WO2021237905A1 - 一种用于喉癌上皮细胞的培养基、培养方法及其应用 - Google Patents

一种用于喉癌上皮细胞的培养基、培养方法及其应用 Download PDF

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WO2021237905A1
WO2021237905A1 PCT/CN2020/103428 CN2020103428W WO2021237905A1 WO 2021237905 A1 WO2021237905 A1 WO 2021237905A1 CN 2020103428 W CN2020103428 W CN 2020103428W WO 2021237905 A1 WO2021237905 A1 WO 2021237905A1
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culture medium
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cell culture
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刘青松
胡洁
任涛
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中科院合肥技术创新工程院
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Priority to EP20937456.0A priority patent/EP4159845A1/en
Priority to JP2022573183A priority patent/JP7503662B2/ja
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  • the present invention belongs to the technical field of medicine. Specifically, it relates to a culture medium and a culture method for culturing or amplifying primary laryngeal cancer epithelial cells in vitro, and also relates to methods and methods for evaluating and screening the therapeutic effects of medicaments on the cells obtained by culture. application.
  • laryngeal cancer accounts for 5.7% to 7.6% of systemic malignant tumors. It ranks third after nasopharyngeal cancer and nasal sinus cancer in the field of otolaryngology.
  • the prevalence age is 50 to 70 years old.
  • the incidence rate is highest in Northeast my country, and most of the patients are male.
  • the cause of the disease is not very clear, but cities with heavy air pollution are higher than those with light pollution.
  • the treatment methods for laryngeal cancer especially for patients with advanced stages, are still limited, resulting in a low five-year clinical survival rate of patients and lack of personalized precise medication guidance.
  • Functional testing refers to a method of testing the sensitivity of anti-tumor drugs on cancer patient cells in vitro.
  • the key to applying this method is to develop a tumor cell model that has a short growth cycle and can represent the biological characteristics of patients with laryngeal cancer.
  • the cell model should be easy to operate and be able to predict the curative effect of clinical medication quickly and efficiently, so as to give cancer patients precise medication guidance in time.
  • the success rate of establishing cell models in vitro with primary tumor cells taken from cancer patients is often very low, the growth cycle is long, and there are problems such as excessive proliferation of interstitial cells such as fibroblasts, which restrict the development of this field.
  • the organoid technology is a technology that embeds the patient’s autologous primary epithelial cells in an extracellular matrix for three-dimensional culture in vitro.
  • the medium of this technology requires the addition of a variety of specific growth factors (such as Wnt protein and R-spondin). Family protein), which is expensive and not suitable for popularization in clinics for large-scale applications.
  • organoids need to embed cells in extracellular matrix glue during the entire culture process. The plating steps of cell seeding, passage and drug sensitivity test are more cumbersome and time-consuming than 2D culture operations.
  • the size of organoids is not easy to control, and some organoids are prone to grow too large and cause internal necrosis.
  • organoid technology compared with 2D culture technology, the operability and applicability of organoid technology is not strong, and it requires professional and technical personnel to operate it. It is not suitable for large-scale and wide application in clinical in vitro drug sensitivity testing (Nick Barker, Nat. Cell Biol., 18 (3): 246-54, 2016).
  • Cell reprogramming technology is a technology that co-cultures patients’ autologous primary epithelial cells and murine-derived feeder cells.
  • the media components reported in the literature are Whether the generation of tumor cells can rapidly expand is still unknown.
  • the cultured laryngeal cancer tumor cells can represent the biological characteristics of patients with laryngeal cancer.
  • the present invention aims to provide a culture medium for culturing primary laryngeal cancer epithelial cells and a method for culturing primary laryngeal cancer epithelial cells using the medium in view of the deficiencies of the prior art.
  • the use of the primary laryngeal carcinoma epithelial cell culture medium and the culture method of the present invention can achieve the purposes of short in vitro culture period, controllable cost, and convenient operation.
  • the technology When the technology is applied to construct a primary laryngeal cancer tumor cell model, it can obtain primary laryngeal cancer tumor cells with the biological characteristics of patients with laryngeal cancer, and can be applied to new drug screening and in vitro drug sensitivity testing.
  • One aspect of the present invention is to provide a primary cell culture medium for culturing primary laryngocarcinoma epithelial cells, which contains an MST1/2 kinase inhibitor, and the MST1/2 kinase inhibitor includes a compound of formula (I) or Its pharmaceutically acceptable salt or solvate.
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independently R 6 Group (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclic group C1-C6 alkyl (the heterocyclic group is selected from, for example, piperidinyl, tetrahydropyran Base, etc.);
  • R 6 is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro) methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro
  • the MST1/2 kinase inhibitor includes a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected C1-C6 alkyl, optionally substituted with 1-2 R 6 independently substituted phenyl, optionally substituted with 1-2 R 6 independently substituted thienyl, and optionally substituted with 1 -2 independently substituted benzyl groups R 6 , R 1 is more preferably phenyl optionally substituted with 1-2 independently R 6 groups;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, and R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is compound 1.
  • the content of the MST1/2 kinase inhibitor in the culture medium is usually 1.25 ⁇ M-10 ⁇ M, preferably 2.5 ⁇ M-10 ⁇ M, and more preferably 5 ⁇ M.
  • the primary cell culture medium of the present invention preferably further contains one or more of the following factors: insulin-like growth factor 1 (IGF-1); fibroblast growth factor 7 (FGF7); insulin-transferrin-selenium complex (ITS); Hepatocyte Growth Factor (HGF); a ROCK kinase inhibitor selected from at least one of Y27632, Fasudil, and H-1152; and selected from A83-01, SB431542, Repsox, SB505124, SB525334 , SD208, LY36494, and SJN2511 TGF ⁇ type I receptor inhibitor.
  • IGF-1 insulin-like growth factor 1
  • FGF7 fibroblast growth factor 7
  • ITS insulin-transferrin-selenium complex
  • HGF Hepatocyte Growth Factor
  • ROCK kinase inhibitor selected from at least one of Y27632, Fasudil, and H-1152
  • the content of the fibroblast growth factor 7 in the culture medium is preferably 5 to 80 ng/ml, more preferably 20 to 80 ng/ml; insulin in the insulin-transferrin-selenium complex
  • the content of /transferrin/sodium selenite is 5 ⁇ 20 ⁇ g/ml-2.5 ⁇ 10 ⁇ g/ml-2.5 ⁇ 10ng/ml respectively; preferably 10 ⁇ 20 ⁇ g/ml-5 ⁇ 10 ⁇ g/ml-5 ⁇ 10ng/ml
  • the content of the insulin-like growth factor 1 is preferably 10 to 80 ng/ml, more preferably 10 to 40 ng/ml;
  • the content of the hepatocyte growth factor is preferably 10 to 80 ng/ml, more preferably 10 to 40 ng/ml ml;
  • the ROCK kinase inhibitor is preferably Y27632, and the content of the ROCK kinase inhibitor is preferably 1.25-20 ⁇ M, more preferably 2.5-10 ⁇ M;
  • the composition of this medium contains MST1/2 kinase inhibitors, but does not contain uncertain components such as serum and bovine pituitary extract, and does not contain Wnt agonists.
  • R-spondin family proteins, BMP inhibitors and other niche factors necessary for organoid culture and do not contain nicotinamide and N-acetylcysteine, which greatly reduces the cost of the culture medium and simplifies the operation of preparing the culture medium
  • the process realizes the in vitro culture of primary laryngeal carcinoma epithelial cells with controllable cost and convenient operation.
  • the primary laryngeal carcinoma epithelial cells may be laryngeal carcinoma tumor cells, normal laryngeal carcinoma epithelial cells, and laryngeal carcinoma epithelial stem cells.
  • One aspect of the present invention is to provide a method for culturing primary laryngeal carcinoma epithelial cells, which includes the following steps:
  • the trophoblast cells may be, for example, irradiated NIH-3T3 cells
  • the irradiation source is X-rays or gamma rays, preferably gamma rays
  • the irradiation dose is 30-50 Gy, preferably 35 Gy.
  • the irradiated NIH-3T3 cells are seeded in a culture container such as a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate or a T25 cell culture flask at 2 ⁇ 10 4 cells/cm 2 and the cells are Standby after sticking to the wall.
  • Primary laryngeal carcinoma epithelial cells can be derived from, for example, laryngeal carcinoma tissue samples and paracancerous tissue samples.
  • Laryngeal cancer tissue samples are, for example, derived from cancer tissue samples surgically removed from patients with laryngeal cancer tumors who have explained and obtained consent, and the para-cancerous tissue samples are collected from laryngeal tissues that are at least 5 cm away from the laryngeal cancer tissue. Collect the above-mentioned tissue samples within half an hour after the patient's surgical resection or biopsy.
  • tissue transport solution a solution that contains 1-2% by volume penicillin/streptomycin, and/or 0.2-0.4% by volume Primocin (hereinafter referred to as tissue transport solution).
  • the streptomycin concentration range is 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL, more preferably 200 ⁇ g/mL, and the penicillin concentration range is 25-400 U/mL, preferably 50- 200 U/mL, more preferably 200 U/mL; when Primocin is used, the concentration range is 25-400 ⁇ g/mL, preferably 50-200 ⁇ g/mL, more preferably 100 ⁇ g/mL.
  • tissue sample In the biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with the transport fluid, and wash off the blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new petri dish, add 1-3 mL of transport fluid, and divide the tissue sample into tissue fragments with a volume less than 3 mm 3 with a sterile surgical blade and surgical forceps.
  • tissue sample fragments to a centrifuge tube, centrifuge with a benchtop centrifuge (Sigma Company 3-18K) at 1000-3000 rpm for 3 to 5 minutes; discard the supernatant, and add tissue transport fluid and tissue digestion at a ratio of 1:1 Solution (The usage amount is about 5mL tissue digestion solution for every 10mg tissue.
  • the preparation method of tissue digestion solution is: 1 ⁇ 2mg/mL collagenase II, 1 ⁇ 2mg/mL collagenase IV, 50 ⁇ 100U/mL deoxyribose Nucleic acid I, 0.5 ⁇ 1mg/mL hyaluronidase, 0.1 ⁇ 0.5mg/mL calcium chloride, 5 ⁇ 10mg/mL bovine serum albumin dissolved in HBSS and RPMI-1640 with a volume ratio of 1:1), label the sample Number, seal with parafilm, digest at 37°C, 200 ⁇ 300 rpm constant temperature shaker (ZQLY-180N), and observe whether the digestion is completed every 1 hour; if there is no obvious tissue mass, stop the digestion, otherwise continue to digest , Until the digestion is complete, the digestion time range is 4 to 8 hours.
  • the cell strainer (cell sieve pore size is for example 70 ⁇ m) to filter out undigested tissue clumps, the tissue clumps on the strainer are washed with tissue transport fluid, the remaining cells are flushed into the centrifuge tube, and the table top centrifuge Centrifuge at 1,000 to 3,000 revolutions per minute for 3 to 5 minutes. Discard the supernatant and observe whether the remaining cell mass contains blood cells. If there are blood cells, add 1-5 mL of blood cell lysate (purchased from Sigma), mix well, lyse at 4°C for 10-20 minutes, shake and mix once for 5 minutes, and the lysis is complete. Then take it out and centrifuge at 1000-3000 rpm for 3 to 5 minutes.
  • blood cell lysate purchased from Sigma
  • the supernatant was discarded, the primary cell culture medium of the present invention was added to resuspend, and the flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) was used for counting to obtain the total number of cells.
  • the flow image counter Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL
  • step (3) Inoculate the primary laryngeal carcinoma epithelial cells isolated in step (3) in a culture vessel pre-inoculated with trophoblast cells, and use the primary cell culture medium in step (1) for culture.
  • a hole of the multi-well plate is pre- inoculated with a density of 2 ⁇ 10 4 to 4 ⁇ 10 4 pieces/cm 2 (for example, 2 ⁇ 10 4 pieces/cm 2 ) after ⁇ -ray irradiation with an irradiation dose of 35 Gy.
  • add to each well 0.5-2mL primary epithelial cell culture medium cultured in a cell incubator at 37°C and 5% CO 2 for 8-16 days. During this period, replace with fresh primary cell culture medium every 4 days.
  • the cancer epithelial cells grow to a cell density of about 80% to 90% of the bottom area of the multiwell plate, they are digested and passaged.
  • this step does not need to mix the primary cells and Matrigel on ice to form glue droplets, and wait for the glue droplets to solidify before adding the culture medium.
  • the amount of expensive extracellular matrix glue is saved, and the operation steps are simplified.
  • the inoculated primary laryngeal carcinoma epithelial cells are cultured for 8-16 days, when the cell clones formed in the culture vessel converge to 80% of the bottom area, discard the supernatant, and add 1 to 2 mL 0.25% pancreatin ( (Purchased from Thermo Fisher) digested for 1 minute, then aspirated 0.25% pancreatin, then added 1 to 2 mL 0.05% pancreatin for cell digestion, incubated at room temperature for 5 to 20 minutes; Bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin culture solution 2 ⁇ 4mL resuspend the digested cells, centrifuge at 1000 ⁇ 3000 rpm for 3 ⁇ 5 minutes, use the primary cell culture medium of the present invention
  • the digested single cells are resuspended, and the obtained cell suspension is placed in a T25 cell culture flask pre-plated with trophoblasts to continue expanding the culture.
  • the expanded laryngocarcinoma epithelial cells grow in 2D, which avoids the uneven size of organoids and the internal necrosis of overgrown organoids caused by the expansion of organoid technology.
  • the laryngeal carcinoma epithelial cells cultured by the primary laryngeal carcinoma epithelial cell culture method of the present invention can be used for the efficacy evaluation and screening of drugs, and the method includes the following steps:
  • the culture method cultivates and expands primary laryngeal cancer epithelial cells (especially primary laryngeal cancer tumor cells) to a cell number of at least 10 5 orders of magnitude, preferably at least 10 6 orders of magnitude.
  • the drug takes its maximum plasma concentration Cmax as a reference, and uses 2-5 times Cmax as the initial concentration to dilute multiple different drug concentration gradients, such as 5-10, preferably 6-8 drug concentration gradients.
  • step (1) Digest the laryngocarcinoma epithelial cells cultured in step (1) into a single cell suspension, count them with a flow image counter, and dilute the single cell suspension with the primary cell culture medium of the present invention. With a density of 2000-4000 holes, the diluted cell suspension is evenly added to the multi-well plate, for example, 50 ⁇ L of cell diluent per well, and adhered overnight.
  • This step avoids the problem of cell reprogramming technology that interferes with the primary cell count and subsequent primary cell viability detection due to the presence of feeder cells, and there is no need to combine the cell suspension with Matrigel on ice like the organoid technology.
  • the cumbersome steps of mixing, embedding and re-laying greatly simplify the operation process and enhance the operability and practicability of the technology. Since the seeded cells are a single-cell suspension rather than a 3D structure like organoids, compared with organoid technology, the number of cells plated is more uniform, and the number of cells in the wells produced by the plating is smaller, and it is more suitable for carrying out Subsequent high-throughput drug screening operations.
  • Adopt a high-throughput automated workstation to add gradient dilutions of selected traditional chemotherapy drugs, targeted drugs, antibody drugs or several drug combinations to the adherent cells obtained in step (4) to perform deal with.
  • the Cell-Titer Glo Luminescence Cell Viability Detection Kit (purchased from Promega) is used to detect the survival rate of laryngeal cancer epithelial cells and screen for drug activity.
  • the action time with the drug is also shorter than the drug detection time of the organoid technology (the average administration time of the organoid technology is 6 days).
  • the medium composition does not contain serum, so it is not affected by the quality and quantity of different batches of serum;
  • the expansion efficiency of laryngeal cancer epithelial cells is high. As long as the number of cells is 10 4 levels, 10 6 orders of laryngeal cancer epithelial cells can be successfully expanded in about two weeks. The expanded laryngeal cancer epithelial cells are still Can be passed down continuously;
  • the passage step does not require operation on ice and dissociation of Matrigel, and the digestion and passage of cells can be completed within 10-15 minutes;
  • the cost of culture is controllable: the primary laryngeal cancer culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and other factors, which saves the cost of cell culture;
  • laryngocarcinoma epithelial cells derived from humans or other mammals can be cultured, including laryngocarcinoma tumor cells, normal laryngocarcinoma epithelial cells, laryngocarcinoma epithelial stem cells, or at least containing these cells Any kind of organization.
  • the culture medium of the technology of the present invention can also be used to develop a kit for the expansion and culture of primary laryngeal cancer cells in vitro.
  • the cells obtained by the culture method of this embodiment can be used in regenerative medicine, basic medical research of laryngeal cancer epithelial cells, screening of drug responses, and development of new drugs derived from laryngeal cancer diseases.
  • Figures 1A-1F are graphs showing the effects of different factors in the culture medium on the proliferation of primary laryngeal cancer cells.
  • Figure 2 is a graph showing the effect of increasing the growth of different factors in the culture medium on the proliferation of primary laryngeal cancer cells.
  • Figures 3A-3G are graphs showing the effect of the concentration of each added factor on the proliferation of primary laryngeal cancer cells.
  • Figures 4A and 4B are the cells isolated from a clinical tissue sample of laryngeal cancer (No. 0S0003) using the culture medium SCM of the present invention, respectively, cultured to the 4th day and the 12th day of the laryngeal cancer tumor cells were photographed under an inverted microscope Photo.
  • Figure 5 is a view showing the comparison of the total number of cells collected from a sample of surgically removed laryngeal cancer (No. 0S0006) cultured in 6 different media for 7 days.
  • Fig. 6 is a comparison chart of cell proliferation effects obtained after cells isolated from 6 surgically resected samples of laryngeal cancer (No. 0S0011, 0S0012, 0S0013, 0S0014, 0S0015, 0S0016) cultured in 6 different medium conditions for 7 days.
  • Fig. 7 is a comparison chart of cell growth curves obtained from cells isolated from a clinical tissue sample of laryngeal cancer (No. 0S0004) cultured in 6 different media conditions.
  • Figures 8A and 8B are the cells isolated from a surgically resected sample of laryngeal cancer (No. 0S0015) after being cultured with the culture medium SCM of the present invention to obtain laryngeal cancer tumor cells, using the non-specific nuclear dye DAPI and the laryngeal cancer specific antibody p63, respectively Stained picture.
  • Figure 9 is a comparison diagram of the immunohistochemical results of the original tissue cells from a surgically excised sample of laryngeal cancer (No. 0S0001) and the laryngeal cancer tumor cells obtained by culturing the cells with the culture medium SCM of the present invention.
  • Figures 10A and 10B respectively show the surgical resection of cancer tissue samples (numbered 0S0020 and numbered 0S0022) from two different patients with laryngeal cancer, the doses of laryngeal cancer tumor cells cultured with the culture medium SCM of the present invention to different chemotherapeutic drugs and targeted drugs -Effect curve graph.
  • epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues.
  • Epithelial stem cells refer to cells that have long-term self-renewal capabilities and differentiate into epithelial cells, and refer to stem cells derived from epithelial tissues.
  • epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)) , Liver, pancreas, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc.
  • the cell culture medium of the present embodiment is preferably used for a culture medium derived from laryngocarcinoma epithelial cells.
  • epithelial tumor cell refers to a cell obtained by tumorigenesis of cells derived from the above-mentioned epithelial tissue.
  • organs refers to a three-dimensional, organ-like cell organization formed by spontaneously organizing and gathering cells at a high density in a controlled space.
  • the MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Since the structures of MST1 and MST2 are similar, the MST1/2 kinase inhibitor may also be, for example, a compound that binds to MST1 or MST2 and reduces its activity.
  • Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): Add 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0g) in a round bottom flask Then methanol (30 mL) was added, and then thionyl chloride (1.2 mL) was added dropwise under an ice bath. The reaction system was reacted overnight at 85°C. After the reaction, the solvent was evaporated to dryness under reduced pressure, and the obtained white solid was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a similar method to compound 1, and their structures and mass spectral data are shown in the following table.
  • Laryngeal cancer tissue samples were derived from three cases of surgically removed cancer tissue samples from patients with laryngeal cancer tumors who had been explained and obtained consent. They were sample numbers 0S0020, 0S0021, and 0S0022. The following is an example of a sample (No. 0S0020) for description.
  • tissue samples within half an hour after the patient's surgical resection or biopsy. More specifically, in a sterile environment, cut a tissue sample from a non-necrotic site with a volume of 0.5 cm 3 or more, and place it in a pre-cooled 4 mL tissue transport solution (see Table 1 for specific preparation). In a 5mL plastic sterile cryopreservation tube with lid (purchased from Guangzhou Jiete Biotechnology), cold chain (0-10°C) transported to the laboratory.
  • Tissue digestive juice composition supplier Final concentration HBSS Gibco 50% (volume) RPMI-1640 Corning 50% (volume) Collagenase II Sigma 2mg/mL Collagenase IV Sigma 2mg/mL Deoxyribonucleic acid I Sigma 50U/mL Hyaluronidase Sigma 0.5mg/mL Calcium chloride Shanghai Shenggong 0.33mg/mL Bovine serum albumin Shanghai Shenggong 10mg/mL
  • tissue sample No. 0S0020
  • tissue transport fluid transfer the tissue sample to a 100mm cell culture dish (purchased from NEST)
  • tissue transport fluid wash away the residual blood on the surface of the tissue sample, and remove the tissue Excess tissue such as fat on the surface of the sample.
  • transfer the rinsed tissue sample to another new 100mm petri dish, add 2 mL of transport fluid, and divide the tissue sample into tissue fragments with a volume less than 3 mm 3 with a sterile scalpel blade and surgical forceps.
  • tissue sample fragments Transfer the tissue sample fragments to a 15mL centrifuge tube, centrifuge at 1500 rpm for 4 minutes in a benchtop centrifuge (Sigma Company 3-18K); discard the supernatant, and add tissue transport fluid and tissue digestion fluid at a ratio of 1:1 (use The amount is about 5mL tissue digestion solution for every 10mg of tissue.
  • tissue digestion fluid For specific preparation, see Table 2), mark the sample number, seal with parafilm, and digest at 37°C, 300 rpm constant temperature shaker (ZQLY-180N ZQLY-180N), with an interval of 1 hour Observe whether the digestion is complete.
  • tissue clumps are filtered out by a 70 ⁇ m filter.
  • the tissue clumps on the filter are washed with tissue transport fluid, and the remaining cells are washed into the centrifuge tube and centrifuged at 1500 rpm for 4 minutes.
  • the cultured NIH-3T3 cells (purchased from ATCC, cultured in DMEM medium containing 10% fetal bovine serum) were digested with 0.25% pancreatin (purchased from Thermo Fisher), and then digested with 5% (v/v) Fetal bovine serum (purchased from Ekosai), 100U/mL penicillin and 100 ⁇ g/mL streptomycin in DMEM medium (purchased from Corning) to terminate the digestion, and collect them in a 15mL centrifuge tube, centrifuge at 1500rpm for 4 minutes, then discard Supernatant.
  • BM basic medium
  • Primocin purchased from Invivogen company, concentration of 50mg/mL
  • DMEM/F-12 medium concentration of 100 ⁇ g/mL
  • the medium of different components was added to a 48-well culture plate pre-plated with NIH-3T3 cells irradiated by gamma rays at a volume of 500 ⁇ l/well.
  • the laryngeal carcinoma tumor cells (numbered 0S0064) isolated from laryngeal carcinoma tissues according to the same method in Example 1 were seeded in the above-mentioned pre-plated NIH-3T3 cells after ⁇ -ray irradiation at a cell number of 4 ⁇ 10 4 cells/well.
  • the surface is disinfected and placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher), so that the same number of freshly isolated laryngeal cancer tumor cells (No.
  • 0S0064 are placed in different medium formula conditions Under cultivation.
  • the culture medium was replaced and the trophoblast cells were supplemented every 4 days after the start of the culture. After culturing for 7 days, the cells were counted.
  • a basal medium (BM) without any additives was used as an experimental control. The results are shown in Figures 1A-F.
  • the ordinate in the figure represents the ratio of the number of cells obtained after culturing in different media to the number of cells obtained after culturing in the basal medium BM.
  • adding different concentrations and different factors in Table 3 has different effects on cell proliferation.
  • B27 additives, N2 additives, insulin-transferrin-selenium complex, hepatocyte growth factor, insulin sample growth factor 1, fibroblast growth factor 7, compound 1, Y27632 and A83-01 It has a certain promotion effect on cell proliferation.
  • the culture medium of different components was added to a 48-well culture plate pre-plated with NIH-3T3 cells irradiated by gamma rays at a volume of 500 ⁇ l/well, and the BM medium was used as an experimental control at the same time.
  • the laryngeal carcinoma tumor cells (numbered 0S0065) isolated from laryngeal carcinoma tissues according to the method of Example 1 were seeded in the above-mentioned pre-plated NIH-3T3 cells after ⁇ -ray irradiation at a cell number of 4 ⁇ 10 4 cells/well.
  • the surface is disinfected and placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher), so that the same number of freshly isolated laryngeal cancer tumor cells (No. 0S0065) are placed in different medium formula conditions Under cultivation. After culturing for 7 days, the cells were counted.
  • the experimental results are shown in Figure 2.
  • No. 7 is the most preferred medium of the patent for culturing and expanding primary cells of laryngeal cancer (hereinafter abbreviated as SCM).
  • SCM laryngeal cancer
  • SCM primary laryngeal carcinoma epithelial cell culture medium
  • FGF7 fibroblast growth factor 7
  • BM basal medium
  • IGF-1 insulin-like growth factor 1
  • HGF hepatocyte growth factor
  • ITS insulin-transferrin-selenium complex
  • laryngeal cancer epithelial cells derived from the cancer tissue were isolated from the cancer tissue (sample number 0S0005) of a patient with laryngeal cancer. Then, the laryngeal cancer tumor cells derived from the cancer tissue were counted with a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. Then, the cells were seeded at a density of 4 ⁇ 10 4 cells/cm 2 into a 12-well plate pre-plated with NIH-3T3 cells irradiated by gamma rays.
  • a flow image counter Joangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL
  • the cell pellet after centrifugation was resuspended in basal medium BM, and counted using a flow image counter (Jiangsu Zhuo Microbe Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells.
  • the obtained cells were used in the following culture experiments.
  • the medium SCM component does not contain fibroblast growth factor 7;
  • Formulation 2 The medium SCM component does not contain insulin-transferrin-selenium complex
  • Formulation 3 The medium SCM component does not contain insulin-like growth factor 1;
  • Formulation 4 The medium SCM component does not contain hepatocyte growth factor
  • Formulation 5 Y27632 is not included in the SCM component of the medium
  • Formulation 6 The medium SCM component does not contain compound 1;
  • Formulation 7 A83-01 is not contained in the SCM component of the medium
  • fibroblast growth factor 7 When using the medium of formula 1, add 250 microliters of prepared fibroblast growth factor 7 to each well of a 48-well plate seeded with primary cells, and the final concentration of fibroblast growth factor 7 is 80ng/ ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and use the medium of formula 1 to set control wells (BC).
  • the medium of formula 2 When using the medium of formula 2, add the prepared insulin-transferrin-selenium complex 250 microliters per well to the 48-well plate inoculated with primary cells, and store the insulin-transferrin-selenium complex.
  • the final concentration of the solution is 1:1600, 1:800, 1:400, 1:200, 1:100, 1:50, 1:25 (corresponding to the final concentration of insulin/transferrin/sodium selenite, respectively 0.3125 ⁇ g/ml-0.15625 ⁇ g/ml-0.15625ng/ml; 0.625 ⁇ g/ml-0.3125 ⁇ g/ml-0.3125ng/ml; 1.25 ⁇ g/ml-0.625 ⁇ g/ml-0.625ng/ml; 2.5 ⁇ g/ml -1.25 ⁇ g/ml-1.25ng/ml; 5 ⁇ g/ml-2.5 ⁇ g/ml-2.5ng/ml; 10 ⁇ g/ml-5 ⁇ g/ml-5ng/ml;
  • the medium of formula 3 When using the medium of formula 3, add 250 microliters of prepared insulin-like growth factor 1 per well to a 48-well plate seeded with primary cells. The final concentration of insulin-like growth factor 1 is 80ng/ml, respectively. 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and use the medium of formula 3 to set control wells (BC).
  • hepatocyte growth factor When using the medium of formula 4, add 250 microliters of prepared hepatocyte growth factor per well to the 48-well plate inoculated with primary cells, and the final concentration of hepatocyte growth factor is 80ng/ml and 40ng/ml respectively. ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml; and use the medium of formula 4 to set control wells (BC).
  • Y27632 When using the medium of formula 5, add 250 microliters of Y27632 per well to the 48-well plate seeded with primary cells.
  • the final concentration of Y27632 is 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.625 ⁇ M; and use the medium of formula 5 to set up control wells (BC).
  • the medium of formula 7 When using the medium of formula 7, add 250 microliters of prepared A83-01 to each well of a 48-well plate seeded with primary cells.
  • the final concentration of A83-01 is 4000nM, 2000nM, 1000nM, 500nM, 250nM, 125nM, 62.5nM, and use the medium of formula 7 to set control wells (BC).
  • the ratio is the ratio of the number of cells obtained by culturing for one generation using each medium to the number of cells obtained by culturing for one generation in the corresponding control wells.
  • a ratio greater than 1 indicates that the formulated medium containing different concentrations of factors or small molecule compounds has a better proliferation effect than the control medium; a ratio less than 1, indicates that the formulated medium containing different concentrations of factors or small molecule compounds promotes proliferation The effect is weaker than that of the control culture medium in promoting proliferation.
  • the content of fibroblast growth factor 7 in the culture medium is preferably 5 to 80 ng/ml, more preferably 20 to 80 ng/ml;
  • the volume concentration of the insulin-transferrin-selenium complex is preferably 1:25 ⁇ 1:100, more preferably 1:25 ⁇ 1:50 (corresponding to the final concentration of insulin/transferrin/sodium selenite respectively 5 ⁇ 20 ⁇ g/ml-2.5 ⁇ 10 ⁇ g/ml-2.5 ⁇ 10ng /ml; preferably 10 ⁇ 20 ⁇ g/ml-5 ⁇ 10 ⁇ g/ml-5 ⁇ 10ng/ml);
  • the content of insulin-like growth factor 1 is preferably 10ng/ml ⁇ 80ng/ml, more preferably 10ng/ml ⁇ 40ng/ml
  • the content of hepatocyte growth factor is preferably 10ng/ml ⁇ 80ng/ml, more preferably 10ng/ml ⁇ 40ng/ml;
  • the content of Y27632 is preferably 1.25 ⁇ M
  • laryngeal cancer epithelial cells derived from the cancer tissue were isolated from the cancer tissue (sample number 0S0003) of patients with laryngeal cancer. Then, the laryngeal cancer tumor cells derived from the cancer tissue were counted with a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. Then, the cells were seeded at a density of 4 ⁇ 10 4 cells/cm 2 into a 12-well plate pre-plated with NIH-3T3 cells irradiated by gamma rays. Add 2 mL of the prepared primary laryngeal carcinoma epithelial cell culture medium SCM to the 12-well plate, and place it in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for culture.
  • Figure 4A is a 12-well plate of NIH-3T3 cells pre-plated with ⁇ -ray irradiated NIH-3T3 cells at a density of 4 ⁇ 10 4 cells/cm 2 in this example. Take pictures with an inverted phase-contrast microscope). Observation under the microscope shows that the primary laryngeal cancer tumor cells derived from the cultured cancer tissues have formed larger clones.
  • Figure 4B is a microscopic photograph of this example from the 12th day of culture after inoculation (photographed with a 100-fold inverted phase-contrast microscope). The cells in the field of view are already overgrown.
  • the primary laryngeal carcinoma cells obtained after isolation can be cultured in vitro for 4 days and can see obvious clonal formation under the microscope, and after 12 days of expansion, the number of cells has been significantly increased.
  • Amplification indicates that the technique of the present invention is an efficient technique for amplifying laryngeal carcinoma epithelial cells in vitro.
  • Example 2 The same method as in Example 2 was used to prepare primary laryngeal carcinoma epithelial cell culture medium SCM and basal medium BM as a control.
  • the medium FM used in the literature of cell conditional reprogramming technology was prepared as another control example.
  • the primary cell culture medium SCM-1 and SCM-2 for laryngeal cancer were prepared. The formula was based on the SCM using 1:50 volume ratio of B27 and 1:100 volume ratio of N2 additives to replace insulin- Transferrin-selenium complex.
  • the commercial medium Defined Keratinocyte SFM was purchased from Gibco Company, also referred to as “KSFM medium” below). The medium formula is shown in Table 6.
  • KSFM Keratinocyte SFM
  • Keratinocyte-SFM Basal Medium Gibco 99% by volume Defined Keratinocyte-SFM Growth Supplement Gibco 1 volume%
  • Example 2 The same method as in Example 1 was used to obtain primary laryngeal carcinoma tumor cells derived from laryngeal carcinoma tissue (No. 0S0006). Then, at the same density (4 ⁇ 10 4 pieces/cm 2 ), they were cultured under the following 6 culture conditions:
  • the technology of the present invention inoculate primary laryngeal cancer tumor cells into a 24-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4 ⁇ 10 4 cells/cm 2 , Using 1 mL of the primary laryngeal carcinoma epithelial cell culture medium SCM of the present invention for culture;
  • Cell conditional reprogramming technology inoculate primary laryngeal cancer tumor cells at a seeding density of 4 ⁇ 10 4 cells/cm 2 on NIH-3T3 cells (purchased from ATCC) that have been irradiated with gamma rays.
  • 1 mL of cell conditional reprogramming technology medium FM was cultured in a 24-well plate (see (Liu et al., Nat. Protoc., 12(2):439-451, 2017) for specific steps);
  • D. Inoculate primary laryngeal cancer tumor cells into a 24-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells (purchased from ATCC) at a seeding density of 4 ⁇ 10 4 cells/cm 2, using 1 mL of The medium SCM-2 is cultivated in a 24-well plate;
  • the primary laryngeal cancer tumor cells were inoculated into a 24-well plate at a seeding density of 4 ⁇ 10 4 cells/cm 2, and cultured in a 24-well plate with 1 mL of commercial medium KSFM.
  • F. Inoculate primary laryngeal cancer tumor cells at a seeding density of 4 ⁇ 10 4 cells/cm 2 into a 24-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells (purchased from ATCC), using 2 mL of The basal medium BM is cultured in a 24-well plate.
  • the cells cultured under the five culture conditions are exchanged every 5 days.
  • the primary laryngeal cancer tumor cells (No. 0S0006) cultured using the technology of the present invention, when the cell growth in the culture plate reaches about 80% of the bottom area, discard the medium supernatant in the 24-well plate, and add 0.5 mL 0.25 Digest with% pancreatin (purchased from Thermo Fisher) for 1 minute, then aspirate 0.25% pancreatin, and then add 0.5 mL 0.05% pancreatin for cell digestion, and incubate at 37°C for 10 minutes until it can be used under the microscope (Invitrogen EVOS M500) It is observed that the cells have been digested completely, that is, 1mL of DMEM/F12 medium containing 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin is used to terminate the digestion, and collected into a 15mL centrifuge tube at 1500rpm After centrifugation for 4 minutes, the supernatant was discarded.
  • the cell pellet after centrifugation was resuspended using the culture medium of the present invention, and counted using a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL), and the total number of cells was 317,000.
  • the cells cultured under the other five culture conditions were digested and counted in the same manner as above.
  • the total number of cells cultured with medium FM, SCM-1, SCM-2, KSFM and BM were 166,700, 266,100, respectively. 277,200, 95,600 and 35,900.
  • Figure 5 is a plot of the total number of cells expanded by the number 0S0006 cell under different conditions.
  • Figure 6 is a sample of 6 patients with laryngeal cancer (numbered 0S0011, 0S0012, 0S0013, 0S0014, 0S0015, 0S0016).
  • the primary laryngeal cancer cells obtained according to Example 1 were cultured under the above six different media conditions for 7 days to obtain the cell proliferation effect
  • ⁇ represents a certain degree of cloning ability and proliferation effect
  • ⁇ represents a more obvious cloning ability and proliferation effect
  • ⁇ represents a very obvious cloning ability and proliferation effect
  • ⁇ It means that a clone cannot be formed. From the figure, it can be confirmed that SCM medium has obvious advantages in cloning ability and promoting cell proliferation in the primary cell culture obtained from laryngeal cancer tissues compared with the other five culture conditions.
  • the primary laryngeal carcinoma epithelial cell culture medium SCM was obtained using the same method as in this example (1), as well as the control culture medium FM, SCM-1, SCM-2, KSFM and BM.
  • the primary laryngeal carcinoma tumor cells (numbered 0S0004) derived from laryngeal carcinoma tissues were cultured in six culture media using the same method as in this example (1), and then digested, passaged and counted.
  • Figure 7 shows the growth curves of the OS0004 cells under six different culture conditions drawn by Graphpad Prism software.
  • the abscissa represents the number of days the cells have been cultured, and the ordinate is the cumulative cell proliferation fold, which represents the multiple of the cell expansion during the culture cycle. The larger the value, the more the cell has been expanded in a certain cycle, that is, the expanded cell The more the number is, the slope represents the rate of cell expansion. From the figure, it can be confirmed that the growth rate of laryngeal carcinoma epithelial cells cultured in the culture medium SCM and SCM-1 and SCM-2 of the present invention is better than that of the other three culture conditions. Continuous training.
  • laryngeal cancer epithelial cells derived from the cancer tissue were isolated from the cancer tissue (sample number 0S0015) of patients with laryngeal cancer. Then, the laryngeal cancer tumor cells derived from the cancer tissue were counted with a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) to obtain the total number of cells. Then inoculate at a density of 4 ⁇ 10 4 cells/cm 2 into a 24-well plate pre-plated with ⁇ -ray irradiated NIH-3T3 cells.
  • round cell fragments for immunofluorescence staining are placed in the 24-well plate in advance ( (Purchased from Thermo Fisher Scientific). Add 1 mL of the prepared primary laryngeal carcinoma epithelial cell culture medium SCM to the 24-well plate, and place it in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for culture.
  • Image under microscope Invitrogen EVOS M500, take pictures and record.
  • Fig. 8A and Fig. 8B are the pictures taken by fluorescence in different fields of view under a 10x objective lens respectively.
  • Fig. 8A is a picture of a cell nucleus stained with the non-specific fluorescent dye DAPI
  • Fig. 8B is a picture stained with a laryngeal cancer specific antibody p63 (located in the nucleus) .
  • the positions of the marked cell nuclei in Fig. 8A are all stained green in Fig. 8B, indicating that the cultured cells are laryngeal squamous cell carcinoma cells, which is consistent with the clinicopathological diagnosis.
  • Example number 0S0001 From a clinical surgical resection sample of a patient with laryngeal cancer, a cancer tissue about the size of a mung bean grain (sample number 0S0001) was taken out and immersed in 1 mL 4% paraformaldehyde for fixation. The remaining cancer tissues were obtained using the same method as in Example 1 to obtain laryngeal cancer epithelial cells (sample number 0S0001). Using the method of Example 3, the sample OS0001 was continuously cultured to the sixth generation using the culture medium SCM of the present invention.
  • Immunohistochemical method was used to detect the expression of important biomarkers related to laryngeal cancer in the original tissue of the sample 0S0001 and the primary cells obtained from continuous culture to the sixth generation.
  • the tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 ⁇ m thick tissue sections with a microtome.
  • the primary antibodies used were Cytokeratin (pCK) (purchased from CST Company), p63 antibody (purchased from CST Company), and Ki67 antibody (purchased from R&D Company).
  • Fig. 9 is a comparison diagram of immunohistochemical results of primitive tissue cells and laryngeal cancer tumor cells obtained by culturing the cells with the culture medium SCM of the present invention. It can be confirmed from Fig. 9 that when the laryngeal cancer tumor cells (sample number 0S0001) cultured using the technology of the present invention are cultured to the 6th generation, the expression of biomarkers related to laryngeal cancer on the cells and the labeling of the cell-derived original tissue sections The expression of objects is basically the same. It shows that the cells cultured by the technique of the present invention maintain the original pathological characteristics of the cancer tissue of patients with laryngeal cancer.
  • laryngeal cancer tumor cells (numbered 0S0003) were isolated from the cancer tissue of a patient with a pathological diagnosis of laryngeal cancer.
  • the culture medium SCM of the present invention was used to culture OS0003.
  • the method of Example 4 was used to digest and collect the laryngeal cancer tumor cells.
  • the volume and growth rate of tumor cells formed by laryngocarcinoma tumor cells in the mouse body were observed every three days on the fat pad of the larynx cancer and the axilla of the right forelimb in deficient mice (NCG) mice (purchased from Nanjing Model Animal Research Institute).
  • the laryngeal cancer tumor cells cultured from a patient-derived laryngeal cancer tumor sample can be used to detect the sensitivity of the patient’s tumor cells to different drugs.
  • Plating of primary laryngeal cancer tumor cells The cell suspension of laryngeal cancer tumor cells (No. 0S0020 and No. 0S0022) obtained according to the method in Example 1 was inoculated at a density of 4 ⁇ 10 4 cells/cm 2 In a 12-well plate covered with NIH-3T3 cells irradiated by gamma rays. Add 2 mL of the prepared primary laryngeal carcinoma epithelial cell culture medium SCM to the 12-well plate, and place it in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher) for culture.
  • the cell pellet after centrifugation was resuspended in SCM medium, and counted using a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL), and the total number of cells was 830,000 and 768,000 respectively. Inoculate in a 384-well plate at a density of 2000-4000 cells/well to allow the cells to adhere overnight.
  • (1) Prepare the drug storage plate by the method of concentration gradient dilution: draw 40 ⁇ L of 10 ⁇ M drug stock solution as the highest concentration, and then draw 10 ⁇ L from it and add it to a 0.5mL EP tube containing 20 ⁇ L of DMSO, and then from the above EP Draw 10 ⁇ L from the tube into the second 0.5mL EP tube containing 20 ⁇ L of DMSO, that is, dilute the drug according to 1:3. Repeat the above method, dilute in sequence, and finally obtain the 7 concentrations required for dosing. Add different concentrations of drugs to the 384-well drug storage plate. In the solvent control group, an equal volume of DMSO was added to each well as a control.
  • the drugs to be tested are bortezomib (purchased from MCE), disulfiram (purchased from MCE), gefitinib (purchased from MCE), and erlotinib (purchased from MCE) .
  • Cell viability detection 72 hours after the administration, use Cell Titer-Glo detection reagent (purchased from Promega) to detect the chemiluminescence value of the cells after the drug is added to the culture.
  • the value of the chemiluminescence value reflects the cell viability and the drug's cell viability
  • cell survival rate (%) chemiluminescence value of dosing hole / chemiluminescence value of control hole * 100%, calculate the cell survival rate of cells after different drugs are applied, and use Graphpad Prism software to map And calculate the half inhibition rate IC 50 , and calculate the cell survival rate corresponding to the maximum blood concentration Cmax of different drugs in the human body at the same time.
  • Figures 10A and 10B respectively show laryngeal cancer tumor cells cultured from surgically removed cancer tissue samples (No. 0S0020 and No. 0S0022) of two different patients with laryngeal cancer, and the treatment of two chemotherapy drugs, bortezomib and disulfiram Sensitivity and sensitivity to targeted drugs gefitinib and erlotinib.
  • 10A is the sensitivity result of the laryngeal cancer cell cultured with the sample number 0S0020 to the four drugs
  • 10B is the sensitivity result of the laryngeal cancer cell cultured with the sample number 0S0022 to the four drugs.
  • the concentration corresponding to the marked line on the abscissa in the figure is the maximum blood concentration Cmax of these four drugs in the human body.
  • the results show that the cells of the same patient have different sensitivity to different drugs at the maximum blood concentration in the human body, and the sensitivity of different patients’ cells to the drug at the maximum blood concentration in the human body is also different. According to the results, laryngeal cancer can be judged. The effectiveness of the patient in clinical use of the drug.
  • the present invention provides a culture medium and culture method for culturing or amplifying primary laryngeal carcinoma epithelial cells in vitro.
  • the cell progeny and organoids cultured using the culture medium and culture method of the present invention can be used to evaluate and evaluate the therapeutic effect of drugs. Screening, toxicity determination and regenerative medicine. Therefore, the present invention is suitable for industrial applications.

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Abstract

提供一种用于培养喉癌上皮细胞的、含有MST1/2激酶抑制剂的原代细胞培养基和使用了该原代细胞培养基的培养方法。所述培养方法中，使用上述原代细胞培养基在预铺有辐照后滋养细胞的培养器皿上培养原代细胞，使得原代细胞快速增殖。由所述的原代细胞培养基和原代细胞培养方法得到的细胞模型，能够用于药物的疗效评估和筛选。

Description

一种用于喉癌上皮细胞的培养基、培养方法及其应用 技术领域
本发明属于医药技术领域，具体而言，涉及用于在体外培养或扩增原代喉癌上皮细胞的培养基及培养方法，还涉及培养得到的细胞在药物的疗效评估和筛选中的方法和应用。
背景技术
喉癌发病率占全身恶性肿瘤的5.7％～7.6％，在耳鼻喉科领域中仅次于鼻咽癌和鼻腔鼻窦癌，居第三位。好发年龄为50～70岁。我国以东北地区发病率最高，患病者中男性居多，其病因尚不十分清楚，但空气污染重的城市高于污染轻的城市。由于喉癌发病的分子机制尚不明确，针对喉癌尤其是中晚期患者的治疗手段仍然有限，导致临床上患者五年生存率较低，缺乏个性化的精准用药指导。
功能性测试是指在体外对抗肿瘤药物在癌症患者细胞上的敏感性进行检测的方法。应用这一方法的关键在于开发生长周期短且能够代表喉癌患者自身生物学特性的肿瘤细胞模型。另外，所述细胞模型应操作便捷且能快速高效地预测临床用药的疗效，从而及时给予癌症患者精准用药指导。然而，取自癌症患者的原代肿瘤细胞在体外建立细胞模型成功率往往很低，生长周期长，并存在成纤维细胞等间质细胞过度增殖等问题，制约着这一领域的发展。目前有两种培养原代上皮细胞/干细胞的技术在肿瘤细胞功能性测试应用领域发展得相对成熟，一种是使用经辐射的滋养细胞和ROCK激酶抑制剂来促进原代上皮细胞的生长以考察个体患者的药物敏感性的技术，即细胞条件重编程技术(Liu等，Am.J.Pathol.，180：599-607，2012)。另一种技术是体外3D培养成体干细胞从而获得类似于组织器官的类器官技术(Hans Clevers等，Cell，11，172(1-2)：373-386，2018)。
然而，类器官技术是将患者自体原代上皮细胞包埋在细胞外基质内进行体外三维立体培养的技术，但是该技术的培养基内需添加多种特定的生长因子(如Wnt蛋白和R-spondin家族蛋白)，成本昂贵，不适于普及到临床进行大规模应用。另外，类器官在整个培养过程中均 需将细胞包埋在细胞外基质胶中，其细胞接种、传代和药物敏感性测试的铺板步骤相较于2D培养操作繁琐费时，且该技术所形成的类器官大小尺寸不好控制，易出现部分类器官生长过大而导致内部发生坏死的情况。因此，类器官技术相较于2D培养技术可操作性和适用性不强，需要专业技术人员操作，不适合大规模广泛应用于临床体外药物敏感性检测(Nick Barker，Nat.Cell Biol.，18(3)：246-54，2016)。
细胞重编程技术是一种将患者自体原代上皮细胞与鼠源性饲养细胞共培养的技术，但是在文献中未见报道针对喉癌样本进行测试，故文献报道的培养基成分对喉癌原代肿瘤细胞是否能够快速的扩增还未可知。
鉴于以上技术的局限性，临床上需要开发一种原代喉癌上皮细胞培养技术，其培养周期短，成本可控，操作便捷，在将该技术应用于构建原代喉癌肿瘤细胞模型时，所培养的喉癌肿瘤细胞能代表喉癌患者自身的生物学特性。通过体外评估抗肿瘤药物在不同癌症患者个体所衍生的细胞模型上的敏感性，来提高临床上抗肿瘤药物的响应率，减少不合适的药物给患者造成的痛苦及医疗资源的浪费。
发明内容
本发明旨在针对现有技术的不足，提供一种用于培养原代喉癌上皮细胞的培养基以及使用该培养基的原代喉癌上皮细胞的培养方法。采用本发明的原代喉癌上皮细胞培养基和培养方法，能够达到体外培养周期短、成本可控、操作便捷的目的。在该技术应用于构建原代喉癌肿瘤细胞模型时，能够获得具有喉癌患者自身生物学特性的原代喉癌肿瘤细胞，并能够应用于新药筛选和体外药物敏感性检测。
本发明的一个方面在于提供一种用于培养原代喉癌上皮细胞的原代细胞培养基，其含有MST1/2激酶抑制剂，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物。
Figure PCTCN2020103428-appb-000001
其中，
R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等)；
R 2和R 3各自独立地选自C1-C6烷基，优选C1-C3烷基，更优选甲基；
R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基(所述杂环基选自例如哌啶基、四氢吡喃基等)；
R 6选自卤素(优选氟和氯，更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
优选的实施方式中，MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
Figure PCTCN2020103428-appb-000002
其中，
R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基，R 1更优选为任选地被1-2个独立地R 6取代的苯基；
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢；
R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基，R 6更优选为氟、甲基或三氟甲基。
优选地，所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。
Figure PCTCN2020103428-appb-000003
Figure PCTCN2020103428-appb-000004
Figure PCTCN2020103428-appb-000005
Figure PCTCN2020103428-appb-000006
Figure PCTCN2020103428-appb-000007
最优选地，本发明的MST1/2激酶抑制剂为化合物1。
在本发明的实施方式中，MST1/2激酶抑制剂在培养基中的含量通常为1.25μM～10μM，优选为2.5μM～10μM，更优选为5μM。
本发明的原代细胞培养基优选还含有以下因子中的一种或多种：胰岛素样生长因子1(IGF-1)；成纤维生长因子7(FGF7)；胰岛素-转铁蛋白-硒复合物(ITS)；肝细胞生长因子(HGF)；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂；和选自A83-01、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511中的至少一种的TGFβI型受体抑制剂。
其中，在优选的实施方式中，所述成纤维细胞生长因子7在培养基中的含量优选为5～80ng/ml，更优选20～80ng/ml；胰岛素-转铁蛋白-硒复合物中胰岛素/转铁蛋白/亚硒酸钠各自的含量分别为5～20μg/ml-2.5～10μg/ml-2.5～10ng/ml；优选10～20μg/ml-5～10μg/ml-5～10ng/ml；所述胰岛素样生长因子1的含量优选为10～80ng/ml，更优选为10～40ng/ml；所述肝细胞生长因子的含量优选为10～80ng/ml，更优选为10～40ng/ml；所述ROCK激酶抑制剂优选为Y27632，且ROCK激酶抑制剂的含量优选为1.25～20μM，更优选为2.5～10μM； 所述TGFβI型受体抑制剂优选A83-01，且TGFβI型受体抑制剂的含量优选为125～1000nM，更优选为125～500nM。
该培养基配方成分与细胞条件重编程培养基和类器官培养基成分相比，添加了MST1/2激酶抑制剂，但不包含血清、牛垂体提取物等不确定成分，也不包含Wnt激动剂、R-spondin家族蛋白、BMP抑制剂等类器官培养所必须的龛因子，并且不包含烟酰胺和N-乙酰半胱氨酸，从而大大降低了培养基的成本，简化了配制培养基的操作流程，实现了成本可控和操作便捷的原代喉癌上皮细胞的体外培养。
本发明中，原代喉癌上皮细胞可以为喉癌肿瘤细胞、正常喉癌上皮细胞、喉癌上皮干细胞。
本发明的一个方面在于提供一种原代喉癌上皮细胞的培养方法，其包括以下步骤：
(1)按上述配方配制本发明的原代细胞培养基。
(2)使用辐照后的滋养细胞预铺培养容器。
具体地，所述的滋养细胞例如可以为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，优选为γ射线，辐照剂量为30～50Gy，优选为35Gy。具体的，将辐照后的NIH-3T3细胞按照2×10 4个/cm 2接种在培养容器例如48孔板、24孔板、12孔板、6孔板或者T25细胞培养瓶中，待细胞贴壁后备用。
(3)从喉癌组织分离得到原代喉癌上皮细胞。
原代喉癌上皮细胞例如可以来源于喉癌组织样本和癌旁组织样本。喉癌组织样本例如来源于进行过说明并获得同意的喉癌肿瘤患者手术切除的癌组织样本，癌旁组织样本采集自离喉癌组织距离至少5cm以上的喉组织。在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的3-5mL DMEM/F12培养基中，培养基盛在塑料无菌带盖离心管内，冰上运输至实验室；其中，DMEM/F12培养基中含有1-2体积％青霉素/链霉素、和/或0.2-0.4体积％Primocin(以下简称组织运输液)。当使用链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为200μg/mL，青霉素浓度范围为25～400U/mL，优选为50～200U/mL，更 优选为200U/mL；当使用Primocin时，浓度范围为25～400μg/mL，优选为50～200μg/mL，更优选为100μg/mL。
在生物安全柜内，将组织样本转移至细胞培养皿内，用运输液润洗组织样本，将组织样本表面的血细胞清洗掉。将润洗后的组织样本转移至另一个新的培养皿内，加入1-3mL运输液，用无菌手术刀片和手术镊将组织样本分割为体积小于3mm 3的组织碎块。
将组织样本碎块转移至离心管内，用台式离心机(Sigma公司3-18K)以1000～3000转/分钟离心3～5分钟；弃上清，按1:1比例加入组织运输液和组织消化液(使用量约为每10mg组织使用5mL组织消化液，其中组织消化液的配制方法为：将1～2mg/mL胶原酶Ⅱ、1～2mg/mL胶原酶Ⅳ、50～100U/mL脱氧核糖核酸Ⅰ、0.5～1mg/mL透明质酸酶、0.1～0.5mg/mL氯化钙、5～10mg/mL牛血清白蛋白溶于体积比1:1的HBSS和RPMI-1640中)，标记样本编号，封口膜密封，以37℃、200～300转恒温摇床(知楚仪器ZQLY-180N)消化，每间隔1小时观察消化是否完成；若未见明显组织块即可终止消化，否则继续消化，直至消化充分，消化时间范围为4～8小时。消化完成后，细胞滤网(细胞筛孔径为例如70μm)过滤掉未消化的组织团块，滤网上的组织团块用组织运输液冲洗，将残留细胞冲入离心管中，用台式离心机以1000～3000转/分钟离心3～5分钟。弃上清，观察剩余细胞团是否含有血细胞，若有血细胞，加1～5mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解10～20分钟，5分钟摇晃混匀一次，裂解结束后取出，以1000～3000转/分钟离心3～5分钟。弃上清，加入本发明的原代细胞培养基重悬，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。
(4)在预接种有滋养细胞的培养器皿内接种步骤(3)中分离得到的原代喉癌上皮细胞，并采用步骤(1)中的原代细胞培养基进行培养。
更具体而言，预先在多孔板的一个孔中按2×10 4～4×10 4个/cm 2(例如2×10 4个/cm 2)的密度接种35Gy辐照剂量γ射线辐照后的NIH-3H3细胞，待细胞贴壁后，按2×10 4～8×10 4个/cm 2(例如4×10 4个/cm 2)的密度接种原代喉癌肿瘤细胞，每孔加入0.5～2mL原代上皮细胞培养基， 在例如37℃、5％CO 2的条件下于细胞培养箱中培养8-16天，期间每4天换成新鲜的原代细胞培养基，在原代喉癌上皮细胞长至占多孔板底面积80％～90％左右的细胞密度时进行消化传代。
该步骤相比类器官技术，无需在冰上将原代细胞和基质胶混匀后形成胶滴，并等待胶滴凝固后加入培养基。此外，还节约了价格昂贵的细胞外基质胶的使用量，简化了操作步骤。
任选地，接种后的原代喉癌上皮细胞在培养8～16天后，当培养容器内形成的细胞克隆汇合达到底面积80％，弃去上清，加入1～2mL0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入1～2mL 0.05％胰酶进行细胞消化，室温下孵育5～20分钟；然后用含有例如5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的培养液2～4mL重悬消化处理后的细胞，以1000～3000转/分钟离心3～5分钟，使用本发明的原代细胞培养基将消化后的单细胞重悬，将所得到的细胞悬液置入预铺有滋养细胞的T25细胞培养瓶中继续扩大培养。T25细胞培养瓶的预处理操作同步骤(2)。
扩增的喉癌上皮细胞呈2D生长，避免了类器官技术扩增出现的类器官大小不均一和生长过大的类器官出现内部坏死等情况。
另一方面，由本发明的原代喉癌上皮细胞的培养方法培养得到的喉癌上皮细胞、特别是喉癌肿瘤细胞，能够用于药物的疗效评估和筛选，所述方法包括以下步骤：
(1)获取原代喉癌上皮细胞，特别优选获取源自喉癌患者的癌组织样本或活检癌组织样本，分离得到原代喉癌上皮细胞，根据如上所述的原代喉癌上皮细胞的培养方法培养并扩增原代喉癌上皮细胞(特别是原代喉癌肿瘤细胞)达至少10 5数量级、优选至少10 6数量级的细胞数目。
(2)选定需要检测的药物。
(3)药物以其最大血浆浓度C max为参考，以2-5倍C max为起始浓度，稀释多个不同的药物浓度梯度，例如5-10个、优选6-8个药物浓度梯度。
(4)将步骤(1)中培养得到的喉癌上皮细胞消化成单细胞悬液，用流式图像计数仪进行计数，用本发明的原代细胞培养基将单细胞悬 液稀释，按每孔2000-4000个的密度将稀释后的细胞悬液均匀地加入到多孔板内，例如每孔50μL细胞稀释液，并进行过夜贴壁。
该步骤避免了细胞重编程技术出现的由于饲养细胞的存在而干扰原代细胞计数和后续的原代细胞活力检测的问题，也无需像类器官技术一样，在冰上将细胞悬液与基质胶混合包埋再铺板的繁琐步骤，从而大大简化了操作流程，增强了技术的可操作性和实用性。由于接种的细胞为单细胞悬液而不是像类器官一样的3D结构，所以该技术与类器官技术相比，铺板的细胞数更加均一，铺板产生的孔间细胞数差异小，也更适合进行后续的高通量药物筛选操作。
(5)采用高通量自动化工作站，对步骤(4)中得到的贴壁细胞添加梯度稀释后的所选定的传统化疗药物、靶向药物、抗体药物或几种药物组合等候选药物，进行处理。
(6)加药处理数小时后，例如72小时后，采用Cell-Titer Glo发光法细胞活力检测试剂盒(购自Promega公司)检测喉癌上皮细胞的存活率，进行药物活性筛选。
具体而言，向每孔加入例如10μL Cell Titer-Glo试剂(购自Promega公司)，均匀震荡后，用荧光酶标仪测量各孔的化学发光强度，根据测得的数值，以药物浓度为横坐标，荧光强度为纵坐标，应用GraphPad Prism软件绘制药物量-效曲线，计算各个药物对所测试细胞的增殖的抑制强度。
在本发明的原代喉癌肿瘤细胞在药物筛选和体外药物敏感性检测的应用中。由于细胞呈2D生长，与药物的作用时间也比类器官技术的药物检测时间短(类器官技术的平均给药时间为6天)。
本发明的有益效果还包括：
(1)提高原代喉癌上皮细胞培养的成功率，成功率达到85％以上；
(2)保证体外原代培养的喉癌上皮细胞能够保持原代细胞来源病人的病理表型和异质性；
(3)培养基成分不含血清，所以不受不同批次血清质量和数量的影响；
(4)扩增喉癌上皮细胞效率高，只要有10 4级别的细胞数量就可在两周左右时间内成功扩增出10 6数量级的喉癌上皮细胞，扩增出的喉 癌上皮细胞还可以连续传代；
(5)传代步骤无需冰上操作和解离基质胶，10-15分钟内即可完成细胞的消化传代；
(6)培养成本可控：原代喉癌癌培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、BMP抑制剂等因子，节约了细胞培养的成本；
(7)操作便捷，该技术相比类器官技术，无需像类器官技术一样将细胞包埋于基质胶内，所述技术操作步骤简便易行；
(9)所述技术培养获得的喉癌上皮细胞数量大，均一化程度高，适合高通量筛选新候选化合物，并为病人提供高通量药物体外敏感性功能测试。
采用本实施方式的细胞培养基，可培养来源于包括人的或其他哺乳动物的喉癌上皮细胞，包括喉癌肿瘤细胞、正常喉癌上皮细胞、喉癌上皮干细胞、或者包含这些细胞中的至少任一种的组织。同时本发明技术的培养基还可以用于开发体外喉癌原代细胞扩增培养的试剂盒。
另外，通过本实施方式的培养方法获得的细胞可应用于再生医疗、喉癌上皮细胞的基础医学研究、药物应答的筛选、以及来源于喉癌疾病的新药研发等。
附图说明
图1A-1F为显示培养基中不同因子对喉癌原代细胞增殖的影响的图。
图2是显示培养基中不同因子递增对喉癌原代细胞增殖的影响的图。
图3A-3G是显示各添加因子的浓度对喉癌原代细胞增殖的影响的图。
图4A和4B是将从1例喉癌临床组织样本(编号0S0003)分离得到的细胞采用本发明的培养基SCM分别培养至第4天和第12天的喉癌肿瘤细胞在倒置显微镜下拍摄的照片。
图5是从1例喉癌手术切除样本分离得到的细胞(编号0S0006)采用6种不同培养基培养7天后收集到的细胞总数比较的视图。
图6是从6例喉癌手术切除样本(编号0S0011、0S0012、0S0013、0S0014、0S0015、0S0016)分离得到的细胞在6种不同培养基条件下培养7天后得到细胞增殖效果的比较图。
图7是将从1例喉癌临床组织样本(编号0S0004)分离得到的细胞分别采用6种不同培养基条件下培养所获得的细胞生长曲线对比图。
图8A和8B是从1例喉癌手术切除样本(编号0S0015)分离得到的细胞采用本发明的培养基SCM培养获得喉癌肿瘤细胞后，分别使用非特异性细胞核染料DAPI和喉癌特异性抗体p63染色的图片。
图9是从1例喉癌手术切除样本(编号0S0001)的原始组织细胞和采用该细胞以本发明的培养基SCM培养而获得的喉癌肿瘤细胞的免疫组化结果对比图。
图10A和10B分别表示从两个不同的喉癌患者的手术切除癌组织样本(编号0S0020和编号0S0022)采用本发明的培养基SCM培养的喉癌肿瘤细胞对不同化疗药物和靶向药物的剂量-效应曲线图。
具体实施方式
本说明书中，上皮细胞包括从上皮组织获取的已分化的上皮细胞及上皮干细胞。“上皮干细胞”是指具有长期的自我更新能力和向上皮细胞分化的细胞，是指来源于上皮组织的干细胞。作为上皮组织，可例举例如角膜、口腔粘膜、皮肤、结膜、膀胱、肾小管、肾脏、消化器官(食道、胃、十二指肠、小肠(包括空肠及回肠)、大肠(包括结肠))、肝脏、胰脏、乳腺、唾液腺、泪腺、前列腺、毛根、气管、肺等。其中，本实施方式的细胞培养基较好是用于来源于喉癌上皮细胞的培养基。
此外，本说明书中，“上皮肿瘤细胞”是指来源于上述的上皮组织的细胞肿瘤化而得的细胞。
本说明书中，“类器官”是指通过使细胞在受控的空间内高密度地自发组织和聚集而成的三维立体的、类似于器官的细胞组织体。
[MST1/2激酶抑制剂的制备实施例]
本说明书中，MST1/2激酶抑制剂是指直接或间接地对MST1/2信 号传导进行负调节的任意的抑制剂。一般来说，MST1/2激酶抑制剂例如与MST1/2激酶结合并降低其活性。由于MST1和MST2的结构具有相似性，MST1/2激酶抑制剂也可以是例如与MST1或MST2结合并降低其活性的化合物。
1.MST1/2激酶抑制剂化合物1的制备
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯 磺酰胺1
Figure PCTCN2020103428-appb-000008
2-氨基-2-(2,6-二氟苯基)乙酸甲酯(A2)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸(2.0克)后加入甲醇(30毫升)，随后冰浴下滴加二氯亚砜(1.2毫升)。反应体系在85℃反应过夜。反应结束后，体系在减压下蒸干溶剂，所得白色固体，直接用于下一步。
2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(A3)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸甲酯(2克)后加入丙酮(30毫升)和碳酸钾(2.2克)，然后用冰盐浴使体系冷却到-10℃，接着缓慢加入2,4-二氯-5-硝基嘧啶(3.1克)的丙酮溶液。反应体系在室温搅拌过夜。反应结束后，过滤，滤液在减压下除去溶剂，残留物经加压硅胶柱层析提纯后得化合物A3。LC/MS：M+H 359.0。
2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(A4)：在圆底烧瓶中加入2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(2.5克)后加入醋酸(50毫升)和铁粉(3.9克)。反应体系在60℃搅拌两小时。反应结束后，体系在减压下蒸干溶剂，所得物用饱和碳酸氢钠中和至碱性。乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A4。LC/MS：M+H 297.0。
2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(A5)：在 圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(2克)和N,N-二甲基乙酰胺(10毫升)，冷却至-35℃，加入碘甲烷(0.9毫升)，随后加入氢化钠(615毫克)，反应体系继续搅拌两小时。反应结束后，加水淬灭，乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A5。LC/MS：M+H 325.0。
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯磺酰胺(1)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(100毫克)、磺胺(53毫克)、对甲苯磺酸(53毫克)和仲丁醇(5毫升)。反应体系在120℃搅拌过夜。反应结束后，过滤，甲醇和乙醚洗涤得化合物1。LC/MS：M+H 461.1。
2.本发明的其他MST1/2抑制剂化合物的制备
本发明的其他MST1/2抑制剂化合物按照与化合物1类似的方法合成，其结构及质谱数据如下表所示。
Figure PCTCN2020103428-appb-000009
Figure PCTCN2020103428-appb-000010
Figure PCTCN2020103428-appb-000011
Figure PCTCN2020103428-appb-000012
Figure PCTCN2020103428-appb-000013
[实施例1]
人原代喉癌上皮细胞的分离
喉癌组织样本来源于三例进行过说明并获得同意的喉癌肿瘤患者手术切除癌组织样本，它们分别是样本编号0S0020、0S0021、0S0022。下面以其中一例样本(编号0S0020)进行说明。
在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在 0.5cm 3以上，将其置于预冷的4mL组织运输液(具体配制见表1)中，运输液盛在5mL塑料无菌带盖冻存管(购自广州洁特生物)内，冷链(0-10℃)运输至实验室。
表1 组织运输液配方
Figure PCTCN2020103428-appb-000014
表2 组织消化液配方
组织消化液成分 供应商 终浓度
HBSS Gibco 50％(体积)
RPMI-1640 康宁 50％(体积)
胶原酶Ⅱ Sigma 2mg/mL
胶原酶Ⅳ Sigma 2mg/mL
脱氧核糖核酸Ⅰ Sigma 50U/mL
透明质酸酶 Sigma 0.5mg/mL
氯化钙 上海生工 0.33mg/mL
牛血清白蛋白 上海生工 10mg/mL
在生物安全柜内，将组织样本(编号0S0020)转移至100mm细胞培养皿(购自NEST公司)内，用组织运输液润洗组织样本，将组织样本表面的残留的血液清洗掉，并剔除组织样本表面的脂肪等多余的组织。将润洗后的组织样本转移至另一个新的100mm培养皿内，加入2mL运输液，用无菌手术刀片和手术镊将组织样本分割为体积小于3mm 3的组织碎块。
将组织样本碎块转移至15mL离心管内，用台式离心机(Sigma公司3-18K)以1500转/分钟离心4分钟；弃上清，按1:1比例加入组织运输液和组织消化液(使用量约为每10mg组织使用5mL组织消化液，具体配制见表2)，标记样本编号，封口膜密封，以37℃、300转恒温摇床(知楚仪器ZQLY-180N)消化，每间隔1小时观察消化是否完成。
消化完成后，70μm滤网过滤掉未消化的组织团块，滤网上的组织 团块用组织运输液冲洗，将残留细胞冲入离心管中，1500转/分钟离心4分钟。
弃上清，观察剩余细胞团是否含有血细胞，若有血细胞，加3mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解15分钟，5分钟摇晃混匀一次，裂解结束后取出，以1500转/分钟离心4分钟。弃上清，得到消化分离后的喉癌原代细胞，加入基础培养基(BM)重悬，其中，基础培养基是由市售的DMEM/F-12培养基中加入0.2体积％Primocin(购自Invivogen公司，浓度为50mg/mL)，以得到100μg/mL的最终浓度。使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数为208万。
另外两例喉癌肿瘤组织样本按照以上同样的方法进行分离，得到的细胞总数分别为197万(0S0021)和232万(0S0022)。
[实施例2]
原代喉癌上皮细胞培养基的优化
(1)不同因子的作用
以0.25％胰酶(购自Thermo Fisher公司)将培养的NIH-3T3细胞(购自ATCC，使用含10％的胎牛血清的DMEM培养液进行培养)消化，用含有5％(v/v)胎牛血清(购自依科赛公司)、100U/mL青霉素和100μg/mL链霉素的DMEM培养液(购自康宁公司)终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用上述含10％的胎牛血清的DMEM培养液重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，用35Gy辐照剂量γ射线进行辐照，随后按2×10 4个/cm 2的密度接种至培养器皿中。在37℃培养箱内培养，待细胞贴壁。接种原代细胞前，移除培养器皿中培养基。
配制基础培养基(缩写为BM)：向市售的DMEM/F-12培养基中加入0.2体积％Primocin(购自Invivogen公司，浓度为50mg/mL)，以得到100μg/mL的最终浓度，配制得到BM。
接着，在基础培养基(BM)中分别加入不同种类和不同浓度的添加剂因子(表3)，配制成含有不同添加成分的喉癌上皮细胞培养基。
表3 不同组分培养基的配制(浓度为终浓度)
Figure PCTCN2020103428-appb-000015
Figure PCTCN2020103428-appb-000016
将不同成分的培养基按500μl/孔体积加入预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内。将按照实施例1相同方法从喉癌组织分离得到的喉癌肿瘤细胞(编号0S0064)以4×10 4个/孔的细胞数量接种在上述预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内，表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)，使相同数量的新鲜分离的喉癌肿瘤细胞(编号0S0064)在不同的培养基配方条件下进行培养。培养开始后每4天进行一次培养基的更换和滋养细胞的补充。培养7天后，进行细胞计数。其中，作为实验对照，使用未添加任何添加剂的基础培养基(BM)。将结果示于图1A-F。
图中纵坐标表示不同培养基培养之后得到的细胞数与基础培养基BM培养之后得到细胞数的比值。如图所示，在BM基础上加入表3中不同浓度不同因子对细胞增殖产生不同的作用。其中，在特定浓度范围下，B27添加剂、N2添加剂、胰岛素-转铁蛋白-硒复合物、肝细胞生长因子、胰岛素样本生长因子1、成纤维细胞生长因子7、化合物1、Y27632和A83-01对细胞增殖有一定的促进作用。
(2)培养基中不同因子递增对本专利方法获得的喉癌原代细胞增殖的影响
向基础培养基BM中分别依次递加不同小分子、添加剂以及生长因子(表4)，配制成含有不同添加成分的喉癌上皮细胞培养基。
表4 不同组分培养基的配制(浓度为终浓度)
培养基 组分
基础培养基/BM DMEM/F12+100μg/mL Primocin
NO.1 BM+5μM化合物1
NO.2 NO.1+1:50胰岛素-转铁蛋白-硒复合物
NO.3 NO.2+10ng/mL肝细胞生长因子
NO.4 NO.3+10μM Y27632
NO.5 NO.4+10ng/mL胰岛素样生长因子1
NO.6 NO.5+40ng/mL成纤维细胞生长因子7
NO.7 NO.6+250nM A83-01
NO.8 NO.7+10ng/mL表皮细胞生长因子
NO.9 NO.8+500nM SB202190
NO.10 NO.9+10μg/mL牛垂体提取物
NO.11 NO.10+1:50B27添加剂
NO.12 NO.11+1:100N2添加剂
NO.13 NO.12+5％体积比胎牛血清
将不同成分的培养基按500μl/孔体积加入预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内，同时将BM培养基作为实验对照。将按照实施例1的方法从喉癌组织分离得到的喉癌肿瘤细胞(编号0S0065)以4×10 4个/孔的细胞数量接种在上述预铺有γ射线辐照过后的NIH-3T3细胞的48孔培养板内，表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)，使相同数量的新鲜分离的喉癌肿瘤细胞(编号0S0065)在不同的培养基配方条件下进行培养。培养7天后，进行细胞计数。将实验结果示于图2。
如图所示，确定NO.7为本专利最优选的培养基用于培养扩增喉癌原代细胞(下文缩写为SCM)。在此基础上再加入一些因子、小分子抑制剂或者一定浓度的血清以及血清替代物N2添加剂和B27添加剂后，均没有更显著的促进细胞增殖的效果。
(3)所添加因子的不同浓度对本专利获得的喉癌原代细胞的增殖作用
配制本专利原代喉癌上皮细胞培养基(缩写为SCM)：向基础培养基(BM)中以最终浓度40ng/ml的条件添加成纤维细胞生长因子7(FGF7)，以最终浓度10ng/ml的条件添加胰岛素样生长因子1(IGF-1)，以最终浓度10ng/ml的条件添加肝细胞生长因子(HGF)，以1:50比例稀释条件添加胰岛素-转铁蛋白-硒复合物(ITS)储液(胰岛素10μg/ml，转铁蛋白在SCM培养基的终浓度为5μg/ml，亚硒酸钠在SCM培养基的终浓度为5ng/ml)，以最终浓度5μM的条件添加化合物1，以最终浓度10μM的条件添加Y27632，以最终浓度250nM条件添加TGFβ1抑制剂A83-01，制备原代喉癌上皮细胞培养基。
使用与实施例1同样的方法从喉癌患者的癌组织(样本编号0S0005)中分离获得癌组织来源的喉癌上皮细胞。接着，将癌组织来源的喉癌肿瘤细胞用流式图像计数仪(江苏卓微生物科技有限公司 JIMBIO FIL)进行计数，得到细胞总数。然后按4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的12孔板内。向12孔板中添加2mL制备好的原代喉癌上皮细胞培养基SCM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。在培养板内细胞生长达到底面积的80％左右时，弃去12孔板内的培养基上清，加入0.5mL 0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入0.5mL 0.05％胰酶进行细胞消化，室温下孵育5～20分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1mL终止消化，并收集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用基础培养基BM重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。所得细胞用于以下培养实验。
接着，配制以下7种配方培养基进行实验：
配方1：培养基SCM组分中不含成纤维细胞生长因子7；
配方2：培养基SCM组分中不含胰岛素-转铁蛋白-硒复合物；
配方3：培养基SCM组分中不含胰岛素样生长因子1；
配方4：培养基SCM组分中不含肝细胞生长因子；
配方5：培养基SCM组分中不含Y27632；
配方6：培养基SCM组分中不含化合物1；
配方7：培养基SCM组分中不含A83-01；
分别使用上述配方1～7来稀释上述消化后的细胞悬液，按照每孔1万细胞，250微升体积种入预铺有γ射线辐照过后的NIH-3T3细胞的48孔板中。
在使用配方1的培养基时，在接种有原代细胞的48孔板中分别添加配制好的成纤维细胞生长因子7每孔250微升，成纤维细胞生长因子7的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时，在接种有原代细胞的48孔板中分别添加配制好的胰岛素-转铁蛋白-硒复合物每孔250微升，胰岛素-转铁蛋白-硒复合物储液的终浓度分别为1:1600、1:800、1:400、1:200、1:100、 1:50、1:25(对应于胰岛素/转铁蛋白/亚硒酸钠的终浓度分别为0.3125μg/ml-0.15625μg/ml-0.15625ng/ml；0.625μg/ml-0.3125μg/ml-0.3125ng/ml；1.25μg/ml-0.625μg/ml-0.625ng/ml；2.5μg/ml-1.25μg/ml-1.25ng/ml；5μg/ml-2.5μg/ml-2.5ng/ml；10μg/ml-5μg/ml-5ng/ml；20μg/ml-10μg/ml-10ng/ml)；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有原代细胞的48孔板中分别添加配制好的胰岛素样生长因子1每孔250微升，胰岛素样生长因子1的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，在接种有原代细胞的48孔板中分别添加配制好的肝细胞生长因子每孔250微升，肝细胞生长因子的终浓度分别为80ng/ml、40ng/ml、20ng/ml、10ng/ml、5ng/ml、2.5ng/ml、1.25ng/ml；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，在接种有原代细胞的48孔板中分别添加配制好的Y27632每孔250微升，Y27632的终浓度分别为40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有原代细胞的48孔板中分别添加配制好的化合物1每孔250微升，化合物1的终浓度分别为40μM、20μM、10μM、5μM、2.5μM、1.25μM、0.625μM；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有原代细胞的48孔板中分别添加配制好的A83-01每孔250微升，A83-01的终浓度分别为4000nM、2000nM、1000nM、500nM、250nM、125nM、62.5nM、；并使用配方7的培养基设置对照孔(BC)。
待细胞扩增至48孔的85％左右消化计数，分别参比对照孔(BC)细胞数计算比值，将结果分别示于图3A～3G。图3A～图3G中，比值为使用各培养基培养一代得到的细胞数与对应的对照孔培养一代得到的细胞数的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基；比值小于1，则说明配 制的含不同浓度的因子或小分子化合物的培养基促增殖效果较对照孔培养基促增殖效果弱。
根据图3A～3G的结果，成纤维细胞生长因子7在培养基中的含量优选为5～80ng/ml，更优选20～80ng/ml；胰岛素-转铁蛋白-硒复合物的体积浓度优选为1:25～1:100，更优选1:25～1:50(对应于胰岛素/转铁蛋白/亚硒酸钠的终浓度分别为5～20μg/ml-2.5～10μg/ml-2.5～10ng/ml；优选10～20μg/ml-5～10μg/ml-5～10ng/ml)；胰岛素样生长因子1的含量优选为10ng/ml～80ng/ml，更优选为10ng/ml～40ng/ml；肝细胞生长因子的含量优选为10ng/ml～80ng/ml，更优选为10ng/ml～40ng/ml；Y27632的含量优选为1.25μM～20μM，更优选为2.5μM～10μM；化合物1的含量优选为1.25μM～10μM，更优选为2.5μM～10μM；A83-01的含量优选为125nM～1000nM，更优选为125nM～500nM。
[实施例3]
人喉癌组织来源的原代喉癌肿瘤细胞的培养
使用与实施例1同样的方法从喉癌患者的癌组织(样本编号0S0003)中分离获得癌组织来源的喉癌上皮细胞。接着，将癌组织来源的喉癌肿瘤细胞用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。然后按4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的12孔板内。向12孔板中添加2mL制备好的原代喉癌上皮细胞培养基SCM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。
图4A是本实施例按4×10 4个/cm 2密度接种预铺有γ射线辐照过后的NIH-3T3细胞的12孔板，自接种开始后培养至第4天的镜下照片(100倍倒置相差显微镜拍照)。镜下观察可见，所培养的癌组织来源的原代喉癌肿瘤细胞已经形成较大克隆。图4B是本实施例自接种后培养第12天的镜下照片(100倍倒置相差显微镜拍照)，视野中细胞已经长满。从图4A和4B两张图可以看出，分离后得到喉癌原代细胞在体外培养4天在镜下就可以看到明显的克隆形成，并且经过12天的扩增，细胞数目得到显著的扩增，提示本发明技术是一种高效的体外扩增喉 癌上皮细胞的技术。
[实施例4]
不同培养基对喉癌组织来源的原代喉癌肿瘤细胞的促增殖效果
(1)不同培养基对初代原代细胞克隆形成的影响和增殖效果的比较
使用与实施例2同样的方法制备原代喉癌上皮细胞培养基SCM，和作为对照的基础培养基BM。另外制备细胞条件重编程技术文献中所用培养基FM作为另一对照例，配制步骤参见(Liu等，Nat.Protoc.，12(2):439-451，2017)，培养基配方见表5。同时，作为另外对照例，制备喉癌原代细胞培养基SCM-1和SCM-2，配方是在SCM基础上分别使用1:50体积比的B27和1:100体积比的N2添加剂替换胰岛素-转铁蛋白-硒复合物。此外，作为另一对照例，自Gibco公司购买商品化培养基Defined Keratinocyte SFM，以下也称“KSFM培养基”)，培养基配方见表6。
表5 细胞条件重编程技术文献中所用培养基(FM)成分
培养基成分 供应商 终浓度
DMEM培养基 Corning 65体积％
胎牛血清 Gibico 10体积％
Ham’s F12营养液 Gibico 25体积％
氢化可的松 Sigma-Aldrich 25ng/ml
表皮生长因子 R&D 0.125ng/ml
胰岛素 Sigma-Aldrich 5μg/ml
两性霉素B Sigma-Aldrich 250ng/ml
庆大霉素 Gibico 10μg/ml
霍乱毒素 Sigma-Aldrich 0.1nM
Y27632 Enzo 10μM
表6 商品化培养基Defined Keratinocyte SFM(KSFM)成分
培养基成分 供应商 终浓度
Defined Keratinocyte-SFM Basal Medium Gibco 99体积％
Defined Keratinocyte-SFM Growth Supplement Gibco 1体积％
使用与实施例1同样的方法获得喉癌组织来源的原代喉癌肿瘤细 胞(编号0S0006)。接着，按照相同的密度(4×10 4个/cm 2)分别在以下6种培养条件下培养：
A.本发明技术：按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的本发明的原代喉癌上皮细胞培养基SCM进行培养；
B.细胞条件重编程技术：按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)上，采用1mL细胞条件重编程技术培养基FM在24孔板中进行培养(具体步骤参见(Liu等，Nat.Protoc.，12(2):439-451，2017)；
C.按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的培养基SCM-1在24孔板中进行培养；
D.按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用1mL的培养基SCM-2在24孔板中进行培养；
E.按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至24孔板内，采用1mL的商品化培养基KSFM在24孔板中进行培养。
F.按4×10 4个/cm 2接种密度将原代喉癌肿瘤细胞接种至预铺有γ射线辐照过后的NIH-3T3细胞(购自ATCC公司)的24孔板内，采用2mL的基础培养基BM在24孔板中进行培养。
上述六种培养中，每5天对五种培养条件下培养的细胞进行换液。同时观察24孔板中各培养基培养下细胞形成克隆和细胞增殖状态，使用显微镜(Invitrogen公司EVOS M500)进行拍照记录细胞生长状况。
对于采用本发明技术培养的原代喉癌肿瘤细胞(编号0S0006)，分别在培养板内细胞生长达到底面积的80％左右时，弃去24孔板内的培养基上清，加入0.5mL 0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入0.5mL 0.05％胰酶进行细胞消化，37℃下孵育10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1mL终止消化，并收 集至15mL离心管内，1500rpm离心4分钟后，弃上清。使用本发明的培养基重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数为31.7万。另五种培养条件下培养的细胞采用如上述同样的操作方式进行消化和计数，使用培养基FM、SCM-1、SCM-2、KSFM和BM培养得到的细胞总数分别是16.67万、26.61万、27.72万、9.56万和3.59万。
图5是编号0S0006细胞在不同条件下扩增的到的细胞总数作图。
图6是6例喉癌病人样本(编号0S0011、0S0012、0S0013、0S0014、0S0015、0S0016)按照实施例1获得的喉癌原代细胞在以上六种不同培养基条件下培养7天后得到细胞增殖效果的比较图，其中√代表有一定的克隆形成能力和促增殖效果，√√代表有较为明显的克隆形成能力和促增殖效果，√√√代表有非常明显的克隆形成能力和促增殖效果，×代表不能形成克隆。从图中可以确认SCM培养基在对喉癌组织来源获得的原代细胞培养中对克隆形成能力和促细胞增殖效果较其他5种培养条件有明显优势。
(2)不同培养基对原代喉癌肿瘤细胞的持续培养和生长曲线的绘制
使用与本实施例(1)中同样的方法获得原代喉癌上皮细胞培养基SCM，以及作为对照的培养基FM、SCM-1、SCM-2、KSFM和BM。
使用与本实施例(1)中同样的方法将喉癌组织来源的原代喉癌肿瘤细胞(编号0S0004)分别在六种培养基培养，并进行消化传代和计数。
当传代后的细胞在培养板内生长再次达到约80％板底面积时，再次按上述操作方法消化收集所培养获得的细胞并计数。同样按4×10 4个/孔密度接种并持续培养。
以下为原代喉癌上皮细胞在不同技术培养条件下细胞的扩增倍数(Population Doubling)的计算公式：
Population Doubling(PD)＝3.32*log 10(消化后细胞总数/初始种入细胞数)，公式参见(Chapman等，Stem Cell Research & Therapy 2014,5:60)。
如图7所示是采用Graphpad Prism软件绘制的六种不同培养条件 下的细胞0S0004的生长曲线。横坐标表示细胞培养的天数，纵坐标是累计的细胞增殖倍数，表示细胞在培养周期内扩增的倍数，数值越大表示细胞在一定周期内扩增的次数越多，即扩增得到的细胞数也就越多，斜率代表的是细胞扩增的速率。从图中可以确认本发明培养基SCM以及SCM-1、SCM-2培养的喉癌上皮细胞的增殖速率优于其他三种培养条件，同时可以确认本发明技术可以对原代喉癌上皮细胞进行持续培养。
[实施例5]
癌组织来源的原代喉癌肿瘤细胞的鉴定
(1)原代喉癌组织和传代培养后的喉癌细胞免疫荧光鉴定
使用与实施例1同样的方法从喉癌患者的癌组织(样本编号0S0015)中分离获得癌组织来源的喉癌上皮细胞。接着，将癌组织来源的喉癌肿瘤细胞用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数。然后按4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的24孔板内，同时24孔板中预先放置用于免疫荧光染色的圆形细胞破片(购自赛默飞公司)。向24孔板中添加1mL制备好的原代喉癌上皮细胞培养基SCM，置于37℃、5％CO 2培养箱(购自赛默飞)中进行培养。
待24孔板中细胞扩增80％底面积时，弃培养液，使用4％甲醛冰上固定细胞30分钟。PBS(购自上海生工)洗5分钟x 3次。弃PBS，加入通透液，避光下，摇床(100rpm左右)破膜30分钟，PBS洗5分钟x 3次。随后使用PBS+0.3％Triton X-100(购自上海生工)配制5％体积浓度的BSA(购自上海生工)溶液用于封闭，37℃封闭30分钟。
提前配制PBS+0.3％Triton X-100用于稀释抗体，按照1:50比例稀释鳞癌特异性抗体p63(购自CST公司)，弃封闭液，加入配制好的一抗稀释液，至4℃冰箱孵育过夜。4℃取出，平衡至室温，37℃继续孵育1小时，PBS洗5分钟x 3次。
提前配制PBS+0.3％Triton X-100用于二抗稀释，按照1:1000比例稀释激发光为488nm且种属为兔的荧光二抗(购自赛默飞公司)， 常温避光孵育1小时，PBS洗5分钟x 3次。
用PBS按照1:1000体积比稀释非特异性荧光染料DAPI(购自Sigma公司)，常温避光染色5分钟，PBS洗5分钟x 3次。显微镜(Invitrogen公司EVOS M500)下成像，拍照记录。
图8A和8B分别为10倍物镜下不同视野荧光拍照的图片，其中图8A是使用非特异性荧光染料DAPI染细胞核的图片，图8B是使用喉癌特异性抗体p63(定位在细胞核)染色的图片。如图所示，在图8A中标记细胞核的位置在图8B中均被染为绿色，表明培养后的细胞为喉鳞癌细胞，与临床病理诊断一致。
(2)原代喉癌组织和传代培养后的喉癌细胞免疫组化鉴定
从一例喉癌患者的临床手术切除样本取出约绿豆粒大小的癌组织(样本编号0S0001)，浸泡在1mL 4％多聚甲醛中固定。剩余癌组织使用与实施例1相同的方法获得喉癌上皮细胞(样本编号0S0001)。使用实施例3的方法采用本发明的培养基SCM将样本0S0001持续培养至第六代。
采用免疫组化法检测样本0S0001原始组织和持续培养至第六代得到的原代细胞中与喉癌相关的重要生物标记物的表达。4％多聚甲醛固定后的组织，经石蜡包埋，用切片机切成4μm厚的组织切片。随后进行常规的免疫组织化学检测(具体步骤参见Li等，Nature Conmunication，(2018)9:2983)。所使用的一抗为细胞角蛋白Cytokeratin(pCK)(购自CST公司)、p63抗体(购自CST公司)、和Ki67抗体(购自R&D公司)。
图9是原始组织细胞和采用该细胞以本发明的培养基SCM培养而获得的喉癌肿瘤细胞的免疫组化结果对比图。由图9可以确认，采用本发明技术培养的喉癌肿瘤细胞(样本编号0S0001)培养至第6代时，细胞上与喉癌相关的生物标记物的表达情况与细胞来源的原始组织切片的标记物表达情况基本一致。说明采用本发明技术所培养的细胞保持了喉癌病人癌组织的原始病理特性。
[实施例6]
癌组织来源的原代喉癌肿瘤细胞在小鼠体内的异种移植成瘤实验
使用与实施例1同样的方法从一例病理诊断为喉癌患者的癌组织中分离获得喉癌肿瘤细胞(编号0S0003)，按照实施例3的方法采用本发明的培养基SCM对0S0003进行培养，待喉癌肿瘤细胞数量达到1×10 7个时，采用实施例4的方法对喉癌肿瘤细胞进行消化，并收集。采用本发明的喉癌肿瘤细胞培养基SCM和
Figure PCTCN2020103428-appb-000017
(购自BD生物科技公司)按照1:1混匀，吸取100μL与Matrigel基质胶混匀后的培养基将5×10 6个喉癌肿瘤细胞重悬，分别注射入6周大的雌性高度免疫缺陷小鼠(NCG)小鼠(购自南京模式动物研究所)的喉癌脂肪垫和右前肢腋下部位，每三天观察一次喉癌肿瘤细胞在小鼠体内形成肿瘤的体积和生长速率。
在肿瘤细胞接种后的第21天可观察到小鼠的两处肿瘤细胞接种部位均有瘤体形成，自第21天起至第40天，小鼠体内肿瘤增殖明显。说明采用本发明的培养方法所培养的癌组织来源的喉癌肿瘤细胞在小鼠体内具有成瘤性。
[实施例7]
癌组织来源的喉癌肿瘤细胞的药物敏感性功能测试
下面以喉癌患者手术切除样本为例，说明由病人来源的喉癌肿瘤样本培养得到的喉癌肿瘤细胞可以用于检测病人肿瘤细胞对不同药物的敏感性。
一、原代喉癌肿瘤细胞的铺板：按照实施例1中方法得到的分离后的喉癌肿瘤细胞(编号0S0020和编号0S0022)细胞悬液，按照4×10 4个/cm 2密度接种至预铺有γ射线辐照过后的NIH-3T3细胞的12孔板内。向12孔板中添加2mL制备好的原代喉癌上皮细胞培养基SCM，置于37℃、5％CO2培养箱(购自赛默飞)中进行培养。在培养板内细胞生长达到底面积的80％左右时，弃去12孔板内的培养基上清，加入0.5mL 0.25％胰酶(购自Thermo Fisher公司)消化1分钟，随后吸出0.25％胰酶，再加入0.5mL 0.05％胰酶进行细胞消化，37℃下孵育10分钟，直至显微镜(Invitrogen公司EVOS M500)下能观察到细胞已经消化完全，即用含有5％(v/v)胎牛血清、100U/mL青霉素和100μg/mL链霉素的DMEM/F12培养液1mL终止消化，并收集至15mL离心管 内，1500rpm离心4分钟后，弃上清。使用SCM培养基重悬离心后的细胞沉淀，使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数，得到细胞总数分别为83万和76.8万。按2000～4000个/孔密度接种于384孔板中，使细胞贴壁过夜。
二、药物梯度实验：
(1)采用浓度梯度稀释的方法配制药物贮存板：分别吸取40μL的10μM待测药物母液作为最高浓度，再分别从中吸取10μL加入到含20μL的DMSO的0.5mL的EP管中，再从上述EP管中吸取10μL到第二个已装有20μL的DMSO的0.5mL的EP管中，即按照1:3稀释药品。重复以上方法，依次稀释，最后得到加药所需的7种浓度。将不同浓度的药物加入384孔药物储存板中。溶剂对照组各孔加入等体积的DMSO作为对照。本实施例中，待测药物为硼替佐米(购自MCE公司)、双硫仑(购自MCE公司)、吉非替尼(购自MCE公司)和厄洛替尼(购自MCE公司)。
(2)使用高通量自动化工作站(Perkin Elmer公司JANUS)将384孔药物贮存板内的不同浓度药物和溶剂对照加入到铺有喉癌肿瘤细胞的384孔细胞培养板中，药物组和溶剂对照组都各设3个复孔。每孔加入药物体积为100nL。
(3)细胞活性检测：给药72小时后，用Cell Titer-Glo检测试剂(购自Promega公司)检测加药培养后细胞的化学发光数值，化学发光数值的大小反映细胞活力以及药物对细胞活力的影响，每孔加入10μL配制好的Cell Titer-Glo检测液，混匀后使用酶标仪(Perkin Elmer公司Envision)检测化学发光数值。
(4)细胞活性检测：按照公式细胞存活率(％)＝加药孔化学发光数值/对照孔化学发光数值*100％，计算得到不同药物作用细胞后的细胞存活率，使用Graphpad Prism软件作图并计算半数抑制率IC 50，同时计算不同药物在人体内最大血药浓度Cmax对应下的细胞存活率。
(5)药物敏感性测试结果如图10所示。
图10A和10B分别表示从两个不同的喉癌患者的手术切除癌组织样本(编号0S0020和编号0S0022)所培养获得的喉癌肿瘤细胞，对两个化疗药物硼替佐米和双硫仑的药物敏感性和对靶向药物吉非替尼和 厄洛替尼的敏感性。其中10A为编号0S0020样本培养获得的喉癌细胞对四种药物的敏感性结果；10B为编号0S0022样本培养获得的喉癌细胞对四种药物的敏感性结果。图中横坐标上标线对应的浓度为这四种药物在人体内最大血药浓度Cmax。结果显示，同一病人的细胞对不同药物在人体内最大血药浓度时的敏感性不同，不同病人的细胞对在人体内最大血药浓度时的药物的敏感性也不同，根据结果可以判断喉癌病人在临床使用该种药物时的有效性。
工业应用性
本发明提供一种用于在体外培养或扩增原代喉癌上皮细胞的培养基及培养方法，使用本发明的培养基和培养方法所培养的细胞后代和类器官可用于药物的疗效评估和筛选、毒性测定和再生医学。因而，本发明适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (12)

  1. 一种用于培养喉癌上皮细胞的原代细胞培养基，其特征在于：
    含有MST1/2激酶抑制剂，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2020103428-appb-100001
    其中，
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基、芳基C1-C6烷基和杂芳基；
    R 2和R 3各自独立地选自C1-C6烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  2. 如权利要求1所述的原代细胞培养基，其中
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基；
    R 2和R 3各自独立地选自C1-C3烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  3. 如权利要求1所述的原代细胞培养基，其中所述MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2020103428-appb-100002
    其中，
    R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基；
    R 5选自氢、C1-C6烷基、和C3-C6环烷基；
    R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。
  4. 如权利要求3所述的原代细胞培养基，其中
    R 1为任选地被1-2个独立地R 6取代的苯基；
    R 5为氢；
    R 6优选为氟、甲基或三氟甲基。
  5. 如权利要求1所述的原代干细胞培养基，其中所述MST1/2激酶抑制剂选自以下化合物或其药学可接受的盐中的至少一种：
    Figure PCTCN2020103428-appb-100003
    Figure PCTCN2020103428-appb-100004
    Figure PCTCN2020103428-appb-100005
    Figure PCTCN2020103428-appb-100006
    Figure PCTCN2020103428-appb-100007
    Figure PCTCN2020103428-appb-100008
  6. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：
    所述MST1/2激酶抑制剂的含量为1.25～10μM，优选为2.5～10μM。
  7. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：
    还含有以下因子中的一种或多种：胰岛素样生长因子1；成纤维生长因子7；胰岛素-转铁蛋白-硒复合物；肝细胞生长因子；选自Y27632、法舒地尔、和H-1152中的至少一种的ROCK激酶抑制剂；和选自A83-01、SB431542、Repsox、SB505124、SB525334、SD208、LY36494、和SJN2511中的至少一种的TGFβI型受体抑制剂。
  8. 如权利要求7所述的原代细胞培养基，其特征在于：
    所述胰岛素样生长因子1的含量为10～80ng/ml，更优选为10～40ng/ml；
    所述成纤维细胞生长因子7的含量为5～80ng/ml，更优选20～80ng/ml；
    所述胰岛素-转铁蛋白-硒复合物中胰岛素/转铁蛋白/亚硒酸钠各自的含量分别为5～20μg/ml-2.5～10μg/ml-2.5～10ng/ml，更优选分别为10～20μg/ml-5～10μg/ml-5～10ng/ml；
    所述肝细胞生长因子的含量为10～80ng/ml，更优选为10～40ng/ml；
    所述ROCK激酶抑制剂的含量优选为1.25～20μM，更优选为2.5～10μM；
    所述TGFβI型受体抑制剂的含量为125～1000nM，更优选为125～500nM。
  9. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：
    不含血清、牛垂体提取物、Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、烟酰胺和N-乙酰半胱氨酸。
  10. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：
    所述喉癌上皮细胞选自喉癌肿瘤细胞、正常喉癌上皮细胞、和喉癌上皮干细胞。
  11. 一种喉癌上皮细胞的培养方法，其特征在于，包括以下步骤：
    (1)配制如权利要求1～10中任一项所述的原代细胞培养基；
    (2)用X射线或者γ射线辐照后的滋养细胞预铺培养器皿；
    (3)在预铺有滋养细胞的培养器皿内接种从喉癌组织分离得到原代喉癌上皮细胞，使用步骤(1)中的所述原代细胞培养基进行培养。
  12. 一种评估或筛选用于治疗喉癌疾病的药物的方法，其特征在于，包括以下步骤：
    (1)根据权利要求11所述的培养方法培养得到喉癌上皮细胞；
    (2)选定需要检测的药物，稀释成不同的药物浓度梯度；
    (3)向步骤(1)培养得到的喉癌上皮细胞中添加梯度稀释后的所述药物，并进行细胞活力检测。
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