CN112143702A

CN112143702A - Neurofibroma cell line and construction method thereof

Info

Publication number: CN112143702A
Application number: CN202010322404.8A
Authority: CN
Inventors: 王智超; 李青峰; 任捷艺; 顾熠辉; 王薇
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2020-12-29
Anticipated expiration: 2040-04-22
Also published as: CN112143702B

Abstract

The invention provides a neurofibroma cell line, in particular to a neurofibroma cell line derived from human type I neurofibromatosis and a construction method thereof. The neurofibroma cell line has a Chinese genetic background and is immortalized.

Description

Neurofibroma cell line and construction method thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a neurofibroma cell line and a construction method and application thereof.

Background

Neurofibromatosis type i (NF 1) is an autosomal dominant genetic disease caused by a mutation in the NF1 gene. The prevalence of NF1 in neonates is about 1/3500-1/3000 (J. American epidemiology, 2000, 151(1): 33-40; J. Neuroscientific history, 2010,19(4): 333-348). Neurofibromas, one of the most common and characteristic symptoms in NF1, have a high proliferation of schwann cells and other nerve fiber supporting components such as perineurium cells, fibroblasts, blood vessels, and mast cells infiltrating therein. Neurofibromas are mainly classified into cutaneous neurofibromas and plexiform neurofibromas, which have no malignant potential, while plexiform neurofibromas mostly exhibit invasive growth, destroy normal tissues and organs around the tumor, and have a malignant tendency, and the surgical efficacy is poor. Therefore, the method becomes a current research hotspot of multiomics and targeted therapy.

However, neurofibromas are benign tumors, and unlike many malignant tumors, neurofibroma cells are not immortalized and hardly survive in vitro for a long time, which makes it difficult to study them. The currently internationally recognized neurofibroma-derived cell lines are all from foreign countries, can be queried and purchased at ATCC (https:// www.atcc.org), and lack neurofibroma cell lines against the genetic background of Chinese.

Therefore, there is a need for a neurofibromatosis cell line, particularly a neurofibromatosis cell line with a genetic background of Chinese, which is immortal, and a method for constructing the same, to advance the research on type I neurofibromatosis.

Disclosure of Invention

The invention aims to provide a neurofibromatosis cell line, in particular to a neurofibromatosis cell line derived from human type I neurofibromatosis, which has a Chinese genetic background and is immortal.

In order to achieve the above objects, according to one aspect of the present invention, there is provided a neurofibromatosis cell line, particularly a neurofibromatosis cell line derived from human type I neurofibromatosis, named neurofibromatosis cell line PNF1-9HOSP-01, with a collection number of CCTCC NO: C202006. The name of the depository unit is: china center for type culture Collection (Wuhan university Collection), the code of the collection unit is CCTCC-China center for type culture Collection, the address of the collection unit is Wuhan, and the collection time is 2020, 4 and 6 days.

According to another aspect of the present invention, there is provided a method for constructing a neurofibroma cell line as described above, which comprises transfecting primary isolated neurofibroma cells with telomerase reverse transcriptase gene and CDK4/m antigen, and performing monoclonal screening to obtain an immortalized neurofibroma cell line.

In some embodiments, in the construction method, primary isolated neurofibroma cells are transfected with telomerase reverse transcriptase gene prior to continued transfection with CDK4/m antigen.

In some embodiments, in the method of constructing, the primary isolated neurofibroma cells are transfected with telomerase reverse transcriptase gene and CDK4/m antigen by a lentiviral suspension and a viral infection enhancing suspension.

In some embodiments, the viral infection enhancing suspension is a polybrene suspension.

In some embodiments, the construction method comprises:

step S1: providing primary neurofibroma cells, and culturing the primary neurofibroma cells to obtain a cell culture to be transfected;

step S2: performing virus infection on a cell culture to be transfected by using the lentivirus suspension and the virus infection enhancement suspension, and performing culture medium replacement in the virus infection process; and the number of the first and second groups,

step S3: monoclonal screening and subculturing cell cultures infected by the viruses to obtain an immortalized neurofibroma cell line; wherein,

in step S2, transfecting the cell culture to be transfected with a telomerase reverse transcriptase gene with a lentivirus suspension and a virus infection enhancing suspension, wherein the MOI value is 30, and medium replacement is performed during virus infection; subsequently, cell cultures transfected with telomerase reverse transcriptase gene were transfected with CDK4/m antigen using lentivirus suspension and virus infection-enhancing suspension with MOI value of 30, and medium replacement was performed during virus infection; and in the step S2, the working concentration of the virus infection enhancing suspension is 5-8 mu g/ml.

In some embodiments, in step S2, the primary neurofibroma cells in the cell culture to be transfected have a concentration of 8 μ g/ml.

In some embodiments, the step S1 includes the steps of:

step S11: placing the sheared neurofibroma tissue into an L15 culture medium containing 10% (v/v) fetal calf serum, 1.25U/ml dispase, 300U/ml collagenase, 100U/ml penicillin and 100ug/ml streptomycin, filtering the tissue through a 30 mu m cell filter screen after overnight at 37 ℃ to collect cells, coating the cells with laminin, and placing the cells into an L15 culture medium containing 15% fetal calf serum, 25ng/ml hNRG-1, 100U/ml penicillin and 100ug/ml streptomycin to obtain primary neurofibroma cells; and the number of the first and second groups,

step S12: coating the culture container with coating solution, spreading the digested cells in the coated culture container uniformly, and culturing at 37 deg.C with 5% CO₂And (4) hermetically culturing to obtain a cell culture to be transfected.

In some embodiments, in the step S12, the primary neurofibroma cells obtained in the step S11 are trypsinized.

In some embodiments, in step S1, the L15 medium does not contain HEPS buffer, and a sealed culture is required.

In some embodiments, the step S2 includes the steps of:

step S21: the cell culture to be transfected is performed at 6X 10 per well⁵The inoculation density of 2ml was inoculated into a well plate at 37 ℃ with 5% CO₂Incubation in the environment;

step S22: replacing a fresh culture medium for the incubated pore plate, and sequentially adding the virus infection enhancement suspension and the lentivirus suspension to transfect a telomerase reverse transcriptase gene for the cell culture to be transfected; wherein the MOI value is 30, the working concentration of the virus infection enhancing suspension is 5-8 mug/ml, and the culture medium replacement is carried out in the virus infection process;

step S23: then cell cultures transfected with telomerase reverse transcriptase gene are transfected with CDK4/m antigen by using lentivirus suspension and virus infection enhancement suspension; wherein the MOI value is 30, the working concentration of the virus infection enhancing suspension is 5-8 mug/ml, and the culture medium replacement is carried out in the virus infection process; and the number of the first and second groups,

step S24: cultures of CDK4/m antigen transfected cells were cultured and passaged.

In some embodiments, in said step S22, a medium replacement is performed every 6 hours.

In some embodiments, in step S22, the transfection time is 72 hours.

In some embodiments, in said step S24, the cell culture transfected with the telomerase reverse transcriptase gene and the CDK4/m antigen is passaged for at least more than 9 passages. .

In some embodiments, the medium used in step S22 and step 23 is Leibovitz' S L-15 medium containing 15% (v/v) fetal bovine serum, 25ng/mL hNRG-1(Human Neuredulin-1, Human Neuregulin-1), 100U/mL penicillin, and 100U/mL streptomycin 100 ug/mL.

In some embodiments, the step S3 includes the steps of:

step S31: the cell culture obtained in step S23 was diluted to a density of 500 cells/ml, and seeded into a well plate by a double dilution method at 37 ℃ with 5% CO₂Culturing in the environment of (1), observing the condition of cell clone formation in the pore plate after one week, and selecting a monoclonal cell pore; and the number of the first and second groups,

step S32: the culture medium is replaced every 24-72 hours, digestion passage is carried out when 70% -80% of the cells grow to fill the bottom of the dish, and the seed separation rate is 1: 2.

In some embodiments, the medium used in steps S31 and S32 is Leibovitz' S L-15 medium containing 15% (v/v) fetal bovine serum, 25ng/mL hNRG-1(Human Neuredulin-1, Human Neuregulin-1), 100U/mL penicillin, and 100U/mL streptomycin 100 ug/mL.

It will be understood by those skilled in the art that the reagents used in the present invention are commercially available products unless otherwise specified.

Compared with the prior art, the invention has the following beneficial effects:

1. the PNF1-9HOSP-01 cell line is a benign tumor cell line which is autonomously established in China and is derived from human type I neurofibromatosis, has the genetic background of Chinese, and has incomparable advantages in the research of the human type I neurofibromatosis compared with the existing cell line pairs in foreign countries.

2. In general, after a benign or malignant tumor is transferred from an in vivo to an in vitro culture, the tumor cell cannot survive easily due to the change of microenvironment, so that the tumor cell is difficult to passage under the general in vitro culture condition, and the research on the primary cell is difficult to advance. The PNF1-9HOSP-01 cell is an immortalized benign cell, can stably survive and passage in vitro according to the culture condition provided by the invention, and provides a good material for the research related to neurofibroma.

3. The construction method of the PNF1-9HOSP-01 cell has good referential property and popularization property, and can effectively and economically establish a benign tumor cell line of the type I neurofibromatosis.

Drawings

FIG. 1: primary cells were passage 5 (20X) under light microscopy.

FIG. 2: PNF1-9HOSP-01 cells (20X) growing adherently in monolayer, more novacells were visible, cells grew in clusters, which were irregular in shape, closely spaced, well defined, and had lost contact inhibition.

Fig. 3A, 3B, 3C: PNF1-9HOSP-01 cell scanning electron microscope (2000X), the cell surface has rugae, and the periphery has microvilli or protrusions.

FIG. 4: PNF1-9HOSP-01 cells growth graph, cell doubling time is 16.16 hours.

FIG. 5: the change of the anchorage rate of PNF1-9HOSP-01 cells after inoculation is plotted along with time, and most of the cells (more than 95 percent) can grow adherently after 6 hours of planting.

FIG. 6: karyotyping of PNF1-9HOSP-01 cells.

FIG. 7: cell type identification flow chart, 92.93% of the cell lines were S100B (+) tumor cells.

FIG. 8: PNF1-9HOSP-01 cell immunofluorescence, green in the figure: S100B, blue: dapi. It can be seen that S100B is mainly expressed in the cytoplasm.

Detailed Description

Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.

Example 1 neurofibroma cell line and its construction

In this example, a neurofibromatosis cell line, particularly a neurofibromatosis cell line derived from human type I neurofibromatosis, designated PNF1-9HOSP-01, with a collection number of CCTCC NO: C202006, is provided.

The neurofibroma cell line is constructed by transfecting primary separated neurofibroma cells with telomerase reverse transcriptase gene (TERT) and CDK4/m antigen, and performing monoclonal screening to obtain an immortalized neurofibroma cell line. And, the construction method comprises the steps of:

step S11: placing the sheared neurofibroma tissue into an L15 culture medium containing 10% (v/v) fetal calf serum, 1.25U/ml dispase, 300U/ml collagenase, 100U/ml penicillin and 100ug/ml streptomycin, filtering the tissue through a 30 mu m cell filter screen after overnight at 37 ℃ to collect cells, coating the cells with laminin, and placing the cells into an L15 culture medium containing 15% fetal calf serum, 25ng/ml hNRG-1, 100U/ml penicillin and 100ug/ml streptomycin to obtain primary neurofibroma cells;

step S12: coating the culture container with coating solution, spreading the digested cells in the coated culture container uniformly, and culturing at 37 deg.C with 5% CO₂Hermetically culturing in an environment to obtain a cell culture to be transfected;

step S23: then cell cultures transfected with telomerase reverse transcriptase gene are transfected with CDK4/m antigen by using lentivirus suspension and virus infection enhancement suspension; wherein the MOI value is 30, the working concentration of the virus infection enhancing suspension is 5-8 mug/ml, and the culture medium replacement is carried out in the virus infection process;

step S24: culturing and passaging a cell culture transfected with CDK4/m antigen;

In the step S12, the primary neurofibroma cells obtained in the step S11 are trypsinized.

In said step S22, a medium replacement is performed every 6 hours.

In step S22, the transfection time was 72 hours.

In step S24, the cell culture transfected with telomerase reverse transcriptase gene and CDK4/m antigen was passaged for at least 9 more passages.

In the steps S22 and S23, the virus infection enhancing suspension is Polybrene (Polybrene).

The culture medium used in the step S22, the step 23, the step S31 and the step S32 is Leibovitz' S L-15 culture medium containing 15% (v/v) fetal bovine serum, 25ng/mL hNRG-1(Human Neuredulin-1, Human Neuregulin-1), 100U/mL penicillin and 100U/mL streptomycin 100 ug/mL.

In one embodiment, the specimen is obtained from yellow XX, male and 18-year-old patients in the rehabilitation surgery of the ninth national hospital affiliated to Shanghai university of transportation, the patients before operation obtain the consent of the patients and guardians, and the specimens of the bushy neurofibroma of the buttocks excised in the operation are signed with an informed consent. The postoperative pathological diagnosis is as follows: neurofibroma (pathological number: 18-19566).

The specific reagents used include: leibovitz's L-15 medium (Gibco, USA), fetal bovine serum (Gibco, USA), hNRG-1(Human Neuredulin-1, Human Neuregulin-1, CST, USA), penicillin (Gibco, USA), streptomycin (Gibco, USA).

The remaining cell culture materials include: 30 μm cell strainer (FALCON, USA); collagenase type XI (Sigma, usa); dispase (Sigma, usa); laminin Laminin (Sigma, USA); 0.25% Trypsin-EDTA (Gibco, USA); closed T25 cell culture flask (Corning, USA), serum-free cell culture medium (Sciencell, USA), Polybrene (Sigma, USA), 6-well plate (Corning, USA), 96-well plate (Corning, USA), inverted microscope (Olympus, Japan).

In the above embodiment, the method for constructing neurofibroma cell line specifically includes:

1. tumor cell acquisition and primary culture

Clinical excised neurofibroma tissue (about 1 cm) was taken³) The excised tissue was rinsed in L15 medium containing 1% double antibody (penicillin 100U/mL, streptomycin 100ug/mL), placed in L-15 medium containing 10% fetal calf serum, and transported in ice boxes. The tissue was sheared in a clean bench with a sterilized cross-surgical scalpel, and the sheared tissue was placed in 10ml of L15 medium containing 10% fetal bovine serum, 1.25U/ml dispase, 300U/ml collagenase, and 1% double antibody overnight at 37 ℃ in an incubator. The cells were filtered through a 30 μm cell strainer into 50ml centrifuge tubes and centrifuged at 500g for 5 min. Collecting cell precipitate, culturing the cells in sealed T25 culture bottle containing complete culture solution of L15 containing 15% fetal calf serum, hNRG-1(25ng/ml) and 1% double antibody after coating with lamin (10 μ g/ml);

preparation before primary culture: the flasks are coated with 1ml of coating solution (10ug/ml of Laminin Laminin diluted in PBS at a dilution ratio of 1: 100; e.g. 10ul of 1mg/ml stock solution diluted in 1ml PBS at a final concentration of 10ug/ml), the incubator is left for 2h, excess supernatant is aspirated off and the PBS is washed gently once.

Passage of primary cells: carefully aspirate the medium and wash with 1 xPBS; after 1xPBS is discarded, a proper amount of pancreatin is added; cells were observed under a microscope and if the cytoplasm retracted, no longer appeared to be connected to each other as pieces, indicating that the cells were digested moderately at this time. Discarding pancreatin, adding a proper amount of preheated fresh culture solution, blowing and dispersing the cells uniformly, sucking the cell suspension into a 15mL centrifuge tube, centrifuging at room temperature at 800rpm for 5min, discarding the culture solution, adding the fresh culture solution, and blowing and dispersing the cells uniformly. Spreading the digested cells in a pre-coated cell bottle uniformly at 37 deg.C with 5% CO₂The culture was carried out using a sealed culture flask.

Remarking: the L15 medium does not contain HEPS buffer solution, and sealed culture is required.

Construction of TERT + Mcdk4 Stable transgenic cell line

Cell preparation-before the establishment of stable cell lines, the cells of interest were kept in good condition, for example, the 5 th passage (20X) of primary cells under light microscope observation as shown in FIG. 1. Carefully aspirate the medium and wash with 1 xPBS; after 1xPBS is discarded, a proper amount of pancreatin is added; cells were observed under a microscope and if the cytoplasm retracted, no longer appeared to be connected to each other as pieces, indicating that the cells were digested moderately at this time. Discarding pancreatin, adding a proper amount of preheated fresh culture solution, blowing and dispersing the cells uniformly, sucking the cell suspension into a 15mL centrifuge tube, centrifuging at room temperature at 800rpm for 5min, discarding the culture solution, adding the fresh culture solution, blowing and dispersing the cells uniformly, and counting by a hemocytometer. At 6X 10⁵A density of 2ml is added into a 6-well plate; incubating the cells in a cell incubator at 37 ℃ and 5% CO2 for 24 h; taking out the 6-hole plate, and replacing with fresh culture solution 2 ml/hole;

transfection of telomerase reverse transcriptase gene: adding 8 μ g/ml Polybrene and 25 μ l/well lentivirus suspension (TERT virus) in sequence, with MOI of 30 and neurofibroma cell concentration of 8 μ g/ml, replacing fresh culture medium every 6 hr, and transfecting for 72 hr;

transfection of CDK4/m antigen: adding 8 μ g/ml Polybrene and 25 μ l/well of lentivirus suspension (CDK4/m virus) in sequence, with MOI of 30 and neurofibroma cell concentration of 8 μ g/ml, replacing fresh medium every 6 hours, and transfecting for 72 hours;

passage: the cell cultures transfected with telomerase reverse transcriptase gene and CDK4/m antigen were passaged for at least 9 passages. The surviving cells were collected, at which point the cells were immortalized.

3. Monoclonal screening of tumor cells

A cell suspension was prepared, diluted to a density of 500 cells/mL, and 200ul of the diluted liquid was pipetted into A1 wells in a 96-well plate. Pipettors added 100ul of media to wells of the 96-well plate, except the a1 well. Suck 100ul of medium from A1 well to B1 well and mix gently to avoid air bubbles. Suck 100ul of the mixed liquid from the B1 well to the C1 well. And so on to H1 hole. 100ul of medium was added to A1-G1 wells. Sucking 100ul of the mixed culture medium from the A1 hole to the A2 hole, and mixing the culture medium and the A2 hole gently to avoid generating bubbles. After mixing, 100ul of medium was aspirated from well A2 to well A3, and so on to well A12. (B1-B12 repeat this step) wells in the 96-well plate with a final volume of less than 200ul were supplemented with cell culture medium. The 96-well plate was incubated at 37 ℃ in an incubator containing 5% CO2, and after one week, the formation of cell clones in the plate was observed. The wells of the single clones were picked and recorded.

4. Subculturing of tumor cells

According to the state of the cells and the change of the color of the culture solution, the L15 complete culture medium is replaced every 2-3 days, 70% -80% of the cells growing on the bottom of the dish are digested and passaged in time, and the seed separation rate is 1:2, continuously culturing 1 flask of the mixed culture medium, and freezing 1 flask of the mixed culture medium for seed preservation.

Verification method

Example 2 verification method

In this example, a method of identifying the neurofibroma cell line PNF1-9HOSP-01 of the present invention is provided. Including the following.

1.1 morphology

1.1.1 general form: after 2 days of cell inoculation, the growth state of the population cells was observed.

1.1.2 light mirror: the morphological structure and the growth characteristics of the cultured living cells are observed under an inverted phase contrast microscope.

1.1.3 Electron microscopy: and taking 21 substituted cells, and observing the ultrastructure of the cells by using a scanning electron microscope.

1.2 cell kinetics

1.2.1 plotting of cell growth curves and cell population doubling time

Cells in good logarithmic growth phase (passage 20) were collected, trypsinized, gently blown, diluted in multiples at 100uL (2X 10) per well³Individual cells) were seeded in 96-well plates to give a final cell concentration of 2 × 10 per well³One per mL, 6 duplicate wells per group. A total of 7 groups were prepared. After 24 hours, the culture medium in one set of 6-well cells was changed to 100uL of serum-free culture medium containing 10% CCK8, while 100uL of serum-free culture medium containing no CCK8 was added to 6 blank wells of a 96-well plate as a blank control, and after incubation at 37 ℃ for 2 hours, absorbance at 450nm was measured with a microplate reader. The cell growth curve was plotted on the horizontal axis of time and on the vertical axis of light absorption for 7 consecutive days. Cell doubling time was calculated by half height method. Calculating the formula: DT ═ T × lg2/lg (Nt/No). Wherein DT is cell doubling time, T is culture time, No is OD value recorded for the first time, and Nt is OD value after T time.

1.2.2 cell adherence Rate

Taking cells in logarithmic growth phase, observing good cell growth, digesting cells with pancreatin, gently blowing uniformly, diluting suspension, counting at 1 × 10 per hole⁴Inoculating the cells into a 24-well plate for 8 wells, slightly washing the cells which are not attached to the wall by using PBS at 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 10h respectively, fixing the attached cells by using 4% paraformaldehyde, photographing under an inverted microscope after 0.1% crystal violet staining, counting the number of the attached cells, and taking 5 random fields for each well. And calculating the cell anchorage rate at 0.25h, 0.5h, 1h, 2h, 4h, 6h and 8h by taking the cell anchorage number at 10h as the final anchorage number, and drawing a time-varying graph of the anchorage rate.

1.3 chromosome analysis

And (3) taking the cells with good growth condition for subculturing, observing the cells growing to 80% fusion degree the next day, adding colchicine to the final concentration of 0.2 mu g/mL, and continuously culturing in an incubator at 37 ℃ for 2 h. After the completion of the culture, the cells were digested with 0.25% trypsin, then the digestion was terminated with complete medium, and then they were transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 8min, and the supernatant was discarded. Gently blow the cell suspension with a pipette. 5mL of hypotonic solution (0.5% KCl solution) was added dropwise, and the cells were uniformly distributed in the suspension and placed in a 37 ℃ water bath for 20 min. 2mL of a freshly prepared fixative (methanol: glacial acetic acid ═ 3: 1) was added and pre-fixed for 2-3 min. Centrifuge at 800rpm for 8min and discard the supernatant. And 5mL of fresh fixing solution is added dropwise, the mixture is re-suspended and uniformly mixed, the mixture is fixed for 40min, then the mixture is centrifuged at 800rpm for 8min, and the supernatant is discarded. This fixing operation was repeated 2 times. After fixation, the cells were gently blown off with a pipette. The cell suspension was aspirated, dropped onto a pre-cooled slide, and the slide was passed through an alcohol lamp several times and dried at room temperature. And taking out the slide, and dropwise adding Giemsa working solution to complete covering, and dyeing for 20-30 min. And (5) washing with tap water to stop dyeing, and performing microscopic examination after natural air drying. The low power lens is used for comprehensive examination to find out the metaphase mitosis phase with uniform chromosome dispersion and moderate length, and then the high power lens (400x) is converted to take a picture after being observed by an oil lens. And calculating the number of chromosomes through pictures, observing the characteristics of the chromosomes, and pairing the karyotypes. Measuring the length values of the long arm and the short arm of the chromosome by imageJ software, and performing corresponding calculation. At least 30 chromosome groups are selected for each sample to be counted, and the normal karyotype ratio is calculated.

1.4 flow identification of cell types

The Schwann cell surface marker antigen S100B (Abnova, MAB14982, 0.5 ug/10) was used⁶Individual cells) PNF1-9HOSP-01 cells were subjected to flow cytometry analysis. 100uL of cell suspension (adjusted to 1X 10 concentration with flow staining buffer after cell counting)⁷and/mL), adding 0.5ug of primary antibody, incubating for 15-30min in a dark place at room temperature, adding 1-2 mL of flow type dyeing buffer solution for washing once, resuspending with 100uL of flow type dyeing buffer solution, adding 0.1uL of mouse-derived fluorescent secondary antibody, incubating for 15-30min in a dark place, adding 1-2 mL of flow type dyeing buffer solution for washing once, centrifuging for 5min at 300g, discarding the supernatant, resuspending with 500uL of flow type dyeing buffer solution, and immediately performing on-machine detection.

1.5 cellular immunofluorescence

Cell crawlers were prepared and fixed in 4% paraformaldehyde followed by staining for cellular immunofluorescence S100B (Sigma, S2532, 1: 500). Permeabilization was performed by incubation with PBS containing 0.1% Triton X-100 for 10 minutes at room temperature. The slide was washed 3 times with PBS for 5 minutes each. Blocking was performed by incubation with 3% BSA PBST (PBS + 0.1% Tween 20) for 30min at room temperature. Antibodies (1: 500) were diluted with freshly prepared blocking solution and incubated in a wet box for 1 hour at 37 ℃. Wash 3 times with PBS for 5min each. The secondary antibody was diluted with blocking solution and incubated at room temperature for 1 hour in the dark. Cells were washed 3 times with PBS in the dark for 5 minutes each. DAPI is added dropwise for nuclear counterstaining. The slides were photographed under an inverted fluorescence microscope and stored at-20 ℃.

Example 3 validation results

In this example, the neurofibroma cell line PNF1-9HOSP-01 of the present invention was verified by the above-described verification method.

Morphology 2..1

2.1.1 general form: as shown in FIG. 2, more novacells were seen, with clustered growth, irregular shape, close arrangement, well defined, and loss of contact inhibition.

2.1.2 light mirror: as shown in FIG. 2, the cultured living cells were observed under an inverted phase contrast microscope, and most of them were polygonal, abundant in cytoplasm, large and round in nucleus, many and distinct in nucleolus, and the division pattern was observed.

2.1.3 Electron microscopy: as shown in FIGS. 3A, 3B and 3C, scanning electron microscopy revealed wrinkles on the cell surface and microvilli or protrusions surrounding the wrinkles.

2.2 cell kinetics

2.2.1 plotting of cell growth curves and cell population doubling times: the 7-day growth curve of the cells is shown in FIG. 4. The cell doubling time was calculated to be 16.16 hours using the half height method.

2.2.2 cell adherence rate: as shown in fig. 5, the cell attachment rate varied with time, and most of the cells (more than 95%) could grow attached to the wall after 6 hours of planting.

2.3 chromosome analysis

As shown in FIG. 6, the karyotype of the neurofibroma cell line PNF1-9HOSP-01 of the present invention is: 56, XY. The abnormal chromosome karyotype not only shows the aneuploidy in the chromosome number, but also is accompanied by abnormal phenomena such as partial deletion of long arm and short arm and total deletion of short arm.

2.4 flow identification of cell types

As shown in FIG. 7, the content of S100B (+) cells of the neurofibroma cell line PNF1-9HOSP-01 of the present invention was 92.93% as determined by flow cytometry.

2.5 cellular immunofluorescence detection of S100B expression

As shown in fig. 8, using the cellular immunofluorescence method, it was detected that schwann cell-specific marker S100B was mainly expressed in the cytoplasm.

The PNF1-9HOSP-01 cell line is a benign tumor cell line which is autonomously established in China and is derived from human type I neurofibromatosis, has the genetic background of Chinese, and has incomparable advantages in the research of the human type I neurofibromatosis compared with the existing cell line pairs in foreign countries. The PNF1-9HOSP-01 cell is an immortalized benign cell, can stably survive and passage in vitro according to the culture condition provided by the invention, and provides a good material for the research related to neurofibroma.

The present invention has been described in relation to the above embodiments, which are only exemplary of the implementation of the present invention. It must be noted that the disclosed embodiments do not limit the scope of the invention. Rather, modifications and equivalent arrangements included within the spirit and scope of the claims are included within the scope of the invention.

Claims

1. A neurofibroma cell line is named PNF1-9HOSP-01, and the preservation number of the preservation center is CCTCC NO: C202006.

2. A method for constructing a neurofibroma cell line is characterized in that a telomerase reverse transcriptase gene and a CDK4/m antigen are transfected into primary isolated neurofibroma cells, and the cells are subjected to monoclonal screening to obtain an immortalized neurofibroma cell line.

3. The method of claim 2, wherein the primary isolated neurofibroma cells are transfected with telomerase reverse transcriptase gene prior to further transfection with CDK4/m antigen.

4. The method of construction according to claim 2, wherein in the method of construction the primary isolated neurofibroma cells are transfected with telomerase reverse transcriptase gene and CDK4/m antigen by a lentiviral suspension and a viral infection enhancing suspension.

5. The method of claim 4, wherein the viral infection enhancing suspension is a polybrene suspension.

6. The construction method according to claim 2, characterized by comprising:

in step S2, transfecting the cell culture to be transfected with a telomerase reverse transcriptase gene with a lentivirus suspension and a virus infection enhancing suspension, wherein the MOI value is 30, and medium replacement is performed during virus infection; subsequently, cell cultures transfected with telomerase reverse transcriptase gene were transfected with CDK4/m antigen using lentivirus suspension and virus infection-enhancing suspension with MOI value of 30, and medium replacement was performed during virus infection; and,

in the step S2, the working concentration of the virus infection enhancing suspension is 5-8 mug/ml.

7. The method of claim 6, wherein in step S2, the primary neurofibroma cell concentration in the cell culture to be transfected is 8 μ g/ml.