Primary Cultures from Human GH-secreting or Clinically Non-functioning Pituitary Adenomas

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Molecular Neurobiology
Sep 2017



Pituitary adenomas are among the more frequent intracranial tumors usually treated with both surgical and pharmacological–based on somatostatin and dopamine agonists–approaches. Although mostly benign tumors, the occurrence of invasive behaviors is often detected resulting in poorer prognosis. The use of primary cultures from human pituitary adenomas represented a significant advancement in the knowledge of the mechanisms of their development and in the definition of the determinants of their pharmacological sensitivity. Moreover, recent studies identified also in pituitary adenomas putative tumor stem cells representing, according to the current hypothesis, the real cellular targets to eradicate most malignancies. In this protocol, we describe the procedure to establish primary cultures from human pituitary adenomas, and how to select, in vitro expand, and phenotypically characterize putative pituitary adenoma stem cells.

Keywords: Pituitary adenoma (垂体腺瘤), Cancer stem cells (癌症干细胞), Primary cultures (原代培养物)


Pituitary adenomas are among the most common intracranial neoplasms (up to 15%) and cross-sectional studies have found a prevalence of around 90 cases per 100,000 inhabitants, with the vast majority being adults over 30 years old. Approximately 10% of unselected pituitaries examined at autopsy (i.e., considering also the pituitaries of subjects without previous diagnosis of pituitary disease) have developed adenomas (Molitch, 2017). Although often benign tumors, the management of pituitary adenomas can be complicated by the clinical syndromes related to hormone hypersecretion, or by the development of aggressive behavior characterized by resistance to treatment, high proliferation rate, rapid recurrence and extrasellar invasion (Carreno et al., 2017). The persistence of stem cells in adult pituitary (Florio, 2011) led to the hypothesis that the development of pituitary adenomas (and possibly of other benign neoplasia) can derive from subpopulations of tumor cells endowed with stem cell properties (mainly self-renewal and differentiation ability), as already established for malignant solid and hematologic tumors.

Recently experimental evidence showed that cancer stem cells (CSC) paradigm also applies to human and mouse pituitary adenomas (Donangelo et al., 2014; Peverelli et al., 2017; Wurth et al., 2017), and it was proposed that oncogenically-transformed CSCs can originate the tumor, clonally. The concept of CSC as tumor-initiating cells (TICs), initially developed for malignant neoplasia, proposes that only a subset of tumor cells, the CSC subpopulations, is responsible of initiating and maintaining tumor growth, causing invasiveness and the formation of metastasis, and conferring drug resistance (Clevers, 2011; Florio and Barbieri, 2012). This theory, leading to a cell hierarchy within a given tumor, replaced the stochastic model of cancerogenesis that proposed the bulk of solid tumors to be composed of cells showing equal tumorigenic potential. A more recent evolution of the CSC model is the ‘dynamic-stemness’ theory, which postulates an interchange between CSCs and more-differentiated (progenitors) cells, determined by epigenetic and microenvironmental signals, transcription factors, miRNAs or the activation of oncogenic pathways (Li and Laterra, 2012).

Besides the relevance from a theoretical point of view, the identification of putative CSCs in pituitary adenomas could represent the basis to identify possible novel pharmacological targets to treat pituitary adenomas, in particular for the more aggressive and poorly responsive subtypes.

Primary cultures from human pituitary adenomas have been used since long to study genetic and pharmacological features of these cells, but the definition of the presence of CSCs within pituitary adenomas raises the issue of their identification and isolation within a non-selected culture, and expansion in vitro to allow genetic, biological and pharmacological studies.

In this protocol, we describe the procedures to establish primary cultures from human pituitary adenomas and to select and expand in vitro putative CSCs (Wurth et al., 2017). Experimental data from their characterization are also reported.

Materials and Reagents

  1. Aluminum foils (Cogepack, catalog number: 30060A )
  2. Petri dishes 60 mm (Eppendorf, catalog number: 0030701119 )
  3. Sterile mono-use scalpel (Paramount Surgimed)
  4. 15 ml centrifuge tubes (EUROCLONE, catalog number: ET5015B )
  5. 50 ml centrifuge tubes (EUROCLONE, catalog number: ET5050B )
  6. 100 µm cell strainer (Corning, Falcon®, catalog number: 352360 )
  7. 24-well plates (Eppendorf, catalog number: 0030722116 )
  8. T-25 culture flask (Eppendorf, catalog number: 0030710029 )
  9. Sterile tips
    10 μl PCR clean/sterile (Eppendorf, catalog number: 022491202 )
    200 μl PCR clean/sterile (Eppendorf, catalog number: 022491296 )
    1,000 μl PCR clean/sterile (Eppendorf, catalog number: 0030077857 )
  10. Autoclave indicator tape (Arintha Biotech, catalog number: TS1950 )
  11. LS Columns (Miltenyi Biotec, catalog number: 130-042-401 )
  12. 8-well Chamber slides (Corning, Falcon®, catalog number: 354118 )
  13. Coverslip (Menzel-Gläser)
  14. pH paper
  15. Membrane filter 0.45 µm (Merck, catalog number: HAWP04700 )
  16. Sterile phosphate buffered saline (PBS) (EUROCLONE, catalog number: ECB4004L )
  17. 70% ethanol
  18. Immune-magnetic sorting (Miltenyi Biotec, catalog number: 130-097-049 )
  19. Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Lonza, catalog number: 10-548E )
  20. Anti-Fibroblast MicroBeads, human (Miltenyi Biotec, catalog number: 130-050-601 )
  21. Normal Goat Serum (NGS) (Sigma-Aldrich, catalog number: G9023 )
  22. CD133 MicroBead Kit–Tumor Tissue, human (Miltenyi Biotec, catalog number: 130-100-857 )
  23. CD133, polyclonal rabbit (Abcam, catalog number: ab28364 )
  24. Notch1 monoclonal mouse (Abcam, catalog number: ab44986 )
  25. CXCR4 monoclonal mouse (R&D Systems, catalog number: MAB21651 )
  26. Oct4, polyclonal rabbit (Abcam, catalog number: ab19857 )
  27. Nestin, polyclonal rabbit (Abcam, catalog number: ab22035 )
  28. D2R, monoclonal mouse (Santa Cruz Biotechnologies, catalog number: sc-5303 )
  29. SSTR2A, polyclonal rabbit (Gramsch Laboratories, catalog number: SS-800 )
  30. AlexaFluor 2nd antibody (488 and 568: Thermo Fisher Scientific, Invitrogen, catalog numbers: A-11008 and A-11004 )
  31. DAPI (Sigma-Aldrich, catalog number: D9542 )
  32. TrypLE-express dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: 12605 )
  33. 0.4% trypan blue solution (Bio-Rad Laboratories, catalog number: 145-0013 )
  34. Bovine serum albumin (BSA) heat shock fraction, pH 7, ≥ 98% (Sigma-Aldrich, catalog number: A7906 )
  35. EDTA (CARLO ERBA Reagents, catalog number: 405497 )
  36. Minimum essential medium (MEM)/HAM’S F12 (1:1, EUROCLONE, catalog numbers: ECB2071L and ECB7502L )
  37. Fetal bovine serum (FBS, Thermo Fisher Scientific, GibcoTM, catalog number: 10270098 )
  38. L-glutamine (EUROCLONE, catalog number: BE17-605E )
  39. Penicillin-streptomycin (EUROCLONE, catalog number: ECB3001D )
  40. B27 (50x, Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  41. Leukemia inhibitory factor (LIF, Sigma-Aldrich, catalog number: L5283 )
  42. bFGF (Miltenyi Biotec, catalog number: 130-093-838 )
  43. EGF (Miltenyi Biotec, catalog number: 130-093-825 )
  44. Paraformaldehyde (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28908 )
  45. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
  46. Glycine (AppliChem, catalog number: A3707 )
  47. Triton X-100 (VWR, catalog number: 437002A )
  48. Glycerol (Sigma-Aldrich, catalog number: 77067 )
  49. Mowiol (Merck, catalog number: 475904 )
  50. Tris buffer (pH 8.5)
  51. Buffer for magnetic cell separator (see Recipes)
  52. Pituitary adenoma stem cell medium (see Recipes)
  53. Standard pituitary adenoma cell medium (see Recipes)
  54. Formaldehyde solution (see Recipes)
  55. 100 mM glycine (see Recipes)
  56. 0.1% Triton X-100 (see Recipes)
  57. Mowiol solution (see Recipes)


  1. Surgical scissors and forceps (Exacta Optech, catalog numbers: 6.236 264 and 9.204 222 )
  2. Autoclave
  3. Pipettes (10, 200, and 1,000 μl, Eppendorf, catalog numbers: 3123000020 , 3123000055 , and 3123000063 )
  4. Tissue culture hood (Euroclone, BIOAIR, model: TopSafe 1.8m Class II )
  5. Water bath (GFL, catalog number: 1083 )
  6. Centrifuge (Eppendorf, models: 5810 R and 5427 R )
  7. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: 3111 )
  8. Light microscope (Leica, model: Leica DMIL HC TYPE 090-135.001 )
  9. MidiMACSTM Separator (Miltenyi Biotec, catalog number: 130-042-302 ) attached to a MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  10. Cell counter (Bio-Rad Laboratories, model: TC20TM )


  1. Statistic software (i.e., PRISM, GraPhad or similar)
  2. ImageJ


  1. Preparation
    1. Wrap clean scissors and forceps in an aluminum foil and autoclave them.
    2. Sterilize the tissue culture hood room by UV light for 30 min.
    3. Prepare two 60 mm dishes, pipette 3 ml cold sterile PBS to each dish and place them in the hood.

  2. Collection of pituitary adenoma tissue
    1. Unselected tumor samples were obtained from patients who underwent surgical treatment at the Neurosurgery IRCCS-AOU San Martino-IST, after patients’ informed consent, and Institutional Ethical Committee approval.
    2. Neurosurgery team placed a portion of the resected tumor in a 15 ml Falcon tube containing 5 ml PBS to be delivered to the lab, to be immediately processed for primary culture’s establishment. Available samples are generally very small (also considering the pathologists’ requirements for diagnosis) but few mm3 are usually enough to isolate putative CSCs.
      Note: The sample has to be collected and maintained at 4 °C.

  3. Tumor tissue dissociation
    Note: All steps should be performed under sterile conditions.
    1. Clean the transport tube with 70% ethanol; transfer the tumor tissue to the 60 mm dishes in the hood (Figure 1).

      Figure 1. Pituitary adenoma tissue dissociation. Representative pictures showing different steps of the protocol aimed to obtain a primary culture from post-surgical human pituitary adenoma samples. From left to right: adenoma samples are initially cut into small pieces with a scalpel, mechanically dissociated in single cells by repeated up-down pipetting, and filtered through 100 μm cell strainer. The last picture on the right represents the morphology of freshly dissociated pituitary adenoma cells (original magnification 10x). Please note that human pituitary adenoma fragments that reach the lab are variable in size from case to case and usually very small.

    2. Using small forceps and the scalpel dissociate the tissue in small fragments (about 2 mm3): sample has to be mechanically dissociated under sterile conditions to obtain single cell suspensions (Figure 1).
      Note: Mechanical dissociation is usually enough to obtain in short-time single cell suspensions (see, for example, Florio et al., 2003). Enzymatic dissociation by trypsin (Schettini et al., 1990) or collagenase (Peverelli et al., 2017) was reported in different studies using both rat and human pituitary adenomas, respectively, but according to our current experience, this is not a necessary step when stem-like cells are isolated.
    3. Filter the cell suspension through a PBS-prewet 100 μm cell strainer placed on the top of a 50 ml tube (Figure 1). Smash any residual clumps with a 1,000 μl tip, and wash with 1x PBS twice (three times, 3 ml each).
    4. Centrifuge the cell suspension at 300 x g for 5 min at room temperature (RT), and carefully discard the supernatant with a 10 ml pipette.
    5. Lysate red blood cells (using Ammonium-Chloride-Potassium (ACK) Lysing Buffer, Lonza); add fresh PBS, and centrifuge the cell suspension at 300 x g for 5 min at room temperature (RT), and discard the supernatant. A representative picture of the obtained cell suspension after tumor dissociation is depicted in Figure 1.
    6. To purify pituitary adenoma cells from fibroblasts, add 80 μl of buffer for magnetic cell separator (Recipe 1), pipette a few times until the cell suspension is homogenous, then add 20 μl Anti-Fibroblast MicroBeads, pipette a few times (Figure 2A) and incubate for 30 min at room temperature. Add 5 ml of fresh buffer for magnetic cell separator and centrifuge at 300 x g for 5 min. In the meantime, clean with 70% ethanol the MACS Separator and arrange it under the biological hood. Place an LS column in the MACS Separator (Miltenyi Biotec, following the manufacturer’s instructions) and apply 2 ml of buffer on top (Figure 2B). As soon as the buffer completely run through, place a new 15 ml tube under the column, resuspend the cell pellet in 1 ml of buffer, and perform the magnetic separation adding the suspension to the column and collecting the effluent. Rinse the column with 1 ml buffer (3 x).

      Figure 2. Removal of fibroblast contamination from pituitary adenoma cell suspension. Add 80 μl of buffer for magnetic cell separator to pituitary adenoma cells, pipette a few times to make cell suspension homogenous, then add 20 μl Anti-Fibroblast MicroBeads, (A). After a brief incubation at room temperature, add 5 ml of fresh buffer and centrifuge at 300 x g for 5 min. In the meantime, clean with 70% ethanol the MACS Separator and arrange it under the biological hood. Place an LS column in the MACS Separator and apply 2 ml of buffer on top. As soon as the buffer completely run through, place a new 15 ml tube under the column, resuspend the cell pellet in 1 ml of buffer, and add to the column to perform the magnetic separation (B). Collect the effluent with the purified cells.

    7. Collect the negative fraction (adenoma cells) in a 15 ml tube, add fresh PBS and centrifuge at 300 x g for 5 min.
      Optional: To recover pituitary adenoma-associated fibroblasts (cell fraction bound to the beads), remove the column from the separator, place it on a new 15 ml tube, add 3 ml of buffer and flush out the magnetically labeled cells using the plunger supplied with the column.
    8. Resuspend cell pellet in 500 μl of pituitary adenoma stem cell medium (pre-warmed at 37 °C in water-bath) (Recipe 2).
    9. Plate each cell suspension (500 μl) in a well of MWs 24.
    10. Culture the cells at 37 °C, in a 5% CO2 incubator.
    11. Carefully replace half of the medium with fresh one every 2 days using 1 ml pipette.
    Plating dispersed cells from post-surgical human pituitary adenoma samples in stem cell-permissive medium promotes the enrichment in the culture of stem-like cells, through selective growth. These cells indeed display cell division ability for several weeks, self-renewal potential (spherogenesis, Figure 3A), and stem marker expression (i.e., Sox2, CD133, CXCR4, nestin, Oct4, Notch1, Figure 4). Importantly, these populations continue to replicate in vitro, although at a low rate, for several weeks.
    Similar results can be reached by CD133-expression cell sorting on whole cell population, using immune-magnetic sorting (Miltenyi Biotec), although this selection may cause the loss of subpopulations of stem-like cells, which do not express CD133. Moreover, in our experience, the percentage of CD133-expressing cells can be very variable among adenomas.
    Conversely, plating cells from the same tumors in standard pituitary adenoma cell medium (containing 10% FBS, Recipe 3), instead, lead to the development of unselected cultures (Figure 3C), mainly containing non-stem cells, characterized by a limited in vitro cell survival. According to previous studies, it was estimated only about a single cell division in vitro.
    1. Post-surgical human pituitary adenoma samples are generally limited in size and therefore the number of viable cells obtained after this procedure could be dramatically small. Moreover, considering the low proliferation rate, the number of assays feasible from each culture is limited.
    2. Besides the in vitro characterization, the only parameter that operationally defines cancer stem cell is the ability to reproduce in vivo the tumors from which they derive. As far as pituitary adenomas, contrasting results were reported for the cells in mice, but recently, engraftment was reported in zebrafish embryos (Gaudenzi et al., 2017; Peverelli et al., 2017; Wurth et al., 2017). 

      Figure 3. Bright-field pictures of primary pituitary adenoma cultures. A. Representative time-course of a primary pituitary adenoma stem-like culture: post-surgical human pituitary adenoma samples, after being dissociated to obtain single cells, are grown in stem cell-permissive medium. After about 1 week, stem-like cell start to duplicate, form large aggregates that eventually organize in cell spheroids. B and C. Representative pictures of primary pituitary adenoma cells, isolated from the same NFPA tissue, after 10 days in vitro in stem cell-permissive medium (B) and in standard pituitary adenoma cell medium (C). Original magnification 10x.

      Figure 4. Pituispheres generated from isolated human non-functioning pituitary adenoma. A. Representative bright-field picture of pituitary adenoma cells cultured in stem cell-permissive medium: cells are growing in suspension as pituispheres. Original magnification 20x. B and C. Representative immunofluorescence pictures showing the expression of stem cell markers (CXCR4, CD133, left, and Sox2, Notch1, right). D and E. Co-expression of stem cell markers (Oct4 and nestin, red) and regulatory pituitary receptors (dopamine receptor 2, D2R, and somatostatin receptor 2, SSTR2, green), suggesting the selection and in vitro expansion of stem-like cells from the original tumors. In all panels, nuclei are counterstained with DAPI.

  4. Characterization of pituitary adenoma stem cell by ICF staining
    1. Resuspend detached cells in culture medium and transfer to 8-well chamber slides (around 10,000 cells/chamber).
    2. After 48 h, rinse the cells with PBS for 5 min and fix in 4% paraformaldehyde (Recipe 4) in PBS for 15 min at RT.
    3. Wash the cells three times in PBS for 5 min each at RT.
    4. Incubate the cells with 100 mM glycine (Recipe 5) in PBS for 10 min at RT.
    5. Permeate cell membranes with 0.1% Triton X-100 (Recipe 6) for 4 min at RT.
    6. Wash the cells twice in PBS each for 5 min each at RT.
    7. Incubate with normal goat serum (NGS, 10% in PBS) for 60 min to minimize the unspecific binding of the following antibodies.
    8. Dilute the primary antibodies in 2% NGS-PBS and incubate the cells overnight at 4 °C.
      Note: According to the analysis required in each individual study different primary antibodies can be used. In our initial characterization (see Wurth et al. 2017), we used the following antibodies: Sox2, polyclonal rabbit (Millipore) dil. 1:100; CD133, polyclonal rabbit (Abcam) dil. 1:100; Notch1 monoclonal mouse (Abcam) dil. 1:100; CXCR4 monoclonal mouse (R&D Systems) dil. 1:250; Oct4, polyclonal rabbit (Abcam) dil. 1:200; nestin, polyclonal rabbit (Abcam) dil. 1:100; D2R, monoclonal mouse (Santa Cruz Biotechnologies) dil. 1:100; SSTR2, polyclonal rabbit (Gramsh Laboratories) dil. 1:100. Examples of markers expression in pituispheres are depicted in Figure 4.
    9. Wash the cells twice in PBS (200 μl) each for 5 min each at RT.
    Note: From here all steps should be performed in the dark.
    1. Incubate the cells with 2nd antibody 1:500 (AlexaFluor) in PBS, 60 min at RT.
    2. Wash the cells twice in PBS (200 μl) for 5 min each at RT.
    3. Add DAPI for 5 min at RT.
    4. Mount with Mowiol (Recipe 7).
    5. Let the slide overnight flat in a horizontal position in the dark at RT. 
    Note: One well without the primary antibodies should be included in all the experiments as a negative control.

  5. Characterization of pituitary adenoma stem cell by self-renewal assay
    1. After one week in culture, collect the cells in a 15 ml tube, centrifuge the cell suspension at 300 x g for 5 min at RT, and discard the supernatant.
    2. Resuspend the pellet with 1 ml of TrypLE-express dissociation reagent and incubate the tube for 5 min at 37 °C.
    3. Add 10 ml of fresh PBS and centrifuge at 300 x g for 5 min.
    4. Determine the viable cell number by adding 1:2 0.4% trypan blue solution and using an automated cell counter (see Equipment).
    5. Seed 1,000 cells/well in three wells of MWs 24 in pituitary adenoma stem cells medium. After 2 min, take pictures from 4 random fields of each well.
    6. Culture the cells at 37 °C, in a 5% CO2 incubator.
    7. Monitor the cell growth every two days, isolated cells will generate spheroid aggregate.
    8. To demonstrate the persistence of self-renewal after long lasting in vitro culturing, isolated cells will generate secondary spheres: dissociate primary spheres and repeat Steps E1-E7.

  6. Representative examples of the described procedures and of data to illustrate the type of results obtained

Data analysis

In order to quantify and monitor the self-renewal potential of human primary pituitary adenoma stem cells among the time (Procedure E), the number of spheres generated every 100 cells plated can be reported as sphere-forming efficiency (SFE) (Wurth et al., 2017). Proliferation activity can be assessed by cell counting using automated cell counter (Equipment #10) and analyzed for significance using statistic software (i.e., PRISM or similar). Differences in marker expression can be quantified using ImageJ software.


  1. Buffer for magnetic cell separator (store at 4-8 °C, use at RT)
    PBS pH 7.2
    0.5% bovine serum albumin (BSA)
    2 mM EDTA
  2. Pituitary adenoma stem cell medium (sterilize with vacuum filter, store at 4 °C)
    Minimum essential medium (MEM)/HAM’S F12 (1:1, EUROCLONE) supplemented with:
    1% of fetal bovine serum (FBS, Gibco)
    2 mM L-glutamine (EUROCLONE)
    1% penicillin-streptomycin (EUROCLONE)
    B27 (50x, Thermo Fisher Scientific, GibcoTM)
    10 ng/ml leukemia inhibitory factor (LIF, Sigma-Aldrich)
    20 ng/ml bFGF (Miltenyi Biotec)
    20 ng/ml EGF (Miltenyi Biotec)
  3. Standard pituitary adenoma cell medium (sterilize with vacuum filter, store at 4 °C)
    Minimum essential medium (MEM) (EUROCLONE) supplemented with:
    10% of fetal bovine serum (FBS, GibcoTM)
    2 mM L-glutamine (EUROCLONE)
    1% penicillin-streptomycin (EUROCLONE)
  4. Formaldehyde solution (store in aliquots at -20 °C)
    1. Dissolve 16 g paraformaldehyde in about 80 ml dH2O by stirring at 70 °C (in a fume cupboard)
    2. Add a few drops of 1 N NaOH to depolymerize the paraformaldehyde
    3. Adjust the pH to about 7.0 and check with pH paper
    4. Cool down to room temperature and bring up to 100 ml
    5. Filter through a 0.45 μm Millipore filter and mix with an equal amount of double strength buffer
    6. Divide into convenient aliquots and store frozen at -20 °C
    7. Discard after thawing
  5. 100 mM glycine (store at 2-8 °C)
    Add 75.07 mg of glycine in 10 ml PBS
  6. 0.1% Triton X-100 (store at 2-8 °C)
    Add 10 μl Triton X-100 in 10 ml PBS
  7. Mowiol solution (store in aliquots at -20 °C)
    1. Mix 6 g of glycerol (analytical grade), and 2.4 g Mowiol in 6 ml dH2O
    2. Stir for at least 6 h
    3. Add 12 ml 0.2 M Tris buffer (pH 8.5) and stir for another 6 h
    4. Let the mix sit for another 2 h before incubate at 50 °C for 10 min
    5. Spin down (5,000 x g, 15 min) to remove undissolved Mowiol
    6. Aliquot and freeze supernatant at -20 °C


This work was supported by the Italian Association for Cancer Research (AIRC). Authors have no conflicts of interest or competing interests to disclose.


  1. Carreno, G., Gonzalez-Meljem, J. M., Haston, S. and Martinez-Barbera, J. P. (2017). Stem cells and their role in pituitary tumorigenesis. Mol Cell Endocrinol 445: 27-34.
  2. Clevers, H. (2011). The cancer stem cell: premises, promises and challenges. Nat Med 17(3): 313-319.
  3. Donangelo, I., Ren, S. G., Eigler, T., Svendsen, C. and Melmed, S. (2014). Sca1+ murine pituitary adenoma cells show tumor-growth advantage. Endocr Relat Cancer 21(2): 203-216.
  4. Florio, T. (2011). Adult pituitary stem cells: from pituitary plasticity to adenoma development. Neuroendocrinology 94(4): 265-277.
  5. Florio, T. and Barbieri, F. (2012). The status of the art of human malignant glioma management: the promising role of targeting tumor-initiating cells. Drug Discov Today 17(19-20): 1103-1110.
  6. Florio, T., Thellung, S., Corsaro, A., Bocca, L., Arena, S., Pattarozzi, A., Villa, V., Massa, A., Diana, F., Schettini, D., Barbieri, F., Ravetti, J. L., Spaziante, R., Giusti, M. and Schettini G. (2003). Characterization of the intracellular mechanisms mediating somatostatin and lanreotide inhibition of DNA synthesis and growth hormone release from dispersed human GH-secreting pituitary adenoma cells, in vitro. Clin Endocrinol 59: 115-128.
  7. Gaudenzi, G., Albertelli, M., Dicitore, A., Wurth, R., Gatto, F., Barbieri, F., Cotelli, F., Florio, T., Ferone, D., Persani, L. and Vitale, G. (2017). Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors. Endocrine 57(2): 214-219.
  8. Li, Y. and Laterra, J. (2012). Cancer stem cells: distinct entities or dynamically regulated phenotypes? Cancer Res 72(3): 576-580.
  9. Molitch, M. E. (2017). Diagnosis and treatment of pituitary adenomas: a review. JAMA 317(5): 516-524.
  10. Peverelli, E., Giardino, E., Treppiedi, D., Meregalli, M., Belicchi, M., Vaira, V., Corbetta, S., Verdelli, C., Verrua, E., Serban, A. L., Locatelli, M., Carrabba, G., Gaudenzi, G., Malchiodi, E., Cassinelli, L., Lania, A. G., Ferrero, S., Bosari, S., Vitale, G., Torrente, Y., Spada, A. and Mantovani, G. (2017). Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors. Int J Cancer 140(8): 1870-1880.
  11. Schettini, G., Florio, T., Meucci, O., Landolfi, E., Grimaldi, M., Lombardi, G., Scala, G. and Leong, D. (1990). Interleukin-1-beta modulation of prolactin secretion from rat anterior pituitary cells: involvement of adenylate cyclase activity and calcium mobilization. Endocrinology 126(3): 1435-1441.
  12. Wurth, R., Barbieri, F., Pattarozzi, A., Gaudenzi, G., Gatto, F., Fiaschi, P., Ravetti, J. L., Zona, G., Daga, A., Persani, L., Ferone, D., Vitale, G. and Florio, T. (2017). Phenotypical and pharmacological characterization of stem-like cells in human pituitary adenomas. Mol Neurobiol 54(7): 4879-4895.


垂体腺瘤是更常见的颅内肿瘤之一,通常用基于生长抑素和多巴胺激动剂手术的手术和药物治疗。 虽然多为良性肿瘤,但侵入性行为的发生常常被检测到,导致预后较差。 来自人类垂体腺瘤的原代培养物的使用代表了对其发育机制的知识以及其药理敏感性决定因素的定义方面的显着进步。 此外,最近的研究也在垂体腺瘤中发现了假定的肿瘤干细胞,根据目前的假设,它代表了根除大多数恶性肿瘤的真实细胞靶标。 在这个协议中,我们描述了从人垂体腺瘤建立原代培养的程序,以及如何选择,体外扩增和表型鉴定推定的垂体腺瘤干细胞。

【背景】垂体腺瘤是最常见的颅内肿瘤之一(高达15%),横断面研究发现每100,000名居民中约有90例发病,其中绝大多数为30岁以上的成年人。大约10%的未经选择的垂体在尸检时进行了检查(即考虑到之前未诊断为垂体疾病的受试者的垂体)( ,Molitch,2017)。尽管通常为良性肿瘤,但垂体腺瘤的处理可因与激素分泌过多相关的临床综合征或发展以治疗抗性,高增殖率,快速复发和绒毛外侵袭为特征的侵袭行为而复杂化(Carreno等人,2017)。成年垂体干细胞的持续存在(Florio,2011)导致垂体腺瘤(以及可能的其他良性瘤形成)的发展可以源自具有干细胞特性(主要是自我更新和分化)的肿瘤细胞的亚群能力),正如已经建立的恶性固体和血液肿瘤一样。

最近的实验证据表明,癌症干细胞(CSC)范例也适用于人和小鼠垂体腺瘤(Donangelo等人,2014; Peverelli等人,2017; Wurth ,et al。,2017),并且有人提出,致癌基因转化的CSCs可以克隆起源肿瘤。最初为恶性肿瘤开发的CSC作为肿瘤起始细胞(TIC)的概念提出,只有一小部分肿瘤细胞CSC亚群负责启动和维持肿瘤生长,引起侵袭和转移的形成,并且赋予耐药性(Clevers,2011; Florio和Barbieri,2012)。这一理论导致了给定肿瘤内的细胞分级结构,取代了癌变发生的随机模型,该模型提出大量实体瘤由具有相同致瘤潜能的细胞组成。 CSC模型最近的发展是'动态干性'理论,它假设CSCs和更多分化(祖细胞)细胞之间的交换,由表观遗传和微环境信号,转录因子,miRNA或致癌途径的激活决定李和Laterra,2012)。




关键字:垂体腺瘤, 癌症干细胞, 原代培养物


  1. 铝箔(Cogepack,目录号:30060A)
  2. 培养皿60毫米(Eppendorf,目录号:0030701119)
  3. 无菌一次性手术刀(派拉蒙手表)

  4. 15 ml离心管(EUROCLONE,产品目录号:ET5015B)

  5. 50 ml离心管(EUROCLONE,目录号:ET5050B)
  6. 100μm细胞过滤器(Corning,Falcon ,目录号:352360)
  7. 24孔板(Eppendorf,目录号:0030722116)
  8. T-25培养瓶(Eppendorf,目录号:0030710029)
  9. 无菌技巧

  10. 高压灭菌指示带(Arintha Biotech,目录号:TS1950)
  11. LS色谱柱(Miltenyi Biotec,产品目录号:130-042-401)
  12. 8孔室幻灯片(Corning,Falcon ,产品目录号:354118)
  13. Coverslip(Menzel-Gläser)
  14. pH纸
  15. 膜过滤器0.45μm(Merck,目录号:HAWP04700)
  16. 无菌磷酸盐缓冲盐水(PBS)(EUROCLONE,目录号:ECB4004L)
  17. 70%乙醇
  18. 免疫磁性分选(Miltenyi Biotec,目录号:130-097-049)
  19. 氯化铵 - 钾(ACK)裂解缓冲液(Lonza,目录号:10-548E)
  20. 抗成纤维细胞微珠,人类(Miltenyi Biotec,目录号:130-050-601)
  21. 正常山羊血清(NGS)(Sigma-Aldrich,目录号:G9023)
  22. CD133微珠试剂盒 - 肿瘤组织,人(Miltenyi Biotec,目录号:130-100-857)
  23. CD133,多克隆兔(Abcam,目录号:ab28364)
  24. Notch1单克隆小鼠(Abcam,目录号:ab44986)
  25. CXCR4单克隆小鼠(R& D Systems,目录号:MAB21651)
  26. Oct4,多克隆兔(Abcam,目录号:ab19857)
  27. 巢蛋白,多克隆兔(Abcam,目录号:ab22035)
  28. D2R,单克隆小鼠(Santa Cruz Biotechnologies,目录号:sc-5303)
  29. SSTR2A,多克隆兔(Gramsch Laboratories,目录号:SS-800)
  30. AlexaFluor 2 nd抗体(488和568:Thermo Fisher Scientific,Invitrogen,目录号:A-11008和A-11004)
  31. DAPI(Sigma-Aldrich,目录号:D9542)
  32. TrypLE-表达解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:12605)
  33. 0.4%台盼蓝溶液(Bio-Rad Laboratories,目录号:145-0013)
  34. 牛血清白蛋白(BSA)热休克片段,pH 7,≥98%(Sigma-Aldrich,目录号:A7906)
  35. EDTA(CARLO ERBA试剂,目录号:405497)
  36. 最低基本培养基(MEM)/ HAM'S F12(1:1,EUROCLONE,产品目录号:ECB2071L和ECB7502L)
  37. 胎牛血清(FBS,Thermo Fisher Scientific,Gibco TM,目录号:10270098)
  38. L-谷氨酰胺(EUROCLONE,目录号:BE17-605E)
  39. 青霉素 - 链霉素(EUROCLONE,目录号:ECB3001D)
  40. B27(50x,Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  41. 白血病抑制因子(LIF,Sigma-Aldrich,目录号:L5283)
  42. bFGF(Miltenyi Biotec,目录号:130-093-838)
  43. EGF(Miltenyi Biotec,目录号:130-093-825)
  44. 多聚甲醛(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28908)
  45. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:71687)
  46. 甘氨酸(AppliChem,目录号:A3707)
  47. Triton X-100(VWR,目录号:437002A)
  48. 甘油(Sigma-Aldrich,目录号:77067)
  49. Mowiol(Merck,产品目录号:475904)
  50. Tris缓冲液(pH 8.5)
  51. 磁性细胞分离器缓冲液(见食谱)

  52. 垂体腺瘤干细胞培养基(见食谱)
  53. 标准垂体腺瘤细胞培养基(见食谱)
  54. 甲醛溶液(见食谱)
  55. 100 mM甘氨酸(见食谱)
  56. 0.1%Triton X-100(见食谱)
  57. Mowiol解决方案(参见食谱)


  1. 外科剪刀和镊子(Exacta Optech,产品目录号:6.236 264和9.204 222)
  2. 高压灭菌器
  3. 移液器(10,200和1,000μl,Eppendorf,产品目录号:3123000020,3123000055和3123000063)
  4. 组织培养罩(Euroclone,BIOAIR,型号:TopSafe 1.8m Class II)
  5. 水浴(GFL,目录号:1083)
  6. 离心机(Eppendorf,型号:5810 R和5427 R)
  7. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:3111)。
  8. 光学显微镜(Leica,型号:Leica DMIL HC TYPE 090-135.001)
  9. 连接到MultiStand(Miltenyi Biotec,目录号:130-042-303)的MidiMACS TM分离器(Miltenyi Biotec,目录号:130-042-302)
  10. 细胞计数器(Bio-Rad Laboratories,型号:TC20 TM)


  1. 统计软件(即,PRISM,GraPhad或类似软件)
  2. ImageJ的


  1. 制备
    1. 将干净的剪刀和镊子包裹在铝箔中并高压灭菌。

    2. 用紫外线对组织培养罩室消毒30分钟
    3. 准备两个60毫米的菜肴,吸管3毫升冷无菌PBS的每盘,并将其放置在引擎盖。

  2. 收集垂体腺瘤组织
    1. 未经选择的肿瘤样本来自在神经外科IRCCS-AOU San Martino-IST接受手术治疗的患者,在患者的知情同意和机构伦理委员会批准后获得。
    2. 神经外科小组将一部分切除的肿瘤置于含有5ml PBS的15ml Falcon管中以送至实验室,立即进行原代培养物的建立。可用的样本通常非常小(同时考虑到病理学家对诊断的要求),但通常少于mm 3通常足以隔离推定的CSC。

  3. 肿瘤组织解离
    1. 用70%乙醇清洁输送管;将肿瘤组织转移到通风橱中的60毫米培养皿(图1)。

      图1.垂体腺瘤组织解离。 代表性图片显示协议的不同步骤旨在从术后人垂体腺瘤样品获得原代培养物。从左至右:腺瘤样品最初用解剖刀切成小块,通过反复上下移液在单个细胞中机械解离,并通过100μm细胞过滤器过滤。右侧的最后一张图片表示新分离的垂体腺瘤细胞的形态(原始放大倍数10倍)。请注意,到达实验室的人体垂体腺瘤碎片的大小各不相同,通常很小。

    2. 使用小钳子和解剖刀将组织分离成小片段(约2mm 3):样品必须在无菌条件下机械分离以获得单细胞悬浮液(图1)。
    3. 通过放置在50毫升管顶部的PBS-预湿100μm细胞过滤器过滤细胞悬液(图1)。用1,000μl吸头粉碎任何残留的团块,并用1x PBS洗涤两次(三次,每次3ml)。
    4. 在室温下(RT)将细胞悬浮液以300gxg离心5分钟,并用10ml移液管小心丢弃上清液。
    5. 裂解红细胞(使用氯化铵 - 钾(ACK)裂解缓冲液,Lonza);加入新鲜的PBS,并在室温(RT)下将细胞悬浮液以300×g离心5分钟,并弃去上清液。图1描述了肿瘤解离后获得的细胞悬液的代表性照片。
    6. 为了从成纤维细胞中纯化垂体腺瘤细胞,加入80μl用于磁性细胞分离器的缓冲液(配方1),吸取几次直至细胞悬液均匀,然后加入20μl抗成纤维细胞微珠,吸取几次(图2A)并在室温下孵育30分钟。加入5ml用于磁性细胞分离器的新鲜缓冲液并在300gxg离心5分钟。同时,用70%乙醇清洗MACS分离器,并将其安置在生物罩下。将LS柱放入MACS分离器(Miltenyi Biotec,按照制造商的说明),并在顶部涂上2ml缓冲液(图2B)。一旦缓冲液完全流过,在柱下放置一个新的15ml试管,将细胞沉淀重悬于1ml缓冲液中,进行磁性分离,将悬浮液加入柱中并收集流出物。用1ml缓冲液(3次)冲洗柱子。


    7. 收集阴性部分(腺瘤细胞)在15毫升管中,加入新鲜的PBS并在300×g下离心5分钟。
    8. 用500μl垂体腺瘤干细胞培养基重悬细胞沉淀(在37℃水浴中预热)(方案2)。
    9. 将每个细胞悬液(500μl)铺在MW24的一个孔中。

    10. 培养细胞在37°C,在5%CO 2培养箱中。

    11. 使用1毫升移液管,每2天仔细更换一次新鲜培养基。
    通过选择性生长,将来自手术后人垂体腺瘤样品的分散细胞在干细胞允许培养基中电镀,促进了干细胞样细胞培养的富集。这些细胞确实显示出数周的细胞分裂能力,自我更新潜能(球形发生,图3A)和干细胞标志物表达(即,Sox2,CD133,CXCR4,巢蛋白,Oct4,Notch1,图4 )。重要的是,这些人群继续在体外复制,尽管速度很低,几周。
    使用免疫磁性分选(Miltenyi Biotec),在整个细胞群体上CD133表达细胞分选可以达到类似的结果,尽管该选择可能导致不表达CD133的干细胞样细胞亚群的丢失。此外,根据我们的经验,表达CD133的细胞在腺瘤中的百分比可能非常不稳定。
    1. 手术后人类垂体腺瘤样品的体积通常受到限制,因此在此过程后获得的活细胞数量可能会非常小。而且,考虑到低增殖率,每种培养物可行的检测数量是有限的。
    2. 除了体外表征之外,操作上限定癌症干细胞的唯一参数是体内再现它们衍生的肿瘤的能力。就垂体腺瘤而言,小鼠中的细胞报道了对比结果,但最近在斑马鱼胚胎中报道了植入(Gaudenzi等,2017; Peverelli等,2017; Wurth等,2017)。

      图3.原代垂体腺瘤培养物的明场照片A.原代垂体腺瘤茎样培养物的代表性时间过程:手术后人垂体腺瘤样本经解离后获得单个细胞在干细胞允许的培养基中生长。大约1周后,干细胞样细胞开始复制,形成最终聚集在细胞球体中的大聚集体。 B和C.在干细胞允许培养基(B)和标准垂体腺瘤细胞培养基(C)中10天后,从相同NFPA组织中分离的原代垂体腺瘤细胞的代表性图片(em) 。原始放大倍数为10倍。

      图4.分离的人体非功能性垂体腺瘤产生的垂体。 :一种。在干细胞允许的培养基中培养的垂体腺瘤细胞的代表性明场照片:细胞以悬浮液形式生长为垂体。原始放大20倍。 B和C.显示干细胞标志物(CXCR4,CD133,左和Sox2,Notch1,右)表达的代表性免疫荧光图片。 D和E.干细胞标记物(Oct4和巢蛋白,红色)和调节性垂体受体(多巴胺受体2,D2R和生长抑素受体2,SSTR2,绿色)的共表达,表明选择和体外从原始肿瘤扩展干细胞样细胞。在所有的小组中,细胞核都用DAPI复染。

  4. 通过ICF染色鉴定垂体腺瘤干细胞

    1. 在培养基中重悬分离的细胞,并转移到8孔腔室载玻片(约10,000个细胞/腔室)。
    2. 48小时后,用PBS冲洗细胞5分钟,并在室温下用PBS中的4%多聚甲醛(方案4)固定15分钟。
    3. 在室温下用PBS洗涤细胞三次,每次5分钟。

    4. 用100mM甘氨酸(配方5)在PBS中孵育细胞10分钟

    5. 用0.1%Triton X-100(配方6)渗透细胞膜4分钟
    6. 每次用PBS洗涤细胞两次,每次5分钟。
    7. 与正常山羊血清(NGS,10%的PBS)孵育60分钟以减少下列抗体的非特异性结合。
    8. 稀释2%NGS-PBS中的一抗,并在4°C孵育细胞过夜。
      注意:根据每个个体研究中所需的分析,可以使用不同的一抗。在我们最初的表征(见Wurth等2017)中,我们使用了以下抗体:Sox2,多克隆兔(Millipore)dil。 1:100; CD133,多克隆兔(Abcam)dil。 1:100; Notch1单克隆小鼠(Abcam)dil。 1:100; CXCR4单克隆小鼠(R& D Systems)dil。 1:250; Oct4,多克隆兔(Abcam)dil。 1:200;巢蛋白,多克隆兔(Abcam)dil。 1:100; D2R,单克隆小鼠(Santa Cruz Biotechnologies)dil。 1:100; SSTR2,多克隆兔(Gramsh Laboratories)dil。 1:100。图4描绘了垂体中标志物表达的实例。
    9. 在室温下用PBS(200μl)洗涤细胞两次,每次5分钟。
    1. 用PBS中的第二抗体1:500(AlexaFluor)孵育细胞,在RT下孵育60分钟。
    2. 在室温下将细胞在PBS(200μl)中洗涤两次,每次5分钟。

    3. 在RT下添加DAPI 5分钟
    4. Mowiol安装(配方7)。

    5. 让幻灯片在黑暗中在水平位置平躺过夜。 

  5. 通过自我更新测定表征垂体腺瘤干细胞
    1. 在培养一周后,将细胞收集在15ml试管中,在室温下将细胞悬浮液在300xg离心5分钟,弃去上清液。
    2. 用1ml TrypLE-express解离试剂重悬沉淀,并在37°C孵育管5分钟。
    3. 加入10毫升新鲜的PBS并在300×g下离心5分钟。
    4. 通过添加1:2 0.4%台盼蓝溶液并使用自动化细胞计数器(参见设备)来确定活细胞数量。
    5. 在垂体腺瘤干细胞培养基中的MW24的三个孔中种植1000个细胞/孔。 2分钟后,从每口井的4个随机区域拍摄照片。

    6. 培养细胞在37°C,在5%CO 2培养箱中。
    7. 每两天监测细胞的生长情况,分离的细胞会产生球状聚集体。
    8. 为了证明长时间持续体外培养后自我更新的持久性,分离的细胞将产生二次球:解离一次球并重复步骤E1-E7。

  6. 所描述的程序和数据的代表性例子来说明所得结果的类型


为了量化和监测人类原代垂体腺瘤干细胞自我更新的潜力(程序E),每100个细胞铺板产生的球体数目可以报告为球形成效率(SFE)(Wurth 等人,,2017)。增殖活性可以通过使用自动化细胞计数器(设备#10)的细胞计数来评估,并使用统计软件(即em.PRISM或类似的)分析显着性。标记物表达的差异可以使用ImageJ软件进行量化。


  1. 磁性细胞分离缓冲液(储存在4-8°C,在室温下使用)
    PBS pH 7.2
    2 mM EDTA
  2. 垂体腺瘤干细胞培养基(用真空过滤器灭菌,在4°C储存)
    最低基本培养基(MEM)/ HAM'S F12(1:1,EUROCLONE)补充:
    2 mM L-谷氨酰胺(EUROCLONE)
    1%青霉素 - 链霉素(EUROCLONE)
    B27(50x,Thermo Fisher Scientific,Gibco TM)
    10ng / ml白血病抑制因子(LIF,Sigma-Aldrich)
    20 ng / ml bFGF(Miltenyi Biotec)
    20 ng / ml EGF(Miltenyi Biotec)
  3. 标准的垂体腺瘤细胞培养基(用真空过滤器灭菌,在4°C储存)
    10%胎牛血清(FBS,Gibco TM)
    2 mM L-谷氨酰胺(EUROCLONE)
    1%青霉素 - 链霉素(EUROCLONE)
  4. 甲醛溶液(在-20°C等分储存)
    1. 通过在70℃下(在通风橱中)搅拌将16g多聚甲醛溶解于约80ml dH 2 O中。
    2. 加几滴1N氢氧化钠解聚多聚甲醛
    3. 将pH值调节到7.0左右,然后用pH试纸检查
    4. 冷却到室温,并带来100毫升
    5. 通过0.45μmMillipore过滤器过滤并与等量的双倍浓度缓冲液混合。
    6. 分成方便的等分试样并在-20°C冷冻保存。
    7. 解冻后丢弃
  5. 100 mM甘氨酸(储存在2-8°C)

  6. 0.1%Triton X-100(2-8°C储存)

    加10μlTriton X-100于10 ml PBS中
  7. Mowiol溶液(在-20°C等分储存)
    1. 将6g甘油(分析级)和2.4g Mowiol在6ml dH 2 O中混合。

    2. 搅拌至少6小时
    3. 加入12ml0.2M Tris缓冲液(pH8.5)并再搅拌6小时
    4. 让混合物再放置2小时,然后在50°C孵育10分钟。
    5. 旋转(5000 em x 15分钟)以除去未溶解的Mowiol
    6. 在-20°C分装并冷冻上清液。




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引用:Würth, R., Pattarozzi, A., Barbieri, F. and Florio, T. (2018). Primary Cultures from Human GH-secreting or Clinically Non-functioning Pituitary Adenomas. Bio-protocol 8(7): e2790. DOI: 10.21769/BioProtoc.2790.