CN114134118B

CN114134118B - Immortalized human laryngeal ring posterior region cell and construction method thereof

Info

Publication number: CN114134118B
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: 2023-05-09
Anticipated expiration: 2041-11-22
Also published as: CN114134118A

Abstract

The invention discloses an immortalized human laryngeal ring posterior region cell which is constructed by introducing a Bmi1 gene into a human laryngeal cavity ring posterior region source cell, wherein the immortalized human laryngeal ring posterior region cell has in-vitro unlimited proliferation or near unlimited proliferation capability. According to the invention, the Bmi1 gene is introduced into primary cells of the posterior region of the laryngeal ring, so that an immortalized human posterior region epithelial cell strain is constructed, and experimental researches show that the constructed immortalized human posterior region epithelial cell strain can slow down the aging of posterior region epithelial cells of the laryngeal ring, maintain the cell activity and provide a stable and reliable cell model for researching the pathogenic mechanism of benign and malignant diseases of the posterior region of the laryngeal ring. Meanwhile, the invention also discloses a method for constructing the cells in the posterior region of the immortalized human laryngeal ring, which has the advantages of convenient material taking, 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 derived from a posterior region of a human laryngeal cavity ring and a construction method thereof.

Background

With the rapid development of society, the living standard of people is continuously improved, and the medical demands are growing. Benign and malignant throat diseases are very common clinically, seriously affect the life quality and physical and mental health of patients, and become a health and social problem which cannot be ignored. However, diagnosis and treatment of benign and malignant diseases of throat are still quite tricky clinically, and the root of the disease is that the research on pathogenic mechanism is insufficient, so that the deep research on molecular pathological mechanism is particularly important. However, since no commercial laryngeal mucosa epithelial cell strain is purchased in the market at present, normal laryngeal mucosa epithelial primary cell culture in vitro is needed to establish laryngeal epithelial cells for research, and an in vitro model is provided for the research of the mechanism of action of different pathogenic factors in inflammatory and malignant diseases of the throat.

Because the throat mucosa belongs to the terminally differentiated tissue of the human body, the proliferation capacity of cells is poor, and the separated cells are fragile and are not easy to survive. And because of the limitation of the condition of the laryngeal cavity, the obtained tissue amount is small, and the tissue cannot be like other tissues of the stomach, the intestine, the mammary gland and the like, a large amount of tissues can be taken as culture raw materials, and if a large amount of subsequent experiments need to be carried out, the materials are obtained for many times, so that the repeatability of the experiments is possibly poor. Thus, there are many subjective and objective difficulties in the primary cell culture of laryngeal epithelium, which greatly limit the progress of the primary cell culture of laryngeal epithelium. Li Dongjun and Korea et al culture the fetal laryngeal epithelium by tissue block culture method, successfully cultivate the primary cells of laryngeal epithelium, but the culture mode is mostly obtained from the fetal laryngeal mucosa tissue of induced labor or dead labor, and the laryngeal mucosa is just differentiated and has high tissue activity, thus improving the success rate of culture. However, fetal laryngeal tissue has a limited source and a complex material taking process. 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 service life in vitro, and the cells which are just cultured can grow well, but when the primary cells are continuously passaged to about 4 generations, the number of aged cells is increased, the proliferation of the cells is slow, and the number of dead cells is increased. In addition to influencing cell proliferation, the appearance of senescent cells slows down the progress of experiments, and changes in numerous molecular biological genes can also occur, which influence the experimental results. Therefore, the inventor hopes to construct a laryngeal epithelium immortalized cell strain capable of stably and continuously passage, establish an ideal cell model and lay a foundation for researching pathogenesis of benign and malignant diseases of the throat.

The first two immortalized laryngeal epithelial cells were established by exogenous introduction of Human Papilloma Virus (HPV) E6/E7 or monkey virus 40 (SV 40) viral oncogenes. However, the direct effects of exogenous viral oncogenes and indirect effects of the production of aneuploidy cells can affect cellular behavior, such as limited differentiation capacity and abnormal changes similar to carcinoma in situ. Therefore, there is a great need to construct immortalized laryngeal epithelial cell lines and provide reliable and stable cell models for comprehensively discussing the study of the pathogenic mechanism of benign and malignant diseases of the throat.

The laryngeal cavity of a person is divided into different parts, including vocal cords, ventricular zones, posterior annuluses, epiglottis and subglottis. There are two different types of mucosal epithelium for different structural epithelium: the vocal cords, posterior annulus and hypopharyngeal epithelium are mainly non-keratinized stratified squamous epithelium, the cell main component is keratinocyte, the subglottal and epiglottic regions are overgrown from pseudo-stratified ciliated columnar epithelium to squamous epithelium, and the cell main component is columnar cells. The squamous epithelium is more closely arranged than the columnar epithelium, and can fall off the stratum corneum, so that the columnar epithelium has a resisting effect on invasive substances, is more easily damaged by exogenous invasive substances than the squamous epithelium in general, and is easy to cause subglottal stenosis. In addition, the different anatomical regions of the throat are different in terms of their molecular composition and tissue composition, and their resistance to exogenous stimuli (e.g., viruses, smoke and alcohol, gastrointestinal reflux) is also different. Such as papillomaviruses, are prone to invasion of the vocal and ventricular tracts, with less chance of papilloma in the posterior annulus. Whereas the posterior loop area of the laryngeal mucosa is more sensitive to laryngopharyngeal reflux (LPR) lesions due to the lack of carbonic anhydrase III components, and is more prone to posterior loop erythema and swelling. Therefore, in order to more comprehensively explore pathogenesis of different lesions of the throat and treatment prognosis conditions, not only is an immortalized laryngeal epithelial cell capable of stably growing, having good state and continuously passaging required to be constructed, a stable molecular platform is provided for cell biology research, but also actions of different pathogenic factors are required to be discussed in regions.

Disclosure of Invention

The invention aims to provide an immortalized cell which has in-vitro unlimited proliferation capacity or nearly unlimited proliferation capacity and is derived from the posterior region of the human laryngeal cavity ring; meanwhile, the invention also provides a construction method of the immortalized loop back region cell.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: an immortalized human laryngeal ring posterior region cell is constructed by introducing a Bmi1 gene into a human laryngeal cavity posterior region source cell, and the immortalized human laryngeal ring posterior region cell has in vitro unlimited proliferation or near unlimited proliferation capability.

The Bmi1 gene is an important component in a multi-comb gene family (a key epigenetic regulator), is involved in proliferation, differentiation and aging of stem cells, and plays an important role in embryonic development, tumorigenesis and maintenance of stem cells. The inventor of the application firstly tries to introduce a Bmi1 gene into primary cells of the posterior region epithelium of the human laryngeal ring, so as to construct an immortalized human posterior region epithelium cell strain, and experiments and researches find that the constructed immortalized human posterior region epithelium strain can slow down the aging of the posterior region epithelium of the laryngeal ring, maintain the cell activity and provide a stable and reliable cell model for researching the pathogenic mechanism of benign and malignant diseases of the posterior region of the laryngeal ring. The application constructs immortalized human laryngeal ring posterior region cells through Bmi1, can avoid unnecessary molecular changes caused by introducing exogenous HPV E6/E7 and SV40 virus oncogenes, and provides a more accurate cell model for later biological mechanism research.

The immortalized human laryngeal ring postregion cell is named as a human immortalized postring Bmi1 (the human immortalized posterior commissure Bmi, hPC-Bmi 1) cell.

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

As a preferred embodiment of the immortalized human laryngeal ring postregion cell, cheng Yong biochemical loop postregion cells are constructed by transfecting recombinant plasmids carrying Bmi1 genes into human laryngeal cavity loop postregion source cells.

As a preferred embodiment of the immortalized human laryngeal ring posterior region cells, the method for transfecting the recombinant plasmid carrying the Bmi1 gene into human laryngeal cavity posterior region-derived cells is a lentiviral vector.

The method for subculturing the immortalized human laryngeal ring posterior region cells (hPC-Bmi 1) comprises the following steps: when each colony grows to be full and has no gap, the old culture solution in the bottle can be passaged by sucking the old culture solution by a liquid-transferring gun, adding 0.5ml of a preheated mixed solution containing 0.25% trypsin and 0.02% EDTA (1:1), placing the mixed solution in a 37 ℃ incubator for digestion for 5 minutes, observing by an inverted microscope, when the cytoplasm is found to shrink and the cell gap is increased, lightly blowing the bottom cells repeatedly by using a 1ml gun head, at the moment, ensuring that the cells fall off in a piece, if the cells still adhere to the bottom, continuously placing the cells into the incubator at 37 ℃ for 1-2 minutes, adding 1ml of 10% DMEM high sugar complete medium to stop digestion, ensuring that the cells fall off to form a cell suspension, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, and adding KBM medium to resuspend the cell plates for culture.

In addition, another object of the present invention is to provide a method for constructing immortalized human laryngeal ring posterior region cells as described above, and to achieve this object, the technical scheme adopted by the present invention is as follows: a method of constructing cells of the posterior region of an immortalized human laryngeal ring, the method comprising the steps of:

(1) Two-step enzymatic digestion with tissue milling human laryngeal ring posterior region epithelial cell primary culture: taking a mucosa tissue in a posterior middle region of a whole-throat specimen, placing the mucosa tissue in a culture dish under a sterile environment, washing surface blood stains and impurities, separating the tissue, and placing the tissue in a centrifuge tube containing dispersive enzyme for soaking; transferring all tissues and culture fluid in the centrifuge tube into a sterile centrifuge tube after soaking for 24 hours, centrifuging, discarding supernatant, adding preheated pancreatin for digestion, adding 10% DMEM high sugar complete culture medium for stopping digestion, and repeatedly blowing the tissues with a gun head to form 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, discarding the supernatant, re-suspending the cell plates by using KBM culture medium for culturing until the cells grow out, and carrying out digestible passage for about 14 days;

(2) Preparation of competent bacteria and plasmid transformation: the adopted Bmi1 plasmid (plVTHM/Bmi 1) contains green fluorescent protein and puromycin resistance marker, the competent bacteria are melted on ice and then are mixed with DNA samples uniformly, the mixture is put into an EP tube, the mixture is put into a water bath kettle at the temperature of 40-45 ℃ for 30-60 seconds after being put into ice for 20-40 minutes, then the mixture is put into ice for 1-2 minutes, a preheated SOC culture medium is added, and shaking culture is carried out after uniform mixing; mixing liquid with gun head in a sterile operation table, fully suspending bacteria, dripping bacteria liquid into ampicillin resistance screening agarose plate, coating uniformly, inverting cell dish, and placing in incubator overnight;

(3) Plasmid extraction: selecting excellent monoclonal bacterial colony, adding the excellent monoclonal bacterial colony into an ampicillin-resistant LB liquid culture medium, shaking uniformly until turbidity appears in the liquid, stopping, adding the turbid liquid into the ampicillin-resistant LB liquid culture medium, shaking overnight, sub-packaging the LB culture solution into a centrifuge tube, centrifuging to precipitate bacteria, and discarding the supernatant; adding Buffer R3 reagent containing RNase into each tube to resuspend bacteria, and blowing to dissolve; adding Buffer L7 into each tube, covering a cover, reversing the centrifuge tube, uniformly mixing, and standing at room temperature; adding Buffer N3 into each tube, rapidly reversing and uniformly mixing, centrifuging at room temperature, transferring supernatant into a Buffer EQl balance filter column for filtering, washing the column, discarding filtrate, adding elution Buffer, filtering, collecting filtrate into an EP tube, adding isopropanol, uniformly mixing, centrifuging, discarding supernatant, adding TE reagent to resuspend plasmid, and measuring plasmid concentration;

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

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

(6) Flow cytometry sorting human laryngeal ring posterior region epithelial cells containing Bmi1 gene: and (3) sorting the cells by a flow cytometry to obtain GFP positive human laryngeal ring posterior region epithelial cells, and obtaining the immortalized human laryngeal ring posterior region cells.

As a preferred embodiment of the method for constructing cells in the posterior region of an immortalized human laryngeal ring, in the step (1), the blood stain and impurities on the surface of the mucosa tissue in the posterior region of the ring in the whole-throat specimen are washed by 95% alcohol and DPBS buffer in sequence; the volume ratio of liquid to tissue in the centrifuge tube after adding the preheated pancreatin is 2:1.

As a preferred embodiment of the method for constructing cells of the posterior region of an immortalized human laryngeal ring according to the present invention, the mixing ratio of competent bacteria to DNA sample in the step (2) is: mix with no more than 10ng of DNA sample per 50. Mu.l.

As a preferred implementation mode of the method for constructing the immortalized human laryngeal ring posterior region cells, the step (3) is to select excellent monoclonal colonies to be added into 8ml of ampicillin-resistant LB liquid medium, shake and shake the liquid uniformly for 8 hours at 37 ℃, stop after turbidity appears, take 50ul to be added into 300ml of ampicillin-resistant LB liquid medium, shake and shake the liquid for overnight at 37 ℃, split-charge the LB liquid into 50ml centrifuge tubes the next day, centrifuge 4000g for 10 minutes to precipitate bacteria, and discard the supernatant; 4ml of Buffer R3 reagent added with RNase is added into each tube to resuspend the bacteria, and the bacteria are blown and dissolved; adding 4ml Buffer L7 into each tube, covering a cover, reversing the centrifuge tube, uniformly mixing, and standing at room temperature for 5min; 4ml Buffer N3 was added to each tube, the mixture was rapidly inverted and homogenized, without swirling, centrifuged at 13000rpm for 10min at room temperature, the supernatant was transferred to a 15ml Buffer EQl equilibrium filter column and slowly filtered, 10mlW wash columns were added for 2 times, the filtrate was discarded, 5ml elution Buffer E4 was added, filtration was performed, the filtrate was collected into 15ml EP tubes, 3.5ml isopropanol was added and homogenized, centrifuged at 13000rpm for 30min at 4 ℃, the supernatant was discarded, 100ul TE reagent was added to resuspend the plasmid, and the plasmid concentration was measured.

As a preferred embodiment of the method for constructing cells of the posterior region of an immortalized human laryngeal ring according to the present invention, the step (4) will be 1.3X10 s 24 hours before transfection ⁶ -1.5×10 ⁶ 293FT cells with good growth state are inoculated into a cell culture dish with 10cm, DMEM complete culture medium containing 10% fetal calf serum is added for culture in advance until the day of transfection, and when the cell density reaches 50% confluence, slow virus packaging is carried out.

As a preferred embodiment of the method for constructing immortalized human laryngeal ring posterior region cells of the present invention, the method for packaging lentiviruses in the step (4) is as follows: 500ul of Opti-MEM medium was added to EP tube (tube A) and mixed with 17ul Lipofectamine 3000 reagent; 500ul of Opti-MEM medium was added to the EP tube (tube B), followed by 16. Mu.g of plVTHM/Bmi1, 12. Mu.g of psPAX2 and 6. Mu.g of pMD2.G were gently mixed to avoid negotiation; adding the liquid in the pipe A into the pipe B, gently mixing, and incubating for 30 minutes at room temperature; the mixed liquid was added dropwise to a 293FT cell culture dish along the culture wall, gently mixed, and after 8 hours in a cell incubator, the cells were removed from the incubator, the medium was carefully aspirated, and the preheated DMEM complete medium was added.

As a preferred implementation mode of the method for constructing the immortalized human laryngeal ring posterior region cells, in the step (5), a 0.45um cell filter is used for filtering virus liquid, the filtered virus liquid can directly infect primary human laryngeal ring posterior region epithelial cells, the virus liquid and KBM-2 culture medium with the volume ratio of 1:1 are added into a cell dish, each time of infection is carried out for 5 hours, and the culture medium is replaced by a virus-free KBM-2 culture medium 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 primary cell of the human laryngeal ring posterior region epithelium, so that an immortalized human laryngeal ring posterior region epithelium cell strain is constructed, 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 epithelium, keep the cell activity, and provide a stable and reliable cell model for the study of the pathogenic mechanism of benign and malignant diseases of the laryngeal ring posterior region. The construction method of 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 the pLVTHM/Bmi1 plasmid;

FIG. 2 is a diagram showing the result of sequencing pLVTHM/Bmi1 plasmid;

FIG. 3 is a graph showing the comparison of human laryngeal mask zona epithelial primary cells with the aging degree of hPC-Bmi1 cells;

FIG. 4 is a graph showing the comparison of human laryngeal mask zona epithelial primary cells with the proliferation potency of hPC-Bmi1 cells;

FIG. 5 is a graph showing cell cycle comparison of human laryngeal mask zona epithelial primary cells with hPC-Bmi1 cells;

FIG. 6 is a graph showing comparison of human laryngeal mask zona epithelial primary cells with hPC-Bmi1 in the tumor formation;

FIG. 7 is a graph showing the effect of Pepsin on IL1 alpha, IL1 beta, IL6, IL8 and caspase1 levels;

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

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

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

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 showing the effects 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 Caspase1, IL 1. Beta. And IL18 transcript levels in Pepsin-treated hPC-Bmi11 cells;

FIG. 16 is a graph comparing the effect of APO on NLRP3, TXNIP, cleaved-Caspase1, clear-IL 1 beta, IL 18;

FIG. 17 is a graph showing the effect of MCC950 on NLRP3, TXNIP, cleaved-Caspase1, clear-IL 1 beta and IL 18;

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

Detailed Description

For better illustrating the objects, technical solutions and advantageous effects of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1

The invention relates to an embodiment of immortalized human laryngeal ring posterior region cells, which is constructed by the following method:

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

cutting the mucosal tissue under the glottis and the ventricular zone in the whole-throat specimen by using a sterile surgical blade, taking the mucosal tissue to the deep layer of the throat as much as possible, putting the mucosal tissue into a centrifuge tube with the size of 0.5-1cm, and immediately placing the mucosal tissue into a centrifuge tube with the size of 1.5ml, wherein the centrifuge tube is filled with a prepared tissue storage solution (DPBS buffer solution and three antibodies), preserving the mucosal tissue at the low temperature of 4 ℃, and transporting the mucosal tissue to a laboratory for treatment as soon as possible, and preferably not more than 24 hours; taking out a throat tissue specimen in a sterile ultra-clean workbench, putting the throat tissue specimen into a 6cm culture dish, firstly sucking up the preservation solution by a pipetting gun, washing for 5 seconds by 95% alcohol, washing twice by using DPBS buffer solution, removing surface blood stains and impurities, separating the tissue by using an ophthalmic scissors, putting the tissue specimen into a 1.5ml centrifuge tube, adding dispersion enzyme, and soaking the tissue specimen at 4 ℃ overnight;

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

(2) Preparation of competent bacteria (E.coli DH 5. Alpha.) and plasmid transformation

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

(3) Plasmid extraction

Selecting excellent monoclonal colonies, adding the excellent monoclonal colonies into 8ml of ampicillin-resistant LB liquid medium, shaking uniformly at 37 ℃ for 8 hours, stopping after turbidity appears on the liquid, adding 50ul of the liquid into 300ml of ampicillin-resistant LB liquid medium, shaking overnight at 37 ℃, subpackaging the LB liquid into 50ml centrifuge tubes the next day, centrifuging 4000g for 10 minutes to precipitate bacteria, and discarding the supernatant; 4ml of Buffer R3 reagent added with RNase is added into each tube to resuspend the bacteria, and the bacteria are blown and dissolved; adding 4ml Buffer L7 into each tube, covering a cover, reversing the centrifuge tube, uniformly mixing, and standing at room temperature for 5min; adding 4ml Buffer N3 into each tube, rapidly reversing and uniformly mixing, rotating, centrifuging at 13000rpm at room temperature for 10min, transferring the supernatant into 15ml Buffer EQl equilibrium filter column, slowly filtering, adding 10mlW to wash the column for 2 times, discarding the filtrate, adding 5ml elution Buffer E4, filtering, collecting the filtrate, adding 3.5ml isopropanol into 15ml EP tube, uniformly mixing, centrifuging at 13000rpm at 4 ℃ for 30min, discarding the supernatant, adding 100ul TE reagent to resuspend plasmid, and measuring plasmid concentration;

(4) Lentivirus package Bmi1 virus

In the embodiment, a Lipofectamine 3000 liposome method is adopted for slow virus packaging; 24h before transfection will be 1.3X10 ⁶ -1.5×10 ⁶ The 293FT cells with good growth state are inoculated into a cell culture dish with the length of 10cm, DMEM complete culture medium containing 10% of fetal calf serum is added for culture in advance until the day of transfection, and when the cell density reaches about 50% of confluence, virus packaging can be carried out; packaging according to Lipofectamine 3000 liposome method: 500ul of Opti-MEM medium was added to EP tube (tube A) and mixed with 17ul Lipofectamine 3000 reagent; 500ul of Opti-MEM medium was added to EP tube (B tube), followed by gentle mixing with 16. Mu.g of plVTHM/Bmi1, 12. Mu.g of psPAX2 (lentiviral packaging plasmid) and 6. Mu.g of pMD2.G (lentiviral vector) to avoid swirling; adding the liquid in the pipe A into the pipe B, gently mixing, and incubating for 30 minutes at room temperature; adding the mixed liquid into a 293FT cell culture dish drop by drop along a culture wall, gently mixing, putting into a cell culture box for 8 hours, taking out cells from the culture box, carefully sucking the culture medium, and adding a preheated DMEM complete culture medium;

(5) Virus 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 KBM-2 culture medium (1:1) into a cell dish, carrying out infection for 5h each time, changing the culture medium into a virus-free KBM-2 culture medium, culturing for 5h, carrying out infection again, and carrying out common infection for 3 times; the virus solution cannot be left for too long at 4 ℃, preferably not more than 24 hours;

(6) Flow cytometry sorting GFP positive cells (laryngeal epithelial cells containing the Bmi1 gene)

Since lentiviral vector plVTHM/Bmi1 carries fluorescent protein GFP and Puromycin resistance marker simultaneously, GFP positive laryngeal epithelial cells are obtained by sorting cells by flow cytometry (Fluorescence Activated Cell Sorter, FACS); the inventors named the obtained cells as human immortalized post-loop Bmi1 (the human immortalized posterior commissure Bmi1, hPC-Bmi 1) cells.

Example 2

Construction and expression identification of pLVTHM/Bmi1 over-expressing Bmi1 plasmid

The constructed Bmi1 plasmid contains GFP fluorescent fragment and puromycin fragment. Amplification and extraction were performed using E.coli, and the identification was performed by colony PCR and DNA sequencing agarose gel electrophoresis to completely agree with the pre-Bmi1 design sequence (see FIGS. 1 and 2). The recombinant plasmid pLVTHM/Bmi1 was suggested to be constructed successfully.

Example 3

hSG-Bmi1 and hV-Bmi1 cell effect validation assays

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

After establishing a primary cell of a human laryngeal ring posterior region stably over-expressing Bmi1, the inventor detects cell aging through a beta-galactosidase staining experiment, and the aging cell can generate a deep blue product under the catalysis of beta-galactosidase. Experiments show that the dark blue product of the epithelial primary cells after the 4 th generation ring is obviously increased compared with the epithelial primary cells after the 2 nd generation ring, the number of the epithelial primary cells in the posterior region of the human laryngeal ring is obviously increased along with the increase of the passage times, the blue product is obviously reduced after the Bmi1 is transfected, the cells in the posterior region of the ring after the transfection are continuously passaged, the increase of the aged cells is not obvious, and the transfected Bmi1 gene is proved to slow down the aging of the epithelial cells in the posterior region of the human laryngeal ring and maintain the cell activity (see the figure 3).

2. Effect of Bmi1 overexpression on epithelial cell proliferation Capacity in the posterior region of the human laryngeal mask

In order to study the influence of the overexpression of Bmi1 on the proliferation capacity of primary cells in the posterior region of the throat ring, the inventor detects the proliferation condition of primary cells in the posterior region of the throat ring, which are not transfected with Bmi1 genes, of the generation 2 and the generation 4, and the proliferation condition of primary cells in the posterior region of the throat ring, which are 5 th and 9 th generation hPC-Bmi1, through EdU flow, and the result shows that the proliferation fluorescence value (PE-A) of primary cells in the posterior region of the throat ring of the generation 2 is 1541, the PE-A value of primary cells in the posterior region of the throat ring of the generation 4 is 668, and the PE-A value of primary cells in the posterior region of the throat ring of the generation 4 shows that the proliferation capacity of cells is remarkably reduced along with the increase of passage, and the PE-A value of cells in the 5 th generation hPC-Bmi1 is 2038, so that the Bmi1 can promote the in vitro growth of the epithelial cells in the posterior region of the throat ring, and the proliferation capacity of the generation 9 hPC-Bmi1 cells is not obviously different from the generation 5 (see shown in fig. 4).

3. Effect of Bmi1 overexpression on epithelial cell cycle in the posterior region of the human laryngeal mask

To further investigate the possible mechanism by which Bmi1 promotes cell proliferation, the inventors examined the cell cycle distribution of the 2 nd and 4 th generation untransfected Bmi1 gene, the posterior pharyngeal ring epithelial primary cells, and the 5 th and 9 th generation hPC-Bmi1 cells, by a flow cell cycle assay kit. The results show that with the increase of the passage times, the primary cell ratio of the epithelial of the posterior region of the 4 th generation throat ring is increased compared with the primary cell ratio of the 2 nd generation in the early stage of division (G1 phase), the cell ratio of the DNA replication stage (S phase) is obviously reduced, and the cell ratio of the posterior stage of division (G2 phase) is reduced. After the Bmi1 gene is transfected into primary cells of epithelium in the posterior region of the throat ring, the proportion of cells in the G1 phase is reduced, the proportion of cells in the S phase is obviously increased, and the proportion of cells in the G2 phase is increased. This result suggests that Bmi1 can promote DNA synthesis and promote differentiation of the epithelial cell cycle from G1 phase to S phase in the posterior pharyngeal wall (see fig. 5).

4. Effect of Bmi1 overexpression on in vivo tumorigenicity of epithelial cells in the posterior region of the human laryngeal ring

The primary cells of the epithelium of the posterior region of the throat ring, hPC-Bmi1 cells and Hep-2 laryngeal carcinoma cells (positive control) with good growth state are randomly inoculated to the left armpit (primary cells of the epithelium of the posterior region of the throat ring), left inguinal (hPC-Bmi 1 cells) and right inguinal (Hep-2 cells of the laryngeal carcinoma) of 10 nude mice respectively, and the number of inoculated cells is 1 multiplied by 10 ⁶ Each group comprised 5. After day 5 inoculation, the right inguinal visible tumor formation was observed with Hep-2 laryngeal cancer cell injection. On day 10 post inoculation, the mass in the right inguinal region increased significantly and the primary cells in the posterior region of the pharyngeal wall and the injection site of hPC-Bmi1 cells were not seen. On day 28 post inoculation, the tumor was visible in the right inguinal region of individual nude mice, which were sent to a small animal MR imager for imaging, showing that two groups of mice had tumor formation only in the right inguinal region (laryngeal carcinoma Hep-2 cells), and no tumor development was seen in the left underarm (posterior pharyngeal region epithelial primary cells) and left inguinal region (hPC-Bmi 1 cells). Nude mice were sacrificed and the mice were further dissected, and two groups of mice had only the right inguinal region with the tumor formed, and other regions were not seen with the tumor, and further tumor was dissected for photographing (see fig. 6).

As shown by the results of the example, the inventor of the application establishes the laryngeal ring posterior region epithelial immortalized cells by transfecting the multi-comb gene family Bmi1 genes into the human laryngeal ring posterior region primary epithelial cells and performs observation and research on the biological characteristics of the laryngeal ring posterior region epithelial immortalized cells, and discovers that the Bmi1 genes can inhibit the aging of the posterior region epithelial primary cells, promote the growth and proliferation of the cells, and the constructed cells can continuously and stably pass but cannot become tumors, so that a stable and reliable in vitro basic model is provided for discussing the mechanism research of the laryngeal ring posterior region benign and malignant diseases.

Example 4

Research and test of pathogenic mechanism of laryngopharynx reflux by using cells in posterior region of immortalized human laryngeal ring

Laryngopharyngeal reflux (1 aryngopharyngeal reflux LPR) is closely related to laryngopharyngeal voice diseases, where pepsin (pepsin) plays an important role. Pepsin is a main invasive component in LPR, and can damage throat mucosa in weak or non-acidic environment of throat, and cause inflammation, which becomes a hot spot for research in the academic community. In recent years, the related research of pepsin is increasing year by year, however, the research on the mechanism of the damage of pepsin to the throat part is still lacking, and the specific inflammatory pathway and mechanism are not clear. To further elucidate the role of pepsin in inflammatory mucosal lesions, the present inventors examined the molecular effects associated with chronic pepsin exposure to the throat mucosa by administering hSG-Bmi1 cells and hV-Bmi1 cells pepsin stimulation at ph=7, observing the role of DNA damage, reactive Oxygen Species (ROS) production, mitochondrial damage, inflammatory factor secretion in the mechanisms of throat epithelial cell damage caused by pepsin.

1. Effects of pepsin on expression levels of immortalized human laryngeal ring posterior region cell pro-inflammatory cytokines, NLRP3, ROS and TXNIP

To study the effect of pepsin on hPC-Bmi1 cells, the inventors used QPCR to detect the transcript levels of several pro-inflammatory cytokines (il1α, il1β, IL2, IL4, IL5, IL6, IL8, IL10, IL 18) following pepsin stimulation. The results show that the transcript levels of il1α, il1β, IL6 and IL8 are significantly elevated (p=0.005, P <0.001, p=0.001, P < 0.001), with the amplitude of il1β and IL8 being most pronounced, whereas IL2, IL4, IL5, IL10 and IL18 are not significantly different compared to the control group. Like IL1 beta, caspase1 transcript levels increased significantly (P < 0.001) (see figure 7).

Subsequently, the inventors performed Western blot detection of Caspase1, IL 1. Beta. And IL 18. The results showed that the levels of clear-IL 1 beta and clear-Casepase 1 were also significantly elevated, with the 1mg/mL pepsin group being more pronounced (see FIG. 8). Compared to the control group, there was no significant difference in the level of clear-IL 18 transcription and protein expression (p=0.616). Fluorescent quantitation showed a significant increase in the relative fluorescence intensity of DHE in hPC-Bmi1 cells following pepsin stimulation, suggesting an elevated intracellular ROS level (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, the 1mg/ml pepsin group had significantly higher intracellular TXNIP levels than the control group (see FIG. 11).

2. Protection of pepsin stimulation by three ROS inhibitors/scavengers

The inventors found that pepsin significantly increased the expression of inflammatory cytokines, NLRP3 and ROS in a dose-dependent manner in hPC-Bmi1 cells. To further investigate their relationship, the inventors first analyzed the role of ROS in inflammatory cytokine release. The inventors pre-cultured NADPH oxidase inhibitor oleandrin (APO)/DPI and ROS scavenger tempol prior to pepsin stimulation and observed effects on inflammatory cytokines, intracellular and mitochondrial ROS levels. The DHE fluorescence quantification experiment results show that the DHE relative fluorescence intensity of the pre-incubated Apocynin, tempol and DPI groups was significantly lower than that of the pepsin alone group, with the APO group reduction most significant. F=86.17, p < 0.01) (see fig. 12). Following pre-incubation with ROS inhibitor/scanner, pepsin stimulation induced a significant decrease in mitochondrial ROS increase, with the strongest effect of APO (f=53.97, p < 0.01) (see fig. 13). Preincubation with different types of ROS inhibitors/scavengers also reduced the transcript levels of pepsin-elevated pro-inflammatory cytokines il1β (f=6.827, p < 0.01) and IL8 (f=5.926, p < 0.01) (see fig. 14). Thus, APO was chosen as the ROS inhibitor in subsequent experiments.

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

To further elucidate the mechanism of pepsin induction of the NLRP3 inflammatory body signaling pathway, the inventors analyzed NLRP3 activity in the presence of ROS scavenger APO and the NLRP3 inhibitor MCC 950. The inventors of the present application first performed QPCR and western blotting to investigate the effect of APO and MCC950 on transcription 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 transcription levels of Caspase1 and IL1 beta 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). APO and MCC950 preincubation significantly reduced the transcript levels of il1β and Caspase1 compared to the 1mg/ml group. There was no significant difference between the groups of IL18 transcript levels (f=0.57, p=0.65) (see fig. 15).

Western blotting results showed that the NLRP3, TXNIP, cleaved-Caspase1 and clear-IL 1 beta levels of pepsin stimulated hPC-Bmi1 cells were significantly increased and the pro-Caspase1/pro-IL1 beta levels were significantly decreased compared to the control group. There was no significant difference in the expression level of IL18 from each group. APO (see fig. 16) and MCC950 (see fig. 17) significantly inhibited expression of NLRP3, TXNIP, cleaved-Caspase1 and clear-il1β, with no significant difference in IL18 expression levels between groups.

LDH assay showed cytotoxicity of 1mg/mL stimulated hV-Bmi11 cells of 36.62.+ -. 3.90. Preincubation of APO (27.83±5.74) and MCC950 (29.23±5.43) effectively reduced pepsin cytotoxicity (f=39.97, p < 0.01) compared to the stimulated group (see fig. 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 common throat and voice diseases, namely laryngopharynx reflux (Laryngopharyngeal reflux LPR). The inventor of the present application observes NLRP3 inflammatory corpuscles, reactive Oxygen Species (ROS) generation, mitochondrial injury and pro-inflammatory cytokine secretion by stimulating the LPR main injury factor pepsin of the hPC-Bmi1 cells under the pH=7 environment, and researches the action mechanism of pharyngeal epithelial cell injury caused by pepsin and potential related molecular effects. The inventors found that as Pepsin concentration increases, hPC-Bmi1 cell ROS production increases, NLRP3 inflammatory small body expression increases, pro-inflammatory cytokines (il1β and IL 8) expression up-regulates, and throat epithelial cell damage increases, suggesting that Pepsin continuously stimulates the throat epithelium, resulting in the production of a large amount of ROS, which contributes to TXNIP binding to TRX for dissociation. As the complex dissociates, TXNIP binds to NLRP3, resulting in activation of the NLRP3 inflammatory bodies. Subsequent cleavage of caspase-1 results in processing of pro-IL-1β into its bioactive 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 throat inflammation.

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

1. An immortalized human laryngeal ring posterior region cell, which is characterized in that the immortalized human laryngeal ring posterior region cell is constructed by introducing a Bmi1 gene into a human laryngeal cavity posterior region source cell, and the immortalized human laryngeal ring posterior region cell has in vitro unlimited proliferation or near unlimited proliferation capacity;

constructing Cheng Yong biochemical loop back region cells by transfecting recombinant plasmids carrying Bmi1 genes into human laryngeal cavity loop back region source cells, wherein the method for transfecting the recombinant plasmids carrying the Bmi1 genes into human laryngeal cavity loop back region source cells is a lentiviral vector;

the construction method of the immortalized human laryngeal ring posterior region cell comprises the following steps:

(2) Preparation of competent bacteria and plasmid transformation: the adopted Bmi1 plasmid plVTHM/Bmi1 contains green fluorescent protein and puromycin resistance marker, the competent bacteria are melted on ice and then are mixed with DNA samples uniformly, the mixture is put into an EP tube, the mixture is put into a water bath kettle at the temperature of 40-45 ℃ for 20-60 seconds after being put into ice for 20-60 minutes, then the mixture is put into ice for 1-2 minutes, a preheated SOC culture medium is added, and shaking culture is carried out after uniform mixing; mixing liquid with gun head in a sterile operation table, fully suspending bacteria, dripping bacteria liquid into ampicillin resistance screening agarose plate, coating uniformly, inverting cell dish, and placing in incubator overnight;

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

2. A method of constructing an immortalized human laryngeal ring posterior region cell according to claim 1, comprising the steps of:

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

3. The method for constructing cells in the posterior region of an immortalized human laryngeal ring according to claim 2, wherein in the step (1), the blood and impurities on the surface of the mucosa tissue in the posterior region of the laryngeal ring in the whole-larynx sample are washed with 95% alcohol and DPBS buffer in sequence; the volume ratio of liquid to tissue in the centrifuge tube after adding the preheated pancreatin is 2:1.

4. The method of constructing cells of the posterior region of an immortalized human laryngeal ring according to claim 2, wherein the ratio of competent bacteria to DNA sample in step (2) is: mix with no more than 10ng of DNA sample per 50. Mu.l.

5. The method for constructing cells in the posterior region of immortalized human laryngeal ring according to claim 2, wherein the step (3) is to select excellent monoclonal colonies, add them into 8ml of ampicillin-resistant LB liquid medium, shake them uniformly for 8 hours at 37 ℃, stop the shaking after the turbidity appears, add 50ul of them into 300ml of ampicillin-resistant LB liquid medium, shake them for overnight at 37 ℃, split-fill the LB liquid into 50ml centrifuge tubes the next day, centrifuge 4000g for 10 minutes to precipitate bacteria, discard the supernatant; 4ml of Buffer R3 reagent added with RNase is added into each tube to resuspend the bacteria, and the bacteria are blown and dissolved; adding 4ml Buffer L7 into each tube, covering a cover, reversing the centrifuge tube, uniformly mixing, and standing at room temperature for 5min; 4ml Buffer N3 was added to each tube, the mixture was rapidly inverted and homogenized, without swirling, centrifuged at 13000rpm for 10min at room temperature, the supernatant was transferred to a 15ml Buffer EQl equilibrium filter column and slowly filtered, 10mlW wash columns were added for 2 times, the filtrate was discarded, 5ml elution Buffer E4 was added, filtration was performed, the filtrate was collected into 15ml EP tubes, 3.5ml isopropanol was added and homogenized, centrifuged at 13000rpm for 30min at 4 ℃, the supernatant was discarded, 100ul TE reagent was added to resuspend the plasmid, and the plasmid concentration was measured.

6. The method of constructing cells of the posterior region of an immortalized human laryngeal ring according to claim 2, wherein the step (4) is performed at 1.3X10 s 24h before transfection ⁶ -1.5×10 ⁶ 293FT cells with good growth state are inoculated into a cell culture dish with 10cm, DMEM complete culture medium containing 10% fetal calf serum is added for culture in advance until the day of transfection, and when the cell density reaches 50% confluence, slow virus packaging is carried out.

7. The method of constructing immortalized human laryngeal ring posterior region cells according to claim 2 or 6, wherein the method of packaging the lentivirus of step (4) comprises: 500ul of Opti-MEM medium is added into the EP tube of the tube A, and 17ul Lipofectamine 3000 reagent is added for mixing; 500ul of Opti-MEM medium was added to the EP tube of the B tube, followed by 16. Mu.g of plVTHM/Bmi1, 12. Mu.g of psPAX2 and 6. Mu.g of pMD2.G were gently mixed to avoid swirling; adding the liquid in the pipe A into the pipe B, gently mixing, and incubating for 30 minutes at room temperature; the mixed liquid was added dropwise to a 293FT cell culture dish along the culture wall, gently mixed, and after 8 hours in a cell incubator, the cells were removed from the incubator, the medium was carefully aspirated, and the preheated DMEM complete medium was added.

8. The method for constructing cells in the posterior region of an immortalized human laryngeal ring according to claim 2, wherein in the step (5), a 0.45um cell filter is used to filter the virus solution, the filtered virus solution can directly infect epithelial cells in the posterior region of the primary human laryngeal ring, the virus solution and KBM-2 medium with the volume ratio of 1:1 are added into a cell dish, each time of infection is carried out for 5 hours, and the culture is replaced by the culture medium without virus KBM-2 for 5 hours, and then the infection is carried out for 3 times.