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Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae
感染淋病奈瑟菌的人宫颈内组织外植体的免疫荧光分析   

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PLOS Pathogens
Apr 2017

Abstract

Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.

Keywords: Neisseria gonorrhoeae (淋病奈瑟菌), Gonorrhea (淋病), Infection (感染), ex vivo (离体), Endocervix (宫颈内), Immunofluorescence staining (免疫荧光染色)

Background

Neisseria gonorrhoeae (GC) infects human genital epithelium causing gonorrhea, a common sexually transmitted infection. Infections in women can lead to severe complications, such as pelvic inflammatory disease, causing fallopian tube scarring and blockage and predisposition to ectopic pregnancy or infertility. Gonorrhea has reemerged as a critical public health issue due to increased prevalence of antibiotic-resistant strains. Because humans are the only host for GC, a lack of an infection model that mimics all aspects of human infections has been a major obstacle to advance our understanding of GC pathogenesis. We have established a human endocervical tissue explant model and immunofluorescence microscopic analysis to examine the mechanism by which GC infect the human endocervix, the primary site for GC infection in women. This ex vivo model maintains the normal cytoarchitecture and tissue integrity of the endocervical epithelium. Using this model and immunofluorescence analysis, we demonstrate that GC colonizes and penetrates into the endocervical tissue, where they potentially cause symptomatic and disseminated gonococcal infection. GC penetration is enabled by the junction disruption and exfoliation of endocervical epithelial cells in response to GC infection. Taken together, our data show that GC infection in endocervical tissue explants resembles GC infection in vivo observed using patients’ biopsies. In combination with immunofluorescent microscopy, this infection model removes an important roadblock to fully understanding the pathogenesis of GC.

Materials and Reagents

  1. Cotton swab (Fisher Scientific, catalog number: 23-400-122 )
    Manufacturer: Thermo Fisher Scientific, catalog number: 16F0024 .
  2. Carbon steel surgical blades #20 (Aspen Surgical, Bard-Parker, catalog number: 371120 )
  3. Petri dishes (VWR, catalog number: 25384-302 )
  4. 6-well tissue culture plates (Corning, Falcon®, catalog number: 353046 )
  5. Sterile Microcentrifuge tubes (1.7 ml) (Sorenson BioScience, catalog number: 16070 )
  6. Sterile 15 ml conical tubes (VWR, catalog number: 21008-216 )
  7. Sterile polyester-tipped applicators (Fisher Scientific, catalog number: 23-400-122 )
  8. Pipette tips (1,000 μl: VWR, catalog number: 83007-382 ; 200 μl: VWR, catalog number: 53509-007 ; 0.1-10 μl: Fisher Scientific, catalog number: 02-717-133 )
  9. PAP pen for immunostaining (Sigma-Aldrich, catalog number: Z377821-1EA )
    Manufacturer: TED PELLA, catalog number: 22309 .
  10. VWR Micro Slides Superfrost Plus (VWR, catalog number: 48311-703 )
  11. Zeiss NO.1.5 cover glasses (ZEISS, catalog number: 474030-9000-000 )
  12. Polysciences flat embedding mold 1.2 x 0.5 x 0.4 cm (Length x Width x Depth) (Polysciences, catalog number: 02615 )
  13. MS11 Neisseria gonorrhoeae strain(s) (kindly provided by Dr. Herman Schneider, Walter Reed Army Institute for Research)
  14. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  15. Liquid nitrogen (Roberts Oxygen Company)
  16. Hoechst 33342, trihydrochloride, trihydrate, 10 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: H3570 )
  17. Alexa Fluor 488 goat anti-mouse IgG1 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21121 )
  18. Mouse anti-human ZO1 (BD, Transduction LaboratoriesTM, catalog number: 610966 )
  19. Goat anti-GC antibody (Lab generated)
  20. DyLight 633 Antibody Labeling Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 53046 )
  21. Aqua Poly/Mount (Polysciences, catalog number: 18606 )
  22. Nail polish (Electron Microscopy Science, catalog number: 72180 )
  23. O.C.T. Compound (SAKURA, Tissue-Tek, catalog number: 4583 )
  24. Penicillin G sodium salt (Sigma-Aldrich, catalog number: P3032-25MU )
  25. Streptomycin sulfate (Sigma-Aldrich, catalog number: S9137 )
  26. Leibovitz’s (1x) L-15 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21083027 )
  27. CMRL medium 1066 (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11530037 )
  28. Hydrocortisone 21-hemisuccinate sodium (Sigma-Aldrich, catalog number: H2270-100MG )
  29. Bovine insulin (Akron Biotech, catalog number: AK8213-0100 )
  30. L-glutamine 200 mM (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  31. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  32. DifcoTM GC medium base (BD, DifcoTM, catalog number: 228950 )
  33. Agar (United States Biological, catalog number: A0930 )
  34. L-glutamine, White Crystals or Crystalline Powder (Fisher Scientific, catalog number: BP379-100 )
  35. Ferric nitrate, nonahydrate (Sigma-Aldrich, catalog number: 254223-10G )
  36. Thiamine pyrophosphate (Sigma-Aldrich, catalog number: C8754-5G )
  37. Glucose, as Dextrose anhydrous (Fisher Scientific, catalog number: BP350-1 )
  38. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-10 )
  39. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  40. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-1 )
  41. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: BP329-1 )
  42. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
    Manufacturer: Thermo Fisher Scientific, catalog number: FLA144212 .
  43. Paraformaldehyde 16% solution (Electron Microscopy Science, catalog number: 15710 )
  44. Sucrose (Merck, catalog number: SX1075-1 )
  45. Gelatin from porcine skin (Sigma-Aldrich, catalog number: G1890-500G )
  46. Gelatin from cold water fish skin (FSG) (Sigma-Aldrich, catalog number: G7765-250ML )
  47. Triton X-100 (Sigma-Aldrich, catalog number: X100-100ML )
  48. 100x penicillin/streptomycin stock (see Recipes)
  49. Cervical tissue culture medium (see Recipes)
  50. GCK agar plate (see Recipes)
  51. 100x Kellogg’s supplement (see Recipes)
  52. 1x phosphate buffer solution (PBS) (see Recipes)
  53. 4% paraformaldehyde (PFA) (see Recipes)
  54. 5% and 15% sucrose solution (see Recipes)
  55. 7.5% and 20% gelatin solution (see Recipes)
  56. Staining solution (see Recipes)

Equipment

  1. Biosafety cabinet (NU-425-600 Class II, A2 Laminar Flow Biohazard Hood) (Nuaire, model: NU-425-600 Class II, A2 , catalog number: 32776)
  2. Spectrophotometer Ultrospec 2000 UV (Pharmacia Biotech, model number: 80-2106-00 )
  3. Heating block (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SP18425Q )
  4. Stainless steel forceps (VWR, Dissecting forceps)
  5. Multi-Purpose rotator (Multi-Purpose Rotator) (Barnstead International Lab-line, Thermo Fisher Scientific, Thermo ScientificTM, model: Model 2309 )
  6. CO2 incubator (Fisher Scientific, model: Isotemp Incubator Model 3530 )
  7. Confocal microscope (Carl Zeiss, model: LSM 710 )
  8. Cryostat (Thermo Fisher Scientific, model: Microm HM 550-388114 )
  9. Slide holder (Newcomer Supply, catalog number: 6841 )

Software

  1. NIH ImageJ and ZEN software
  2. GraphPad Software (La Jolla, CA)

Procedure

  1. Sample collection and shipping
    Cervical tissues (~5 g/piece) from patients undergoing voluntary hysterectomy are obtained in sterile DMEM with 1x penicillin/streptomycin (see Recipes) at 4 °C within 24 h post-surgery (Merbah et al., 2011), coordinated by National Disease Research Interchange (NDRI) (an institutional review board must approve the research protocol involving human tissues. Please verify with your own institutional review board for the regulation).

  2. Tissue preparation
    The tissue explants were cultured based on a method described by Schürch et al. (1978).
    1. Rinse tissues three times with cold HBSS.
    2. Rinse tissues three times with cold Leibovitz’s (1x) L-15 medium.
    3. Remove mucus gently using a cotton swab.
    4. Remove muscle tissues using a carbon steel surgical blade (Figure 1A, black dashed line).
    5. Separate the endocervical region from the rest of the cervix tissue explant using a carbon steel surgical blade (Figure 1A). Store the endocervix for the next steps of the protocol while the rest of the cervix is discarded.
    6. Rinse endocervical tissue explants three times with cervical tissue culture medium.
    7. Incubate endocervical tissue explants in a 6-well tissue culture plate, one piece per well, in cervical tissue culture medium at 37 °C with 5% CO2 for 24 h.
    8. Take a picture of tissue along with its well.
    9. Determine the area of the tissue luminal surface (dotted line in Figure 1B) by measuring the tissue to well size ratio using NIH ImageJ software.
    10. Wash the endocervical tissue explants three times with antibiotic-free cervical tissue culture medium (see Recipes), and incubate the tissue in the antibiotic-free media at 37 °C with 5% CO2 for 24 h before bacterial inoculation.


      Figure 1. Endocervical tissue explant in culture. The endocervical tissues (A), after removing mucus, muscle tissue and other parts of the cervix, are trimmed into the desired size (B) and cultured in the cervical tissue culture medium in a six-well plate. The black dashed lines indicate the cutting line on tissues and the yellow dash line circles the luminal surface of the endocervix. Scale bar = 10 mm.

  3. Infection of the endocervix with GC
    1. Grow GC on GCK agar plate (see Recipes) for 16-18 h.
    2. Collect GC by using a sterile applicator to swab GC from the plate and re-suspend GC in pre-warmed antibiotic-free cervical tissue culture media. Determine the number of bacteria by spectrophotometry at a wavelength of 650 nm (an OD650 of 1 = 1 x 109 CFU/ml).
    3. Estimate the number of epithelial cells by dividing the luminal surface area of a tissue explant by the average luminal surface area of individual endocervical epithelial cells (25 µm2) (Figure 3D).
    4. Add an aliquot of the GC suspension (1 ml) directly onto each tissue piece to make an MOI = 10 bacteria to 1 epithelial cell and incubated at 37 °C with 5% CO2 for 3 min.
    5. Add 1 ml of antibiotic-free cervical tissue culture medium (see Recipes) to each of the above tissue pieces.
    6. Incubate tissue explants at 37 °C with 5% CO2 with gentle shaking for 24 h. Rinse infected tissue explants 6 times with 3 ml antibiotic-free cervical tissue culture medium at 6 and 12 h to remove un-adhered bacteria and replace with 2 ml of fresh media without bacteria.

  4. Tissue preparation for cryosection–Fixation
    1. Rinse tissue explants three times with 3 ml PBS (see Recipes).
    2. Fix tissue explants with 3 ml 4% paraformaldehyde (in PBS) (see Recipes) for 1 h at room temperature.
    3. Rinse tissue explant three times with PBS.
    4. Cut tissues into a desired size, approximately 1.0 x 0.5 x 0.2 cm (Length x Width x Depth), using a razor blade.

  5. Tissue preparation for cryosection–Infiltration & Embedding
    1. Incubate tissue explants in 5 ml of 5% sucrose in PBS (see Recipes) at room temperature for 30 min until the explants settle to the bottom of the tube.
    2. Carefully remove the 5% sucrose and resuspend the tissue in 5 ml of 15% sucrose (in PBS) (see Recipes) and incubated overnight at 4 °C.
    3. Carefully remove the sucrose and resuspend tissue explants in 7.5% porcine skin gelatin in PBS (see Recipes) for 4 h at 37 °C.
    4. Carefully remove the gelatin solution and resuspend tissue explants in 5 ml pre-warmed 20% porcine skin gelatin in PBS (see Recipes) and incubated for 2 h at 37 °C.
    5. Pre-warm a flat embedding mold (Figure 2A) for each piece of tissues on a heating block at 37 °C.
    6. Fill the bottom 1/3 of molds with 20% gelatin and let it solidify at room temperature.
    7. Put one tissue piece per mold and top with 20% gelatin at 37 °C. Arrange the orientation of the tissue using pipette tips as shown in Figure 2A to have luminal surface at the top. Make sure the tissue piece is fully embedded in gelatin (Figure 2B).
    8. Let samples sit for 5 min at room temperature and then on ice block for 10 min.
    9. Use long forceps to place the mold containing tissues into liquid nitrogen vapor until the gelatin turns white.
    10. Dip the mold into liquid nitrogen for a few seconds till the samples are all frozen.
    11. Push sample blocks out of molds by warming the bottom of the mold at 42 °C on a heating block for 30 sec.
    12. Store the embedded samples in conical tubes at -80 °C.

  6. Cryosection
    1. Place a sample block on the stage of a cryostat in an orientation, which enables sectioning through both the luminal surface and the subepithelium (Figure 2C).
    2. Section sample blocks in 40 μm thickness until reaching the tissue.
    3. Switch to desired section thickness ranging from 10-40 μm.
    4. Collect sample sections by attaching them to glass slides (Figure 2C).
    5. Store cryosections on glass slides at 4 °C for the same day processing or -20 or -80 °C for future processing.


      Figure 2. Embedding and sectioning of endocervical tissues. A-B. Endocervical tissues are embedded with the luminal side up in 20% gelatin. Black arrow, the direction that the tissue is positioned. C. Endocervical tissue blocks are sectioned transversally through both the luminal surface and the subepithelial tissue. Endocervical tissue sections are collected using super frost plus slides for immunofluorescence staining.

  7. Immunofluorescence staining for imaging
    1. Incubate tissue sections on glass slides in a slide holder with PBS at 42 °C for 10 min to remove gelatin.
    2. Wash tissue sections three times with PBS.
    3. Aspirate excess PBS and draw an aqueous barrier circle round tissue sections using a pap pen.
    4. Fill the circle with the staining solution (see Recipes) and incubate at room temperature for 1 h for permeabilizing and blocking.
    5. Incubate tissue sections with a primary antibody in the staining solution at room temperature for 1 h. (Table 1)

      Table 1. The antibodies/chemicals and the final concentration used in Figures 2 and 3

      Note: Fluorescent phalloidin and immunofluorescence E-cadherin staining can also be included to assess the cellular location of GC.

    6. Rinse tissue sections three times with the staining solution.
    7. Incubate tissue sections with a secondary antibody in the staining solution at room temperature for 1 h. (Table 1)
    8. Rinse tissue sections three times with the staining solution.
    9. Incubate tissue sections with Hoechst 33342 (0.2 μg/ml) in the staining solution at room temperature for 10 min. (Table 1)
    10. Rinse tissue sections three times with the staining solution.
    11. Mount tissue sections using Aqua/poly mounting media and No.1.5 or No. 1 coverslip glass. Dry for 5 min before sealing the edge of coverslip using nail polish.
    12. Slides are now ready for imaging or can be stored at 4 °C for up to 2 months.

  8. Imaging
    1. Acquire images using a ZEISS LSM 710 confocal microscope (an equivalent microscope can be used).
    2. Acquire Z-stack of 0.37 μm slices from the top to the bottom of a tissue section (Figure 3A).
    3. Reconstitute Z-stack images into 3D-images using ImageJ or ZEN software (Figure 3B), which allows obtaining both the transversal (Figure 3C) and luminal views (Figure 3D) of the endocervical epithelium.


      Figure 3. 3D-reconstruction of fluorescence images. Cryo-sections of endocervical tissue explants were stained for ZO-1 (green), GC (red), and DNA (blue). Z-stack images were taken using a confocal fluorescent microscope. Shown are series of Z-stack images (A) and their 3D reconstruction (B). Images of XZ view (C) and XY view (D) of the luminal surface are then generated for publication. The red dashed line circles the luminal surface of an epithelial cell (~25 µm2). Scale bar = 10 µm.

Data analysis

In our published studies, we quantitatively analyzed the penetration of GC into the subepithelium of the endocervix and the exfoliation of endocervical epithelial cells.

  1. To estimate the level of GC penetration into the subepithelium, the number of non-exfoliated endocervical epithelial cells (in visible epithelial monolayers) with GC staining at the basal side and the total number of GC-associated epithelial cells in randomly acquired image are visually counted to calculate the percentage of infected epithelial cells with GC penetration into the epithelium (Figure 4A).
  2. To quantify epithelial exfoliation, the number of endocervical epithelial cells localized on the top of the monolayer (exfoliated) and the total number of endocervical epithelial cells were counted by visual inspection in each randomly acquired image to determine the percentage of exfoliated cells (Figure 4B).
  3. Quantified values are plotted into column graphs, and statistical significance is assessed using the Student’s t-test and one-way ANOVA by Prism (GraphPad Software, La Jolla, CA).


    Figure 4. Fluorescent image analysis of GC infection in the endocervical tissue explant. Cryo-sections of cervical tissue explants were stained for ZO-1 (green), GC (red), and DNA (blue). Shown are representative images that cross both the luminal and basal surfaces of epithelial cells (Scale bars = 10 μm). A. GC subepithelial penetration was quantified using immunofluorescence images as the percentage of epithelial cells with basal GC staining (arrows) among the total number of GC-associated epithelial cells. B. The average percentage of exfoliated epithelial cells was determined by the number of epithelial cells localizing above the endocervical epithelium (white dash lines) versus the total number of epithelial cells, based on the nuclear staining.

Recipes

  1. 100x penicillin/streptomycin (P/S) stock
    10,000 UI/ml penicillin
    10,000 μg/ml streptomycin
    Phenol Red-free DMEM/F12 medium
    Store at -20 °C
  2. Cervical tissue culture medium
    CMRL-1066 medium
    0.1 μg/ml hydrocortisone 21-hemisuccinate sodium
    1 μg/ml bovine insulin
    2 mM L-glutamine
    5% FBS
    1x penicillin-streptomycin (P/S)
    Store at 4 °C
  3. GCK agar plate (for 1 L)
    35 g Difco GC medium base
    6 g agar
    10 ml 100x Kellogg’s supplement
    Store at 4 °C
  4. 100x Kellogg’s supplement (for 1 L)
    5 g L-glutamine Crystalline Powder
    0.5 g ferric nitrate, anhydrous
    0.02 g thiamine pyrophosphate
    400 g glucose
    Store at 4 °C
  5. 1x phosphate buffer solution (PBS) (for 1 L)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Adjust pH to 7.4 with HCl
    Store at 4 °C
  6. 4% paraformaldehyde (PFA)
    30 ml 16% paraformaldehyde stock
    10 ml 1x PBS
    Store at -20 °C
  7. 5% and 15% sucrose solution
    5 g (for 5%) or 15 g (for 15%) sucrose
    100 ml 1x PBS
    Store at -20 °C
  8. 7.5% and 20% gelatin solution
    7.5 g (for 7.5%) or 20 g (for 20%) porcine skin gelatin
    100 ml 1x PBS
    Heat to solubilize
    Store at -20 °C
  9. Staining solution
    1x PBS
    0.66% Fish skin gelatin (FSG)
    0.2% Triton X-100
    Store at 4 °C

Acknowledgments

We would like to thank NDRI for tissue collection. This work was supported by NIH grants (RO1 AI068888, R21 AI103797, and RO1 AI123340 to WS and DCS). This protocol was partially adapted from previously published work by Merbah et al. (2011) and Schürch et al. (1978). The authors declare no conflicts of interest within this work.

References

  1. Merbah, M., Introini, A., Fitzgerald, W., Grivel, J. C., Lisco, A., Vanpouille, C. and Margolis, L. (2011). Cervico-vaginal tissue ex vivo as a model to study early events in HIV-1 infection. Am J Reprod Immunol 65(3): 268-278.
  2. Schürch, W., McDowell, E. M. and Trump, B. F. (1978). Long-term organ culture of human uterine endocervix. Cancer Res 38(11 Pt 1): 3723-3733.

简介

上皮的定植和穿透是粘膜病原体的感染启动途径。 上皮细胞通过引发宿主细胞应答来抵抗感染,同时维持粘膜屏障功能。 专性人类性传播细菌淋病奈瑟氏球菌或淋球菌属(GC)主要从宫颈内膜上皮感染女性生殖道。 由于缺乏模仿女性生殖道人类感染的各个方面的感染模型,GC发病机制知之甚少。 该方案利用培养物中繁殖的人宫颈组织的生存力和功能完整性来产生体外感染模型。 这种组织模型保持了感染目标和环境的性质,而没有任何操作,如病毒永生化上皮细胞。 使用免疫荧光显微镜,分析GC与子宫颈上皮细胞的相互作用。


【背景】淋病奈瑟菌(GC)感染人类生殖器上皮,引起淋病,一种常见的性传播感染。女性感染可导致严重的并发症,如盆腔炎,引起输卵管瘢痕和堵塞,易发生异位妊娠或不孕。由于抗生素耐药菌株的流行率增加,淋病又成为重要的公共卫生问题。由于人类是GC的唯一宿主,因此缺乏模仿人类感染各个方面的感染模型是推进我们对GC发病机理的理解的主要障碍。我们建立了人宫颈组织外植体模型和免疫荧光显微镜分析来检测GC感染妇女GC感染的主要部位人间宫颈细胞的机制。这种体外模型保持了宫颈上皮细胞的正常细胞构筑和组织完整性。使用这种模型和免疫荧光分析,我们表明,气相色氨酸定居和渗透到子宫颈组织,在那里他们可能导致症状和播散性淋球菌感染。 GC渗透是由于宫颈上皮细胞响应GC感染的交界处破裂和剥落而实现的。总之,我们的数据显示,在宫颈组织外植体中的GC感染类似于使用患者活组织检查观察到的体内GC感染。结合免疫荧光显微镜,这种感染模型消除了一个重要的路障,充分了解GC的发病机制。

关键字:淋病奈瑟菌, 淋病, 感染, 离体, 宫颈内, 免疫荧光染色

材料和试剂

  1. 棉签(Fisher Scientific,目录号:23-400-122)
    制造商:Thermo Fisher Scientific,产品目录号:16F0024。
  2. 碳钢手术刀#20(Aspen Surgical,Bard-Parker,目录号:371120)
  3. 培养皿(VWR,目录号:25384-302)
  4. 6孔组织培养板(Corning,Falcon,目录号:353046)
  5. 无菌微量离心管(1.7ml)(Sorenson BioScience,目录号:16070)
  6. 无菌15毫升锥形管(VWR,目录号:21008-216)
  7. 无菌聚酯涂抹器(Fisher Scientific,目录号:23-400-122)
  8. 移液管吸头(1,000μl:VWR,目录号:83007-382;200μl:VWR,目录号:53509-007;0.1-10μl:Fisher Scientific,目录号:02-717-133)
  9. 用于免疫染色的PAP笔(Sigma-Aldrich,目录号:Z377821-1EA)
    制造商:TED PELLA,产品目录号:22309。
  10. VWR Micro Slides Superfrost Plus(VWR,目录号:48311-703)
  11. 蔡司NO.1.5盖玻片(蔡司,目录号:474030-9000-000)
  12. Polysciences平包埋模具1.2 x 0.5 x 0.4厘米(长x宽x深)(Polysciences,目录号:02615)
  13. MS11淋病奈瑟氏球菌菌株(由沃尔特•里德陆军研究所的Herman Schneider博士提供)
  14. Hank's平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14025092)
  15. 液氮(罗伯茨氧气公司)
  16. Hoechst 33342,三盐酸盐,三水合物,10mg / ml(Thermo Fisher Scientific,Invitrogen TM,目录号:H3570)
  17. Alexa Fluor 488山羊抗小鼠IgG 1(Thermo Fisher Scientific,Invitrogen,目录号:A-21121)
  18. 小鼠抗人ZO1(BD,Transduction Laboratories TM,目录号:610966)
  19. 山羊抗GC抗体(实验室产生)
  20. DyLight 633抗体标记试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:53046)
  21. Aqua Poly / Mount(Polysciences,产品目录号:18606)
  22. 指甲油(电子显微镜科学,目录号:72180)
  23. O.C.T.化合物(SAKURA,Tissue-Tek,目录号:4583)
  24. 青霉素G钠盐(Sigma-Aldrich,目录号:P3032-25MU)
  25. 硫酸链霉素(Sigma-Aldrich,目录号:S9137)
  26. Leibovitz's(1×)L-15培养基(Thermo Fisher Scientific,Gibco TM,目录号:21083027)
  27. CMRL培养基1066(1x)(Thermo Fisher Scientific,Gibco TM,目录号:11530037)
  28. 氢化可的松21-半琥珀酸钠(Sigma-Aldrich,目录号:H2270-100MG)
  29. 牛胰岛素(Akron Biotech,目录号:AK8213-0100)
  30. L-谷氨酰胺200mM(100x)(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  31. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  32. DifcoTM GC培养基(BD,Difco TM,产品目录号:228950)
  33. 琼脂(美国生物,目录号:A0930)
  34. L-谷氨酰胺,白色晶体或结晶粉末(Fisher Scientific,目录号:BP379-100)
  35. 硝酸铁,九水合物(Sigma-Aldrich,目录号:254223-10G)
  36. 硫胺素焦磷酸(Sigma-Aldrich,目录号:C8754-5G)
  37. 葡萄糖,如无水葡萄糖(Fisher Scientific,目录号:BP350-1)
  38. 氯化钠(NaCl)(Fisher Scientific,目录号:S671-10)
  39. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  40. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-1)
  41. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:BP329-1)
  42. 盐酸(HCl)(Fisher Scientific,目录号:A144-212)
    制造商:Thermo Fisher Scientific,产品目录号:FLA144212。
  43. 多聚甲醛16%溶液(电子显微镜科学,目录号:15710)
  44. 蔗糖(Merck,目录号:SX1075-1)
  45. 来自猪皮的明胶(Sigma-Aldrich,目录号:G1890-500G)
  46. 来自冷水鱼皮(FSG)的明胶(Sigma-Aldrich,目录号:G7765-250ML)
  47. Triton X-100(Sigma-Aldrich,目录号:X100-100ML)
  48. 100倍青霉素/链霉素库存(见食谱)
  49. 宫颈组织培养基(见食谱)
  50. GCK琼脂平板(见食谱)
  51. 100倍凯洛格的补充(见食谱)
  52. 1x磷酸缓冲液(PBS)(见食谱)
  53. 4%多聚甲醛(PFA)(见食谱)
  54. 5%和15%蔗糖溶液(见食谱)
  55. 7.5%和20%的明胶溶液(见食谱)
  56. 染色液(见食谱)

设备

  1. 生物安全柜(NU-425-600 Class II,A2层流生物危害罩)(Nuaire,型号:NU-425-600 Class II,A2,目录号:32776)
  2. 分光光度计Ultrospec 2000 UV(Pharmacia Biotech,型号:80-2106-00)
  3. 加热块(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SP18425Q)
  4. 不锈钢钳(VWR,解剖钳)
  5. 多功能旋转器(多功能旋转器)(Barnstead International Lab-line,Thermo Fisher Scientific,Thermo Scientific TM,型号:Model 2309)
  6. CO 2培养箱(Fisher Scientific,型号:Isotemp培养箱,型号3530)。
  7. 共焦显微镜(卡尔蔡司,型号:LSM 710)
  8. Cryostat(Thermo Fisher Scientific,型号:Microm HM 550-388114)
  9. 幻灯片架(新人供应,目录号:6841)

软件

  1. NIH ImageJ和ZEN软件
  2. GraphPad软件(La Jolla,CA)

程序

  1. 样品收集和运输
    使用1x青霉素/链霉素的无菌DMEM(参见食谱)在手术后24小时内在4℃下获得子宫颈切除术(5g /块) ,2011),由国家疾病研究交流中心(NDRI)协调(机构审查委员会必须批准涉及人体组织的研究协议,请与您自己的机构审查委员会核实有关规定)。

  2. 组织准备
    基于Schürch等人描述的方法培养组织外植体。 (1978)。
    1. 用冷HBSS冲洗纸巾三次。
    2. 用冷的Leibovitz(1x)L-15培养基冲洗纸巾3次。

    3. 使用棉签轻轻地去除粘液

    4. 使用碳钢手术刀去除肌肉组织(图1A,黑色虚线)
    5. 使用碳钢外科刀片将子宫颈内区域与子宫颈组织外植体的其余部分分开(图1A)。存储endocervix协议的下一个步骤,而宫颈的其余部分被丢弃。

    6. 用宫颈组织培养基冲洗宫颈组织外植体三次
    7. 在宫颈组织培养基中,在37℃,5%CO2下孵育宫颈内组织培养板6孔组织培养板,每孔一个,24小时。

    8. 拍摄组织的照片
    9. 通过使用NIH ImageJ软件测量组织对大小的比例来确定组织腔表面的面积(图1B中的虚线)。
    10. 用无抗生素的宫颈组织培养基清洗宫颈组织外植体三次(见食谱),并将组织在无抗生素的培养基中于37℃用5%CO 2孵育24小时细菌接种。


      图1宫颈组织外植体的培养将子宫颈组织(A)取出宫颈粘液,肌肉组织及其他部位后,剪成所需的大小(B),在颈椎将组织培养基置于六孔板中。黑色的虚线表示组织上的切割线,黄色的虚线表示宫颈管腔的表面。比例尺= 10毫米。

  3. 气雾剂感染endocervix
    1. 在GCK琼脂平板上生长GC(见食谱)16-18小时。
    2. 用无菌涂药器收集GC,从平板上擦拭GC,在预热的无抗生素宫颈组织培养基中重新悬浮GC。通过分光光度法在650nm波长(1×10 9 CFU / ml的OD 650)下测定细菌的数量。
    3. 通过将组织外植体的腔表面积除以各个宫颈上皮细胞(25μm2)的平均腔表面积(图3D)来估计上皮细胞的数量。
    4. 直接在每个组织片上加入等分试样的GC悬浮液(1ml)以制备MOI = 10个细菌至1个上皮细胞,并在37℃下用5%CO 2孵育3分钟。 br />
    5. 每个上述组织片添加1毫升无抗生素宫颈组织培养基(见食谱)。
    6. 在37℃下用5%CO 2孵育组织外植体,轻轻摇动24小时。用3ml无抗生素宫颈组织培养基在6和12小时清洗感染组织外植体6次,以去除未粘附的细菌,并用2ml新鲜培养基替换,而不含细菌。

  4. 冷冻固定的组织准备
    1. 用3毫升的PBS冲洗组织外植体三次(见食谱)。
    2. 用3ml 4%多聚甲醛(在PBS中)固定组织外植体(参见食谱),室温下1小时。

    3. 用PBS冲洗组织外植体三次
    4. 使用剃刀将组织切成所需尺寸,大约1.0×0.5×0.2cm(长×宽×深)。

  5. 冷冻切片的组织准备 - 渗透&嵌入
    1. 将组织外植体在室温下在5ml含5%蔗糖的PBS(参见食谱)中室温孵育30分钟,直到外植体沉降到管底。
    2. 小心地取出5%的蔗糖,并悬浮在5毫升15%蔗糖(在PBS中)的组织(见食谱),并在4°C孵育过夜。
    3. 小心取出蔗糖,并在PBS中7.5%猪皮明胶重新悬浮组织外植体(见食谱)在37°C 4小时。
    4. 小心地取出明胶溶液,并重新悬浮组织外植体5毫升预热20%猪皮明胶在PBS中(见食谱),并在37°C温育2小时。

    5. 在一个加热块上预热一个扁平包埋模具(图2A)

    6. 用20%的明胶填充模具的底部1/3,并在室温下固化。
    7. 每个模具放上一块薄纸,顶部放上20%的明胶,37℃。如图2A所示使用移液管尖端来排列组织的取向,以在顶部具有腔表面。确保组织块完全嵌入明胶(图2B)。
    8. 让样品在室温下放置5分钟,然后在冰块上放置10分钟。
    9. 使用长镊子将含有组织的霉菌放入液氮蒸气中直至明胶变白。
    10. 将模具浸入液氮中几秒钟,直到样品全部冷冻。
    11. 通过在42°C的加热块上加热模具底部30秒,将样品块从模具中推出。
    12. 将嵌入的样品储存在-80°C的锥形管中。

  6. 冰冻切片

    1. 在低温恒温器的台面上放置一个样品块,使其能够穿过管腔表面和上皮下层(图2C)。
    2. 切片样本块40微米厚,直到到达组织。

    3. 切换到所需的部分厚度从10-40微米
    4. 收集样品部分,将其连接到载玻片上(图2C)。
    5. 将玻片上的冷冻切片放置在4°C的同一天处理或-20或-80°C以备将来处理。


      图2.宫颈内膜组织的嵌入和切片图A-B。将宫颈内侧组织以20%明胶包埋。黑色箭头,组织定位的方向。 C.宫颈组织块横向穿过腔表面和上皮下组织。宫颈组织切片收集使用超级霜加幻灯片进行免疫荧光染色。

  7. 免疫荧光染色成像

    1. 用42℃的PBS在载玻片上的玻片上孵育组织切片10分钟,以去除明胶。

    2. 用PBS清洗组织切片三次
    3. 吸取多余的PBS,并用纸笔在组织切片上画出水性屏障圈。
    4. 用染色溶液填充圆圈(参见食谱)并在室温下孵育1小时进行透化和封闭。
    5. 在染色溶液中室温孵育组织切片与第一抗体1小时。 (表1)

      表1.图2和3中使用的抗体/化学物质和最终浓度

      注:荧光鬼笔环肽和免疫荧光E-钙粘蛋白染色也可以包括在内以评估GC的细胞定位。


    6. 用染色液冲洗组织切片三次
    7. 在染色溶液中用二次抗体在室温孵育组织切片1小时。 (表1)
    8. 用染色液冲洗组织切片三次。
    9. 用Hoechst33342(0.2μg/ ml)在染色溶液中室温孵育组织切片10分钟。 (表1)

    10. 用染色液冲洗组织切片三次
    11. 使用Aqua /聚安装媒体和1.5号或1号盖玻片安装组织切片。干燥5分钟,然后用指甲油密封盖玻片的边缘。
    12. 幻灯片现在可以进行成像,或者可以在4°C下保存2个月。

  8. 成像
    1. 使用蔡司LSM 710共聚焦显微镜(可使用等效显微镜)获取图像。

    2. 从组织切片的顶部到底部采集0.37μm切片的Z-叠层(图3A)
    3. 使用ImageJ或ZEN软件(图3B)将Z-Stack图像重建为三维图像,从而获得宫颈上皮细胞的横向(图3C)和管腔视图(图3D)。


      图3:荧光图像的三维重建对宫颈组织外植体的低温切片进行染色, , GC(红色) DNA(蓝色) 。使用共焦荧光显微镜拍摄Z-堆叠图像。所示为一系列的Z-堆叠图像(A)及其三维重建(B)。然后生成管腔表面的XZ视图(C)和XY视图(D)的图像用于出版。红色虚线环绕上皮细胞的腔表面(〜25μm2)。比例尺= 10微米。

数据分析

在我们发表的研究中,我们定量分析了气管上皮细胞在宫颈上皮内的渗透和宫颈上皮细胞的脱落。

  1. 为了估计GC渗透到上皮下的水平,目视统计在基底侧具有GC染色的非脱落的宫颈上皮细胞(在可见的上皮单层中)和随机获取的图像中的GC相关上皮细胞的总数目计算GC渗透到上皮细胞中的感染上皮细胞的百分比(图4A)。
  2. 为了量化上皮剥脱,在每个随机获取的图像中通过目视检查计数位于单层顶部(剥落的)上的宫颈上皮细胞的数量和宫颈上皮细胞的总数,以确定剥落细胞的百分比(图4B) 。
  3. 将量化值绘制成柱状图,使用Prism(GraphPad Software,La Jolla,CA)的Student's t检验和单向ANOVA评估统计显着性。


    图4.在宫颈组织外植体中GC感染的荧光图像分析。对宫颈组织外植体的低温切片进行染色,以显示出对照的结果。 ZO-1(green) DNA(blue) =“color:#E53333;”> GC(红色)

食谱

  1. 100x青霉素/链霉素(P / S)股票
    10,000 UI / ml青霉素
    10,000μg/ ml链霉素
    酚红无DMEM / F12培养基
    在-20°C储存
  2. 宫颈组织培养基
    CMRL-1066中等
    0.1μg/ ml氢化可的松21-半琥珀酸钠
    1μg/ ml牛胰岛素
    2mM L-谷氨酰胺
    5%FBS
    1x青霉素 - 链霉素(P / S)
    在4°C储存
  3. GCK琼脂平板(1升)
    35克Difco GC中等基地
    6克琼脂
    10毫升100克凯洛格的补充
    在4°C储存
  4. 100倍凯洛格的补充(1升)
    5克L-谷氨酰胺结晶粉
    0.5克硝酸铁,无水
    0.02克焦磷酸硫胺素
    400克葡萄糖
    在4°C储存
  5. 1x磷酸缓冲液(PBS)(1L)
    8克NaCl
    0.2克KCl
    1.44克Na 2 HPO 4 4 0.24克KH 2 PO 4 4 用HCl调节pH至7.4
    在4°C储存
  6. 4%多聚甲醛(PFA)
    30毫升16%多聚甲醛储备
    10毫升1x PBS
    在-20°C储存
  7. 5%和15%的蔗糖溶液
    5克(5%)或15克(15%)蔗糖
    100毫升1x PBS
    在-20°C储存
  8. 7.5%和20%明胶溶液
    7.5克(7.5%)或20克(20%)猪皮明胶
    100毫升1x PBS
    加热溶解
    在-20°C储存
  9. 染色解决方案
    1x PBS
    0.66%鱼皮明胶(FSG)
    0.2%Triton X-100
    在4°C储存

致谢

我们要感谢NDRI的纸巾收集。这项工作得到了NIH资助(RO1 AI068888,R21 AI103797和RO1 AI123340到WS和DCS)的支持。该协议部分改编自以前由Merbah等人(2011年)和Schürch等人(1978年)发表的工作。作者声明在这项工作中没有利益冲突。

参考

  1. Merbah,M.,Introini,A.,Fitzgerald,W.,Grivel,J.C。,Lisco,A.,Vanpouille,C.和Margolis,L。(2011)。 宫颈阴道组织离体作为研究早期事件的模型HIV-1感染。 65(3):268-278。
  2. Schürch,W.,McDowell,E.M。和Trump,B.F。(1978)。 人类子宫内膜异位症的长期器官培养。
    38(11 Pt 1):3723-3733。
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引用:Wang, L., Yu, Q., Stein, D. C. and Song, W. (2018). Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae. Bio-protocol 8(3): e2720. DOI: 10.21769/BioProtoc.2720.
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