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Aug 2020
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Isolation of Primary Leydig Cells from Murine Testis
从小鼠睾丸中分离出原发性睾丸间质细胞   

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Abstract

In males, Leydig cells are the primary source of testosterone, which is necessary for testis development, masculinization, and spermatogenesis. Leydig cells are a valuable cellular model for basic research; thus, it is important to develop an improved method for isolation and purification of Leydig cells from testes. The available methods for Leydig cell isolation have some drawbacks, including the need for sophisticated instruments, high cost, tediousness, and time consumption. Here, we describe an improved protocol for isolation of primary Leydig cells from testicular tissue by digestion with collagenase IV.

Keywords: Primary Leydig cell (原发性淋巴细胞), Testis (睾丸), Testosterone (睾酮), Collagenase IV (胶原蛋白酶IV), Late-onset Hypogonadism (晚发性腺功能减退症)

Background

Leydig cells, located in the interstitial tissue of testis, are steroidogenic cells that contribute to testosterone synthesis and secretion. Leydig cell steroidogenesis is strictly regulated by luteinizing hormone (LH) secreted by the pituitary gland. LH binds luteinizing hormone receptor (LHCGR) located on the surface of Leydig cells to activate adenylyl cyclase and increase the production of cyclic adenosine monophosphate (cAMP). The cAMP signaling cascade stimulates the expression of steroidogenic acute regulatory protein (StAR), which transports cholesterol from cytoplasm into the inner membrane of mitochondria. Cholesterol is converted to pregnenolone by a P450 side-chain cleavage enzyme (CYP11A1) and translocated to the endoplasmic reticulum. Finally, pregnenolone is converted to testosterone by a series of lyase reactions. Male hypogonadism is characterized by testosterone deficiency and affects approximately 6% of males, with an increasing incidence and prevalence in recent years. However, the mechanisms of testosterone deficiency are far from clear. Immortalized cell lines provide useful tools to investigate the molecular mechanisms underlying testosterone deficiency. Characterized Leydig cell lines, such as MA-10, BLTK1, and TM3 (Engeli et al., 2018), are unable to produce testosterone due to a lack or low expression of testosterone synthase genes. In contrast, the expression level of these genes in primary Leydig cells is closer to the cells in vivo. Furthermore, primary Leydig cells feature a complete signaling pathway of testosterone synthesis and are widely used to investigate late-onset hypogonadism (LOH) and diseases of the reproductive system. Additionally, Leydig cell and stem Leydig cell transplantation have been developed as alternative therapies for the treatment of testicular damage, LOH, and sexual dysfunction, achieving good curative effects (Artyukhin et al., 2007; Sun et al., 2009; Jiang et al., 2014). Therefore, procedures for isolation and enrichment of Leydig cells from testes are vital for establishing the role of Leydig cells in hypogonadism. In recent years, various approaches have been used to isolate Leydig cells, but gradient centrifugation is the most frequently used (Conn et al., 1977; Gale et al., 1982). Therefore, these methods are complicated, costly, time-consuming, and provide low yield. Here, we describe a cost-effective and time-saving protocol for isolating Leydig cells via digestion with collagenase VI.

Materials and Reagents

  1. Sterile 50 ml centrifuge tubes (Corning, catalog number: 430829)

  2. 40 μm cell strainers (Falcon, catalog number: 352340)

  3. Sterile 100 × 20 mm cell culture dish (Corning, catalog number: 430167)

  4. 0.22 μm filters (Millipo, catalog number: GNWP02500)

  5. 8-week-old healthy Kunming (KM) mice

  6. DMEM basic (1×) (Gibco, catalog number: C11995500BT)

  7. Fetal bovine serum (FBS) (ExCellBio, catalog number: FSD500)

  8. Collagenase IV (Biosharp, catalog number: C-5138)

  9. NaCl (Guangzhou Chemical Reagent Factory, catalog number:7647-14-5)

  10. KCl (Guangzhou Chemical Reagent Factory, catalog number: 7447-40-7)

  11. Na2HPO4·12H2O (Guangzhou Chemical Reagent Factory, catalog number: 7558-79-4)

  12. KH2PO4 (Guangzhou Chemical Reagent Factory, catalog number: 7778-77-0)

  13. Penicillin G Potassium Salt (MDBio, catalog number: W627099)

  14. Streptomycin Sulfate (Diamond, catalog number: G821BA0034)

  15. 100% ethyl alcohol (Sinopharm, catalog number: 64-17-5)

  16. Nitrotetrazolium Blue chloride (NTB) (Sigma-Aldrich, catalog number: N6876-50G)

  17. DHEA (Sigma-Aldrich, catalog number: 252805-10MG)

  18. DMSO(Sigma-Aldrich, catalog number: D2650-100ML)

  19. β-NAD (Sigma-Aldrich, catalog number: N1511-250MG)

  20. I125-testosterone Coat-A-Count RIA kits (Beijing North Institute of Biological Technology, catalog number: 201202)

  21. Luteinizing Hormone (Sigma-Aldrich, catalog number: 869003)

  22. HSD3B antibody (Santa Cruz Biotechnology, catalog number: sc-515120)

  23. StAR antibody (CST, catalog number: D1Z2A)

  24. SF-1 antibody (CST, catalog number: D10H12)

  25. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Abcam, catalog number: ab150077)

  26. Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (Abcam, catalog number: ab150115)

  27. Primary & secondary antibody diluent for immunostaining (Yeasen, catalog number: 36323ES60)

  28. Paraformaldehyde (Shanghai Lingfeng, catalog number: 30525-89-4)

  29. 1× PBS (see Recipes)

  30. D-PBS (see Recipes)

  31. Penicillin/streptomycin solution (see Recipes)

  32. 0.75 mM KCl hypotonic buffer (see Recipes)

  33. 75% ethyl alcohol (see Recipes)

  34. 10 mg/ml Collagenase IV solution (see Recipes)

  35. HSD3B staining solution (see Recipes)

  36. Complete medium (see Recipes)

  37. 4% paraformaldehyde solution (see Recipes)

  38. StAR antibody dilution solution (see Recipes)

  39. HSD3B antibody dilution solution (see Recipes)

  40. SF-1 antibody dilution solution (see Recipes)

Equipment

  1. Surgical Instruments:

    Scissors (Yuyan instruments, catalog number: Y15103)

    4× forceps (Yuyan instruments, catalog number: Y15201)

    Pointed forceps (Fine Science Tools, catalog number: 11252-00)

  2. Low-speed centrifuge (Zonkia, model: SC-3610)

  3. Biological safety cabinet (Heal Force, model: HFsafe-I200LC)

  4. Water bath kettle (Jing Hong, model: XMTD-8222)

  5. Cell incubator (Thermo Fisher, model: 371)

  6. Fully automatic autoclave sterilizer (STIK, model: MJ-78A)

  7. Inverted microscope (Nikon, model: TS100)

  8. Electronic analytical balance (Sartorius, model: BSA124S-CW)

  9. Gamma radioimmunoassay counter (Anhui ustc zonkia scientific, model:GC -1200γ)

  10. Hemacytometer (Marienfeld, catalog number: 0650030)

Procedure

The protocol described in this manuscript is summarized in Figure 1.



Figure 1. Protocol work flow.

Schematic summarizing the major steps of the protocol.


See Video 1 for procedure of Leydig cell isolation.


Video 1. Testis isolation, wash, and decapsulation.

  1. Sacrifice ten 8-week-old healthy Kunming (KM) mice in a desiccator using CO2 (Leary, 2013). Wait until breathing stops, soak the mice in 75% alcohol for 10 min to sterilize them, and place them on the operating table (Figure 2A).

  2. Pull up the skin in the lower abdomen region, cut it horizontally with sterile surgical scissors, locate and remove the testes with sterile forceps, and transfer them into a 50 ml tube containing 30 ml of precooled PBS.

  3. Move the testes to the biosafety cabinet, pour testes and PBS into an empty clean cell dish, add 20 ml of PBS (with 1× penicillin/streptomycin solution) to each of the four dishes in advance, and wash the testes four times (Figure 2B).

  4. Clamp the testes one by one to an empty cell dish using forceps and puncture the testis with another pointed forceps. Squeeze out the content and place it into a new cell dish containing 20 ml of PBS (Figure 2C). Finally, collect all into a 50 ml tube with 10 ml of collagenase IV (1 mg/ml) solution.

  5. Put the tube in a shaking water bath to digest for 7 min at 37°C.

  6. Add 20 ml of complete medium to the tube to terminate the digestion at room tempreture (figure 2D).

  7. Let the contents settle for 5 min until the seminiferous cords sink to the bottom of the centrifuge tube.

  8. Transfer the supernatant to a new 50 ml centrifuge tube, filter it on a 40 μm cell strainer, and centrifuge at 250 × g for 10 min.

  9. After centrifugation, discard the supernatant and add 2 ml of complete medium to resuspend the cells in a tube.

  10. Seed the cell suspension evenly into two 100 mm culture dishes containing 5 ml of complete medium.

  11. Incubate the dishes in 5% CO2 incubator at 37°C for 1 h (Figure 2E).

  12. One hour later, remove the non-adherent cells, and add 6 ml of fresh complete medium to the dish to continue culturing for 24 h (Figure 2F).

  13. Twenty-four hours later, discard the original medium, treat the cells with 4 ml of KCl hypotonic solution for 5 min to further remove the myoid cells, and wash with PBS three times.

  14. Add 6 ml of fresh complete medium per dish and continue culturing for 48 h (Figure 2G).

  15. After 48 h of culture, harvest the Leydig cells for further experiments.

  16. Stain for HSD3B enzyme to identify the purity of Leydig cells (figure 2H). Perform total cell counts using a hemacytometer. Approximately 2 million cells can be obtained from ten mice. Harvest approximately 20,000 cells, resuspend them in PBS, smear on the slides, and leave to dry. Add 50-100 μl HSD3B enzyme staining solution to cover the cells and incubate for 30 min at 37°C away from light. Discard the staining solution and wash the slide twice with PBS. Use a TS100 Inverted microscope to capture the images, and perform analysis with ImageJ.

  17. Add complete medium (with 1ng/ml LH) to the Leydig cells, and continue to culture for 48 h, following on from step 15. Then collect the supernatants to measure testosterone with I125-testosterone Coat-A-Count RIA kits by radioimmunoassay (RIA) (Figure 2I) according to the manufacturer’s protocol.

  18. Perform immunostaining for Leydig cell markers StAR, HSD3B, and SF-1 (Figures 2J-2L). Fix the cells in 4% paraformaldehyde solution for 10 min, wash the cells three times with PBS, permeabilize cells in 0.5% Triton-X for 10 min, and then block nonspecific adhesion sites using 5% BSA for 1 h at room temperature. Dilute the primary and second antibodies in the antibody diluton solution. Incubate cells with primary antibody solution (such as StAR, HSD3B, and SF-1) overnight at 4°C. Wash with PBS three times and then incubate cells with secondary antibody for 1 h at room temperature. Wash with PBS three times and stain nuclei with DAPI. Use a LSM710 confocal microscope (Zeiss) to capture the images.



    Figure 2. Isolation and identification of primary Leydig cell from mouse.

    A. The KM mice are sacrificed and disinfected in 75% alcohol. B. Testis are taken from the sacrificed mice. C. Testis with tunica albuginea removed. D. Testis after digestion by collagenase IV. E. Cells adhere for 1 h (Scale bar: 100 μm). F. Cells adhere for 24 h (Scale bar: 100 μm). G. Cells adhere for 72 h (Scale bar: 100 μm). H. The isolated cells stained for HSD3B enzyme (purple) (Scale bar: 100 μm). I. Testosterone levels of cell supernatants contein 1 ng/ml LH (LH) and without LH (control). J. Immunostaining of Leydig cell marker StAR (Scale bar: 20 μm). K. Immunostaining of Leydig cell marker HSD3B (Scale bar: 10 μm). L. Immunostaining of Leydig cell marker SF-1 (Scale bar: 20 μm).

Data analysis

  1. Five random microscopic fields were examined per slide. The positive rate of HSD3B enzyme staining is calculated with ImageJ using the Analyze Particles function. Set 1,000 as the minimum particle size.

  2. Only the isolated cells with more than 85% positive rate are used in further experiments.

Recipes

  1. 1× PBS (pH = 7.4)

    8 g NaCl

    0.2 g KCl

    3.63 g Na2HPO4·12H2O

    0.24 g KH2PO4

    Add 1 L ddH2O

    Sterilize it at 121°C for 30 min.

  2. D-PBS (pH = 7.4)

    8 g NaCl

    0.2 g KCl

    1.15 g Na2HPO4·12H2O

    0.2 g KH2PO4

    Add 1 L ddH2O

  3. Penicillin/streptomycin solution (10×)

    3.1 g Penicillin G Potassium Salt

    5 g Streptomycin Sulfate

    50 ml 1× PBS (pH = 7.4)

    Perform filter sterilization through 0.22 μm filters.

  4. 0.75 mM KCl hypotonic buffer

    14 g KCl

    Add 250 ml ddH2O

  5. 75% alcohol

    750 ml 100% ethyl alcohol

    250 ml ddH2O

  6. 10 mg/ml Collagenase IV solution

    100 mg Collagenase IV

    10 ml 1× PBS (pH = 7.4)

  7. HSD3B staining solution

    1. Solution A:

      1 mg NTB

      0.6 mg DHEA

      0.6 ml DMSO

    2. Solution B:

      10 mg β-NAD

      9.5 ml D-PBS

      Add 0.30 ml of solution A to 4.75 ml of solution B and mix thoroughly.

  8. Complete medium

    10% FBS

    90% DMEM basic (1×)

  9. 4% paraformaldehyde solution

    4.0 g paraformaldehyde

    Add 100 ml 1×PBS

  10. StAR antibody dilution solution

    5 μl StAR antibody

    Add 5 ml Primary & secondary antibody diluent for immunostaining

  11. HSD3B antibody dilution solution

    5 μl HSD3B antibody

    Add 5 ml Primary & secondary antibody diluent for immunostaining

  12. SF-1 antibody dilution solution

    5 μl SF-1 antibody

    Add 5 ml Primary & secondary antibody diluent for immunostaining

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 81871150, 31801235, and 82071634), Natural Science Foundation of Guangdong Province (No. 2018A030313473), and The Science and Technology Plan Project of Guangzhou (No. 201508020001, 2016A020214013, 201803010044, 201704YG066, 2020A1515011203). This protocol was adapted from previous work (Yang et al., 2020).

Competing interests

The authors declare that they have no competing interests.

References

  1. Artyukhin, A. A. and Zaraiskii, E. I. (2007). A new method of culturing Leydig cells and prospects of their practical use in andrology. Bull Exp Biol Med 143(1): 155-159.
  2. Conn, P. M., Tsuruhara, T., Dufau, M. and Catt, K. J. (1977). Isolation of highly purified Leydig cells by density gradient centrifugation. Endocrinology 101(2): 639-642.
  3. Engeli, R. T., Fürstenberger, C., Kratschmar, D. V. and Odermatt, A. (2018). Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis. Heliyon 4(2): e00527.
  4. Gale, J. S., Wakefield, J. S. and Ford, H. C. (1982). Isolation of rat Leydig cells by density gradient centrifugation. J Endocrinol 92(2): 293-302.
  5. Jiang, M. H., Cai, B., Tuo, Y., Wang, J., Zang, Z. J., Tu, X., Gao, Y., Su, Z., Li, W. and Li, G. (2014). Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction. Cell Res 24(12): 1466-1485.
  6. Leary, S., (2013). AVMA Guidelines for the Euthanasia of Animals. 2013 Edition. University of alaska anchorage..
  7. Sun, J., Xi, Y. B., Zhang, Z. D., Shen, P., Li, H. Y., Yin, M. Z., Li, W. Y. and Shi, C. R. (2009). Leydig cell transplantation restores androgen production in surgically castrated prepubertal rats.
  8. Yang, Y., Zhou, C., Zhang, T., Li, Q., Mei, J., Liang, J., Li, Z., Li, H., Xiang, Q. and Zhang, Q. (2020). Conversion of fibroblast into functional leydig-like cell using defined small molecules. Stem Cell Reports 15(2): 408-423.

简介

[摘要]在男性中,Leydig 细胞是睾丸激素的主要来源,睾丸激素是睾丸发育、男性化和精子发生所必需的。Leydig 细胞是基础研究的宝贵细胞模型;因此,开发一种从睾丸中分离和纯化 Leydig 细胞的改进方法非常重要。Leydig 细胞分离的可用方法有一些缺点,包括需要复杂的仪器、成本高、繁琐和耗时。在这里,我们描述了一种通过胶原酶 IV 消化从睾丸组织中分离原代 Leydig 细胞的改进方案。

[背景] 睾丸间质组织中的间质细胞是类固醇生成细胞,有助于睾酮的合成和分泌。Leydig 细胞类固醇生成受到垂体分泌的促黄体激素 (LH) 的严格调控。LH 结合位于 Leydig 细胞表面的促黄体激素受体 (LHCGR) 以激活腺苷酸环化酶并增加环磷酸腺苷 (cAMP) 的产生。cAMP 信号级联刺激类固醇生成急性调节蛋白 (StAR) 的表达,该蛋白将胆固醇从细胞质转运到线粒体内膜。胆固醇通过 P450 侧链裂解酶 (CYP11A1) 转化为孕烯醇酮并转移到内质网。最后,孕烯醇酮通过一系列裂解酶反应转化为睾酮。男性性腺功能减退症的特点是睾酮缺乏,影响大约 6% 的男性,近年来发病率和患病率不断增加。然而,睾酮缺乏的机制尚不清楚。永生化细胞系为研究睾酮缺乏症的分子机制提供了有用的工具。特征性 Leydig 细胞系,例如 MA-10、BLTK1 和 TM3(Engeli等,2018),由于缺乏或低表达睾酮合酶基因而无法产生睾酮。相比之下,这些基因在原代 Leydig 细胞中的表达水平更接近体内细胞。此外,原代 Leydig 细胞具有完整的睾酮合成信号通路,被广泛用于研究迟发性性腺机能减退 (LOH)和生殖系统疾病。此外,Leydig 细胞和 Leydig 干细胞移植已被开发作为治疗以下疾病的替代疗法 睾丸损伤、LOH、性功能障碍,取得了良好的疗效(Artyukhin等,2007;Sun等,2009;Jiang等,2014)。因此,从睾丸中分离和富集 Leydig 细胞的程序对于确定 Leydig 细胞在性腺机能减退中的作用至关重要。近年来,已使用各种方法来分离 Leydig 细胞,但最常用的是梯度离心(Conn等,1977;Gale等,1982)。因此,这些方法复杂、昂贵、费时且产率低。在这里,我们描述了一种通过胶原酶 VI 消化来分离 Leydig 细胞的经济高效且省时的协议。

关键字:原发性淋巴细胞, 睾丸, 睾酮, 胶原蛋白酶IV, 晚发性腺功能减退症

材料和试剂

 

1.     无菌 50 ml 离心管(Corning,目录号:430829

2.     40 μm 细胞过滤器(Falcon,目录号:352340

3.     无菌 100 × 20 mm 细胞培养皿(Corning,目录号:430167

4.     0.22 μm 过滤器(Millipo,目录号:GNWP02500

5.     8 周龄健康昆明 (KM) 小鼠

6.     DMEM 基本型()(Gibco,目录号:C11995500BT

7.     胎牛血清(FBS)(ExCellBio,目录号:FSD500

8.     胶原酶 IVBiosharp,目录号:C-5138

9.     NaCl(广州化学试剂厂,目录号:7647-14-5

10.  KCl(广州化学试剂厂,目录号:7447-40-7

11.  Na HPO ·12H O(广州化学试剂厂,目录号:7558-79-4

12.  KH PO (广州化学试剂厂,目录号:7778-77-0

13.  青霉素 G 钾盐(MDBio,目录号:W627099

14.  硫酸链霉素(钻石,目录号:G821BA0034

15.  100%乙醇(国药集团,目录号:64-17-5

16.  氯化硝基四唑蓝(NTB)(Sigma-Aldrich,目录号:N6876-50G

17.  DHEASigma-Aldrich,目录号:252805-10MG

18.  DMSOSigma-Aldrich,目录号: D2650-100ML

19.  β-NADSigma-Aldrich,目录号:N1511-250MG

20.  125-睾酮 Coat-A-Count RIA 试剂盒(北京北方生物技术研究所,目录号:201202

21.  促黄体激素(Sigma-Aldrich,目录号:869003

22.  HSD3B抗体(Santa Cruz Biotechnology,目录号:sc-515120

23.  StAR抗体(CST,目录号:D1Z2A

24.  SF-1抗体(CST,目录号:D10H12

25.  山羊抗-IgG HLAlexa® 488)(Abcam公司,目录号:ab150077

26.  山羊抗小鼠IgG HLAlexa® 647)(Abcam公司,目录号:ab150115

27.  用于免疫染色的一抗和二抗稀释剂(Yeasen,目录号:36323ES60

28.  多聚甲醛(上海凌峰,目录号:30525-89-4

29.  1× PBS(见食谱)

30.  D-PBS(见食谱)

31.  青霉素/链霉素溶液(见食谱) 

32.  0.75 mM KCl 低渗缓冲液(见配方)

33.  75% 乙醇(见配方)

34.  10 mg/ml 胶原酶 IV 溶液(见配方)

35.  HSD3B 染色溶液(见配方)

36.  完全培养基(见食谱)

37.  4% 多聚甲醛溶液(见配方)

38.  StAR 抗体稀释溶液(见配方)

39.  HSD3B 抗体稀释溶液(见配方)

40.  SF-1 抗体稀释溶液(见配方)

 

设备

 

1.     手术器械:

剪刀(玉燕仪器,目录号:Y15103

镊子(玉燕仪器,目录号:Y15201

尖头钳(Fine Science Tools,目录号:11252-00

2.     低速离心机(Zonkia,型号:SC-3610 

3.     生物安全柜(Heal Force,型号:HFsafe-I200LC

4.     水浴壶(景红,型号:XMTD-8222

5.     细胞培养箱(Thermo Fisher,型号:371

6.     全自动高压灭菌器(STIK,型号:MJ-78A

7.     倒置显微镜 (尼康,型号:TS100

8.     电子分析天平(Sartorius,型号:BSA124S-CW

9.     伽马放射免疫测定计数器(安徽科大中科,型号:GC -1200γ

10.  血细胞计数器(Marienfeld,目录号:0650030

 

程序

 

本手稿中描述的协议在图 1中进行了总结。

 

 

1. 协议工作流程。

示意图总结了协议的主要步骤。

 

有关 Leydig 细胞分离的过程,请参见视频 1

 

 

视频 1. 睾丸分离、清洗和脱囊。

 

1.     在干燥器中使用 CO 2处死 10 8 周大的健康昆明 (KM) 小鼠(Leary2013 年)。等到呼吸停止,将小鼠在 75% 酒精中浸泡 10 分钟以对其进行消毒,然后将它们放在手术台上(图 2A)。

2.     拉起下腹部皮肤,用无菌手术剪水平剪开,用无菌手术剪定位并取出睾丸。 镊子,和 将它们转移到含有 30 ml 预冷 PBS 50 ml 管中。

3.     将睾丸移至生物安全柜,将睾丸和PBS倒入空的干净细胞培养皿中,四个培养皿中分别加入20ml PBS(含青霉素/链霉素溶液),冲洗睾丸四次(图) 2B)

4.     使用镊子将睾丸一个一个夹在一个空的细胞盘上,并用另一个尖头镊子刺破睾丸。挤出内容物并将其放入含有 20 ml PBS 的新细胞培养皿中(图 2C)。最后,将所有样品收集到装有 10 ml 胶原酶 IV (1 mg/ml) 溶液的 50 ml 管中。

5.     将试管置于摇晃的水浴中,在 37°C 下消化 7 分钟。

6.     向管中加入 20 ml 完全培养基,以在室温下终止消化(图 2D)。

7.     让内容物静置 5 分钟,直到生精索沉入离心管底部。

8.     将上清液转移到新的 50 ml 离心管中,用 40 μm 细胞滤网过滤,250 × g离心10 分钟。

9.     离心后,弃去上清液,加入 2 ml 完全培养基,将细胞重悬于管中。

10.  将细胞悬液均匀地接种到两个含有 5 ml 完全培养基的 100 mm 培养皿中。

11.  5% CO 2培养箱中在 37°C 下孵育菜肴1 小时(图 2E)。

12.  一小时后,去除非贴壁细胞,向培养皿中加入 6 ml 新鲜完全培养基,继续培养 24 小时(图 2F)。

13.  24小时后弃去原培养基,用4ml KCl低渗溶液处理细胞5分钟,进一步去除肌样细胞,PBS3次。

14.  每道菜添加 6 ml 新鲜完全培养基,并继续培养 48 小时(图 2G)。

15.  培养 48 小时后,收获 Leydig 细胞以进行进一步实验。

16.  HSD3B染色以鉴定 Leydig 细胞的纯度(图 2H)。使用一个执行总细胞计数血细胞计数器从十只小鼠中可以获得大约 200 万个细胞。收获大约 20,000 个细胞,将它们重悬在 PBS 中,涂抹在载玻片上,然后晾干。加入50-100 μl HSD3B酶染液覆盖细胞,37°C避光孵育30分钟。丢弃染色溶液并用 PBS 清洗载玻片两次。使用TS100 倒置显微镜捕获图像,并使用 ImageJ 进行分析。

17.  将完全培养基(含 1ng/ml LH)加入 Leydig 细胞,继续培养 48 小时,从第 15 步开始。然后收集上清液,用125-睾酮涂层 A-Count RIA 试剂盒通过放射免疫测定法测量睾酮(RIA) ( 2I) 根据制造商的协议。

18.  Leydig 细胞标记 StARHSD3B SF-1 进行免疫染色(图 2J-2L)。将细胞在 4% 多聚甲醛溶液中固定 10 分钟,用 PBS 清洗细胞 3 次,在 0.5% Triton-X 中透化细胞 10 分钟,然后在室温下使用 5% BSA 封闭非特异性粘附位点 1 小时。稀释抗体稀释溶液中的一抗和二抗。将细胞与一抗溶液(如 StARHSD3B SF-1)在 4°C 下孵育过夜。用 PBS 洗涤 3 次,然后用二抗在室温下孵育细胞 1 小时。用 PBS 洗涤 3 次,用 DAPI 染色细胞核。使用 LSM710 共聚焦显微镜 (蔡司) 捕获图像。

 

2. 小鼠原代 Leydig 细胞的分离和鉴定。

A. KM 小鼠被处死并在 75% 酒精中消毒。B. 睾丸取自处死的小鼠。C. 去除白膜的睾丸。D. 胶原酶 IV 消化后的睾丸。E. 细胞粘附 1 小时(比例尺:100 μm)。F. 细胞粘附 24 小时(比例尺:100 μm)。G. 细胞粘附 72 小时(比例尺:100 μm)。H. 针对 HSD3B 酶(紫色)染色的分离细胞(比例尺:100 μm)。I. 细胞上清液的睾酮水平含有 1 ng/ml LH (LH) 和不含 LH(对照)。J. Leydig 细胞标记物 StAR 的免疫染色(比例尺:20 μm)。K. Leydig 细胞标记物 HSD3B 的免疫染色(比例尺:10 μm)。L. Leydig 细胞标记物 SF-1 的免疫染色(比例尺:20 μm)。


数据分析

 

1.     每张载玻片检查五个随机显微镜视野。HSD3B 酶染色的阳性率是通过 ImageJ 使用分析粒子功能计算的。设置 1,000 作为最小粒径。

2.     只有阳性率超过 85% 的分离细胞才能用于进一步的实验。

 

食谱

 

1.     1 × PBS (pH = 7.4)

8 克氯化钠

0.2 克氯化钾

3.63 Na HPO ·12H O

0.24 KH PO 4

添加 1 L ddH O

121度消毒 30 分钟。

2.     D-PBS (pH = 7.4)

8 克氯化钠

0.2 克氯化钾

1.15 Na HPO ·12H O

0.2 KH PO 4

添加 1 L ddH O

3.     青霉素/链霉素溶液(10×

3.1 克青霉素 G 钾盐

5克硫酸链霉素

50 毫升 1× PBSpH = 7.4

通过 0.22 μm 过滤器进行过滤灭菌。

4.     0.75 mM KCl 低渗缓冲液

14 克氯化钾

添加 250 毫升 ddH O

5.     75% 酒精

750 毫升 100% 乙醇

250 毫升 ddH O

6.     10 毫克/毫升胶原酶 IV 溶液

100 毫克胶原酶 IV

10 毫升 1× PBSpH = 7.4

7.     HSD3B染色液

a.     解决方案一:

1 毫克 NTB

0.6 毫克 DHEA

0.6 毫升二甲基亚砜

b.     解决方案B

10 毫克 β-NAD

9.5 毫升 D-PBS

0.30 ml 溶液 A 加入 4.75 ml 溶液 B 中并充分混合。

8.     完全培养基

10% 胎牛血清

90% DMEM 基础 (1×)

9.     4%多聚甲醛溶液

4.0 克多聚甲醛

添加 100 ml 1×PBS

10.  StAR抗体稀释液

5 μl StAR 抗体

添加 5 ml 一抗和二抗稀释剂用于免疫染色

11.  HSD3B抗体稀释液

5 μl HSD3B 抗体

添加 5 ml 一抗和二抗稀释剂用于免疫染色

12.  SF-1抗体稀释液

5 μl SF-1 抗体

添加 5 ml 一抗和二抗稀释剂用于免疫染色

 

致谢

 

这项工作得到了国家自然科学基金(818711503180123582071634)、广东省自然科学基金(2018A030313473)和广州市科技计划项目(2015080200216104A)的支持201803010044201704YG0662020A1515011203)。该协议改编自以前的工作(Yang等人2020)。

 

利益争夺

 

作者声明他们没有竞争利益。

 

参考

 

1.      Artyukhin, AA Zaraiskii, EI (2007)一种培养 Leydig 细胞的新方法及其在男科学中的实际应用前景。Bull Exp Biol Med 143(1): 155-159

2.     Conn, PMTsuruhara, T.Dufau, M. Catt, KJ (1977)通过密度梯度离心分离高度纯化的 Leydig 细胞。内分泌学1012):639-642

3.      Engeli, RTFürstenberger, C.Kratschmar, DV Odermatt, A.2018 年)。目前可用的鼠 Leydig 细胞系可用于研究类固醇生成的早期步骤,但不能用于研究睾酮合成。太阳系4(2)e00527

4.     Gale, JS, Wakefield, JS 和福特, HC (1982)通过密度梯度离心分离大鼠 Leydig 细胞。J Endocrinol 92(2): 293-302

5.     Jiang, MH, Cai, B., Tuo, Y., Wang, J., Zang, ZJ, Tu, X., Gao, Y., Su, Z., Li, W., Li, G., et al . (2014)将巢蛋白阳性干 Leydig 细胞表征为治疗睾丸 Leydig 细胞功能障碍的潜在来源。细胞研究24(12)1466-1485               

6.      Leary, S. (2013)AVMA 动物安乐死指南。2013 年版。阿拉斯加大学锚地。

7.      Sun, J., Xi, YB, Zhang, ZD, Shen, P., Li, HY, Yin, MZ, Li, WY Shi, CR (2009)Leydig 细胞移植可恢复手术阉割的青春期前大鼠的雄激素生成。

8.     Yang, Y., Zhou, C., Zhang, T., Li, Q., Mei, J., Liang, J., Li, Z., Li, H.,Q., Zhang, Q.,(2020)使用定义的小分子将成纤维细胞转化为功能性 leydig 样细胞。干细胞报告15(2):408-423

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引用:Liang, J., Tang, Y., Li, H., Mei, J., Cao, Z., Xia, H., Huang, R., Yang, Y. and Huang, Y. (2021). Isolation of Primary Leydig Cells from Murine Testis. Bio-protocol 11(22): e4223. DOI: 10.21769/BioProtoc.4223.
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