CN114134118A

CN114134118A - Immortalized human laryngeal ring posterior region cell and construction method thereof

Info

Publication number: CN114134118A
Application number: CN202111385406.2A
Authority: CN
Inventors: 李湘平; 谭嘉杰; 鲁娟; 戴远锋; 莫婷婷
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-03-04
Anticipated expiration: 2041-11-22
Also published as: CN114134118B

Abstract

The invention discloses an immortalized human laryngeal ring posterior region cell, which is constructed by introducing Bmi1 gene into a human laryngeal ring posterior region source cell, and the immortalized human laryngeal ring posterior region cell has the capacity of in vitro unlimited proliferation or nearly unlimited proliferation. The invention introduces Bmi1 gene into epithelial primary cells of the posterior region of the human laryngeal ring, thereby constructing an immortalized epithelial cell strain of the posterior region of the human laryngeal ring, and experimental research shows that the constructed immortalized cell strain of the posterior region of the human laryngeal ring can slow down the aging of epithelial cells of the posterior region of the laryngeal ring, maintain the activity of cells and provide a stable and reliable cell model for the research of pathogenesis of benign and malignant diseases of the posterior region of the laryngeal ring. Meanwhile, the invention also discloses a construction method of the immortalized human laryngeal ring posterior cell, which has the advantages of convenient material acquisition, strong repeatability and convenient operation.

Description

Immortalized human laryngeal ring posterior region cell and construction method thereof

Technical Field

The invention discloses an immortalized cell and a construction method thereof, in particular to an immortalized cell from a human laryngeal cavity ring posterior region and a construction method thereof.

Background

With the rapid development of society, the living standard of people is continuously improved, and the medical requirements are increasingly increased. Throat benign and malignant diseases are common in clinic, seriously affect the life quality and physical and psychological health of patients, and become a health and social problem which cannot be ignored. However, the clinical diagnosis and treatment of benign and malignant diseases of the throat are still quite troublesome, and the root of the diagnosis and treatment lies in the insufficient research on pathogenic mechanisms, so that the intensive research on molecular pathological mechanisms is particularly important. However, no commercial laryngeal mucosal epithelial cell strain is purchased in the market at present, and the primary cell culture of the normal laryngeal mucosal epithelium needs to be carried out in vitro to establish the laryngeal epithelial cells for research, so that an in vitro model is provided for comprehensively discussing the action mechanisms of different pathogenic factors in inflammatory and malignant diseases of the larynx.

Because the throat mucosa belongs to the terminal differentiation tissue of a human body, the cell proliferation capability is poor, and the separated cells are fragile and difficult to survive. And because the condition of the laryngeal cavity is limited, the obtained tissue quantity is small, the tissue can not be like other tissues of parts such as stomach, intestine, mammary gland and the like, a large quantity of tissues can be taken as culture raw materials, and if a large quantity of subsequent experiments need to be carried out, the repeated times of the experiments can cause poor repeatability. Therefore, there are many subjective and objective difficult factors in primary cell culture of laryngeal epithelium, which greatly limits the progress of primary cell culture of laryngeal epithelium. The tissue block culture method for the human beings such as Lidongjun and Kechaoyang successfully cultures the fetal laryngeal epithelial tissue to culture primary laryngeal epithelial cells, but most of the culture methods are derived from fetal laryngeal mucosal tissue of induced labor or dead labor, and because the fetal laryngeal mucosal tissue is just differentiated, the tissue activity is high, and the culture success rate can be improved. However, the fetal throat tissue has limited sources and the material taking process is complicated. And the primary culture process is complex, the required culture time is long, the spirit and physical strength of a large number of researchers are required to be consumed, and the cost is high. In addition, the primary cells are limited by the limited life span in vitro, the cells cultured at the beginning can grow well, but when the cells are continuously passaged to about 4 generations, the number of aged cells of the primary cells is increased, the cell proliferation is slow, and the number of dead cells is increased. Besides affecting cell proliferation and slowing down the experiment progress, the appearance of aging cells also changes a plurality of molecular biological genes and affects the experiment result. Therefore, the inventor hopes to construct the immortalized cell line of the laryngeal epithelium which can be stably and continuously passaged, establish an ideal cell model and lay a foundation for the research of pathogenesis of benign and malignant diseases of the throat.

The first two immortalized laryngeal epithelial cells were established by exogenous introduction of the Human Papilloma Virus (HPV) E6/E7 or the monkey virus 40(SV40) viral oncogene. However, the direct effects of exogenous viral oncogenes and the indirect effects of producing aneuploid cells can affect cellular behavior, such as limited differentiation capacity and abnormal changes similar to carcinoma in situ. Therefore, the establishment of immortalized laryngeal epithelial cell lines is urgently needed, and a reliable and stable cell model is provided for the comprehensive research on pathogenic mechanisms of benign and malignant diseases of the throat.

The human laryngeal cavity is divided into different parts, including the vocal cords, the ventricular girdle, the posterior ring, the epiglottis and the subglottal. Epithelia of different structure there are two different types of mucosal epithelia: the vocal cords, posterior annulus and hypopharynx epithelium are mainly non-keratinized stratified squamous epithelium, the main component of the cell is keratinocyte, the subglottal and epiglottic region is transited from pseudostratified ciliated columnar epithelium to squamous epithelium, and the main component of the cell is columnar cell. The squamous epithelium is more closely arranged than the columnar epithelium, and can shed the stratum corneum to resist the invaders, so that the columnar epithelium is more easily damaged by the exogenous invaders than the squamous epithelium in general and is easy to have subglottic stenosis. In addition, different anatomical regions of the larynx have different resistance to exogenous stimuli (such as viruses, smoke, alcohol, gastrointestinal reflux) due to different embryo sources, different molecular components and different tissue structures. If papilloma virus easily invades vocal cords and ventricular cords, papilloma is less likely to appear in the posterior region of the annulus. The laryngeal mucosa of the posterior ring is more sensitive to throat reflux (LPR) injury due to the lack of the carbonic anhydrase III component, and is more prone to posterior erythema and swelling. Therefore, in order to more comprehensively explore the pathogenesis of different pathological changes of the throat and the treatment prognosis situation, it is necessary to construct immortalized laryngeal epithelial cells which can grow stably, have a good state and can be continuously passaged, provide a stable molecular platform for cell biology research, and also discuss the effects of different pathogenic factors in different regions.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an immortalized cell which has the capability of unlimited proliferation or nearly unlimited proliferation in vitro and is derived from the posterior region of the human laryngeal cavity; meanwhile, the invention also provides a construction method of the immortalized ring posterior region cell.

In order to achieve the purpose, the invention adopts the technical scheme that: the immortalized human laryngeal ring posterior region cell is constructed by introducing Bmi1 gene into human laryngeal ring posterior region source cell, and the immortalized human laryngeal ring posterior region cell has the capacity of in vitro unlimited proliferation or nearly unlimited proliferation.

The Bmi1 gene is an important component in a polycomb gene family (a key epigenetic regulatory factor), participates in proliferation, differentiation and senescence of stem cells, and plays an important role in embryonic development, tumorigenesis and maintenance of stem cells. The inventor of the application tries to introduce the Bmi1 gene into primary epithelial cells in the rear region of the human laryngeal ring for the first time, so that an immortalized epithelial cell strain in the rear region of the human laryngeal ring is constructed, and experimental research shows that the constructed immortalized cell strain in the rear region of the human laryngeal ring can slow down the aging of epithelial cells in the rear region of the laryngeal ring, maintain the activity of cells, and provide a stable and reliable cell model for research on pathogenesis of benign and malignant diseases in the rear region of the laryngeal ring. According to the application, the Bmi1 is used for constructing the immortalized human laryngeal ring posterior region cell, so that unnecessary molecular changes caused by introducing exogenous HPV E6/E7 and SV40 virus oncogenes can be avoided, and a more accurate cell model is provided for later-stage biological mechanism research.

The immortalized human posterior laryngeal ring cells described herein are designated human post-immortalized spatial admixture Bmi1(the human immortalized spatial admixture Bmi1, hPC-Bmi1) cells).

The Bmi1 gene described herein is commercially available directly, including but not limited to, from GeneCopoeia, Inc., and is designated plVTHM/Bmi1 and contains a Green Fluorescent Protein (GFP) and puromycin resistance marker.

As a preferred embodiment of the immortalized human posterior laryngeal ring cell of the present invention, the immortalized posterior laryngeal ring cell is constructed by transfecting a recombinant plasmid carrying Bmi1 gene into a human posterior laryngeal ring source cell.

As a preferred embodiment of the immortalized human posterior laryngeal ring cell of the present invention, the method of transfecting the recombinant plasmid carrying the Bmi1 gene into the human posterior laryngeal ring derived cell is a lentiviral vector.

The subculturing method of the immortalized human laryngeal ring posterior region cell (hPC-Bmi1) comprises the following steps: when each colony grows up and has no gap, the passage can be carried out, an old culture solution in the bottle is sucked by a pipette, 0.5ml of a preheated mixed solution containing 0.25 percent of trypsin and 0.02 percent of EDTA (1:1) is added, the mixed solution is placed in an incubator at 37 ℃ for digestion for 5 minutes, an inverted microscope is used for observation, when the cytoplasm is retracted and the cell gap is increased, a 1ml of a gun head is used for blowing and beating the cells at the bottom repeatedly, the cells are seen to be flaked and shed at the moment, if the cells are still adhered to the bottom, the mixed solution can be continuously placed in the incubator at 37 ℃ for prolonging the digestion time for 1 to 2 minutes, 1ml of 10 percent DMEM high-glucose complete culture medium is added for stopping digestion, the cells are shed into cell suspension, the cell suspension is centrifuged at 1000rpm for 5 minutes, the supernatant is discarded, and KBM culture medium heavy suspension cells are added for plating for culture.

In addition, another object of the present invention is to provide a method for constructing cells in the posterior region of the immortalized human laryngeal ring, which comprises the following steps: a construction method of immortalized human posterior laryngeal ring cells, comprising the following steps:

(1) two-step enzyme digestion method and primary culture of epithelial cells in the posterior region of human laryngeal ring by tissue grinding: taking mucosa tissue in the posterior region of the larynx specimen, placing the larynx specimen into a culture dish in a sterile environment, washing surface bloodiness and impurities, separating the tissue, and soaking the tissue in a centrifugal tube containing dispase; after soaking for 24 hours, transferring all tissues and culture solution in the centrifugal tube into a sterile centrifugal tube, centrifuging, removing supernatant, adding preheated pancreatin for digestion, adding 10% DMEM high-sugar complete culture medium to stop digestion, and repeatedly blowing and beating the tissues by using a gun head to form a single-cell suspension; pouring the single cell suspension into a screen for filtering, collecting filtrate, putting the rest tissues on the screen into a culture dish, repeatedly grinding, adding 10% DMEM high-sugar complete culture medium, uniformly mixing the tissues, further filtering by using the screen, centrifuging, removing supernatant, and carrying out culture by using a KBM (KBM) culture medium for re-suspending cells and plating until the cells grow out, wherein the cells can be digested and passaged for about 14 days;

(2) preparation of competent bacteria and plasmid transformation: the adopted Bmi1 plasmid (plVTHM/Bmi1) contains green fluorescent protein and puromycin resistance markers, competent bacteria are melted on ice and then are uniformly mixed with DNA samples to be filled into an EP tube, the mixture is placed in ice for 20-40 minutes and then is placed in a water bath kettle at the temperature of 40-45 ℃ for 30-60 seconds, then is placed in ice for 1-2 minutes, a preheated SOC culture medium is added, and the mixture is uniformly mixed and then is subjected to shaking culture; uniformly mixing the liquid in a sterile operating platform by using a gun head, taking the bacterium liquid after the bacterium is fully suspended, dripping the bacterium liquid on a screening agarose plate for ampicillin resistance, uniformly coating the bacterium liquid on the agarose plate, inverting a cell dish, and placing the cell dish in an incubator overnight;

(3) plasmid extraction: selecting excellent monoclonal colonies, adding the excellent monoclonal colonies into an ampicillin-resistant LB liquid culture medium, shaking uniformly until the liquid is turbid, stopping shaking, adding the turbid liquid into an ampicillin-resistant LB liquid culture medium, shaking overnight, subpackaging the LB liquid culture medium into centrifuge tubes the next day, centrifuging to precipitate bacteria, and discarding the supernatant; adding Buffer R3 reagent containing RNase into each tube to resuspend bacteria, blowing and dissolving; adding Buffer L7 into each tube, covering the cover, reversing the centrifuge tube, mixing uniformly, and standing at room temperature; adding Buffer N3 into each tube, quickly reversing and uniformly mixing without whirling, centrifuging at room temperature, transferring the supernatant into a Buffer EQl balance filter column for filtration, washing the column, discarding the filtrate, adding an elution Buffer solution, filtering, collecting the filtrate for an EP tube, adding isopropanol, uniformly mixing, centrifuging, discarding the supernatant, adding a TE reagent to resuspend plasmids, and measuring the plasmid concentration;

(4) lentivirus packaging Bmi1 virus: adding Opti-MEM culture medium into an EP tube (tube A), and adding a Lipofectamine 3000 reagent for mixing; adding Opti-MEM medium to the EP tube (B tube), then adding plVTHM/Bmi1, psPAX2 and pMD2.G, gently mixing to avoid vortexing; adding the liquid in the tube A into the tube B, gently mixing uniformly, and incubating at room temperature; adding the mixed liquid into a 293FT cell culture dish along the culture wall drop by drop, gently mixing uniformly, putting the mixed liquid into a cell culture box for culture, taking out cells, absorbing a culture medium, and adding a preheated DMEM complete culture medium;

(5) virus collection and cell infection: adding a preheated DMEM complete culture medium into the step (4) to culture for 72 hours, collecting virus supernatant, filtering virus liquid by using a cell filter, directly infecting primary laryngeal epithelial cells by using the filtered virus liquid, adding the virus liquid and a KBM-2 culture medium into a cell dish, infecting the cells for 5 hours each time, replacing the virus liquid with a virus-free KBM-2 culture medium to culture for 5 hours, and infecting the cells for 3 times;

(6) flow cytometry is used for sorting the epithelial cells of the posterior region of the human laryngeal ring containing the Bmi1 gene: and (3) sorting the cells by a flow cytometer to obtain GFP positive epithelial cells of the posterior region of the human laryngeal ring, so as to obtain the immortalized cells of the posterior region of the human laryngeal ring.

As a preferred embodiment of the construction method of the immortalized human retrolaryngeal ring cell, the blood stain and impurities on the surface of the mucosal tissue of the retrolaryngeal ring in the whole laryngeal specimen are washed by 95% alcohol and DPBS buffer solution in sequence in the step (1); after the preheated pancreatin is added, the volume ratio of liquid to tissue in the centrifugal tube is 2: 1.

As a preferred embodiment of the method for constructing the immortalized human posterior laryngeal ring cell of the present invention, the mixing ratio of the competent bacteria to the DNA sample in the step (2) is: each 50. mu.l was mixed with not more than 10ng of DNA sample.

As a preferred embodiment of the construction method of the immortalized human posterior laryngeal ring cell, in the step (3), excellent monoclonal colonies are selected and added into 8ml of LB liquid culture medium containing ampicillin resistance, shaking and shaking are carried out for 8 hours at 37 ℃, the liquid is stopped after turbidity appears, 50ul of the liquid is added into 300ml of LB liquid culture medium containing ampicillin resistance, shaking is carried out overnight at 37 ℃, the next day, LB culture solution is subpackaged into 50ml of centrifuge tubes, bacteria are precipitated by centrifuging for 10 minutes at 4000g, and the supernatant is discarded; adding 4ml of Buffer R3 reagent added with RNase into each tube to resuspend the bacteria, blowing and dissolving; adding 4ml of Buffer L7 into each tube, covering the cover, reversing the centrifuge tube, mixing uniformly, and standing at room temperature for 5 min; adding 4ml of Buffer N3 into each tube, quickly reversing and uniformly mixing the mixture without rotating, centrifuging the mixture at the room temperature of 13000rpm for 10min, transferring the supernatant into a 15ml of Buffer EQl balance filter column for slow filtration, adding 10mlW8 to wash the column for 2 times, discarding the filtrate, adding 5ml of elution Buffer E4, filtering the mixture, collecting the filtrate in a 15ml EP tube, adding 3.5ml of isopropanol for uniformly mixing the mixture, centrifuging the mixture at the temperature of 13000rpm for 30min at 4 ℃, discarding the supernatant, adding 100ul of TE reagent to resuspend the plasmids, and measuring the plasmid concentration.

As a preferred embodiment of the method for constructing the immortalized human posterior laryngeal ring cell of the present invention, the step (4) is 1.3X 10 hours before transfection is carried out⁶-1.5×10⁶The 293FT cells with good growth state are inoculated into a 10cm cell culture dish, DMEM complete culture medium containing 10% fetal calf serum which is preheated in advance is added for culture until the day of transfection, and when the cell density reaches 50% confluence, lentivirus packaging is carried out.

As a preferred embodiment of the construction method of the immortalized human posterior laryngeal ring cell of the present invention, the lentivirus packaging method of step (4) comprises: add 500ul Opti-MEM medium to EP tube (tube A), and add 17ul Lipofectamine 3000 reagent and mix; add 500ul Opti-MEM medium to EP tube (B tube) and then add 16 μ g plVTHM/Bmi1, 12 μ g psPAX2 and 6 μ g pMD2.G gently mixed to avoid vortexing; adding the liquid in the tube A into the tube B, gently mixing uniformly, and incubating for 30 minutes at room temperature; and adding the mixed liquid into a 293FT cell culture dish along the culture wall dropwise, mixing the mixed liquid gently and uniformly, putting the mixed liquid into a cell culture box for 8 hours, taking out the cells from the culture box, carefully sucking the culture medium, and adding preheated DMEM complete culture medium.

As a preferred embodiment of the construction method of the immortalized human posterior laryngeal ring cell, in the step (5), a 0.45um cell filter is used for filtering the virus liquid, the filtered virus liquid can directly infect primary human posterior laryngeal ring epithelial cells, the virus liquid with the volume ratio of 1:1 and the KBM-2 culture medium are added into a cell dish, each infection lasts for 5 hours, the virus-free KBM-2 culture medium is replaced for culturing for 5 hours, and then the infection is carried out for 3 times.

According to the immortalized human laryngeal ring posterior region cell, the Bmi1 gene is introduced into the epithelial primary cell in the human laryngeal ring posterior region, so that an immortalized human laryngeal ring posterior region epithelial cell strain is constructed, and experimental research shows that the constructed immortalized human laryngeal ring posterior region cell strain can slow down the aging of the laryngeal ring posterior region epithelial cell, maintain the cell activity, and provide a stable and reliable cell model for research on a benign and malignant disease pathogenesis in the pharyngeal laryngeal ring posterior region. The construction method of the immortalized human laryngeal ring posterior region cells has the advantages of convenient material acquisition, strong repeatability and convenient operation.

Drawings

FIG. 1 is a schematic diagram of the structure of pLVTHM/Bmi1 plasmid;

FIG. 2 is a diagram showing the sequencing result of plasmid pLVTHM/Bmi 1;

FIG. 3 is a graph comparing the degree of senescence of primary epithelial cells and hPC-Bmi1 cells in the posterior region of the human laryngeal ring;

FIG. 4 is a graph comparing the proliferation capacities of primary epithelial cells and hPC-Bmi1 cells in the posterior region of the human laryngeal ring;

FIG. 5 is a comparison of cell cycle of primary epithelial cells and hPC-Bmi1 cells in the posterior region of the human laryngeal ring;

FIG. 6 is a graph comparing the effect of primary epithelial cells on the posterior region of the human laryngeal ring on the formation of tumors by hPC-Bmi 1;

FIG. 7 is a graph of the effect of Pepsin on the levels of IL1 α, IL1 β, IL6, IL8 and caspase 1;

FIG. 8 is a graph of the effect of Pepsin on Caspase1, IL1 β and IL18 levels;

FIG. 9 is a graph of the effect of Pepsin on ROS levels using fluorescence quantification;

FIG. 10 is a graph of the effect of Pepsin on ROS levels using flow cytometry;

FIG. 11 is a graph of the effect of Pepsin on TXNIP levels;

FIG. 12 is a graph of the effect of Tempol, DPI and APO on intracellular ROS following pepsin stimulation;

FIG. 13 is a graph of the effect of Tempol, DPI and APO on mitochondrial ROS following pepsin stimulation;

FIG. 14 is a graph of the effect of Tempol, DPI and APO on pro-inflammatory factors following pepsin stimulation;

FIG. 15 is a graph comparing the transcript levels of Caspase1, IL1 β and IL18 in Pepsin treated hPC-Bmi11 cells;

FIG. 16 is a graph comparing the effect of APO on NLRP3, TXNIP, cleared-Caspase 1, cleared-IL 1 β, IL 18;

FIG. 17 is a graph comparing the effect of MCC950 on NLRP3, TXNIP, cleared-Caspase 1, cleared-IL 1 β, IL 18;

FIG. 18 is a graph comparing the effect of APO and MCC950 on pepsin cytotoxicity;

Detailed Description

To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1

In an embodiment of the present invention, the immortalized posterior laryngeal ring cell is constructed by the following method:

(1) two-step enzyme digestion method and primary culture of tissue grinding laryngeal epithelium:

cutting the mucosa tissue under the ventricular zone and glottis in the whole larynx specimen with a sterile scalpel blade, taking the mucosa tissue to the deep layer of the larynx as much as possible, taking the mucosa tissue with the size of 0.5-1cm, immediately putting the mucosa tissue into a 1.5ml centrifuge tube filled with prepared tissue storage liquid (DPBS buffer solution and three antibodies), storing the mucosa tissue at low temperature of 4 ℃, transporting the mucosa tissue to a laboratory as soon as possible, and preferably not exceeding 24 hours; taking out the laryngeal tissue specimen in a sterile super-clean workbench, putting the laryngeal tissue specimen in a 6cm culture dish, firstly sucking the preservation solution by using a liquid transfer gun, washing the laryngeal tissue specimen for 5 seconds by using 95% alcohol, then washing the laryngeal tissue specimen twice by using DPBS buffer solution, removing surface bloodiness and impurities, separating the tissue by using an ophthalmic scissors, putting the tissue into a 1.5ml centrifuge tube, adding dispase, and soaking the tissue at 4 ℃ overnight;

transferring all tissue blocks and culture solution to a 15ml sterile centrifuge tube every other day, centrifuging at 1500rpm for 5 minutes, discarding supernatant, adding preheated pancreatin (2:1 ═ liquid: tissue) for digesting at 37 ℃ for 5 minutes, adding 10% DMEM high-sugar complete culture medium to stop digesting, repeatedly blowing and beating the tissue by using a gun head to form single cell suspension, pouring the cell suspension into a 100um screen mesh, filtering, and collecting filtrate; placing the tissues on the rest screens into a culture dish, repeatedly grinding the tissues by using the bottom end of a 5ml syringe, further grinding the tissues, adding 10% DMEM high-sugar complete culture medium, uniformly mixing the tissues, further filtering the tissues by using a 100-micron screen, centrifuging the tissues at 1000rpm for 5 minutes, discarding the supernatant, using a KBM culture medium to resuspend cells, paving the cells for culturing, preferably using a 12-hole plate, preferably adding 0.5ml of liquid into each hole, changing the liquid every other day, increasing the amount of the culture medium at the moment, wherein 1ml of the culture medium is added into each hole, the cells can grow out in 2-3 days generally, and the cells can be digested in about 14 days;

(2) preparation of competent bacteria (e.coli DH5 α) and plasmid transformation

Bmi1 was purchased from GeneCopoeia, designated pVTHM/Bmi 1, and contained a Green Fluorescent Protein (GFP) and puromycin resistance marker; melting competent bacteria on ice (avoiding repeated freeze thawing), uniformly mixing 50 microliter of the competent bacteria with a DNA sample (less than or equal to 10ng recommended), putting the mixture into a 1.5ml EP tube, placing the mixture in ice for about 30 minutes, then placing the mixture into a 42 ℃ water bath kettle for 45 seconds, then placing the mixture in ice for 1 to 2 minutes, adding 1ml of SOC culture medium preheated at 37 ℃, shaking and culturing the mixture for 1 hour at 37 ℃ after uniformly mixing; mixing the liquid uniformly in a sterile operating platform by using a gun head, taking about 20ul of bacteria liquid after the bacteria are fully suspended, dripping the bacteria on a screening agarose plate for ampicillin resistance, uniformly coating the bacteria on the surface by using a glass tube, inverting a cell dish, and placing the cell dish in an incubator at 37 ℃ for overnight;

(3) plasmid extraction

Selecting excellent monoclonal colonies, adding the excellent monoclonal colonies into 8ml of an ampicillin-resistant LB liquid culture medium, shaking and shaking uniformly at 37 ℃ for 8 hours, stopping when the liquid is turbid, adding 50ul of the excellent monoclonal colonies into 300ml of an ampicillin-resistant LB liquid culture medium, shaking overnight at 37 ℃, subpackaging the LB liquid culture medium into 50ml of centrifuge tubes the next day, centrifuging the mixture for 10 minutes at 4000g, precipitating bacteria, and discarding the supernatant; adding 4ml of Buffer R3 reagent added with RNase into each tube to resuspend the bacteria, blowing and dissolving; adding 4ml of Buffer L7 into each tube, covering the cover, reversing the centrifuge tube, mixing uniformly, and standing at room temperature for 5 min; adding 4ml of Buffer N3 into each tube, quickly reversing and uniformly mixing the mixture without rotating, centrifuging the mixture at 13000rpm for 10min at room temperature, transferring the supernatant into a 15ml of Buffer EQl balance filter column for slow filtration, adding 10mlW8 for washing the column for 2 times, discarding the filtrate, adding 5ml of elution Buffer E4, filtering, collecting the filtrate in a 15ml EP tube, adding 3.5ml of isopropanol for uniformly mixing the filtrate, centrifuging the mixture at 13000rpm4 ℃ for 30min, discarding the supernatant, adding 100ul of TE reagent for resuspending plasmids, and measuring the plasmid concentration;

(4) lentivirus packaging Bmi1 virus

In the present example, Lipofectamine 3 was usedPackaging the lentivirus by a 000 liposome method; will be 1.3X 10 h 24h before transfection⁶-1.5×10⁶Inoculating 293FT cells with good growth state into a 10cm cell culture dish, adding a DMEM complete culture medium preheated in advance and containing 10% fetal calf serum, and culturing until the transfection day, wherein when the cell density reaches about 50% confluence, virus packaging can be carried out; packaging according to Lipofectamine 3000 liposome method steps: add 500ul Opti-MEM medium to EP tube (tube A), and add 17ul Lipofectamine 3000 reagent and mix; add 500ul Opti-MEM media to EP tube (B tube) and then add 16 μ g plVTHM/Bmi1, 12 μ g psPAX2 (lentiviral packaging plasmid) and 6 μ g pMD2.G (lentiviral vector) gently mixed to avoid vortexing; adding the liquid in the tube A into the tube B, gently mixing uniformly, and incubating for 30 minutes at room temperature; adding the mixed liquid into a 293FT cell culture dish along the culture wall drop by drop, gently mixing uniformly, placing the mixed liquid into a cell culture box for 8 hours, taking out cells from the culture box, carefully absorbing a culture medium, and adding a preheated DMEM complete culture medium;

(5) viral collection and cell infection

Collecting virus supernatant after 72h, filtering virus liquid by using a 0.45um cell filter, directly infecting primary laryngeal epithelial cells by the filtered virus liquid, adding the virus liquid and a KBM-2 culture medium (1:1) into a cell dish, infecting for 5h each time, replacing the virus-free KBM-2 culture medium for culturing for 5h, and infecting again, wherein the infection is generally carried out for 3 times; the virus liquid can not be stored for too long at 4 ℃, and preferably does not exceed 24 h;

(6) flow cytometry for sorting GFP positive cells (laryngeal epithelial cells containing Bmi1 Gene)

Since the lentiviral vector plVTHM/Bmi1 carries both fluorescent protein GFP and Puromycin resistance markers, GFP positive laryngeal epithelial cells were obtained by sorting (FACS) the cells by flow cytometry; the present inventors named the obtained cells as human immortalized postapopore commisure Bmi1(the human immortalized poriferor commisure Bmi1, hPC-Bmi1) cells).

Example 2

Construction of pLVTHM/Bmi1 of overexpression Bmi1 plasmid and expression identification

The constructed Bmi1 plasmid contained a GFP fluorescent fragment and a puromycin fragment. The DNA fragment was amplified and extracted by E.coli, and was identified by colony PCR and DNA sequencing agarose gel electrophoresis to be completely identical to the designed sequence of pre-Bmi1 (see FIG. 1 and FIG. 2). The success of the construction of the recombinant plasmid pLVTHM/Bmi1 was suggested.

Example 3

hSG-Bmi1 and hV-Bmi1 cell effect verification test

1. Effect of Bmi1 overexpression on the degree of senescence of epithelial cells in the posterior region of the human laryngeal ring

After establishing primary cells of the human throat ring posterior region stably overexpressing Bmi1, the inventor of the application detects the cell senescence condition through a beta-galactosidase staining experiment, and the senescent cells can generate dark blue products under the catalysis of beta-galactosidase. Experiments show that the dark blue product of the epithelial primary cell after the 4 th generation ring is obviously increased compared with the epithelial primary cell after the 2 nd generation ring along with the increase of the passage number, the number of the epithelial primary cell senescent cells in the posterior region of the human laryngeal ring is increased along with the increase of the passage number, the blue product is obviously reduced after the Bmi1 is transfected, the cells in the posterior region of the transfected ring are continuously subjected to passage finding, the senescent cells are not obviously increased, and the Bmi1 gene is transfected to slow down the senescence of the epithelial cells in the posterior region of the human laryngeal ring and maintain the activity of the cells (see the attached figure 3).

2. Effect of Bmi1 overexpression on the proliferative Capacity of epithelial cells in the posterior region of the human laryngeal Loop

In order to study the effect of Bmi1 overexpression on proliferation capacity of primary epithelial cells in the posterior pharyngeal ring region, the inventors of the present application examined proliferation of primary epithelial cells in the posterior pharyngeal ring region of generation 2 and generation 4 without transfection of Bmi1 gene and generation 5 and generation 9 of hPC-Bmi1 cells by EdU flow, and as a result, it was suggested that fluorescence value (PE-a) of primary epithelial cells in the posterior pharyngeal ring region of generation 2 was 1541, primary PE-a value of primary epithelial cells in the posterior pharyngeal ring region of generation 4 was 668, which suggested that cell proliferation decreased significantly with increasing number of passages, and PE-a value of primary hPC-Bmi1 cells in generation 5 after infection of Bmi1 was 2038, which suggested that Bmi1 could promote epithelial cell growth in vitro in the posterior pharyngeal ring region, and that hPC-Bmi1 cells in generation 9 did not have significant difference in proliferation capacity compared with generation 5 (see fig. 4).

3. Effect of Bmi1 overexpression on the cell cycle of human posterior laryngeal Loop epithelial cells

In order to further research the possible mechanism of the Bmi1 for promoting cell proliferation, the inventor of the application detected the cell cycle distribution of 2 nd and 4 th generation epithelial primary cells of the posterior pharyngeal-laryngeal ring untransfected with the Bmi1 gene and 5 th and 9 th generation hPC-Bmi1 cells by a flow cell cycle detection kit. The results show that with the increase of the number of passages, the proportion of epithelial primary cells in the posterior region of the 4 th generation of the laryngo pharynx ring is increased compared with the proportion of cells in the prophase division (G1 phase) of the 2 nd generation of the cells, the proportion of cells in the DNA replication phase (S phase) is obviously reduced, and the proportion of cells in the anaphase division (G2 phase) is reduced. After the Bmi1 gene is transfected into primary epithelial cells in the rear region of the laryngopharynx, the proportion of G1 cells is reduced, the proportion of S cells is obviously increased, and the proportion of G2 cells is increased. This result suggests that Bmi1 promotes DNA synthesis and promotes differentiation of the epithelial cell cycle from the G1 phase to the S phase in the posterior region of the pharyngeal-laryngeal ring (see fig. 5).

4. Effect of Bmi1 overexpression on the ability to form tumors in vivo in epithelial cells of the posterior region of the human laryngeal Ring

Epithelial primary cells, hPC-Bmi1 cells and Hep-2 laryngeal cancer cells (positive control) in the posterior pharyngeal-laryngeal area were randomly inoculated to the left axilla (primary epithelial cells in the posterior pharyngeal-laryngeal area), the left groin (hPC-Bmi1 cells) and the right groin (Hep-2 cells) of 10 nude mice, respectively, and the number of the inoculated cells was 1 × 10⁶Each group comprised 5. After 5 days of inoculation, tumor formation was observed in the right groin injected with Hep-2 laryngeal cancer cells. On the 10 th day after inoculation, the tumor mass in the right inguinal region is obviously increased, and the formation of the tumor mass in the primary epithelial cells in the posterior region of the pharyngeal-laryngeal ring and the injection part of the hPC-Bmi1 cells is not seen. After 28 days after inoculation, the right inguinal region of each nude mouse was broken, and the nude mice were taken to a small animal MR imager for photographing, which revealed that only the right inguinal region (throat cancer Hep-2 cells) had mass generation in both groups of mice, and that the left axilla (epithelial primary cells in the posterior region of the pharyngeal-laryngeal ring) and the left inguinal region (hPC-Bmi1 cells) had no mass development. Nude mice were sacrificed and further dissected, and two groups of mice had tumor formation only in the right inguinal region and no tumor was seen in other regions, and further tumor was dissected and photographed (see fig. 6).

The results of the embodiment show that the inventor of the application establishes epithelial immortalized cells in the posterior region of the laryngeal ring by transfecting a polycomb gene family Bmi1 gene into primary epithelial cells in the posterior region of the laryngeal ring, and observes and researches the biological characteristics of the cells, finds that the Bmi1 gene can inhibit the primary epithelial cell senescence in the posterior region of the laryngeal ring, promotes the cell growth and proliferation, and the established cells can be continuously and stably passaged but cannot form tumor, thereby providing a stable and reliable in vitro basic model for researching the mechanism of benign and malignant diseases in the posterior region of the laryngeal ring.

Example 4

Research and test of using the immortalized human laryngeal ring posterior region cell to the laryngopharyngeal reflux pathogenesis

Throat reflux (1 arygopharyngeal reflux LPR) is closely related to throat voice diseases, in which pepsin (pepsin) plays an important role. Pepsin is a main invasive component in LPR, can damage laryngeal mucosa in a weak acid or non-acid environment of the laryngeal, generates inflammation, and becomes a hot point of research in the academia. Related studies on pepsin have been increasing year by year in recent years, however, studies on mechanisms of pepsin to damage the throat are still lacking, and specific inflammatory pathways and mechanisms thereof are not clear. To further elucidate the role of pepsin in inflammatory mucosal lesions, the inventors of the present application observed the role of DNA damage, Reactive Oxygen Species (ROS) production, mitochondrial damage, and inflammatory factor secretion in the mechanism of pepsin-induced injury of pharyngeal epithelial cells by administering hSG-Bmi1 cells and hV-Bmi1 cells pepsin stimulation at pH 7 to study the relevant molecular effects of chronic pepsin on exposure of pharyngeal mucosa.

1. Effect of pepsin on expression levels of proinflammatory cytokines, NLRP3, ROS and TXNIP in immortalized human laryngeal loop posterior region cells

To investigate the effect of pepsin on hPC-Bmi1 cells, the inventors used QPCR to measure the transcriptional levels of several pro-inflammatory cytokines (IL1 α, IL1 β, IL2, IL4, IL5, IL6, IL8, IL10, IL18) following pepsin stimulation. The results show that the transcriptional levels of IL1 α, IL1 β, IL6 and IL8 were significantly increased (P ═ 0.005, P <0.001, P ═ 0.001, P <0.001), with the amplitudes of IL1 β and IL8 being most pronounced, whereas IL2, IL4, IL5, IL10 and IL18 were not significantly different compared to the control group. Like IL1 β, caspase1 transcript levels were significantly increased (P <0.001) (see figure 7).

Subsequently, the present inventors performed western blot assays for Caspase1, IL1 β, and IL 18. The results showed that the levels of clear-IL 1 β and clear-Casepase 1 were also significantly increased, with the 1mg/mL pepsin group being more significant (see FIG. 8). There was no significant difference in the level of clear-IL 18 transcription and protein expression compared to the control group (P ═ 0.616). Quantitative fluorescence measurements showed a significant increase in relative fluorescence intensity of DHE in hPC-Bmi1 cells following pepsin stimulation, suggesting increased intracellular ROS levels (P <0.01) (see figure 9). Flow cytometry results showed a significant increase in ROS for mitochondrial pepsin stimulated cells (P <0.01) (see figure 10). In addition, intracellular TXNIP levels were significantly higher in the 1mg/ml pepsin group than in the control group (see FIG. 11).

2. Protective effects of three ROS inhibitors/scavengers on pepsin stimulation

The present inventors found that pepsin significantly increased the expression of inflammatory cytokines, NLRP3 and ROS in hPC-Bmi1 cells in a dose-dependent manner. To further investigate the relationship between them, the present inventors first analyzed the role of ROS in inflammatory cytokine release. The present inventors pre-incubated NADPH oxidase inhibitor oleandrin (APO)/DPI and ROS scavenger tempol prior to pepsin stimulation and observed the effects on inflammatory cytokines, intracellular and mitochondrial ROS levels. The results of the DHE fluorescence quantification experiment show that the relative fluorescence intensity of DHE of the preincubated Apocynin, Tempol and DPI groups is significantly lower than that of the pepsin group alone, with the most significant decrease in APO group. F86.17, P <0.01) (see fig. 12). The increase in mitochondrial ROS induced by pepsin stimulation was significantly reduced after pre-incubation with ROS inhibitor/scanner, with APO acting most strongly (F53.97, P <0.01) (see figure 13). Preincubation with different types of ROS inhibitor/scavenger also reduced the transcriptional levels of pepsin-elevated pro-inflammatory cytokines IL1 β (F6.827, P <0.01) and IL8 (F5.926, P <0.01) (see fig. 14). Therefore, APO was chosen as ROS inhibitor in subsequent experiments.

3. Role of ROS and NLPR3 in pepsin-induced inflammatory injury

To further elucidate the mechanism by which pepsin induces the NLRP3 inflammasome signaling pathway, the inventors analyzed NLRP3 activity in the presence of ROS scavenger APO and the NLRP3 inhibitor MCC 950. The present inventors first performed QPCR and western blots to investigate the effect of APO and MCC950 on the transcriptional and protein levels of TXNIP and NLRP3 inflammatory bodies and pro-inflammatory cytokines. In addition, LDH tests were also performed to investigate the effect of APO and MCC950 on pepsin-induced cytotoxicity.

The transcript levels of Caspase1 and IL1 β in 1mg/ml pepsin treated hPC-Bmi11 cells were significantly higher than in the control group (F ═ 72.71, P < 0.01; F ═ 117.66, P < 0.01). Pre-incubation with APO and MCC950 significantly reduced the transcript levels of IL1 β and Caspase1 compared to the 1mg/ml group. The level of IL18 transcript was not significantly different between groups (F0.57 and P0.65) (see figure 15).

Western blot results showed that the level of NLRP3, TXNIP, cleared-Caspase 1 and cleared-IL 1 beta was significantly increased and the level of pro-Caspase1/pro-IL1 beta was significantly decreased in pepsin-stimulated hPC-Bmi1 cells compared to the control group. There was no significant difference in the expression level of IL18 among the groups. APO (see figure 16) and MCC950 (see figure 17) can significantly inhibit the expression of NLRP3, TXNIP, cleared-Caspase 1 and cleared-IL 1 beta, and the expression level of IL18 between groups is not significantly different.

The LDH assay showed that 1mg/mL stimulated group hV-Bmi11 cells had a cytotoxicity of 36.62. + -. 3.90. Pre-incubation of APO (27.83 ± 5.74) and MCC950(29.23 ± 5.43) was effective in reducing pepsin cytotoxicity (F39.97, P <0.01) compared to the stimulated group (see figure 18).

After the hPC-Bmi1 cells are successfully constructed, the inventor of the application utilizes the hPC-Bmi1 cells to carry out mechanism research on the common disease of throat voice, namely throat regurgitation (Larynggorrygeal reflux LPR). The inventor of the application observes NLRP3 inflammasome, Reactive Oxygen Species (ROS) generation, mitochondrial damage and proinflammatory cytokine secretion by administering hPC-Bmi1 cells under the condition of pH 7 and stimulating LPR (lipoprotein lipase) as a main injury factor, and researches an action mechanism and potential related molecular effects of the pepsin-induced pharyngeal epithelial cell injury. The inventors of the present application found that with increased Pepsin concentration, ROS production by hPC-Bmi1 cells is increased, NLRP3 inflammasome expression is increased, expression of pro-inflammatory cytokines (IL1 β and IL8) is up-regulated, and throat epithelial cell damage is increased, suggesting that Pepsin continuously stimulates the larynx epithelium, resulting in the production of large amounts of ROS, which contribute to TXNIP binding to TRX for dissociation. As the complex dissociates, TXNIP binds to NLRP3, resulting in activation of NLRP3 inflammasome. Subsequent cleavage of caspase-1 results in the processing of pro-IL-1 β into its biologically active form, which then promotes the release of other inflammatory cytokines and recruits a large number of inflammatory cells, ultimately amplifying the inflammatory cascade, leading to the continued progression of chronic inflammation of the throat.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The immortalized human laryngeal ring posterior region cell is constructed by introducing Bmi1 gene into a human laryngeal ring posterior region source cell, and the immortalized human laryngeal ring posterior region cell has the capacity of in vitro unlimited proliferation or nearly unlimited proliferation.

2. The immortalized human posterior laryngeal ring cell according to claim 1, wherein the immortalized posterior laryngeal ring cell is constructed by transfecting a recombinant plasmid carrying the Bmi1 gene into a human posterior laryngeal ring source cell.

3. The immortalized human posterior laryngeal ring cell according to claim 2, wherein the recombinant plasmid carrying the Bmi1 gene is transfected into the human posterior laryngeal ring-derived cell as a lentiviral vector.

4. A method for constructing the immortalized human posterior laryngeal ring cell according to any one of claims 1 to 3, comprising the following steps:

5. The method for constructing immortalized human cells in the posterior laryngeal ring region according to claim 4, wherein the blood stain and impurities on the surface of the mucosal tissue in the posterior laryngeal ring in the whole laryngeal specimen are washed with 95% ethanol and DPBS buffer solution in sequence in step (1); after the preheated pancreatin is added, the volume ratio of liquid to tissue in the centrifugal tube is 2: 1.

6. The method for constructing immortalized human posterior laryngeal ring cell according to claim 4, wherein the mixing ratio of the competent bacteria to the DNA sample in the step (2) is: each 50. mu.l was mixed with not more than 10ng of DNA sample.

7. The method for constructing the immortalized human posterior laryngeal ring cell according to claim 4, wherein the step (3) is that the excellent monoclonal colony is selected and added into 8ml of LB liquid culture medium containing ampicillin resistance, shaking and shaking are carried out for 8 hours at 37 ℃, the liquid is stopped after turbidity appears, 50ul of the liquid is added into 300ml of LB liquid culture medium containing ampicillin resistance, shaking and shaking are carried out overnight at 37 ℃, the next day, the LB culture solution is subpackaged into 50ml centrifuge tubes, bacteria are precipitated by centrifugation for 10 minutes at 4000g, and the supernatant is discarded; adding 4ml of Buffer R3 reagent added with RNase into each tube to resuspend the bacteria, blowing and dissolving; adding 4ml of Buffer L7 into each tube, covering the cover, reversing the centrifuge tube, mixing uniformly, and standing at room temperature for 5 min; adding 4ml of Buffer N3 into each tube, quickly reversing and uniformly mixing the mixture without rotating, centrifuging the mixture at the room temperature of 13000rpm for 10min, transferring the supernatant into a 15ml of Buffer EQl balance filter column for slow filtration, adding 10mlW8 to wash the column for 2 times, discarding the filtrate, adding 5ml of elution Buffer E4, filtering the mixture, collecting the filtrate in a 15ml EP tube, adding 3.5ml of isopropanol for uniformly mixing the mixture, centrifuging the mixture at the temperature of 13000rpm for 30min at 4 ℃, discarding the supernatant, adding 100ul of TE reagent to resuspend the plasmids, and measuring the plasmid concentration.

8. The method for constructing immortalized human posterior laryngeal ring cell according to claim 4, wherein step (4) is carried out 24 hours before transfection at 1.3X 10⁶-1.5×10⁶The 293FT cells with good growth state are inoculated into a 10cm cell culture dish, DMEM complete culture medium containing 10% fetal calf serum which is preheated in advance is added for culture until the day of transfection, and when the cell density reaches 50% confluence, lentivirus packaging is carried out.

9. The method for constructing immortalized human posterior laryngeal ring cell according to claim 4 or 8, wherein the lentivirus packaging in step (4) is performed by: add 500ul Opti-MEM medium to EP tube (tube A), and add 17ul Lipofectamine 3000 reagent and mix; add 500ul Opti-MEM medium to EP tube (B tube) and then add 16 μ g plVTHM/Bmi1, 12 μ g psPAX2 and 6 μ g pMD2.G gently mixed to avoid vortexing; adding the liquid in the tube A into the tube B, gently mixing uniformly, and incubating for 30 minutes at room temperature; and adding the mixed liquid into a 293FT cell culture dish along the culture wall dropwise, mixing the mixed liquid gently and uniformly, putting the mixed liquid into a cell culture box for 8 hours, taking out the cells from the culture box, carefully sucking the culture medium, and adding preheated DMEM complete culture medium.

10. The method for constructing immortalized human posterior laryngeal ring cells according to claim 4, wherein in the step (5), the virus solution is filtered by a 0.45um cell filter, the filtered virus solution can directly infect primary human posterior laryngeal ring epithelial cells, the virus solution and the KBM-2 culture medium in a volume ratio of 1:1 are added into a cell dish, each infection lasts for 5h, the virus-free KBM-2 culture medium is replaced for 5h, and then the infection is carried out for 3 times.