CN109486765B

CN109486765B - NF2-/-Method for establishing Schwann cell line of vestibular nerve sheath tumor and cell line thereof

Info

Publication number: CN109486765B
Application number: CN201710822683.2A
Authority: CN
Inventors: 刘丕楠; 赵赋; 何琪杨; 张晶; 陈阳
Original assignee: Individual
Current assignee: Beijing Neurosurgical Institute
Priority date: 2017-09-13
Filing date: 2017-09-13
Publication date: 2021-11-19
Anticipated expiration: 2037-09-13
Also published as: CN109486765A

Abstract

The invention discloses human NF2^‑/‑A method for establishing a schwann cell line of vestibular nerve sheath tumor and a cell line thereof. The establishing method comprises the following steps: A. schwann cells (Schwann cell) in tumors were isolated to obtain human NF2^‑/‑Primary schwann cells (human NF2)^‑/‑Schwann cell) for primary culture; B. establishment of immunodeficient sciatic nerve human NF2 in mouse^‑/‑Primary Schwann cell transplantation tumor model; C. screening transplanted tumors with the diameter of more than 1cm from sciatic nerves of mice; D. separating Schwann cells, and performing primary cell culture; E. carrying out continuous subculture for more than 5 generations; F. obtained by screening with human NF2^‑/‑The cell morphology and the main biological characteristics of the primary Schwann cells and the primary transplantation tumor cell strains are consistent, and the NF2 can be subjected to unlimited passage^‑/‑Vestibular nerve Schwann cell. The cell has a success rate of over 95 percent to grow out of the transplanted tumor.

Description

NF2-/-Method for establishing Schwann cell line of vestibular nerve sheath tumor and cell line thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to NF2^-/-A method for establishing a schwann cell line of vestibular nerve sheath tumor and a cell line thereof.

Background

The schwann cell is a main functional cell of the peripheral nervous system, not only has the functions of migration, adhesion, extracellular matrix production, myelination and peripheral nerve repair, but also can secrete a plurality of nerve factors and bioactive substances, promote the survival, inhibit the apoptosis and promote the regeneration of neurons and glial cells, thereby repairing the central nerve injury. The tumorous proliferation of schwann cells results in the formation of schwannoma, a benign tumor that extends the growth of peripheral nerves and can occur at various ages and in different sexes. When schwannoma presses the nerve tissue, sensory and motor dysfunction may occur, resulting in pain and numbness in the corresponding area, and loss of motor function. Schwannomas occur more frequently in cranial nerves than in peripheral nerves, with the most common type being vestibular schwannomas occurring in the vestibulocochlear nerve, commonly referred to as auditory neuroma. Most vestibular nerve sheath tumors are sporadic, can cause nerve function symptoms such as deafness and facial paralysis of patients, and can cause serious symptoms such as hydrocephalus and disturbance of consciousness when tumors grow, thereby being dangerous to life.

Neurofibromatosis Type 2 (NF 2) is an autosomal dominant genetic disease caused by mutations in the NF2 tumor suppressor gene on autosome 22q 12. The disease can cause multiple schwannoma in the whole body of a patient, while bilateral vestibular schwannoma (auditory neuroma) is a NF2 marker, and more than 95 percent of patients with NF2 can have bilateral vestibular schwanoma. The tumor can grow along with the tumor to cause serious symptoms such as progressive bilateral hearing impairment, facial paralysis, balance disorder and the like, endanger the life of a patient, cause social disorder of the patient and induce depression. The current main methods for treating NF2 vestibular nerve sheath tumor are surgical resection and radiation therapy, both of which cause cranial nerve damage, resulting in serious complications. Targeted drug therapy is the focus of current research, but to date, there is a lack of effective drug therapy. In addition, the NF2 gene is also an important gene which causes the formation of Sporadic schwannomas (Sporadic schwannomas), meningiomas, and the like.

In the pathological and pharmaceutical research process, the good nerve sheath tumor cell line can rapidly and deeply analyze the functions of tumor molecules and cells, screen potential therapeutic compounds and also be the basis for establishing a nerve sheath tumor animal model. However, only 1 cell line of NF2 vestibular schwannoma is currently available for cytology and laboratory animals worldwide, and was created by Gene Hung doctor et al, the U.S. House ear institute. They obtained tumor tissue from a 56 year old NF2 patient, cultured primary Schwann cell tumor, and succeeded in immortalizing the cells by introducing the E6-E7 gene of Human Papilloma Virus (HPV), a cell line called HEI193[1 ]. However, transfection of oncogenes resulted in changes in the molecular characteristics and cell morphology of the HEI193 cell line compared to primary cells, limiting its role as a model of human vestibular schwannoma [1 ]. In addition, the HEI193 cell line is taken from a 56-year-old NF2 patient, the NF2 gene is mutated into a 15-exon splicing site region, partial function deletion of the encoding protein Merl in is caused, and most NF2 patients are mutated into nonsense mutation or large fragment deletion to form truncated protein (truncating protein). Therefore, this cell line cannot be fully used to achieve the full biological characteristics of NF2 schwannomas.

There are also some laboratories that use primary culture cells of vestibular schwannoma to study in vitro drug experiments by improving the primary cell culture method, but they still have the following problems:

1) primary cells grow slowly, typically 7-14 days to 90% confluence.

2) After 5 generations of primary cells, the cell morphology is changed, the protein expression of S100 (Schwann cell characteristic expression protein) is reduced, part of cells are aged, and the key protein expression of main signal pathways is changed.

3) The number of cells in each generation is small, the tumorigenic rate in nude mice is low, and the number of cells required by animal experiments related to drug screening cannot be met.

Still other methods involve inoculating primary tumor cells cultured in vitro into immunodeficient mice in situ, and after a period of time the tumor grows to a certain volume, the tumor is removed and cultured again to establish a stable cell line.

Using the above method of transplantation tumor animal model, different molecular subtype cell lines of medulloblastoma have been successfully established. The method can avoid the long-time in-vitro culture medium screening process of the tumor cells, and provide a tumor microenvironment required by cell growth to the maximum extent. The screened transplanted tumor cells have no obvious difference from primary cells in the aspects of histopathology and related gene expression. However, in order to successfully implement this method, it is necessary to establish a good growth state of primary cells required for transplantation of tumor model and to have a certain tumorigenic rate. Moreover, for the transplantation of some tumor cells in situ, the operation is difficult.

Therefore, the prior art has yet to be developed.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a NF2^-/-An establishing method of a vestibular nerve Schwann cell line and cells thereof, aiming at solving the problem of NF2 in the prior art^-/-The vestibular nerve Schwann cell line cannot be well applied to experimental research.

In order to achieve the purpose, the invention adopts the following technical scheme:

NF2^-/-A method for establishing a vestibular nerve Schwann cell line. Wherein, the method comprises the following steps:

A. NF2 was paired with Advance DMEM/F12 medium supplemented with 10% fetal bovine serum and growth factors^-/-Primary culture is carried out on the schwann cell tumor of the vestibular nerve;

B. b, inoculating the primary cells obtained in the step A in a BALB/c immune deficiency mouse, and establishing a sciatic nerve transplantation tumor model;

C. taking transplanted tumor of mouse with tumor diameter larger than 1cm 28-42 days after tumor inoculation

D. Performing primary culture on the transplanted tumor cells obtained in the step C by using an advance DMEM/F12 culture medium added with 10% fetal bovine serum and growth factors;

E. after the growth rate of the transplanted tumor cells is stable, replacing with an advance DMEM/F12 culture medium added with 10% fetal calf serum for continuous subculture;

F. in the process of continuous subculture, NF2 which is consistent with the cell morphology 1 and the molecular biological shape of the primary cell strain and can be subjected to infinite passage is obtained by screening^-/-Vestibular nerve Schwann cell.

The method described in the above, wherein the step a specifically includes:

a1 taking pretreated vestibular nerve sheath tumor sample, and placing into a container156U/ml collagenase 1, 1.25U/ml dispase type 1, 10,000IU penicillin, 10,000IU streptomycin and 50mg/ml gentamycin in DMEM high sugar medium at 37 ℃ with 5% CO₂Digesting for a predetermined time under conditions of (a);

a2, adding the cell sample digested in the step A1 into a sample solution with the volume ratio of 1: 1, stopping the reaction by DMEM mixed solution of 10% fetal calf serum, and centrifuging to remove the supernatant;

a3, resuspending the cell pellet from step A2 in an Advance DMEM/F12 complete nutrient medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin double antibody, 0.5. mu.M forskolin, 10ng/ml beta-human neuregulin, 2.5ul/ml insulin;

a4, spreading the resuspended cell suspension on 35mm petri dish coated with polylysine/laminin, placing at 37 deg.C and 5% CO₂And (5) culturing.

A5, when the cultured cells in A4 reached 80-90% confluency, subcultured at a ratio of 1:2 until a predetermined number of primary cells were obtained.

The method described in the above, wherein the step B specifically includes:

b1, selecting a plurality of BALB/c immunodeficient mice with the age of 4-8 weeks and separating sciatic nerves of the mice;

b2, injecting the culture medium containing the primary cells obtained in the step A into sciatic nerve;

b3, suture wound and injecting antibiotics.

The method described in the above, wherein the step B2 specifically includes: 5 mul of culture medium containing primary cells is injected into sciatic nerve, the total cell concentration is 1 multiplied by 105 cells/m, and the injection time is 15-20 s.

The method described in the above, wherein the step F specifically includes: taking cell photos of each generation of cells at different growth densities, and observing whether the tumor cells grow in a bipolar form or not, the diameters and the volumes of the cells, and differences between the forms and the sizes of the cells of each generation;

after the growth speed of the cells is stable, extracting DNA of the cells to carry out gene sequencing of NF2 genes;

detecting the expression intensity of the S100 protein of the cells by a protein immunofluorescence staining method;

extracting intracellular protein every 5 generations, and identifying the expression condition of vestibular nerve sheath tumor related protein through an immunoblotting test;

cell line nodulation experiments were performed using BALB/c immunodeficient mice 4-8 weeks old and corresponding cell line growth curves were plotted.

NF2^-/-A vestibular nerve Schwann cell line obtained by the method according to any one of the above-mentioned methods.

NF2^-/-The vestibular nerve Schwann cell line is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 14310.

The cell, wherein the function of the Merl in protein of the cell is deleted.

The cell has a tumor-causing rate of 95% or more in BALB/c immunodeficient mice of 4-8 weeks old.

The cell has stable growth speed after 3 generations of subculture, and can be stably subcultured for more than 2 generations.

Has the advantages that: the invention provides NF2^-/-The establishment method of the vestibular nerve Schwann cell line and the cells thereof optimize the primary culture method of the vestibular nerve Schwann cell tumor, ensure that the primary cells have good state and higher tumorigenicity rate when the tumor transplantation method is used. The cell line obtained by the method has no exogenous gene transfection and retains NF2^-/-The cell morphology and the molecular biology characteristics of the Schwann cell tumor of the vestibular nerve. The gene mutation mode is more typical, the growth speed of the cell line is consistent after 3 generations, and the cell line can be stably passaged to more than 20 generations. In the process of continuous subculture, the Schwann cell specific protein S100 and the NF2 vestibular nerve sheath tumor related protein are stably expressed in the body of the immunodeficient mouse. In addition, the cell line can be proliferated in a large amount in a short time, the tumor causing rate of the immunodeficiency mice is 95 percent, and the requirements of related experiments such as in vitro drug screening and the like are met.

Drawings

FIG. 1 is NF2 according to an embodiment of the present invention^-/-A method flow chart of a method for establishing a vestibular nerve sheath tumor Schwann cell line.

FIG. 2 is a schematic diagram of the cell morphology of the transplanted tumor cells during passaging according to an embodiment of the present invention.

FIG. 3 is a growth curve of transplanted tumor cells according to an embodiment of the present invention.

FIG. 4 is a schematic diagram showing the sequencing alignment of the NF2 gene in the tumor sample of the patient with the transplanted tumor cell line according to an embodiment of the present invention.

FIG. 5 is a graph showing the immunofluorescence staining results for S100 protein of a transplanted tumor in accordance with an embodiment of the present invention.

FIG. 6 is a graph showing the results of an immunoblot assay according to an embodiment of the present invention.

FIG. 7 is a plot of cell line growth according to an embodiment of the present invention.

FIG. 8 is a schematic diagram of an experiment for tumor formation in immunodeficient mice according to an embodiment of the present invention.

Fig. 9 shows STR identification results of the embodiment of the present invention.

Fig. 10 shows STR identification results of the embodiment of the present invention.

Detailed Description

The invention provides NF2^-/-In order to make the object, technical scheme and effect of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

FIG. 1 shows NF2 according to an embodiment of the present invention^-/-A method for establishing a schwann cell line of vestibular nerve sheath tumor. As shown in fig. 1, the method comprises the steps of:

100. NF2 was paired with Advance DMEM/F12 medium supplemented with 10% fetal bovine serum and growth factors^-/-Primary culture is carried out on the Schwann cell tumor of the vestibular nerve.

200. And (3) inoculating the primary cells obtained in the step A into BALB/c immunodeficient mice, and establishing a sciatic nerve transplantation tumor model.

300. After 28-42 days of tumor inoculation, transplanted tumors of mice with tumor diameter larger than 1cm are taken.

400. The transplanted tumor cells obtained in step 300 were subjected to primary culture using an advance DMEM/F12 medium to which 10% fetal bovine serum and growth factors were added.

500. After the growth rate of the transplanted tumor cells is stabilized, continuous subculture is performed by using an advance DMEM/F12 medium added with 10% fetal bovine serum.

600. In the process of continuous subculture, NF2 which is consistent with the cell morphology and the molecular biological shape of the primary cell strain and can be subjected to infinite passage is obtained by screening^-/-Vestibular nerve Schwann cell.

Specifically, the screening and cell detection methods may specifically include the following:

1. and (3) taking cell photos of each generation of cells at different growth densities, and observing whether the tumor cells grow in a bipolar form or not, the cell diameters and the volumes of the tumor cells, and the difference between the form and the size of each generation of cells.

2. After the cell growth rate is stable, cell DNA is extracted for gene sequencing of NF2 gene.

3. The expression intensity of S100 protein of the cells was examined by protein immunofluorescence staining.

4. Extracting intracellular protein every 5 generations, and identifying the expression condition of vestibular nerve sheath tumor related protein through an immunoblot test.

5. Cell line nodulation experiments were performed using BALB/c immunodeficient mice 4-8 weeks old and corresponding cell line growth curves were plotted.

The establishment method is described in detail below with reference to specific examples, and is performed based on a method of transplanting a tumor, and can be roughly divided into the following 3 parts:

primary culture of schwannoma cell tumor of vestibular nerve sheath tumor:

firstly, a vestibular nerve sheath tumor sample is taken and placed in a DMEM medium under the aseptic condition, and is washed for 3 times by PBS, and then impurities such as blood clots, cystic changes and the like are removed. Cutting the tumor into 1mm with an ophthalmic scissors³Size ofAfter the fragments were placed in DMEM high-sugar medium containing 156U/ml collagenase type 1, 1.25U/ml dispase type 1, 10,000IU penicillin/streptomycin double antibody and 50mg/ml gentamicin at 37 ℃ with 5% CO₂Digesting for 18-20 hours under the condition of (1).

The digested cell sample was then added to a volume ratio of 1: after the reaction was terminated with 1 DMEM mixture of 10% fetal bovine serum, the supernatant was centrifuged. The cell pellet after supernatant removal was resuspended in an advance DMEM/F12 complete nutrient medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin diabody, 0.5. mu.M forskolin, 10ng/ml β -human neuregulin, 2.5ul/ml insulin.

The resuspended cell suspension was then plated on 35mm dishes coated with polylysine/laminin and placed at 37 ℃ with 5% CO₂And (5) culturing. After 24 hours of culture, the cells and tissue blocks were observed to adhere to each other, and the solution was changed every 2-3 days.

Finally, when cultured cells reached 80-90% confluency, subculture was performed at a ratio of 1:2 until a predetermined number of primary cells were obtained. Sampling the primary culture method described above can amplify up to 60 million primary cells in 7-14 days.

Secondly, establishing an immunodeficient mouse transplantation tumor model:

firstly, 5 BALB/c immunodeficient mice of 4-8 weeks old are selected, 8% chloral hydrate (0.4 ml/100 g) is used for carrying out intraperitoneal injection and anesthesia on nude mice, the thigh area of the right back is preserved and disinfected, and the left lateral decubitus is taken. After the right femoral position was determined, a parallel femoral incision was made 0.3-0.5cm caudal to the femur. The muscles were bluntly separated in the direction of the white line using vascular clamps, and the sciatic nerve between 2 muscles was seen.

The sciatic nerve can then be picked up using the toothless forceps. After the sciatic nerve was isolated, a micro-syringe needle was inserted parallel to the nerve, and 5. mu.l of a culture medium containing primary cells was injected into the sciatic nerve at a total cell concentration of 1X 10⁵One cell per m, and the cell injection time is kept between 15 and 20 s.

And finally, after the operation is finished and the wound is sutured, injecting antibiotics into the abdominal cavity of the nude mouse to prevent infection, and keeping the temperature until the nude mouse revives.

After inoculation, tumor growth was observed every 7 days. In this example, after about 28-42 days of tumor inoculation, 2 nude mice had a tumor bulge on the caudal side of the right femur, with a tumor-causing rate of 40%. At about 28-35 days, nude mice were sacrificed when the maximum diameter of the tumor exceeded 1cm, the skin was incised along the original surgical incision, tumor samples were separated into DMEM medium, and transported to the laboratory for cell culture at 4 ℃.

Thirdly, primary tumor cell transplantation culture:

and (4) washing the transplanted tumor sample obtained in the second step with PBS (phosphate buffer solution) for 3 times to remove impurities such as blood clots. First, the tumor was cut to 1mm with an ophthalmic scissors³The fragments were placed in 20ml of DMEM high-sugar medium containing 156U/ml collagenase type 1, 1.25U/ml dispase type 1, 10,000IU penicillin/streptomycin diabase and 50mg/ml gentamicin at 37 ℃ with 5% CO₂Digestion was carried out under conditions for 12 hours.

After digestion was complete, the volume 1: 1 DMEM mixture containing 10% fetal bovine serum was added to stop the reaction. Centrifuging at 800r/min for 5min to remove supernatant. Then, the cell pellet was suspended in an advanced DMEM/F12 complete nutrient medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin double antibody (Gibco), 0.5. mu.M forskolin, 10ng/ml β -hereugin (β -human neuregulin) and 2.5ul/ml insulin.

Finally, the resuspended cell suspension was plated on a polylysine/laminin coated 100mm dish and placed at 37 ℃ with 5% CO₂And (5) culturing.

After 18-24 hours of culture, cell and tissue block adherence was seen. Fluid was changed every 2 days, 1:2 passages when cells reached 80% -90% confluence. After passage to 6 th generation, 10% fetal calf serum and 1% penicillin/streptomycin double-antibody advance DMEM/F12 medium are changed to continue culture to screen and establish NF2^-/-Schwann cell line of vestibular schwann nerve sheath tumor.

Fourthly, cell identification:

the cells can be evaluated or identified as a whole using different methods and at different angles of judgment to determine whether they can be used for experiments.

It may specifically include:

1) and taking cell pictures of each generation of cells at different growth densities, observing whether the tumor cells grow in a bipolar form or not, and observing the cell diameter and the cell volume, and comparing the difference between the form and the size of each generation of cells.

2) And after the cell growth speed is stable, extracting cell DNA and carrying out gene sequencing of NF2 gene.

3) And detecting the expression intensity of the S100 protein of the cells by a protein immunofluorescence staining method.

4) And extracting intracellular protein every 5 generations, and identifying the expression condition of the vestibular nerve sheath tumor related protein through an immunoblotting test.

5) And performing a cell line tumorigenic experiment by using BALB/c immunodeficient mice of 4-8 weeks old, and drawing a corresponding cell line growth curve.

In this example, specific cell identification results are as follows:

1) as shown in fig. 2, the transplanted tumor cells grow in bipolar form, have long cell diameter and small volume during passage, and have no obvious change in cell form and size of each generation. As shown in FIG. 3, the cell growth curve is shown. From this curve, it can be seen that the growth rate of tumor cells was stable after 3 passages.

2) As shown in FIG. 4, the C > T nonsense mutation exists between the transplanted tumor cell line and 169bp position of NF2 gene No. 2 EXON (EXON2) of a patient tumor sample.

3) As shown in fig. 5, the results of immunofluorescence staining of S100 protein for primary cells, 5 generations, 10 generations, and 15 generations of the transplanted tumor sample respectively show that: the expression of the primary cell S100 is strongest, and the expression intensity of the S100 protein of the 5-15 generation cells is consistent.

4) FIG. 6 shows the results of immunoblotting (western blot) performed on each 5 th generation of extracted proteins, which can be used to identify the expression of vestibular schwannoma-associated proteins. Wherein, the samples in lanes 1-10 are respectively: 1. primary tumor cells; 2-5 are respectively: NF2 using growth factor supplemented media^-/-Schwann cell lines primary (2), passage 5 (3), passage 10 (4) and passage 15 (5); 6-9 are respectively: culturing with growth factorRadical NF2^-/Vestibular schwannomas schwann cell lines passage 1 (6), passage 5 (7), passage 10 (8), passage 15 (9); 10. NF2^-/-Primary tumor sample is transplanted to schwann cell line of vestibular nerve sheath tumor. As shown in FIG. 5, the degree of expression of primary transplanted tumor cells was consistent with that of Merl in, PDGFR-beta, YAP, Erk1/2, p-S6 and cyclin in tumor samples, and the in vitro culture had a greater effect on the expression of related proteins

5) As shown in FIGS. 7 and 8, the cell line growth curves are shown schematically. In this experiment, 20 BALB/c immunodeficient mice 4-8 weeks were selected for the cell line oncosis experiment and the tumor growth volume was measured every 7 days. After 14 days, the right sciatic nerve of 19 nude mice can see tumor growth, and the tumor formation rate is 95% after entering logarithmic growth phase.

6) As shown in FIGS. 9 and 10, the results of the identification of the cell line STR (short repeat). By aligning the identification results in the DSMZ database, the matching degree is 72%.

In summary, compared with the existing HEI193 cell line, the cell line established by the method provided by the embodiment of the invention has the following advantages:

first, no transfection of foreign genes, NF2 was retained^-/-The cell morphology and the molecular biology characteristics of the Schwann cell tumor of the vestibular nerve. Moreover, the mode of gene mutation is more typical, and the function of Merlin protein is lost. In addition, the growth speed of the cell line is consistent after 3 generations, and the cell line can be stably passaged to more than 20 generations; the Schwann cell specific protein S100 is stably expressed, and the NF2 vestibular nerve sheath tumor related protein is stably expressed in the body of an immunodeficient mouse. And the cell line can be proliferated in a large amount in a short time, the tumor causing rate of the immunodeficiency mice is 95 percent, and the requirements of related experiments such as in vitro drug screening and the like are met.

^-/-The biological material of NF2 vestibular nerve sheath tumor Schwann cell line established by the embodiment of the invention is preserved in Chinese microorganism General microbiological center of the Committee for culture Collection;

the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North;

^-/and (3) classification and naming: human NF2 schwann cell line;

the preservation date is as follows: year 2017, month 07, 03

The preservation number is: 14310.

it should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Claims

1. NF2^-/-The vestibular nerve Schwann cells are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC 14310.

2. The cell of claim 1, wherein the cell has a loss of Merlin protein function.

3. The cell of claim 1, wherein the cell has an oncogenic rate of 95% or greater in BALB/c immunodeficient mice of 4-8 weeks of age.

4. The cell according to claim 1, wherein the cell grows stably after 3 passages and can be stably subcultured for more than 2 passages.