CN118147082A - Immortalized epithelial cells of leucorrhea with white spots of human voice and culture method and application thereof - Google Patents

Immortalized epithelial cells of leucorrhea with white spots of human voice and culture method and application thereof Download PDF

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CN118147082A
CN118147082A CN202410400512.0A CN202410400512A CN118147082A CN 118147082 A CN118147082 A CN 118147082A CN 202410400512 A CN202410400512 A CN 202410400512A CN 118147082 A CN118147082 A CN 118147082A
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epithelial cells
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周水洪
傅子铭
王勤瑛
鲍洋洋
陈哲
钟江涛
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First Affiliated Hospital of Zhejiang University School of Medicine
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Abstract

The invention relates to a human vocal cord white spot immortalized epithelial cell and a culture method and application thereof, belonging to the technical field of cell culture. The invention provides a human vocal cord white spot immortalized epithelial cell: the product is named hVCL-MSDEP01 and is preserved in China Center for Type Culture Collection (CCTCC) in 2024, 1 month and 15 days, wherein the preservation number is CCTCC NO: c202432, the preservation address is Chinese, wuhan, university of Wuhan, is a new cell. The cell state is good, the appearance presents squamous epithelial appearance, the cell size is not obviously different, and the uniformity is good. The immortalized epithelial cells of the vocal cords and the white spots have good epithelial cell characteristics, can express immortalized characteristic proteins, have lower growth speed and higher apoptosis rate compared with the epithelial cell lines of the laryngeal squamous cell carcinoma, can be used as a cell model for basic research of the vocal cords and the white spots, and provides an extremely important experimental material for basic research of the occurrence and development of the vocal cords and the white spots into the laryngeal carcinoma.

Description

Immortalized epithelial cells of leucorrhea with white spots of human voice and culture method and application thereof
Technical Field
The invention relates to the technical field of cell culture, in particular to a human vocal cord white spot immortalized epithelial cell and a culture method and application thereof.
Background
The leucorrhea is a common precancerous lesion of laryngeal carcinoma, is clinically mostly a white plaque which is not easy to wipe off on the vocal cord tissue under a laryngoscope, has a plurality of histological types and comprises simple squamous epithelial hyperplasia, mild, moderate and severe abnormal hyperplasia and carcinoma in situ. The mechanism of the occurrence and development of the leucorrhea to the laryngeal carcinoma is important for the prevention of the laryngeal carcinoma. Because of the small lesion volume of the leucorrhea tissue and the lack of a proper in vitro immortalized leucorrhea epithelial cell line, the related research is limited to the tissue level and lacks a good immortalized epithelial cell model. The vocal cord white spot epithelial cells which are not subjected to immortalization generally have the problems of quick aging and short culture period, and the phenomena of cell aging such as slow cell growth, volume increase, intracellular vacuole increase, nucleus increase, cell spacing increase and the like are usually generated within 10 generations. The number of epithelial cells obtained in vitro is still insufficient to perform multiple molecular experiments, increasing the cost and cycle time of the experiments. The leucorrhea epithelial cells have primary cells, but since not tumor cells do not have in vitro immortality, there has been no stable leucorrhea epithelial cell line. Meanwhile, the pathological tissues of the leucorrhea are usually very small, the average tissue weight is 0.3-0.4g, and the sample size left after the tissues of the clinical department are planed off is about 0.1-0.2g, so that the cell culture method in other discipline fields has great difficulty in extracting and culturing the leucorrhea epithelial cells. The applicant successfully builds a human leucoderma epithelial cell three-dimensional culture model (CN 110484488A) in vitro in the earlier stage, but uses the building method of the human leucoderma epithelial cell three-dimensional culture model to culture leucoderma primary cells obtained by leucoderma cells, but does not carry out immortalization treatment, and has lower obtaining rate of the leucoderma primary cells, relatively more sample size is needed for primary culture, the separation of the leucoderma epithelial layer and basal layer is difficult in the primary culture process, the time is longer, and the situation that the growth of the epithelial cells is inhibited by the fibroblasts is easy to occur in the later stage, so that the culture success rate is reduced, and the establishment of an immortalized leucoderma epithelial cell line is further limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a human vocal cord white spot immortalized epithelial cell, and a culture method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
In a first aspect, the present invention provides a human vocal cord white spot immortalized epithelial cell named hVCL-MSDEP01, which was preserved in China Center for Type Culture Collection (CCTCC) at 1 month 15 of 2024, with a preservation number of CCTCC NO: c202432, the preservation address is China, university of Wuhan.
The invention provides a novel human vocal cord white spot immortalized epithelial cell hVCL-MSDEP01, and the identification results of STR sites and Amelogenin sites show that the human vocal cord white spot immortalized epithelial cell hVCL-MSDEP01 cannot be matched with all cells in a cell bank, and is a novel cell line. The immortalized epithelial cells hVCL-MSDEP of human vocal cords have good states, no obvious vacuoles in the cells, the appearance of the immortalized epithelial cells presents squamous epithelial-like appearance, the boundaries among the cells are clear, the sizes of the immortalized epithelial cells are not obviously different, and the uniformity is good. Identified, human vocal cord white spot immortalized epithelial cells hVCL-MSDEP01 of the invention are capable of expressing epithelial cell specific proteins (Keratin 17/19, E-cadherin, keratin 8/18) and immortalized proteins (SV 40 LT ANTIGEN); and the growth curve shows no apparent growth plateau.
The invention successfully obtains an immortalized vocal cord white spot epithelial cell line and provides a good research material for researching the vocal cord white spots. The immortalized epithelial cells hVCL-MSDEP01 of the vocal cords of the human are non-tumor epithelial cell lines derived from the vocal cords, which are in the range of precancerous lesions, and have part of normal cells and cells which tend to become cancerous inside. Compared with the laryngeal squamous cell carcinoma epithelial cell line SNU-899, the hVCL-MSDEP01 ensures the epithelial cell characteristics of cells, has lower growth speed and higher apoptosis rate, has a difference with a tumor cell model, can be used as a research model, and provides an important epithelial cell model for basic research of the vocal cords and the leucoma.
In a second aspect, the invention provides a method for culturing the human vocal cord white spot immortalized epithelial cells, comprising the following steps:
s1, digesting the leucorrhea leucoma tissues, and culturing the leucoma tissues by using PneumaCult TM Ex culture medium to obtain primary epithelial cells of the leucoma;
S2, continuously culturing and passaging the primary epithelial cells of the vocal cords with PneumaCult TM Ex culture medium to 2-3 generations, then transfecting with SV40 slow virus, screening out cells positive and stable in SV40 expression, continuously culturing for 1-2 generations to obtain transfected stable cells, and culturing with serum-free culture medium to obtain immortalized epithelial cells of the vocal cords with the leucoma.
The invention utilizes PneumaCult TM Ex culture medium to culture cells after the vocal cord white spot tissue digestion, further cultures primary epithelial cells of the vocal cord white spot and cells after SV40 lentivirus transfection, and finally successfully cultures human vocal cord white spot immortalized epithelial cells hVCL-MSDEP01.PneumaCult TM Ex culture medium is a serum-free culture medium, and PneumaCult Ex culture medium is used, so that the culture medium is more suitable for growth of epithelial cells, can inhibit growth of fibroblasts, further ensures singleness of the epithelial cells, and can be used for successfully culturing human vocal cords white spots immortalized epithelial cells hVCL-MSDEP, and can not be replaced by conventional serum-containing culture medium such as DMEM/F12.
As a preferred embodiment of the method for culturing human vocal cords white spot immortalized epithelial cells of the present invention, the composition of the digestive juice used for the digestion is: ADVANCED DMEM/F12, 2-2.4U/mL of dispase II (Dispase II), 40-45 mug/mL of collagenase I, 2-3% of Fetal Bovine Serum (FBS) and 1-1.5% of green streptomycin.
The invention cultures primary cells by utilizing the CN110484488A patent and carries out immortalization treatment, so that immortalized epithelial cells can not be obtained basically, and the primary cells have lower yield. The invention adjusts the digestive system (digestive juice): the ADVANCED DMEM/F12 and 2 to 3 percent of FBS are utilized to provide nutrition for the tissue blocks, so that the tissue blocks and the cell viability in the tissue blocks are prevented from being reduced, and the 1 to 1.5 percent of the blue chain mycin prevents the cell pollution during the shake table digestion; the enzyme amount of digestive enzyme is strictly controlled, the using amount of disperse enzyme II is lower than 2.4U/mL, and collagenase is I type, so that IV type with stronger digestion force is avoided. The invention utilizes the digestive juice to carry out preliminary sorting on cells of the vocal cord white spot tissue, thereby ensuring that epithelial cells are fully resolved and dissociated, avoiding the dissociation of fibroblasts and the damage of the epithelial cells and retaining as many primary epithelial cells as possible; the primary epithelial cells have higher yield, and the immortalized epithelial cells are successfully cultured.
As a preferred embodiment of the method for culturing human vocal cords white spot immortalized epithelial cells of the present invention, the composition of the digestive juice used for the digestion is: ADVANCED DMEM/F12, dispase II (Dispase II) -2.4U/mL, collagenase I40-45 mug/mL, fetal Bovine Serum (FBS) 2% and green streptomycin 1%.
As a preferred embodiment of the method for culturing human leucorrhea-immortalized epithelial cells of the present invention, in step S1, the leucorrhea tissue is washed with a PBS solution containing penicillin-streptomycin and then sheared into tissue pieces of 0.8-1.2 mm 3 before being digested with a digestive juice.
Before digestion, the vocal cords white spot tissues are washed by the PBS solution containing the double antibodies, so that the problem of cell pollution is avoided, the vocal cords white spot tissues are sheared into tissue blocks with the thickness of 0.8-1.2 mm 3, the digestion speed of the vocal cords white spot tissues is accelerated, and the digestion time is saved. The sheared tissue block is easier to digest, and dissociation of fibroblast caused by overlong digestion time can be avoided, so that the singleness of epithelial cells is ensured.
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells of the present invention, the vocal cord white spot tissue is washed with PBS solution containing penicillin-streptomycin in 1% by volume and then sheared into 1mm 3 tissue pieces before being digested with the digestive juice.
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells of the present invention, the digestion time is 60 to 120min; preferably, the time of digestion is 100min. The invention can ensure the sufficient dissociation digestion of the epithelial cells and avoid the dissociation digestion of the fibroblasts by controlling the digestion time, and can further ensure the singleness of the epithelial cells.
As a preferred implementation mode of the culture method of the human vocal cord white spot immortalized epithelial cells, the vocal cord white spot tissues are digested by using digestive juice, the digestion is stopped by using a DMEM/F12 complete culture medium containing 10% of FBS by volume percent, the cell suspension is obtained by filtering after the digestion is stopped, the cell precipitation is obtained by centrifugation, the cell precipitation is resuspended by using PneumaCult TM Ex culture medium and then is cultured to cell attachment, and the culture medium is removed after the cell attachment, so that the vocal cord white spot primary epithelial cells are obtained.
As a preferred embodiment of the method for culturing human vocal cords white spot immortalized epithelial cells according to the present invention, in step S1, the digestion temperature is 35 to 37 ℃. At the digestion temperature, the activity of digestive enzymes in digestive juice can be ensured, and the good activity of cells can be ensured.
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells, in the step S2, the primary vocal cord white spot epithelial cells are cultured by using fresh PneumaCult TM Ex medium, the medium is replaced every 1-2 days, and subculture is performed after the cell density reaches 70% -80%. The subculture is as follows: removing the culture medium after the cell density reaches 70% -80%, then washing by using a PBS solution containing penicillin-streptomycin, digesting the washed cells by using trypsin digestion liquid, filtering after stopping digestion to obtain a cell suspension, centrifuging, collecting cell sediment, re-suspending the cell sediment by using PneumaCult TM Ex culture medium, and then carrying out the steps of: culturing according to the passage ratio of (1-3).
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells according to the present invention, the trypsin digestion solution is a digestion solution containing 0.02% EDTA and 0.25% trypsin; the digestion temperature is 35-37 ℃, and the digestion time is 3-5min.
As a preferred embodiment of the method for culturing human leucorrhea immortalized epithelial cells according to the present invention, in step S2, the SV 40-utilizing lentivirus transfection is performed by culturing primary leucorrhea epithelial cells in PneumaCult Ex medium containing SV40 lentivirus and a transfection enhancer (3 days), then culturing the primary leucorrhea primary epithelial cells in PneumaCult Ex medium containing 1-1.5% penicillin-streptomycin, and detecting SV40 expression after 2-3 passages of subculture.
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells of the present invention, in step S2, the serum-free medium is composed of the following components by volume percent: 98.5 to 99 percent of mixed culture medium and 1 to 1.5 percent of penicillin-streptomycin; the mixed culture medium is a mixture of PneumaCult Ex culture medium and ADVANCED DMEM/F12 culture medium, and the volume ratio of PneumaCult Ex culture medium to ADVANCED DMEM/F12 culture medium is (2.8-3.2): 1.
As a preferred embodiment of the method for culturing human vocal cord white spot immortalized epithelial cells of the present invention, in step S2, the serum-free medium is composed of the following components by volume percent: 99% of mixed culture medium and 1% of penicillin-streptomycin; the mixed culture medium is a mixture of PneumaCult Ex culture medium and ADVANCED DMEM/F12 culture medium, and the volume ratio of PneumaCult Ex culture medium to ADVANCED DMEM/F12 culture medium is 3:1.
In a third aspect, the invention provides the use of the human vocal cord white spot immortalized epithelial cells described above in a vocal cord white spot research model.
The human vocal cord white spot immortalized epithelial cells hVCL-MSDEP01 have immortalization, can be used as a cell model/research model for basic research in the related field of vocal cord white spots, and provide research materials for research of vocal cord white spots.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides an immortalized human vocal tract white spot epithelial cell: the human vocal cord white spot immortalized epithelial cells hVCL-MSDEP01 are good in cell state, have no obvious vacuoles in the cells, are in squamous epithelial-like appearance, are clear in cell demarcation, have no obvious difference in size and are good in uniformity. The immortalized epithelial cells of the human vocal cords and white spots have good epithelial cell characteristics, can express immortalized characteristic proteins, have low growth speed and higher apoptosis rate, are different from tumor cell models, and can be used as research models. The invention fills the blank of immortalized leucorrhea epithelial cell line, the obtained cell line has the characteristics closest to the inside of the body, and can stably provide the cell source of subsequent experiments, thereby providing an extremely important experimental material for generating and developing leucorrhea to laryngeal cancer, laying a better research foundation for establishing a stable leucorrhea cell in-vitro model and further exploring the related functions of the leucorrhea cell line, and having great significance for researching the formation mechanism of leucorrhea and the treatment method of the leucorrhea.
Drawings
FIG. 1 shows the cell morphology of the immortalized epithelial cells of the vocal cords of example 2 after 1 day of subculturing;
FIG. 2 shows cell morphology after 2 days of subculture of the immortalized epithelial cells of vocal cords leucoma in example 2 of the present invention, wherein A represents a 40-fold magnification and B represents a 100-fold magnification;
FIG. 3 shows the cell morphology of the immortalized epithelial cells of the vocal cords of example 2 after 4 days of subculturing;
FIG. 4 is a genotyping map of the immortalized epithelial cells of the leucorrhea with white discharge in example 2 of the invention;
FIG. 5 is a genotyping map of sample 1 cells in example 3 of the present invention;
FIG. 6 is a genotyping map of sample 2 cells in example 3 of the present invention;
FIG. 7 shows cell morphology of the immortalized epithelial cells of the vocal cords of the comparative example after 1 day of subculturing with a serum-containing medium;
FIG. 8 is a cell morphology of the immortalized epithelial cells of the vocal cords of the comparative example after 3 days of subculturing with a serum-containing medium;
FIG. 9 shows cell morphology of the immortalized epithelial cells of the vocal cords of the comparative example after 5 days of subculturing with a serum-containing medium;
FIG. 10 shows the result of Western Blot detection of cell-characteristic proteins of example 4 of the invention, wherein SV40-hVCL P8/P16/P20/P24/P28 refers to the immortalized epithelial cells of the vocal cords of passage 8/16/20/24/28, respectively;
FIG. 11 is a graph showing the results of SV40-hVCL cellular immunofluorescence in example 4 of the present invention;
FIG. 12 shows the cell cycle flow results for SV40-hVCL and SNU-899 in example 5 of the present invention, wherein A represents SV40-hVCL and B represents SNU-899;
FIG. 13 shows the apoptosis results of SV40-hVCL and SNU-899 in example 6 of the present invention, wherein A represents SV40-hVCL and B represents SNU-899;
FIG. 14 shows the results of the cell growth curve assay in example 7 of the present invention.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Other materials, reagents, etc. used in the examples are commercially available unless otherwise specified.
PneumaCult TMExMedium(PneumaCultTM Ex medium), STEMCELL Technologies.
EXAMPLE 1 culture of vocal cord white spot immortalized epithelial cells
1. Culture of primary epithelial cells of the vocal cords leukoplakia
The leucorrhea tissue obtained from a white plaque specimen on the leucorrhea tissue obtained by clinical surgery of a patient with leucorrhea was stored at 4℃in DMEM/F12 medium containing 1% penicillin-streptomycin.
(1) Digestion of digestive juice: 3mLGibco Advanced DMEM/F12+1mL 8U/mL Dispersion enzyme II+80. Mu.L 2. Mu.g/. Mu.L collagenase I+80. Mu. LFBS +1% Green streptomycin.
The digestive juice of the invention meets the following conditions: ADVANCED DMEM/F12, dispase II (Dispase II), collagenase I, fetal Bovine Serum (FBS) and green streptomycin, wherein the concentration of the dispase II (Dispase II) in the whole system is 2-2.4U/mL, the concentration of the collagenase I is 40-45 mug/mL, the concentration of the Fetal Bovine Serum (FBS) is 2-3%, and the concentration of the green streptomycin is 1-1.5%.
Transferring the leucorrhea tissue into a PBS solution containing 1% penicillin-streptomycin in a sterile operation table, cleaning for 2-3 times, trimming the tissue by using an ophthalmic scissors and an ophthalmic forceps, removing blood clots on a tissue sample, cutting the blood clots into tissue blocks with the size of about 1mm 3, transferring the tissue blocks into a digestive juice, placing the digestive juice on a horizontal constant temperature shaking table, digesting for 100min at the temperature of 37 ℃ at the rotating speed of 70rpm, and adding the same volume of DMEM/F12 complete culture medium containing 10% FBS into the digestive juice to stop digestion; after the digestion was terminated, the tissue mass in the digested solution was blown off with a pipetting gun, cells adhering to the tissue mass were blown off, the resulting mixture was filtered with a 100 μm sieve to give a cell suspension, the cell suspension was centrifuged at 1100rpm for 5min, and the supernatant was discarded to collect cell pellets.
(2) Primary culture
Resuspension is carried out on the cell sediment by PneumaCult TM Ex culture medium (STEMCELL), the obtained cell suspension is inoculated in a six-hole plate, the six-hole plate is placed in a 37 ℃ and 5% CO 2 culture box for culture, the cell adherence condition is observed the next day after inoculation, the fresh PneumaCult TM Ex culture medium is replaced after the cell adherence, and cell fragments and dead cells in the culture medium are removed, thus obtaining the primary epithelial cells of the vocal cords white spots; then the culture medium is replaced every 1-2 days, and the record is photographed under an inverted fluorescence microscope every 3 days; and after the cell density is increased to 70% -80% or the cell growth in a certain area is inhibited by contact, the cell can be passaged.
(3) Subculture
After the cell density is increased to 70% -80% or the cell proliferation is inhibited, removing the culture medium by a pipetting gun, washing 2-3 times by a PBS solution containing 1% penicillin-streptomycin, adding 0.25% trypsin digestion solution containing 0.02% EDTA, digesting for 3-5min at 37 ℃ by 5% CO 2, adding the same volume of DMEM/F12 culture medium containing 10% FBS into the trypsin digestion solution, and stopping digestion to obtain a cell suspension; the cell suspension was centrifuged at 1000rpm for 5min, the supernatant was discarded, the cell pellet was collected, resuspended in PneumaCult Ex medium and the resuspended cell suspension was re-seeded in culture flasks/dishes at a 1:2 passage ratio.
2. Culture of immortalized epithelial cells of the vocal cords leukoplakia
SV40 lentiviral transfection may be considered when passaging the primary epithelial cells of the vocal cords to passage 2-3. To avoid senescence of non-tumor cells during in vitro culture, immortalized viruses should be transfected as early as possible.
The primary epithelial cells of the vocal cords with the growth density of 50 to 60 percent and passaged to the generation 2 are taken out from the incubator, the culture medium is removed by a pipetting gun, the cells are washed for 2 to 3 times by adding PBS without penicillin-streptomycin, and the process is as gentle as possible to avoid blowing off the adherent cells.
SV40 lentivirus (and Meta Biotechnology (Shanghai) Co., ltd., H20635, pLenti-CMV-SV40gp 6-WPRE) was added to each 1: 1mLPneumaCult Ex medium with 10. Mu.LSV 40 lentivirus (titer: 5.38X10 8 TU/mL) and 1. Mu.L transfection enhancer (and Meta Biotechnology (Shanghai) Co., ltd.), the medium and lentivirus were mixed to give a transfection medium, added to a primary epithelial cell flask with a growth density of about 60% -70%, shaken and placed in a 37℃5% CO 2 cell incubator overnight for culturing. The replacement was performed daily with a new configuration of transfection medium for 3 consecutive days. After 3 days of transfection, the transfection medium was changed to PneumaCult Ex medium containing 1% penicillin-streptomycin, and the cell status was changed once a day and observed to avoid contamination in the medium or vacuoles in the cytoplasm.
T25 culture flasks can be full of primary epithelial cells of vocal cords white spots 3-4 days after SV40 virus transfection, and after the cells are full, passage is carried out according to the ratio of 1:2. After 2-3 generations of subculture, SV40 expression in cells is detected by using Western Blot, qPCR and other technologies, the expression is positive and stable, after 1-2 generations of continuous culture are subjected to slow virus transfection and stabilization, a cell culture medium (PneumaCultEx culture medium containing 1% penicillin-streptomycin) is replaced by a serum-free culture medium (74% PneumaCult Ex culture medium+25% ADVANCED DMEM/F12+1% penicillin-streptomycin) and conventional culture is carried out, so that the vocal cord white spot immortalized epithelial cells are obtained.
Cryopreserved cells are considered when the vocal cord white spots immortalized epithelial cells grow to a density of about 70% -80%. Discarding the culture medium, washing with PBS containing 1% penicillin-streptomycin for 2-3 times, adding 0.25% trypsin digestion solution containing 0.02% EDTA, digesting for 3-5min at 37deg.C with 5% CO 2, adding trypsin solution, and stopping digestion with 10% FBS-containing DMEM/F12 culture medium to obtain cell suspension; centrifuging the cell suspension at 1000rpm for 5min, discarding the supernatant, collecting cell precipitate, re-suspending the cell precipitate by using cell freezing solution, transferring into a cell freezing tube, marking, and placing into a programmed cooling freezing box; the freezing box is placed in a refrigerator at the temperature of minus 80 ℃ overnight, and the freezing tube is taken out and transferred into a liquid nitrogen tank for long-term storage on the 2 nd day; the cell cryopreservation solution is 90% PneumaCult Ex culture medium+10% DMSO or serum-free cell cryopreservation solution of neoacemet.
Example 2 identification of vocal cord white spot immortalized epithelial cells
1. Morphological identification of vocal cord leukoplakia immortalized epithelial cells
The vocal cord white spot immortalized epithelial cells obtained in example 1 were cultured in a serum-free medium (74% PneumaCult Ex medium+25% ADVANCED DMEM/F12+1% penicillin-streptomycin) for 1 generation, and the cell morphology was observed by an inverted microscope.
The cell morphology of the immortalized epithelial cells of the vocal cords is shown in the figure 1 after 1 day of subculture, and the results show that the immortalized epithelial cells of the vocal cords are good in state, have no obvious vacuoles in the cells, are in squamous-like appearance, have clear boundaries between cells, have no obvious difference in size, have good cell growth speed, and can ensure that culture flasks grow full 3-4 days after subculture.
Cell morphology after 2 days of subculture of the vocal cord leukoplakia immortalized epithelial cells is shown in FIG. 2, wherein A represents a 40-fold magnification and B represents a 100-fold magnification. The results show that after 2 days of subculture, the cell number is significantly increased compared with the cell number seen in the previous day, the whole cell presents squamous epithelial-like characteristics, no obvious vacuoles exist in the cell, and no cell aging occurs.
The cell morphology of the immortalized epithelial cells of the vocal cords after 4 days of subculture is shown in FIG. 3, and the results show that the cells have grown into pieces, squamous epithelium-like, without obvious vacuoles in the cells after 4 days of subculture, and can be subjected to the next passage.
2. Identification of Short tandem repeat (Short TANDEM REPEAT, STR) of the immortalized epithelial cells of the leucorrhea from example 1 was carried out in the cell bank/stem cell bank of the national academy of sciences of China Committee for culture Collection.
DNA from the vocal cord leucoma immortalized epithelial cells was extracted, 20 Short Tandem Repeat (STR) sites and Amelogenin sex determination sites were amplified using MicroreaderTM ID System, PCR product detection was performed using an ABI 3730xl type genetic Analyzer, and the detection results were analyzed using GENEMAPPERID-X software (Applied Biosystems) and compared with ATCC, DSMZ and ExPASy databases.
The spectrogram of the immortalized epithelial cells of the leucorrhea after DNA amplification is clear, the parting result is good, and the genotyping spectrogram of the immortalized epithelial cells of the leucorrhea is shown in figure 4.
Genotyping results of STR site and Amelogenin site of the vocal cord leukoplakia immortalized epithelial cells and comparison results with database are shown in Table 1, table 1
Table 1 shows that the Amelogenin sites of the vocal cord white spot immortalized epithelial cells are Amelogenin, and the STR sites include D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX and CSF1PO. The data matching rate of the vocal cord white spot immortalized epithelial cells (detection samples) of the invention and UACC-732Breast Adenocarcinoma Human cells in ATCC, 1987-05-01:00:00 in DSMZ and AIDHC-00138 in ExPASy is lower than 80%, which indicates that the vocal cord white spot immortalized epithelial cells of the invention are novel cells.
The vocal cord white spot immortalized epithelial cells are named as human vocal cord white spot immortalized epithelial cells hVCL-MSDEP, and are preserved in China Center for Type Culture Collection (CCTCC) in 1 month 15 of 2024, and the preservation number is CCTCC NO: c202432, the preservation address is China, university of Wuhan.
EXAMPLE 3 identification of the homology of immortalized epithelial cells of the vocal cords leukoplakia
Cell pellet of the vocal cord white spot immortalized epithelial cells of example 2 was collected and designated as test material 1; collecting blood of a patient corresponding to the vocal cord white spot tissue from the vocal cord white spot immortalized epithelial cells, and marking the blood as a detection material 2. And (5) identifying whether the detection material 1 and the detection material 2 are of the same source or not, and whether cross contamination exists or not.
A proper amount of detection material is taken, microread Genomic DNA Kit is used for extracting DNA, microreaderTM ID System is used for amplifying 20 STR sites and sex identification sites, ABI 3730xl type genetic analyzer is used for detecting PCR products, and GENEMAPPERID-X software (Applied Biosystems) is used for analyzing detection results.
The patterns of the amplified DNA of the cells of the detection material 1 and the detection material 2 are clear, the parting result is good, and the genotyping pattern of the cells of the detection material 1 is shown in figure 5; the genotyping map of the test material 2 cells is shown in FIG. 6.
Genotyping results of STR site and Amelogenin site of test material 1 are shown in Table 2,
TABLE 2
Genotyping results of STR site and Amelogenin site of test material 2 are shown in Table 3,
TABLE 3 Table 3
In tables 2 and 3, markers are listed as detection loci (sites), and Allle 1/2/3 is the detection locus typing. More than three loci appear, three of which are typed, possibly with other sources of human cell contamination.
The results in tables 2 and 3 show that the match rate of sample 1 and sample 2 was 100% in 20 loci, and both can be considered as the same source. There are no three genotyping cases of more than three loci, no contamination of human cells.
Comparative example
The vocal cord white spot immortalized epithelial cells obtained in example 1 were cultured in a serum-containing medium (DMEM/f12+10% fbs+1% penicillin-streptomycin) for 1 generation, and the cell morphology was observed by an inverted microscope.
The initial deterioration of the state of the immortalized epithelial cells of the vocal cords was observed after day 2 of subculture: clear areas are visible in cells, cells lose squamous epithelial-like morphology, cells start to become narrow and deformed, boundaries among cells are no longer clear, and cell size change is large, which is an expression that cells tend to age. Meanwhile, the cell growth speed is slowed down, only aged and apoptotic cells are seen by changing liquid every day, and cell growth is not seen.
The cell morphology of the immortalized epithelial cells of the vocal cords was shown in FIG. 7 after 1 day of subculturing with the serum-containing medium, and the results showed that the cell growth rate was slowed down, the cell morphology became long and narrow, and the cytoplasm was transparent, indicating that the cell state was poor.
The cell morphology of the immortalized epithelial cells of the vocal cords was shown in FIG. 8 after 3 days of subculturing with the serum-containing medium, and the results showed that the cell proliferation rate was slower, the cell morphology became long and narrow, and the cytoplasm was clear, indicating that the cell state was poor, compared with the day 1.
The cell morphology of the immortalized epithelial cells of the vocal cords is shown in fig. 9 after 5 days of subculturing by using a serum-containing medium, and the results show that the cells are obviously increased, the cell fusion phenomenon starts to appear between adjacent cells, the boundary is not clear, and squamous epithelial-like characteristics are lost, which indicates that the cells approach to aging and growth limit.
In conclusion, the culture effect of the immortalized epithelial cells of the vocal cords and the white spots is poor by using a serum-containing culture medium, and the culture effect is better by using a serum-free culture medium.
Example 4 cell-characteristic protein detection
1. Detection of vocal cords white spot immortalized epithelial cells (SV 40-hVCL) and characteristic proteins of vocal cord white spot primary epithelial cells (Pri-hVCL) of example 1 by Western Blot SV40-hVCL and Pri-hVCL, respectively, were transferred to 5mL centrifuge tubes and centrifuged at 1000rpm for 5min. The supernatant was discarded, 1mLPBS resuspension was added to each tube, the cell suspension was transferred to 1.5mLEP tubes and centrifuged at 1000rpm again for 5min. The supernatant was discarded, and 150. Mu. LRIPA lysate and 1.5. Mu.L protease inhibitor cocktail (100X) were added to each EP tube, and after thoroughly mixing, the mixture was allowed to stand on ice for 20min. After completion of lysis, the supernatant was transferred to a fresh EP tube and labeled by centrifugation at 12000rpm for 5min, and the protein concentration was quantified using the BCA protein concentration detection kit. After calibrating the protein concentration of each tube, 1/4 of the 5 Xprotein loading buffer was added according to the final volume and gently mixed by a pipette. Placing into metal heating bath, and heating at 100deg.C for 5min to denature protein. The obtained samples were directly used for Western Blot electrophoresis and the results were analyzed using software ImageJ software.
Immortalized related proteins: SV40 LT ANTIGEN (90 kDa); epithelial cell related protein: E-Cadherin (120 kDa), keratin 17/19 (48/41 kDa); internal reference protein: GAPDH (36 kDa).
The detection result of the cell characteristic protein Western Blot is shown in FIG. 10, wherein SV40-hVCL P8/P16/P20/P24/P28 respectively refers to the vocal cord white spot immortalized epithelial cells of passage 8/16/20/24/28; the results show that the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) can express the epithelial cell specific protein Keratin 17/19, E-cadherein, immortalized protein SV40 LT ANTIGEN; whereas primary epithelial cells of the vocal cords (Pri-hVCL) only express the epithelial cell-specific protein Keratin 17/19, E-cadherein, and do not express the immortalized protein SV40 LT ANTIGEN.
2. The proteins characteristic of the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) of example 1 were detected using immunofluorescence experiments.
The digested growth state is good, SV40-hVCL is stably passaged, the product is passaged into a 12-hole plate, and the product is fully and evenly shaken and then placed into a 37 ℃ 5% CO 2 cell incubator for overnight culture. Culturing until the cell density in a 12-well plate reaches 70% -80% by normal liquid exchange, discarding the culture medium, washing the cells for 2-3 times by using PBS containing 1% of streptomycin, adding a proper amount of 4% of paraformaldehyde universal tissue fixing solution, fixing for 15-20min at room temperature, and then washing the cells for 2-3 times by using PBS containing 0.1% of Triton X-100 for 5min each time. Adding immunofluorescence rapid blocking solution into each hole, and blocking for 30min at room temperature by shaking table. The cells were washed 3 times with PBS for 5-10min each. The primary antibody was incubated overnight at 4 ℃. The cells were washed 3 times with PBS for 5-10min each. Incubate in a shaking table at room temperature for 1h. The cells were washed again with PBS 3 times, 5-10min each. Treatment was performed for 10min at room temperature using an anti-fluorescence decay capper (containing DAPI). The images of 100X of the bright field, the FITC field and the DAPI field are observed and shot sequentially under an inverted fluorescence microscope.
Epithelial cell-specific protein: E-Cadherin, keratin/18; fibroblast-related protein: vimentin.
The SV40-hVCL cell immunofluorescence results are shown in FIG. 11, which shows that the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) can express the epithelial cell specific protein Keratin8/18, E-cadherein; the fibroblast-associated protein Vimentin was not expressed.
Example 5 cell growth Rate assay
The cell cycle distribution of the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) of example 1 was examined by flow cytometry. The laryngeal squamous cell carcinoma epithelial cells (SNU-899) were used as a comparison.
Approximately 2X 10 5-1×106 well-grown, stable passaged SV40-hVCL and SNU-899 were separately digested and collected, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Cells were washed 1 time with PBS, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Adding into cell cycle flow detection kit 1mLDNA Staining solution and 10 μ LPermeabilization solution, and mixing with vortex oscillation for 5-10 s. Incubate at room temperature for 30min in the dark. The lowest loading rate was selected and the cell PI dye staining was detected on a flow cytometer.
The cell cycle flow results of SV40-hVCL and SNU-899 are shown in FIG. 12, wherein A represents SV40-hVCL and B represents SNU-899;
The results showed that the growth rate of the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) was lower than that of the laryngeal squamous cell carcinoma epithelial cells (SNU-899).
EXAMPLE 6 apoptosis assay
The degree of apoptosis of the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) of example 1 was examined by flow cytometry. The laryngeal squamous cell carcinoma epithelial cells (SNU-899) were used as a comparison.
Approximately 1X 10 6-3×106 stable passaged SV40-hVCL and SNU-899 cells were collected by digestion, washed twice with pre-chilled PBS, and the supernatants discarded and tested for apoptosis using the Annexin V-FITC/PI apoptosis kit. Add 500 μ LApoptosis Positive Control Solution to resuspend and incubate on ice for 30min. The supernatant was discarded after centrifugal washing with pre-chilled PBS. An appropriate amount of pre-chilled 1 Xbinding Buffer was added for resuspension and the same amount of untreated living cells were added and mixed with it. Adding pre-cooling 1×binding Buffer to 1.5mL, and equally dividing into three tubes, wherein one tube is a blank reference tube, and two tubes are single-dyeing tubes. Mu. LAnnexin V-FITC or 10. Mu.LPI was added to the single-stained tube and incubated at room temperature for 5 minutes in the dark. On a flow cytometer, the voltages of FSC, SSC and fluorescent channels are adjusted with a blank tube, and under this voltage condition, the compensation of the fluorescent channels is adjusted with a single dye tube. Annexin V-FITC staining was detected by FITC detection channel (ex=48nm; em=530 nm) and PI staining was detected by PI detection channel (ex=535 nm; em=615 nm).
The apoptosis results of SV40-hVCL and SNU-899 are shown in FIG. 13, wherein A represents SV40-hVCL and B represents SNU-899;
the results showed that the apoptosis of the vocal cord leukoplakia immortalized epithelial cells (SV 40-hVCL) was significantly higher than that of the laryngeal squamous cell carcinoma epithelial cells (SNU-899).
Example 7 cell growth Curve assay
Cell count experiments (cell counting kit-8assay, CCK 8) examined the cell growth curves of the vocal cord white spot immortalized epithelial cells (SV 40-hVCL) and the vocal cord white spot primary epithelial cells (Pri-hVCL) of example 1.
The digested growth state is good, SV40-hVCL or Pri-hVCL is stably passaged, the product is passaged into a 96-well plate, blank holes of 4 independent culture mediums are additionally prepared, and the culture medium is fully shaken and then placed in a 5% CO 2 cell incubator at 37 ℃ for overnight culture. 4-hole cells and 4-hole blank holes are selected 24h after the plating, 10% of CCK8 reagent with the volume of the culture medium is added, so that bubbles are avoided, and the cells are fully shaken and cultured in a 37 ℃ 5% CO 2 cell incubator for 3h. The absorbance (OD) at 450nm was measured using a microplate reader.
The results of the cell growth curve assay are shown in FIG. 14, and the results show that the primary epithelial cells of the vocal cords leucoma (Pri-hVCL) have a distinct growth plateau, whereas the immortalized epithelial cells of the vocal cords leucoma (SV 40-hVCL) have no distinct growth plateau.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (9)

1. The immortalized epithelial cells of the leucorrhea of a human voice are named hVCL-MSDEP and are preserved in China Center for Type Culture Collection (CCTCC) in the year 2024, month 1 and 15, and the preservation number is CCTCC NO: c202432, the preservation address is China, university of Wuhan.
2. The method for culturing human vocal cords white spot immortalized epithelial cells according to claim 1, comprising the steps of: s1, digesting the leucorrhea leucoma tissues, and culturing the leucoma tissues by using PneumaCult TM Ex culture medium to obtain primary epithelial cells of the leucoma; s2, continuously culturing and passaging the primary epithelial cells of the vocal cords with PneumaCult TM Ex culture medium to 2-3 generations, then transfecting with SV40 slow virus, screening out cells positive and stable in SV40 expression, continuously culturing for 1-2 generations to obtain transfected stable cells, and culturing with serum-free culture medium to obtain immortalized epithelial cells of the vocal cords with the leucoma.
3. The method according to claim 2, wherein in step S1, the composition of the digestion solution used for digestion is: ADVANCED DMEM/F12, 2-2.4U/mL of dispersing enzyme II, 40-45 mug/mL of collagenase I, 2-3% of fetal bovine serum and 1-1.5% of green streptomycin.
4. The culture method according to claim 2, wherein in step S1, the temperature of the digestion is 35 to 37 ℃; and/or, the digestion time is 60-120min.
5. The culture method according to claim 2, wherein in step S1, the leucorrhea tissue is washed with a PBS solution containing penicillin-streptomycin and then sheared into a tissue mass of 0.8 to 1.2mm 3 before being digested with a digestive fluid.
6. The culture method according to claim 2, wherein in step S2, the primary epithelial cells of the vocal cords are cultured with fresh PneumaCult TM Ex medium, the medium is replaced every 1-2 days, and subculture is performed after culturing until the cell density reaches 70% -80%;
and/or, the ratio of said passages is 1: (1-3).
7. The method of claim 2, wherein in step S2, the primary epithelial cells of the vocal cords are cultured using PneumaCult Ex medium containing SV40 lentivirus and a transfection enhancer, and then further cultured using PneumaCult Ex medium containing 1 to 1.5% penicillin-streptomycin.
8. The culture method according to claim 2, wherein in step S2, the serum-free medium consists of the following components in volume percent: 98.5 to 99 percent of mixed culture medium and 1 to 1.5 percent of penicillin-streptomycin; the mixed culture medium is a mixture of PneumaCult Ex culture medium and ADVANCED DMEM/F12 culture medium, and the volume ratio of PneumaCult Ex culture medium to ADVANCED DMEM/F12 culture medium is (2.8-3.2): 1.
9. Use of the immortalized epithelial cells of human vocal cords with white spots according to claim 1 in a model of vocal cords white spots study.
CN202410400512.0A 2024-04-03 2024-04-03 Immortalized epithelial cells of leucorrhea with white spots of human voice and culture method and application thereof Pending CN118147082A (en)

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