CN115433718A

CN115433718A - Immortalized oral epithelial cells and preparation method and application thereof

Info

Publication number: CN115433718A
Application number: CN202211085437.0A
Authority: CN
Inventors: 朱京山
Original assignee: Beijing H&j Novomed Co ltd
Current assignee: Beijing H&j Novomed Co ltd
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2022-12-06

Abstract

The application relates to the technical field of immortalized cells, and particularly discloses an immortalized oral epithelial cell and a preparation method and application thereof. The application discloses a preparation method of immortalized oral epithelial cells, which specifically comprises the following steps: and (3) carrying out cell infection on the oral epithelial cells by using SV40LT over-expressed lentivirus, and obtaining the immortalized oral epithelial cells through cell screening, identification and passage. The preparation method provided by the application can be used for quickly obtaining the immortalized oral epithelial cells, and the immortalized oral epithelial cells have higher cell density and cell activity.

Description

Immortalized oral epithelial cells and preparation method and application thereof

Technical Field

The application relates to the technical field of immortalized cells, in particular to an immortalized oral epithelial cell and a preparation method and application thereof.

Background

The anti-perinuclear factor is one of the related autoantibodies of the rheumatoid arthritis, and when the anti-perinuclear factor antibody shows a positive result, the possibility of suffering from the rheumatoid arthritis is higher. The oral epithelial cells are used as target antigens of the anti-perinuclear factor antibody and can be used for detecting the anti-perinuclear factor antibody, thereby providing early specific diagnosis indexes for the rheumatoid arthritis and having good prompting function on the judgment and prognosis of diseases. Therefore, the oral epithelial cells have important significance for the research of rheumatoid arthritis.

The oral epithelium is required to be used for the separation and culture of the oral epithelium every time, the preparation procedure is complicated, time and labor are wasted, and the difference exists among cells of each batch, so that the stability of the physiological indexes of the cells cannot be maintained; in addition, the cell state is not easy to control, and the cell state contains hybrid cells, the difference between batches is large, and the cytopathy is not obvious. Therefore, it is necessary to obtain oral epithelial cells capable of being stably passaged, however, oral epithelial cells are terminally differentiated cells, and it is very difficult to culture oral epithelial cells in vitro, and the number of passages is very limited, and after the cells are passaged for a certain number of times, the cells enter the aging stage and cannot continue to proliferate, thus greatly limiting the research and application of oral epithelial cells.

At present, immortalized cells are capable of conferring a property of a cell to acquire the ability to grow continuously and proliferate. However, few studies have been reported on immortalized oral epithelial cells.

Disclosure of Invention

In order to obtain the immortalized oral epithelial cells and simultaneously improve the cell density and the cell activity of the immortalized oral epithelial cells, the application provides the immortalized oral epithelial cells and a preparation method and application thereof.

In a first aspect, the present application provides a method for preparing immortalized oral epithelial cells, which adopts the following technical scheme:

a preparation method of immortalized oral epithelial cells specifically comprises the following steps:

and (3) infecting oral epithelial cells by using SV40LT over-expressed lentiviruses, and screening, identifying and passaging the cells to obtain the immortalized oral epithelial cells.

Preferably, the oral epithelial cells are human oral epithelial cells.

The application takes the human oral epithelial cells as host cells, and uses SV40LT over-expressed lentiviruses to carry out cell infection on the human oral epithelial cells, so that SV40LT genes and the host cells are stably integrated and recombined, and then the oral epithelial cells after cell screening and identification are subjected to subculture, and the stably passaged immortalized oral epithelial cells can be obtained. In the process, the cancer suppressor gene in the oral epithelial cell is combined with the related combination region on the SV40LT antigen fragment, so that the cancer suppressor gene in the oral epithelial cell is invalid and can not be expressed, thereby preventing cell division arrest and leading the cell to be capable of unlimited proliferation; meanwhile, a plurality of differentiation phenotypes of the original cells can be kept, relatively stable proliferation characteristics and functional states are achieved, and the obtained immortalized oral epithelial cells are high in cell density and cell activity.

Preferably, the method of cell infection is: and (2) incubating the oral epithelial cells at the temperature of 10-20 ℃ for 20-40min, and then adding SV40LT recombinant lentivirus and an infection enhancer into the oral epithelial cells to infect the cells.

In a particular embodiment, the temperature may be: 10 deg.C, 15 deg.C, 20 deg.C.

In some specific embodiments, the temperature may also be: 10-15 deg.C, 15-20 deg.C.

In a particular embodiment, the incubation time may be: 20min, 30min and 40min.

In some specific embodiments, the incubation time may also be: 20-30min and 30-40min.

According to experimental analysis, compared with the method of directly infecting cells by using recombinant lentiviruses in the preparation process of the immortalized cells, the method has the advantages that the oral epithelial cells are placed at the temperature of 10-20 ℃ and incubated for 20-40min before the cells are infected, so that the infection efficiency of the cells can be effectively improved, and the immortalized oral epithelial cells can be rapidly obtained.

Further, the SV40LT overexpression lentivirus is a pBABE-Puro SV40LT lentivirus.

According to experimental analysis, when telomerase containing an hTERT gene is selected as a lentivirus to infect cells, primary cells cannot be converted into immortalized oral epithelial cells; when cells were selected to be infected with a lentivirus containing both the pBABE-Puro SV40LT gene and the hTERT gene, the conversion power of immortalized oral epithelial cells was low. The application selects pBABE-Puro SV40LT lentivirus, and can obtain immortalized oral epithelial cells with higher conversion power. Thus, the present application selects for infection of cells with pBABE-Puro SV40LT lentivirus.

Further, the multiplicity of infection of the cell infection is MOI = (5-15): 1.

further, the infection enhancer is polybrene with the concentration of 4-8 mug/mL.

The polybrene can neutralize the electrostatic repulsion between sialic acid on the surface of the oral epithelial cells and the recombinant lentivirus particles, and promote the adsorption between the recombinant lentivirus and the oral epithelial cells, so that the infection efficiency of the recombinant lentivirus on the oral epithelial cells is obviously improved.

In a particular embodiment, the concentration of polybrene may be: 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL.

In some specific embodiments, the concentration of the polybrene may also be: 4-6. Mu.g/mL, 6-8. Mu.g/mL.

As can be seen from the experimental analysis, when the concentration of polybrene is controlled within the above range, the infection efficiency of cells can be further improved. Therefore, the present application controls the concentration of polybrene within the above range.

Preferably, the cell screen comprises a primary screen and a secondary screen.

According to the application, the killing curve in cell screening is determined, and the use concentration of puromycin is 1.5 mu g/mL, and the action time is 3d; therefore, if only one selection of infected oral epithelial cells is performed, it takes 3d to obtain successfully infected oral epithelial cells. The infected cells are screened twice, the action time of the screening is 12-16h in total, and the successfully infected oral epithelial cells can be obtained.

In addition, test analysis shows that when infected oral epithelial cells are screened only once, the obtained immortalized oral epithelial cells have lower cell density and cell activity; the infected cells are screened twice, so that the cell density and the cell activity of the immortalized oral epithelial cells can be obviously improved.

Further, the primary screening method comprises the following steps: the oral epithelial cells obtained by infection are placed in a culture medium containing 0.5-1.5 mu g/mL puromycin for primary screening, and the screening time is 6-10h.

In a specific embodiment, the concentration of puromycin in the primary screening can be: 0.5. Mu.g/mL, 1.0. Mu.g/mL, 1.5. Mu.g/mL.

In some specific embodiments, the concentration of puromycin in the primary screening can also be: 0.5-1.0. Mu.g/mL, 1.0-1.5. Mu.g/mL.

As is apparent from the experimental analysis, when the puromycin concentration in one screening is controlled to be within the above range, the cell density and cell activity of the cells can be further improved. Therefore, the present application controls the puromycin concentration in the primary screening to the above range.

Further, the secondary screening method comprises the following steps: and placing the oral epithelial cells obtained by the primary screening in a culture medium containing 1.5-2.0 mu g/mL puromycin for secondary screening, wherein the screening time is 6-10h.

In a specific embodiment, the concentration of puromycin in the secondary screening may be: 1.5. Mu.g/mL, 1.75. Mu.g/mL, 2.0. Mu.g/mL.

In some specific embodiments, the concentration of puromycin in the secondary screening can also be: 1.5-1.75. Mu.g/mL, 1.75-2.0. Mu.g/mL.

As is apparent from the experimental analysis, when the concentration of puromycin in the secondary screening is controlled within the above range, the cell density and cell activity of the cells can be further improved. Therefore, the present application controls the puromycin concentration in the secondary screening to the above range.

In a second aspect, the present application provides immortalized oral epithelial cells prepared by the above-described method.

According to the method, the growth curve of the oral epithelial cells is drawn, so that the proliferation growth curve of the oral epithelial cells is S-shaped and accords with the growth rule of the epithelial cells. In addition, the activity of the immortalized cells is higher than that of the primary cells at the beginning of 3d, which shows that the obtained immortalized cells have strong proliferation capacity in the in vitro culture process and are better than the primary cells, and the result shows that the immortalized oral epithelial cells are successfully prepared by the application.

In a third aspect, the application provides the application of the immortalized oral epithelial cells in the field of detecting anti-perinuclear factor antibodies.

The immortalized oral epithelial cells obtained by the application have good and stable proliferation characteristics and functional states, can be used as target antigens of anti-perinuclear factor antibodies, are used for detecting the anti-perinuclear factor antibodies, and have important significance for researching rheumatoid arthritis diseases.

To sum up, the technical scheme of this application has following effect:

according to the application, the SV40LT overexpression lentivirus is used for carrying out cell infection on the oral epithelial cells to obtain the stably passaged immortalized oral epithelial cells, and the immortalized oral epithelial cells have relatively stable proliferation characteristics and functional states.

According to the method, the oral epithelial cells are incubated at 10-20 ℃ before cell infection, so that the infection efficiency of the oral epithelial cells is effectively improved, and the method is favorable for quickly obtaining the immortalized oral epithelial cells.

The application screens infected oral epithelial cells twice, controls the concentration of puromycin in the two screening processes, and obviously improves the cell density and the cell activity of the immortalized oral epithelial cells.

Drawings

FIG. 1 shows the growth curves of primary oral epithelial cells (P0), 30 th generation cells (P30) and 60 th generation cells (P60) in example 3 of the present application.

Detailed Description

In a first aspect, the present application provides a method for preparing immortalized oral epithelial cells, comprising the following steps:

s1: digesting the oral epithelial tissue by using protease to obtain the oral epithelial primary cells.

S2: transforming pBABE-Puro SV40LT plasmid (purchased from Addgene company) into DH5 alpha competent cells to obtain pBABE-Puro SV40LT recombinant lentiviral vector; fully and uniformly mixing a DMEM culture solution, a pBABE-Puro SV40LT recombinant lentiviral vector and an infection reagent Lipofectamine, and incubating the obtained mixture for 20min at room temperature; the mixture was then added dropwise to HEK-293T cells to obtain SV40LT recombinant lentiviruses.

S3: primary oral epithelial cells were cultured at 1 × 10 ⁵ Inoculating the cells to a 6-well plate at a density of each well, wherein the cell fusion rate of the next day reaches 70-80%; then placing the oral epithelial cells at the temperature of 10-20 ℃ for incubation for 20-40min, and then adding SV40LT recombinant lentivirus and an infection enhancer into the oral epithelial cells for cell infection.

S4: placing the oral epithelial cells obtained by infection in a DMEM/F12 culture medium containing puromycin of 0.5-1.5 mu g/mL for screening for 6-10h, so that the uninfected cells die massively and the surviving cells start to grow; placing the human oral epithelial cells obtained by primary screening in a DMEM/F12 culture medium containing 1.5-2.0 mu g/mL puromycin for screening for 6-10h; and during the screening, observing the survival proportion of the cells every 4h, and obtaining the cells which survive after the screening is finished, namely the human oral epithelial cells which are successfully infected.

S5: when large positive cells are cloned, selecting the monoclonal cells to passage to a 48-well plate, adding a DMEM/F12 culture medium to continue culturing, and changing the culture solution and passing once every 2 d. After more than 30 generations of culture, immortalized oral epithelial cells were obtained.

Wherein the oral epithelial cells are human oral epithelial cells.

further, the infection enhancer is polybrene 4-8 μ g/mL.

In a second aspect, the present application provides immortalized oral epithelial cells prepared by the above-described preparation method.

In a third aspect, the application provides an application of the immortalized oral epithelial cells in the field of detecting anti-perinuclear factor antibodies.

The present application is described in further detail below in connection with examples 1-23, comparative examples 1-2, and performance testing tests, which are not to be construed as limiting the scope of the claimed application.

Examples

Examples 1 to 9

Examples 1-9 provide a method for preparing immortalized oral epithelial cells, respectively.

The difference between the above embodiments is that: incubation temperature and incubation time before cell infection in step S3. The details are shown in Table 1.

The preparation method of the immortalized oral epithelial cells in each embodiment specifically comprises the following steps:

s1: isolation and culture of oral epithelial cells

The redundant oral mucosa tissue blocks discarded in the surgical operation of the hospital are brought back to the laboratory, and the test is carried out on an intercellular superclean bench; the tissue mass was washed with PBS buffer containing 100U/mL penicillin, 100U/mL streptomycin, and pH 7.2, and the human oral mucosal tissue mass was trimmed to 2mm × 2mm size with sterilized ophthalmic scissors.

Placing mucosa tissue in a mixed solution containing 0.2% trypsin and 0.01% EDTA, digesting at 4 deg.C for 10 hr, and removing epidermis with sterilized forceps to obtain oral mucosa epithelial tissue; after being washed by PBS, the epithelial tissues of the oral mucosa are placed in Dispase containing 0.4 percent of neutral protease, and then are subjected to constant-temperature shaking digestion for 20min at 37 ℃ and 80rpm, and the neutralizing solution of DMEM containing 10 percent of serum is mixed according to the weight ratio of 1:1 to stop digestion; gently blowing and beating by using a pipettor to form cell suspension, and filtering the cell suspension by using a 200-mesh sterile filter screen; centrifuging the filtrate at 1200rpm for 4min, and discarding the supernatant to obtain cell precipitate; add K-sfm medium to resuspend the cells and count them using a hemocytometer counting plate, stain viable cells with trypan blue, and count cell viability and number.

Pressing the cell to 10 ⁴ -10 ⁵ Cell density of one/mL was inoculated into DMEM/F12 serum-free medium and placed at 37 ℃ with 5% CO ₂ Culturing in an incubator, changing the culture solution the next day to remove the non-adherent cells, and changing the culture solution every other day later. The formation of clones was observed under a microscope at around 7-14d, and when the cell fusion rate was 80% -90%, primary cells P0 were obtained by 0.125% trypsinization of the cells.

S2: preparation of recombinant lentiviruses

Taking out DH5 alpha competent cells frozen at-80 ℃, and placing the cells in an ice bath for thawing; then taking 1 mu L of plasmid LpBABE-Puro SV40LT (purchased from Addgene company) to be placed in 50 mu L of DH5 alpha competent cells, uniformly mixing, and placing in an ice bath for 30min; then placing the mixture in a water bath kettle at 42 ℃ for incubation for 70s; adding 0.5mL of LB culture medium at 37 ℃, and performing shake culture for 1h at 37 ℃ and 220 rpm; coating 0.2mL of bacterial liquid in an LB solid culture medium containing kanamycin, and culturing overnight at 37 ℃; and selecting colonies, adding the colonies into an LB liquid culture medium containing kanamycin for overnight culture, amplifying bacteria, extracting plasmids, performing sequencing identification, and obtaining a pBABE-Puro SV40LT recombinant lentiviral vector after correct identification.

Fully and uniformly mixing 1mL of DMEM culture solution, 5 mu g of pBABE-Puro SV40LT plasmid and 100 mu g of infection reagent Lipofectamine, and incubating the obtained mixture for 20min at room temperature; then, the mixture is dropwise added to HEK-293T cells with the fusion rate of 70%, after infection for 48 hours, virus cell supernatant is harvested, low-speed centrifugation is carried out at 100rpm, cell fragments are taken out, and then filtration is carried out by using a filter membrane with the diameter of 0.45 mu m, so as to obtain recombinant lentivirus liquid. The titer of the obtained recombinant lentivirus was determined using Clontech P24 quantitative determination kit, and the titer of the recombinant lentivirus was 2X 10 ⁶ PFU/mL。

S3: infection of cells

Primary oral epithelial cells were cultured at 1 × 10 ⁵ Inoculating the cells to a 6-well plate at a density of each well, wherein the cell fusion rate of the next day reaches 70-80%; the oral epithelial cells were incubated according to the incubation temperature and time shown in table 1. Then LiThe recombinant lentivirus solution was diluted one-fold with DMEM/F12 medium containing 10% FBS, and polybrene was added at a concentration of 6. Mu.g/mL to give a recombinant lentivirus dilution having a titer of 1X 10 ⁶ PFU/mL; then adding 1mL of virus diluent into a 6-well plate per well to infect cells; after 12h of infection, the DMEM/F12 culture medium containing 6 mug/mL polybrene is replaced for continuous culture; and after the culture is continued for 12 hours, the DMEM/F12 culture medium containing 6 mu g/mL polybrene is replaced for continuous culture, and after the culture is continued for 12 hours, the infected human oral epithelial cells are screened.

S4: cell screening

Determination of the killing curve in cell screening: uninfected human oral epithelial cells were cultured at 1X 10 ⁵ Each well was plated in a 24-well plate, incubated overnight, the next day, the old medium was removed from the 24-well plate, fresh medium containing different concentrations of puromycin (0.5. Mu.g/mL, 1. Mu.g/mL, 1.5. Mu.g/mL, 2. Mu.g/mL, 2.5. Mu.g/mL, 3. Mu.g/mL) was added to the 24-well plate with the plated cells, fresh selection medium was changed every 2 days, the survival rate of the cells was observed daily, and the minimum puromycin use concentration was the lowest selection concentration at which all cells were killed within 1-4 days from puromycin selection.

As a result: puromycin was used at a concentration of 1.5. Mu.g/mL for a duration of 3 days.

And (3) cell screening: placing the human oral epithelial cells obtained by infection in a DMEM/F12 culture medium containing puromycin of 0.5-1.5 mug/mL for screening for 8 hours, so that a large amount of uninfected cells die and the surviving cells start to grow; placing the human oral epithelial cells obtained by primary screening in a DMEM/F12 culture medium containing 2-3 mu g/mL puromycin for screening, wherein the screening time is 8h; and during the screening, observing the survival proportion of the cells every 4h, and obtaining the cells which survive after the screening is finished, namely the human oral epithelial cells which are successfully infected.

S5: acquisition of immortalized oral epithelial cells

When large positive cells are cloned, selecting the monoclonal cells to passage to a 48-well plate, adding a DMEM/F12 culture medium to continue culturing, and changing the culture solution and passing once every 2 d. After more than 30 generations of culture, immortalized oral epithelial cells were obtained.

TABLE 1 incubation temperature and incubation time before cell infection in examples 1-9

Example 10

Example 10 provides a method of preparing immortalized oral epithelial cells.

The present embodiment is different from embodiment 3 in that: the step of cell infection in step S3 is different. The method comprises the following specific steps:

s3: infection of cells

Human oral epithelial cells were cultured at 1X 10 ⁵ Inoculating the cells to a 6-well plate at a density of each well, wherein the cell fusion rate of the next day reaches 70-80%; the recombinant lentivirus solution was diluted one-fold using DMEM/F12 medium containing 10% FBS, and polybrene was added at a concentration of 6. Mu.g/mL to give a recombinant lentivirus dilution having a titer of 1X 10 ⁶ PFU/mL; then adding 1mL of virus diluent into a 6-well plate per well to infect the cells; after 12h of infection, the DMEM/F12 medium containing 6 mu g/mL polybrene is replaced to continue culturing; and after the culture is continued for 12 hours, the DMEM/F12 culture medium containing 6 mu g/mL polybrene is replaced for continuous culture, and after the culture is continued for 12 hours, the infected human oral epithelial cells are screened.

Examples 11 to 14

Examples 11-14 each provide a method for preparing immortalized oral epithelial cells.

The above embodiments are different from embodiment 3 in that: the concentration of polybrene varied. Specifically, as shown in table 2.

TABLE 2 concentration of polybrene in examples 3, 11-14

Examples	Concentration of polybrene (μ g/mL)
		3	6
11	2
		12	4
13	8
		14	10

Performance test

1. Performance Effect of infection conditions on the infection efficiency of oral epithelial cells

The cell infection efficiency was examined by using the preparation methods provided in examples 1 to 14 as examination subjects.

The detection method comprises the following steps: and (4) observing the oral epithelial cells infected in the step (S3) under a fluorescence microscope, and detecting the EGFP fluorescence rate to calculate the infection efficiency.

And (3) detection results: as shown in table 3.

TABLE 3 efficiency of cell infection in examples 1-14

With reference to table 3, it can be seen by analyzing the detection results of examples 1 to 14 that, in the present application, before cell infection, the infection efficiency of the human oral epithelial cells can be effectively improved by incubating the human oral epithelial cells at 10 to 20 ℃ for 20 to 40min, and then infecting the cells with the recombinant lentiviral fluid containing 4 to 8 μ g/mL polybrene, thereby facilitating to rapidly obtain immortalized oral epithelial cells.

By comparing the detection results of example 3 and example 10, when the cells are not incubated before the infection of the cells, the infection efficiency of the cells is 68.4%, and the cells are incubated for 30min at 15 ℃ before the infection of the cells, the infection efficiency of the oral epithelial cells can be effectively improved.

According to the detection results of comparative examples 1-5, the infection efficiency of the oral epithelial cells is as high as more than 83.8% when the incubation temperature is controlled within the range of 10-20 ℃. The incubation temperature is controlled within the range, so that the infection efficiency of the oral epithelial cells is further improved.

According to the detection results of comparative examples 3 and 6-9, when the incubation time is controlled within the range of 20-40min, the infection efficiency of the oral epithelial cells is up to more than 82.6%. The application shows that the incubation time is controlled within the range, which is beneficial to further improving the infection efficiency of the oral epithelial cells.

According to the detection results of comparative examples 3 and 11 to 15, when the concentration of polybrene is controlled within the range of 4 to 8 mu g/mL, the infection efficiency of the oral epithelial cells is up to more than 83.7 percent. The method has the advantages that the concentration of polybrene is controlled in the range, and the infection efficiency of the human oral epithelial cells is further improved.

Examples 15 to 23

Examples 15-23 provide a method for preparing immortalized oral epithelial cells, respectively.

The above embodiments are different from embodiment 3 in that: the step and conditions of cell screening in step S4 are different. The details are shown in Table 4.

TABLE 4 procedures and conditions for cell selection in examples 3, 15-23

Comparative example

Comparative example 1

This comparative example provides a method of preparing immortalized oral epithelial cells.

This comparative example differs from example 3 in that: telomerase containing hTERT gene was used as lentivirus. The rest steps are the same.

Comparative example 2

The comparative example differs from example 3 in that: telomerase containing both the pBABE-Puro SV40LT gene and the hTERT gene was used as a lentivirus. The rest steps are the same.

Performance test

2. Performance effects of screening conditions on cell density and cell activity of immortalized oral epithelial cells

The preparation methods provided in examples 3 and 15 to 23 were used as the test subjects, and the immortalized oral epithelial cells obtained in the 30 th generation were cultured for 5 days, and the cell density and cell activity were measured to examine the effect of the screening conditions on the performance of the culture effect of human oral epithelial cells. Wherein, counting cells by using a blood counting chamber, and measuring the cell density; the cell activity was measured by trypan blue staining,

and (3) detection results: as shown in table 5.

TABLE 5 cell Density and cell Activity of immortalized oral epithelial cells P30 in examples 3, 15-23

With reference to table 5, it is understood by analyzing the detection results of examples 3 and 15 to 23 that the cell density and cell activity of the immortalized oral epithelial cells can be effectively increased by twice screening infected cells.

By comparing the results of example 3 with those of example 23, when infected oral epithelial cells were screened only once, the cell density of the 30 th generation cells was 2.8X 10 ⁵ cells/mL, cell viability of passage 30 cells was 91.4%; in the application, infected cells are selected and screened twice, and the cell density of 30 th generation cells is higher than 3.2X 10 ⁵ cells/mL, cell activity of 30 th generation cells higher than 94.4%; the method shows that the infected cells are screened twice, so that the cell density and the cell activity of the cells can be obviously improved.

By comparing the results of the tests of examples 3 and 15 to 18, the cell density and cell activity of the cells can be further improved by controlling the puromycin concentration in the primary screening to be in the range of 0.5 to 1.5. Mu.g/mL. Therefore, the present application controls the concentration of puromycin in one screening to the above range.

By comparing the results of the tests of examples 3 and 19 to 22, the cell density and cell activity of the cells can be further improved by controlling the puromycin concentration in the secondary screening to be in the range of 1.5 to 2.0. Mu.g/mL. Therefore, the present application controls the concentration of puromycin in the secondary screening to the above range.

3. Immortalization assay for oral epithelial cells

The oral epithelial cells of examples 3, 10 and 23 and comparative examples 1 to 2 were used as test subjects, and the conversion of the oral epithelial cells into immortalized oral epithelial cells was examined.

And (3) detection results: as shown in table 6.

TABLE 6 number of strains transformed into immortalized cell lines in examples 3, 10, 23 and comparative examples 1-2

Combining the detection results in table 6, it can be seen that when the oral epithelial cells are infected by the telomerase containing hTERT gene in comparative example 1, the number of strains converted into immortalized cell lines is 0, i.e., the primary cells die, which results in the failure of establishment of immortalized oral epithelial cell lines. When the oral epithelial cells were infected with the lentivirus containing both the SV40LT gene and hTERT gene in comparative example 2, the number of strains converted into immortalized cell lines was only 1, i.e., the conversion power of immortalized oral epithelial cells was low. When example 10 was not incubated at low temperature before cell infection, the number of strains transformed into immortalized cell lines was 4; example 23 infected cells were screened only once and the number of strains transformed into immortalized cell lines was 6, indicating that the success rate of immortalized oral epithelial cells obtained by the two preparation methods was low. In the application, pBABE-Puro SV40LT lentivirus is selected to infect oral epithelial cells, low-temperature incubation is carried out before cell infection, and the infected cells are screened twice, so that the success rate of transforming the oral epithelial cells into immortalized cell lines is higher, the immortalized cell lines can be continuously passaged in vitro, the cell state is not obviously different from that of primary cells, the cell lines are vigorous, the cell lines are high in proliferation speed, and the successful preparation of the immortalized oral epithelial cells with higher transformation rate is shown.

4. Determination of growth curves of oral epithelial cells

The primary cells (P0), 30 th generation cells (P30) and 60 th generation cells (P60) of example 3 were cultured at 1.0X 10, respectively ⁴ Cell density of individual cells/well was inoculated into 96-well plates and cultured, setting 6 replicates per plate, setting at 37 ℃,5% CO ₂ The cells were cultured in the cell culture chamber for 7 days. During the incubation period, only one well plate was removed per day for measurement, 10. Mu.L of CCK8 solution was added per well, incubated in an incubator at 37 ℃ for 2 hours, absorbance was measured at a wavelength of 450nm using a microplate reader,then, the culture time was plotted on the abscissa and the mean value of OD values was plotted on the ordinate, thereby obtaining a growth curve of the cells.

And (3) detection results: as shown in fig. 1.

By analyzing the detection result of the growth curve of the oral epithelial cells with reference to fig. 1, it can be known that the oral epithelial primary cells and the prepared immortalized oral epithelial cells are in the latent period within 1-2d of the continuous culture period; at 3-5d, the cells were in logarithmic growth phase; at 6-7d, the cells enter a plateau phase; the detection result shows that the proliferation and growth curve of the oral epithelial cells is S-shaped and conforms to the growth rule of the epithelial cells. In addition, the activity of the immortalized cells is higher than that of the primary cells at the beginning of 3d, which indicates that the obtained immortalized cells have strong proliferation capacity in the in vitro culture process and are better than the primary cells, and the result further indicates that the immortalized oral epithelial cells are successfully prepared by the application.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A preparation method of immortalized oral epithelial cells is characterized by comprising the following steps:

and (3) carrying out cell infection on the oral epithelial cells by using SV40LT over-expressed lentivirus, and obtaining the immortalized oral epithelial cells through cell screening, identification and passage.

2. The method for preparing immortalized oral epithelial cells according to claim 1, wherein said method of cell infection is: and (2) incubating the oral epithelial cells at the temperature of 10-20 ℃ for 20-40min, and then adding SV40LT recombinant lentivirus and an infection enhancer into the oral epithelial cells to infect the cells.

3. The method for producing immortalized oral epithelial cells according to claim 2, wherein said SV40LT overexpressing lentivirus is a pBABE-Puro SV40LT lentivirus.

4. The method for producing immortalized oral epithelial cells according to claim 2, wherein said cell is infected at a multiplicity of infection of MOI = (5-15): 1.

5. the method for producing immortalized oral epithelial cells according to claim 2, wherein said infection enhancer is polybrene at 4 to 8 μ g/mL.

6. The method for producing immortalized oral epithelial cells according to claim 1, wherein the cell selection comprises a primary selection and a secondary selection.

7. The method for preparing immortalized oral epithelial cells according to claim 6, wherein the primary screening method comprises: the oral epithelial cells obtained by infection are placed in a culture medium containing 0.5-1.5 mu g/mL puromycin for primary screening, and the screening time is 6-10h.

8. The method for preparing immortalized oral epithelial cells according to claim 6, wherein said secondary screening is performed by: and placing the oral epithelial cells obtained by the primary screening in a culture medium containing 1.5-2.0 mu g/mL puromycin for secondary screening, wherein the screening time is 6-10h.

9. Immortalized oral epithelial cells produced by the production method according to any one of claims 1 to 8.

10. Use of immortalized oral epithelial cells as defined in claim 9, in the field of detection of antibodies against perinuclear factors.