CN113969259A

CN113969259A - Separation method of Leydig cells in rat testis stroma

Info

Publication number: CN113969259A
Application number: CN202111218183.0A
Authority: CN
Inventors: 陈浩林; 赵星仪; 季敏鹏; 温馨; 陈丹
Original assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Current assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2022-01-25

Abstract

The invention discloses a method for separating Leydig cells from rat testis stroma, which comprises the following steps: obtaining a rat testis and preparing a rat testis cell suspension; marking the testis cells of the rat; screening positive cells by a magnetic bead sorting (MACS) method; and (4) repeatedly purifying the positive cells by a magnetic bead sorting method. The separation method of the invention has simple and easy operation, high cell yield, high purity and high activity, and does not need expensive equipment.

Description

Separation method of Leydig cells in rat testis stroma

Technical Field

The invention relates to a biological medicine, in particular to a separation method of Leydig cells of rat testicular stroma.

Background

The primary function of Leydig Cells (LCs) is to produce testosterone (T), a male hormone that is essential for reproductive function and development and maintenance of male characteristics. Defects in the number or function of LCs can lead to complex symptoms including changes in body composition, increased fatigue, sexual dysfunction, depressed mood, decreased cognitive function, and decreased immune response. Because of their critical role in male reproduction and overall well-being, LCs have been studied extensively for many years in both in vivo and in vitro settings, including in the field of toxicology. In vitro studies of LCs require isolation and culture of cells under the best possible conditions to minimize interference from other testicular cells in order to examine cell function under optimal physiological conditions. Over the years, various methods have been developed to isolate LCs from rodent testes. However, due to the complexity of tissue cells, obtaining large quantities of high purity, high viability LCs remains challenging.

Early 50 years ago, researchers began attempting to separate LCs, most studies using a single step method, namely separation of LCs from other testicular cells by Percoll centrifugation. To improve the low purity of the cells isolated by this method, a two-step procedure, centrifugal elutriation and Percoll gradient centrifugation, was developed. This two-step process can yield LCs of very high purity (about 95%), but with relatively low cell yields and time and labor consuming. To increase recovery, the separation procedure was modified to include a unit gravity precipitation method instead of a filtration step to increase recovery of the LCs. In addition to these classical separation methods based on different cell sizes and/or densities, a recent study has been directed to the separation of LCs from mouse testes using flow sorting (FACS) techniques based on the difference in autofluorescence of the LCs population from other intratesticular cells.

While these currently available methods of separating LCs are capable of obtaining cells of high purity (about 95%), they either have low cell recovery or low cell viability or are time consuming and, more importantly, require special equipment such as elutriation equipment or flow cytometers with sorting capabilities.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the method for separating the Leydig cells of the rat testicular stroma, which has the advantages of simple operation, time saving, high cell yield, high purity, high activity and no need of expensive equipment.

In order to achieve the purpose, the invention provides the following technical scheme: a method for separating Leydig cells from rat testis stroma comprises the following steps:

obtaining a rat testis and preparing a rat testis cell suspension;

marking the testis interstitial cells of the rat;

screening positive cells by a magnetic bead sorting (MACS) method;

and (4) repeatedly purifying the positive cells by a magnetic bead sorting method.

Preferably, the marker for rat testicular interstitial cells is labeled with a prolactin receptor (PRLR) antibody.

Preferably, the preparation process of the rat testicular cell suspension is as follows:

placing testis parenchyma in a culture medium for digestion;

the digested tissue suspension was allowed to stand at room temperature and the supernatant was filtered.

Specifically, the preparation process of the rat testicular cell suspension is as follows:

the testicular parenchyma was digested in DMEM/F12 medium containing 1mg/ml collagenase IV (supplemented with 0.1% BSA) and shaken slowly in a shaker bath at 34 ℃ for 30min (90 rpm);

standing the digested tissue suspension at room temperature for 1min, and filtering the supernatant with a 70 μm-pore nylon net.

Preferably, the filtered rat testicular cells are washed by DMEM/F12 medium; and rat testicular cells were resuspended in ice-cold BD IMag^TM(BI) buffer;

and/or, when the positive cells are repeatedly purified by the magnetic bead sorting method, the magnetic bead sorting is carried out after the positive cell part is resuspended by 900 mu L of BI buffer solution each time.

Preferably, bead sorting is performed after resuspending the positive cell fraction with 900. mu.L of BI buffer for each repeated purification of positive cells, and incubation is performed for at least 4 minutes for each resuspension.

Preferably, rat testicular cells are resuspended in ice-cold BD IMag^TM(BI) buffer, cell density was also adjusted to 2-8X10⁷Individual cells/ml.

AsPreferably, the BD IMag is labeled when rat testicular interstitial cells are labeled^TM(BI) buffer Add mouse-PRLR primary antibody (1: 150) incubate for 30min at 4 ℃; the cells were then washed twice with BI buffer, labeled with anti-mouse IgG1 magnetic particles (1: 20), and incubated for 30min at 4 ℃.

Preferably, in the case of screening positive cells by the magnetic bead sorting method, the incubation is further performed for 8 minutes, and then the supernatant containing negative cells is discarded.

The invention has the beneficial effects that:

1. the separation method is simple to operate and saves time;

2. the separated cells have high yield, high purity and high activity;

3. low cost, no need of expensive equipment, and capability of completing separation in various conventional laboratories.

Drawings

FIG. 1 shows the specific expression of PRLR protein in mesenchymal cells. Adult rat leydig cells specifically express PRLR protein, with the seminiferous tubule boundaries marked by white arrows and a few weakly stained germ cells indicated by gray arrows. C. There was no anti-staining negative control.

FIG. 2 shows co-expression of PRLR and CYP11A1 protein in adult rat testis. Both PRLR (A, A1) and CYP11a1(B, B1) are expressed only by leydig cells. The two stains (C, C1) combined showed complete overlap, confirming that the LCs specifically expressed PRLR.

FIG. 3 is a comparison of the isolation of stromal cells using the MACS and E/Percoll programs. A. Unstained testicular cells contained almost no PRLR + cells. 6.7% of cells stained with PRLR antibody before MACS were positive. C. After MACS, PRLR + cells were enriched to more than 95%. Cell suspension stained with PRLR antibody before macs. Cell suspension stained with PRLR antibody after macs. F.E/Percoll program with HSD3B enzymatic activity staining cell suspension. G.E/Percoll program followed by cell suspension stained with HSD3B enzymatic activity. Scale bar: 100 μm.

FIG. 4 shows the co-expression of PRLR and CYP11A1 proteins in testis cell suspensions before and after MACS. A-C: prior to MACS, the testis cell suspension contained a small number of PRLR (A) and CYP11A1(B) positive cells, completely overlapping (C). D-F: after MACS, almost all cells were positive for prlr (d) and CYP11a1(E), with complete overlap (F). Scale bar: 100 μm.

FIG. 5 is a graph showing the effect of LH on the production of both steroid and non-steroid hormone function by MACS or LCs isolated from E/Percoll. Lh dose-dependently stimulates testosterone production by LCs isolated by MACS or E/Percoll programs. LH on MACS in the separation of LCs Cyp2r1, Insl3 gene expression effect. LH exerts an effect on MACS separation of LCs in the range of 25-OH-VD. Data are presented as mean ± standard deviation (group a n 4, group B, group C n 3). P < 0.05 or 0.01, respectively, with significant differences from the control group (0ng/ml LH).

FIG. 6 the effect of LH and DEX on the secretion of androgens by MACS isolated LCs. Time and LH dependent effects of dex on LCs T production. Effects of dex on testosterone (T), Dihydrotestosterone (DHT) and 3 α -androstanediol (3 α -DIOL) production. Data are presented as mean ± standard deviation (group a n 4, group B n 3). Denotes P < 0.05, 0.01 or 0.001, respectively, with significant differences from the control group (0 μ g/ml DEX).

Figure 7 is the effect of DEX on steroid pathway gene expression: LCs were treated with DEX (0.1. mu.g/ml) and LH (0.2ng/ml) for 24 hours. And (3) taking Rps16 as an internal reference, and detecting the expression of testosterone synthesis and metabolism genes by QPCR. Data are expressed as mean ± standard deviation (n ═ 3). P < 0.05, which is significantly different from the control group.

Figure 8 is the effect of DEX on steroid pathway protein expression: LCs were treated with DEX (0.1. mu.g/ml) and LH (0.2ng/ml) for 24 hours. B-ACTIN is used as an internal reference, and WB detects the expression of testosterone synthetic and metabolic proteins. Data are expressed as mean ± standard deviation (n ═ 3). P < 0.05 or 0.01, respectively, with significant differences from the control group.

Detailed Description

The invention will be further described in detail with reference to the following examples, which are given in the accompanying drawings.

As shown with reference to figures 1-8,

example 1

A method for separating Leydig cells from rat testis stroma comprises the following steps:

obtaining a rat testis and preparing a rat testis cell suspension;

marking the testis interstitial cells of the rat;

screening positive cells by a magnetic bead sorting (MACS) method;

Firstly, preparing a cell suspension from rat testis, wherein the preparation method of the suspension mainly comprises the steps of placing testicular parenchymal tissue into a culture medium for digestion; and standing the digested tissue suspension at room temperature, and filtering supernatant to obtain testis cell suspension for separation. One innovation of the method is that a magnetic bead sorting method is adopted to screen the rat testicular interstitial cells, so that magnetic labels are matched, magnetic particle labels are marked on the surfaces of the rat testicular interstitial cells, then positive cells are screened, the cells marked by the magnetic beads are transferred into a collecting pipe, the collecting pipe is immediately placed on a cell separation magnet fixer, the magnetic bead sorting step is carried out, negative cells and positive cells are separated, then supernatant containing the negative cells is discarded, the positive cells are resuspended, then the magnetic bead sorting mode is repeated for 3 times, the positive cells are repeatedly purified, and finally the separation is finished, and the separated cells are obtained.

The method has the advantages of low operation cost, simple steps and simple operation, and is convenient for laboratories with various specifications to implement. And high yield, purity and activity, which are verified as described in the examples below.

As another innovation point of the method, the method adopts the PRLR antibody to mark the leydig cells, can be used as the optimal mark of the leydig cells based on the specificity expression of the PRLR, is well matched with the magnetic bead sorting method to sort the leydig cells, and can be used as the separation base condition for the separation method.

Specifically, when rat mesenchymal testicular cells were labeled, BD IMag was detected^TM(BI) buffer Add mouse-PRLR primary antibody (1: 150) incubate for 30min at 4 ℃; cells were then washed twice with BI buffer and then anti-minicarMouse IgG1 magnetic particles (1: 20) were labeled and incubated at 4 ℃ for 30 minutes.

Through the incubation conditions, the PRLR antibody and the magnetic bead can be better combined on the rat testicular interstitial cells, and on the basis, the cell purity and the yield are improved through repeated sorting.

the testicular parenchymal tissue was digested in DMEM/F12 medium containing 1mg/ml collagenase IV (supplemented with 0.1% BSA) and shaken slowly in a shaker bath at 34 ℃ for 30min (90 rpm);

In addition, the rat testicular cells passed through the filtration were washed with DMEM/F12 medium; and rat testicular cells were resuspended in ice-cold BD IMag^TM(BI) buffer;

and/or when the positive cells are repeatedly purified by the magnetic bead sorting method, the magnetic bead sorting is carried out after the positive cell part is resuspended by 900 mu L of BI buffer solution each time.

The sorted positive cells are resuspended in the same buffer each time, allowing the magnetic beads to have sorted carriers for sorting. And the buffer solution is kept consistent, so that a stable environment can be kept, and the test variables are less.

To facilitate yield and activity, magnetic bead sorting was performed after resuspending the positive cell fraction with 900 μ L BI buffer for each replicate purification, and incubation for at least 4 minutes for each resuspension.

To enhance the effect of magnetic bead sorting, rat testicular cells were resuspended in ice-cold BD IMag^TM(BI) buffer, cell density was also adjusted to 2-8X10⁷Individual cells/ml.

Preferably, when the magnetic bead sorting method is used for screening positive cells, the incubation is performed for 8 minutes, and then the supernatant containing the negative cells is discarded, so that the separation effect of the negative cells and the positive cells can be better, and the yield and the activity of the positive cells can be ensured.

Example 2

Reagent

DMEM/F12 cell culture medium, Bovine Serum Albumin (BSA), collagenase type IV, testosterone-d 3, penicillin/streptomycin double antibody was purchased from Sigma-Aldrich (St. Louis, Mo.). Trypan blue solution (0.4%) was from Thermo Fisher (waltham, massachusetts). The reverse transcription kit was from Promega (madison, wisconsin).

480

Green I Master from Roche (Switzerland, Basel). Anti-mouse IgG1 magnetic particles and BD IMag^TMBuffer (10X) was from BD biosciences (franklin lake, new jersey). LH from MyBiosource (California, san Diego). Dihydrotestosterone (DHT) and 5 alpha-androstane, 3 alpha, 17 alpha-diol (3 alpha-diol) and 25-hydroxy-vitamin D (25-OH-VD) ELISA kits were purchased from Jianglai Biotechnology Ltd (China, Shanghai). Dexmedetomidine (DEX) was obtained from Yangzhou pharmaceutical industries, Inc. (China, Tay.). Acetonitrile and methanol were from Merck corporation (new jersey ).

Laboratory animal

Adult male SD rats of 2-3 months of age were purchased from the Shanghai laboratory animal center. Rats were housed in the laboratory animal room of the second hospital affiliated to Wenzhou medical university, with light (12 h: 12h) and temperature (22 ℃), and were given free access to drinking water and feed. These animals were not subjected to any chemical or procedural treatment and were used only for the separation of LCs. The experimental procedures were approved by the animal care and use committee of the university of medical Wenzhou, and were in accordance with the American national institutes of health ("guidelines for laboratory animal care and use").

Preparation of testis cell suspension

After one week of acclimation, animals were acclimated with CO₂The rats were euthanized by asphyxiation and the testes removed. Removing testis envelope, digesting testis parenchyma in DMEM/F12 medium containing 1mg/ml collagenase IV (with 0.1% BSA), and shaking slowly in a 34 deg.C water bath shaker for 30min (90 rpm)). The digested tissue suspension was allowed to stand at room temperature for 1min and the supernatant was filtered through a 70 μm pore nylon mesh. After washing with DMEM/F12 medium, the LCs were isolated from testis cell suspensions by two different methods.

Magnetic bead sorting (MACS) purification of LCs

Testis cells were resuspended in ice-cold BD IMagTM (BI) buffer and cell density adjusted to 2-8X10⁷Cells/ml, mouse-PRLR primary antibody (1: 150) was added and incubated at 4 ℃ for 30 minutes. The cells were then washed twice with BI buffer, labeled with anti-mouse IgG1 magnetic particles (1: 20), and incubated for 30min at 4 ℃. Magnetic bead sorting was used for positive selection of PRLR + cells. The magnetic bead-labeled cells were transferred to a collection tube, and the collection tube was immediately placed on a cell separation magnet holder (BD Biosciences, usa). After 8 minutes of incubation, the supernatant containing the negative cells was discarded and the fraction of positive cells adhering to the tube wall was resuspended in 900. mu.l of BI buffer. The magnetic bead separation step was repeated two more times, each for 4 minutes, to further purify the positive cells.

0.4% trypan blue was mixed 1: 1 with the cell suspension and the viability of the finally isolated cells was determined. After waiting 3min, the number of viable cells was counted under the microscope. Phycoerythrin (PE) -labeled goat anti-mouse IgG secondary antibody (1: 500) was added to the final isolated cell suspension, and after 1 hour of light-shielding staining, the percentage of PRLR + cells was analyzed by Flow Cytometry (FCM). For the assay of steroidogenic function, cells were suspended in DMEM/F12 medium and plated in 96-well plates (10)⁵Individual cells/well), the concentration of testosterone (T) in the culture medium is detected or inoculated in a 24-well plate (5X 10)⁵Individual cells/well) for QPCR or WB.

Centrifugal elutriation/Percoll gradient centrifugation method for purifying LCs

By way of comparison, the LCs were also isolated by classical methods including elutriation and Percoll density gradient centrifugation. The testis cell suspension after collagenase (1mg/ml) digestion was first enriched by centrifugal elutriation (flow rate 20ml/min, rotor speed 2000 rpm). The enriched cell suspension was then centrifuged through a Percoll gradient (60% Percoll, 27000g, 1h, automatically generating a density gradient during centrifugation)And (5) purifying the LCs. The fraction collected heavier than the 1.068g/ml density layer was LCs. The purity of the final isolated cells was checked by HSD3B staining. Cell viability was determined with trypan blue (0.4%). The remaining cells were suspended in DMEM/F12 medium and seeded in 96-well plates (10)⁵Individual cells/well, 3h) and detect T.

Culturing and treating purified LCs with LH and/or DEX

Purified LCs were plated in 96-well or 24-well plates in DMEM/F12 medium supplemented with 2.2g/L NaHCO3, 2.4g/L HEPES, 0.1% BSA and penicillin: streptomycin (100 IU: 100. mu.g/ml), pH 7.4. Cells in 96 wells (10)⁵One cell/well) culture plate, adding LH (0-100 ng/ml) for culturing for 3h, collecting culture solution and performing T measurement. In addition, cells were plated in 24-well plates (5X 10)⁵One cell/well), adding 0.2ng/ml LH and DEX (0-0.5 mu g/ml), treating, and collecting culture medium at 3h, 24h and 48h respectively to measure androgen. At the end of the culture, the cells are lysed to extract the RNA or protein.

Immunofluorescence

Adult rat testes were first fixed with 4% paraformaldehyde for 48 hours. After washing, dehydration with sucrose (20% sucrose solution for 12h, 30% sucrose solution for 12h) followed by OCT embedding at-20 ℃. Frozen sections 8 μm thick were cut. After 3 washes with PBS, the tissue sections were incubated overnight at 4 ℃ with two primary antibodies (PRLR, mouse monoclonal 1: 500; CYP11A1, rabbit monoclonal 1: 500) and then stained with two fluorescent secondary antibodies (PRLR: goat anti-mouse IgG-PE 1: 1000; CYP11A 1: goat anti-rabbit IgG-Dylight 488, 1: 1000) in the dark at room temperature for 1 h. To co-stain fresh LCs isolated by MACS, purified LCs were cultured for 3 hours until the cells were adherent. After washing with PBS, cells were co-stained with PRLR and CYP11a1 primary antibody in the same manner as for testis tissue sections. Before microscopic observation, a mounting medium containing DAPI was added dropwise to visualize the nuclei.

Flow cytometry analysis of PRLR positive cells

The cells from MACS were analyzed for percent PRLR + cells using flow cytometry. As a control group, no PRLR staining or PRLR staining was also performedCells that were not subjected to MACS were analyzed. Unsorted and sorted cells were incubated with fluorescent secondary antibody (PE-conjugated goat anti-mouse IgG) for 1h in the dark, respectively. After 3 PBS washes, the mixture was washed with Atture^TMNxT flow cytometry (Thermo Fisher, USA) analyzed cells.

Liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) or enzyme-linked immunosorbent assay (ELISA) for the detection of androgen and 25-hydroxy-vitamin D (25-OH-VD)

The testosterone (T) working solution was diluted from the T stock with methanol. And (3) diluting the working solution by using a cell culture medium gradient without T to prepare a calibration standard substance and a quality control sample. The Internal Standard (IS) working solution IS prepared from a T-d3 stock solution. IS working solution (10. mu.l) was added to the collected cell culture medium (100. mu.l) and mixed with 200. mu.l acetonitrile. After shaking, centrifuge at 12000g for 15 min. The supernatant (10. mu.l) was placed in an autosampler for detection. The acceptance of the validation test for intra-assay and inter-assay variability was set at ≦ ± 15%.

The assay of Dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha-DIOL) and 25-hydroxy-vitamin D (25-OH-VD) was performed using an ELISA kit. Briefly, the medium used for the assay was 100. mu.l. To avoid inter-assay variation, all samples tested for DHT, 3 α -DIOL, and 25-OH-VD were done in one assay. The detection sensitivity of the DHT kit is 0.5ng/L, and the coefficient of variation in detection is 10%. The detection sensitivity of the 3 alpha-DIOL kit is 1ng/ml, and the coefficient of variation in detection is less than 10%. The detection sensitivity of the 25-OH-VD kit is 1ng/ml, and the coefficient of variation in detection is less than 10%.

RNA extraction and Quantitative Polymerase Chain Reaction (QPCR)

By using

The Mini Kit (Qiagen, Germany) Kit, total RNA was extracted and cDNA was synthesized for QPCR gene detection. All major T synthesis and metabolism related genes were analyzed, including Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, Srd5a1, and Akrc 14. The mRNA level of ribosomal protein S16(Rps16) was used as an internal reference, and the Ct value of the target gene was normalized to the Ct value of Rps 16.

Immunoblotting (WB)

Extracting total cell protein with RIPA lysate (Biyun, China), and determining protein concentration with enhanced BCA protein concentration determination kit (Biyun, China). Sample proteins (30. mu.g) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The proteins analyzed included LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3, SRD5A1, AKRC14 and β -ACTIN. The β -ACTIN was used as an internal reference protein, and the gray level of each target protein was quantitatively analyzed based on the gray level.

Data analysis

Data are presented as mean ± standard deviation of 3-4 independent experiments. And verifying whether the two experiments have significant difference by using a t test. Significant differences between groups were determined using one-way analysis of variance (ANOVA) and SNK test. P < 0.05, 0.01 or 0.001, indicating a significant difference.

Results

Specific expression of PRLR in LCs in rodent testis

To confirm that the PRLR protein is expressed specifically by LCs, adult rat testis sections were immunofluorescent stained (fig. 1A-1C). PRLR is specifically expressed by mesenchymal cells, but is not clearly expressed in seminiferous tubule cells, with very slight staining of only a few germ cells (grey arrows, fig. 1A, 1B). Seminiferous tubule boundaries are marked with white arrows (fig. 1B). As a negative control, when incubation was performed with buffer instead of primary antibody, no staining was detected throughout the section (fig. 1C). To further confirm that these PRLR + cells were indeed LCs, a co-staining of PRLR and CYP11a1 was performed (fig. 2). CYP11a1 marked cells in the interstitial chamber very clearly (fig. 2A-2C). Under high power, it was clear that 2 stains matched perfectly (fig. 2a1-2C1), confirming that the PRLR antibody did specifically label the LCs.

Magnetic bead sorting (MACS) and centrifugal elutriation/Percoll gradient centrifugation (E/Percoll) comparison of LCs

Testis cell suspensions were stained with mouse-derived PRLR primary antibody, labeled with anti-mouse IgG magnetic beads, and LCs were isolated using MACS. The stained cell suspension was positively sorted 3 times with a cell separation magnet. PRLR + cells were then stained with PE-labeled goat anti-mouse IgG antibody and analyzed by flow cytometry (fig. 3). To correct for interference of cellular autofluorescence, FITC channel was introduced as reference (x-axis). In general, autofluorescence is not wavelength specific and can be emitted at similar intensities through multiple channels. With FITC and PE channels as the x-axis and y-axis, respectively, the autofluorescent cells were distributed along the diagonal (FIG. 3A). Cells not stained with PRLR antibody had few cells in the PE-positive triangular region (fig. 3A). After PRLR antibody staining without MACS procedure, approximately 6.7% of the cells fell within the PE positive region (fig. 3B). After 3 rounds of MACS, PE positive cells were enriched to around 95.5% (fig. 3C).

Fluorescence microscopy also confirmed that MACS significantly enriched PRLR + cells (fig. 3D, 3E). Prior to MACS, there were fewer PRLR positive cells (fig. 3D). PRLR staining was positive for nearly all cells after MACS (fig. 3E). Similarly, approximately 5% of the cellular HSD3B stained positive prior to the E/Percoll program (fig. 3F), and almost all of the cellular HSD3B stained positive after the E/Percoll program (fig. 3G), indicating that the E/Percoll program was also significantly enriched for LCs.

To further confirm that the isolated PRLR + cells were indeed LCs, the isolated cells were co-stained: PRLR and CYP11a1 (fig. 4). Some cells stained double-positive before MACS (FIGS. 4A-4C), while almost all cells were double-positive after MACS (FIGS. 4D-4F). Staining results from both fluorescence channels were pooled to match cells well before (figure 4C) or after MACS (figure 4F). PRLR staining was mainly distributed in the peri-cellular region (fig. 4D), whereas CYP11a1 staining was concentrated inside the cells (fig. 4E). These staining patterns are consistent with the distribution of these two proteins in the cell, i.e., PRLR is located at the cell membrane and CYP11a1 is located in the cytoplasm (mitochondria).

Table 1 compares in detail the cells obtained by MACS and E/Percoll isolation. The cell purity and viability of the two methods were similar. However, MACS is more advantageous in terms of cell yield, time required and whether special equipment is required.

Table 1: magnetic bead sorting (MACS) and centrifugal elutriation/Percoll gradient centrifugation (E/Percoll) comparison of LCs

Data are presented as the mean of 3 independent experiments.

Functional comparison of MACS-isolated cells with E/Percoll-isolated cells

To compare the steroidogenic function of cells isolated by two different methods, the same number of cells were cultured in vitro, LH concentration was increased, cultured for 3 hours, and T was detected in the culture medium (fig. 5A). In both cell populations there was a clear dose-dependent increase in T production of LH. At 0.5ng/ml LH, T production reaches half maximal amount, and at 5-10ng/ml, T reaches maximal amount. There was no significant difference in T production detected between the two cells at all LH doses, indicating that the two methods isolated cells were equally capable of producing T in response to LH stimulation.

To further test whether the non-steroid hormone function of LCs isolated by MACS protocol responded to LH stimulation, the expression of 2 non-steroid hormone related genes inst 3 and Cyp2r1 was tested by QPCR (fig. 5B). At LH concentrations corresponding to half maximal effector concentrations (0.5ng/ml) and maximal effector concentrations (10ng/ml) of stimulated T production, both genes increased slightly but significantly. In addition, a slight but significant increase in the non-steroid formed product, 25-OH-VD, also occurred (FIG. 5C).

Application of LCs separated by MACS method in toxicology research

In addition to their responsiveness to LH stimuli, to assess whether these cells isolated by MACS are suitable for toxicology studies, they were tested using Dexmedetomidine (DEX), a sedative drug that is more commonly used clinically. With increasing DEX concentration (0.01-0.5. mu.g/ml), cells were incubated for 48 hours and T production exhibited a dose-dependent decrease in DEX (FIG. 6A). The effect was visible as early as 3 hours and continued for up to 48 hours. Since the inhibitory effect did not accumulate over time, it was suggested that the effect could be acute or that the drug could be rapidly metabolized by the LCs.

To elucidate the mechanism of the reduction of T secretion, two T metabolites, DHT and 3 α -DIOL, were tested (FIG. 6B). DEX (0.1 μ g/ml, 24 hours) treated cells had a decrease in T, while both metabolites were significantly upregulated, suggesting that one of the causes of T downregulation may be an increase in T metabolism.

Effect of DEX on LCs steroid-related Gene and protein expression

To further investigate the effect of DEX on expression of genes and proteins associated with the steroid pathway of LCs, total RNA and proteins were extracted from cells treated with DEX at 0.1. mu.g/ml for 24h for QPCR and WB assays. QPCR results showed that none of the 7 genes involved in T synthesis were significantly altered, including 5 genes directly involved in steroid production (Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3) and 2 genes involved in cholesterol transport (Scarb1 and Star), while 2 genes associated with T metabolism (Srd5a1 and Akr1c14) were significantly up-regulated (fig. 7). Furthermore, immunoblot analysis (WB) of whole cell proteins was in complete agreement with QPCR results (fig. 8). DEX (0.1. mu.g/ml, 24h) treatment did not affect 7 proteins/enzymes involved in T synthesis, while 2 were responsible for a significant increase in T-metabolizing enzyme expression.

To sum up the above

This protocol describes a novel method for the preparation of high purity leydig cells from adult rat testis based on the magnetic bead sorting (MACS) method. The method is based on the specific expression of membrane protein PRLR by adult testis interstitial cells (ALCs). Rat LCs purified by the novel method retain good activity and function. These cells show a good LH dose response curve in T production, with a 10-fold response to LH stimulation at maximal concentration. These reactions are similar to cells isolated by the classical E/Percoll procedure.

MACS has several advantages over classical LCs separation methods. First, since LCs specifically express PRLR in large quantities, very high purity (95%) cells can be obtained. Compared with LHCGR, the expression of PRLR of Leydig cells is more stable and the expression is more uniform among single cells, so that the protein can be easily and consistently detected by using a commercial antibody. Based on this unique marker, purification can be achieved with high purity, high activity (93%) and high yield (about 10)⁶Individual cells/testis). Since MACS does not require centrifugation of cells at very high speeds (27000g) as in E/Percoll, or flow sorting FACSThe high pressure nozzle sprays the cells, so that the vitality of the cells can be better preserved.

In addition to the better quality of the cells isolated, this new method has other advantages. For example, it does not require high-end equipment such as elutriators or flow cytometers with sorting capabilities, and thus the method is applicable to any common laboratory that only owns the infrastructure. Furthermore, this process is simpler and less technically demanding than conventional methods, which is an advantage for new people with less laboratory experience. In addition, MACS has flexibility in terms of scale of operation, and can meet various experimental requirements. For example, for a typical T-test experiment, the method can be scaled down to extract LCs from a single rat testis that can produce enough cells (about 10)⁶One) for a plurality of wells of a 96-well culture plate. On the other hand, for experiments requiring large numbers of cells, such as experiments involving mitochondrial isolation, exosome production or proteomic work, the protocol can be easily scaled up to 20 testes with a cell yield of 2 × 10⁷。

PRLR is one of the members of the cytokine/prolactin/growth hormone receptor family and is expressed in at least 8 organs/tissues including breast, ovary, testis, liver, etc. Early studies using in situ hybridization and RT-PCR techniques in the testis indicated that PRLR mRNA is expressed predominantly by mesenchymal and germ cells. However, recent studies of scRNA-seq technology indicate that PRLR is expressed predominantly by LCs. Although this new technology does not detect PRLR mRNA in germ cells, in addition to the universal and strong expression of PRLR protein in LCs, weak positive PRLR protein was also found in few germ cells. However, PRLR almost perfectly isolates LCs using MACS protocols, despite the presence of a small number of weakly positive germ cells.

LCs can also produce non-steroidogenic products, such as INSL3 and 25-OH-VD, in addition to T. Unlike T, these two products are not strongly regulated by LH. They can be produced by LCs and are not dependent on steroidogenesis, but can still be considered as functional markers for LCs. In the present study, the synthesis and/or expression of the genes Cyp2r1 and inst 3 involved in these two products was also examined. After 24 hours of LH treatment, these three parameters were slightly but significantly upregulated. This finding not only confirms that these two products are not as strongly regulated by LH as T, but also extends the previous conclusions, suggesting that long-term maintenance of these two products may still be dependent on LH. These results indicate that MACS isolated LCs are not only useful for the study of T production, but also useful for studying the modulation of non-steroid function.

DEX is widely used for clinical sedation due to its unique sedative and analgesic properties, and does not interfere with respiration. Short-term use of DEX has been found to regulate secretion of rat immature mesenchymal T cells, but its potential toxicity to adult mesenchymal cells (ALCs) has not been detected. As an example of the use of the isolated cells for toxicological applications, the potential side effects of the drug on ALCs prepared by MACS were tested. DEX is able to suppress the production of LCs T. The low concentration (0.01. mu.g/ml) is comparable to the typical blood concentration achieved when a human is under anesthesia. Moreover, this effect can be produced quickly in as little as 3 hours, and can last for 48 hours.

To elucidate the mechanism, the concentrations of two major T metabolites, DHT and 3 α -DIOL, were determined. Both metabolite concentrations increased after DEX treatment, while the concentration of T decreased. These results strongly suggest that increased metabolism may be one of the major causes of decreased T output. To further investigate how DEX affects T production and metabolism, steroid pathway-associated mRNA and protein levels were analyzed using QPCR and WB. The mRNA and protein results are consistent showing: DEX has no effect on 7 major proteins involved in T synthesis, including 5 steroidogenic proteins (LHCGR, CYP11a1, HSD3B1, CYP17a1, HSD1783) and 2 cholesterol transporters (SCARB1 and STAR). While DEX significantly increased the expression of two major T-metabolizing enzymes, SRD5a1 and AKR1C 14. SRD5A1 is a unidirectional enzyme responsible for converting T to DHT, while AKR1C14 is a bidirectional enzyme responsible for converting DHT to 3 α -DIOL. Upregulation of these two enzymes demonstrated that DEX can increase the metabolic activity of T. These results are in full agreement with hormonal changes, supporting the conclusion that DEX significantly reduces T output by increasing metabolism of T and DHT.

It is well known that in the testis that produces T, the concentration of T is much higher than in surrounding tissues, including blood (in rats, T concentration in testis is 20 times higher than in blood, in humans, T concentration in testis is 100 times higher than in blood; testosterone is the main androgen that regulates spermatogenesis in testis; however, in many androgen-targeted tissues outside testis, such as prostate, epididymis and skin, DHT is the main androgen form, binds to Androgen Receptor (AR) and activates androgen-dependent signaling pathways; DHT is 10 times stronger in binding to and activating AR; the conversion of T to DHT in peripheral tissues is an important cascade of limited local activation of androgen AR; therefore, DEX upregulates DHT synthase (D5A 1) and metabolic enzyme (SRR 1C14) may have a far different effect on the physiology of systemic androgen, than whether DEX also affects two other major androgen-targeted tissues, further research is needed. Just like LCs, the action of both enzymes should be tested on human cells or human patients. Finally, the molecular mechanisms of upregulation of these two enzymes should also be studied.

Therefore, the scheme establishes a novel testis interstitial cell separation method based on the cell surface specific marker PRLR. The procedure involves enzymatic digestion of testicular tissue, staining the cells of interest with PRLR antibody, and then sorting the cells of interest using MACS. The method is simple and rapid, and can obtain high purity and high activity cells without expensive equipment. Cells isolated by the novel method further demonstrate that the anesthetic DEX increases the conversion of T produced by LCs to DHT and then to 3 alpha-DIOL by up-regulating two metabolic enzymes, SRD5A1 and AKR1C14, at clinically relevant concentrations in humans, thereby reducing T output. These results demonstrate that the novel cell isolation procedure is a good tool to provide high quality stromal cells for endocrinology, pharmacology and toxicology studies.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Claims

1. A method for separating Leydig cells from rat testis mesenchyme is characterized by comprising the following steps: obtaining a rat testis and preparing a rat testis cell suspension;

marking the testis interstitial cells of the rat;

screening positive cells by a magnetic bead sorting (MACS) method;

2. The method of claim 1, wherein the marker for rat leydig cells is labeled with a prolactin receptor (PRLR) antibody.

3. The method of claim 1, wherein the rat testicular cell suspension is prepared by:

placing testis parenchyma in a culture medium for digestion;

4. The method of claim 3,

washing the filtered rat testis cells with DMEM/F12 culture medium; and resuspending rat testicular cells in ice-cold BDIMag^TM(BI) buffer;

5. The method of claim 4, wherein the magnetic bead sorting is performed after resuspending the positive cell fraction with 900 μ L of BI buffer for each replicate of the purified positive cells, and incubating for at least 4 minutes for each resuspension.

6. The method of claim 4, wherein the rat testis is selected from the group consisting of rat testisPellet cells resuspended in ice-cold BD IMag^TM(BI) buffer, cell density was also adjusted to 2-8X10⁷Individual cells/ml.

7. The method of claim 6, wherein the labeling of rat testicular cells is performed on BD IMag^TM(BI) buffer Add mouse-PRLR primary antibody (1: 150) incubate for 30min at 4 ℃; the cells were then washed twice with BI buffer, labeled with anti-mouse IgG1 magnetic particles (1: 20), and incubated for 30min at 4 ℃.

8. The method of claim 3, wherein the rat testicular cell suspension is prepared by:

9. The method of claim 1, wherein the magnetic bead sorting method is performed for positive cells by incubating for 8 minutes before discarding the supernatant containing negative cells.