell aging marker and therapeutic target CCN5. Detecting the CCN5 level in testicular Leydig cells can truly reflect the aging of Leydig cells; targeted reduction of CCN5 expression levels in the testis can effectively improve the aging characteristics of Leydig cells and testicular reproductive endocrine function.

Claims

1. Use of a preparation for detecting the expression level of CCN5 protein with an amino acid sequence as shown in SEQ ID No. 1 in the preparation of a product for evaluating the aging of testicular Leydig cells.

2. Use of a preparation for detecting the expression level of the CCN5 protein with an amino acid sequence such as that shown in SEQ ID No. 1 in the preparation of a product for assisting in the diagnosis of diseases caused by testicular Leydig cell aging.

3. The application according to claim 2, characterized in that: The disease caused by Leydig cell aging is late-onset hypogonadism.

4. Use of a preparation for inhibiting the expression of CCN5 protein with an amino acid sequence as shown in SEQ ID No. 1 in the preparation of a product for improving testicular Leydig cell aging and / or testicular reproductive endocrine function.

5. Use of a preparation for inhibiting the expression of the CCN5 protein having an amino acid sequence as shown in SEQ ID No. 1 in the preparation of a product for treating diseases caused by testicular Leydig cell aging.

6. The use according to claim 5, characterized in that: The disease caused by testicular Leydig cell aging is late-onset hypogonadism.

7. The use according to claim 4 or 5, characterized in that: The preparation is an RNAi molecule and a carrier thereof for targeted inhibition of CCN5 protein expression.

8. The use according to claim 7, characterized in that: The RNAi molecule is shRNA.

9. The use according to claim 7, characterized in that: The vector is selected from adeno-associated virus, lentivirus or liposome.

10. The use according to claim 9, characterized in that: The vector is selected from adeno-associated virus.

Images