CN109486765A - A kind of NF2-/-The method for building up and its cell line of vestibular schwannomas schwann cell system - Google Patents
A kind of NF2-/-The method for building up and its cell line of vestibular schwannomas schwann cell system Download PDFInfo
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Abstract
The invention discloses people NF2‑/‑The method for building up and its cell line of vestibular schwannomas schwann cell system.The method for building up includes: A, separates to the schwann cells (Schwann cel l) in tumour, obtains people NF2‑/‑Primary schwann cells (human NF2‑/‑Schwann cel l) carry out originally culture；B, immunodeficient mouse sciatic nerve people NF2 is established‑/‑Primary Schwann cell transplantation tumor model；C, the transplantable tumor that diameter is greater than 1cm is screened from mouse sciatic nerve；D, schwann cells are separated, primitive cell culture is carried out；E, the continuous 5 generation above secondary culture is carried out；F, it is obtained and people NF2 in screening‑/‑Primary schwann cells, transplantable tumor primary cell strain cellular morphology and main biological property NF2 that is consistent, and can infinitely passing on‑/‑Vestibular nerve schwann cell.The cell has 95% or more success rate to grow transplantable tumor.
Description
Technical field
The present invention relates to technical field of cell culture more particularly to a kind of NF2-/-Vestibular schwannomas schwann cell system
Method for building up and its cell line.
Background technique
Schwann cell is the chief functional cells of peripheral nervous system, it not only has migration, adhesion, generates extracellular base
Matter forms myelin, participates in the function of Peripheral nerve repair, can also secrete a variety of neural factors and bioactive substance, promotes mind
Through first and Deiter's cells survival, inhibit its apoptosis and promote its regeneration, so that Central nervous damage is repaired.It applies
The generation neoplastic hyperplasia of prosperous cell will form neurinoma (also known as schwann's cell tumor, schwannoma), which is good
Property tumour, prolong peripheral nerve growth, can various ages, different sexes can occur.When neurinoma pressuring nerve tissue, then
Feeling and dyskinesia can occur, cause corresponding position that pain and numb and loss of motor function occurs.Neurolemma
Tumor betides that cranial nerve is more common compared with peripheral nerve person, and one of the most common type is the vestibular mind for betiding vestibulocochlear nerve
Through sheath tumor, commonly known as acoustic neurinoma.Most of vestibular schwannomas are to distribute, and can cause the nerves such as patient's deafness, facial paralysis
Function symptom can cause the serious symptoms such as hydrocephalus, the disturbance of consciousness, crisis life when tumour increases.
2 type neurofibromatosises (Neurofibromatosis Type 2, NF2) are a kind of autosomal dominants
Disease is caused by the NF2 tumor suppressor gene mutation on autosome 22q12.The nerve that the disease can cause entire patient multiple
Sheath tumor, and bilateral vestibular schwannomas (acoustic neurinoma) is NF2 symbolic characteristic, 95% or more NF2 patient can occur double
Side vestibular schwannomas.The tumour with tumour growth, can cause the hearing impairment of the bilateral of progressive, facial paralysis, paralysis and flat
Weigh the serious symptoms such as obstacle, jeopardizes patient vitals, and cause patient human communication disorders occur and induce depression.Treatment NF2 at present
The main method of vestibular schwannomas only operation excision and radiotherapy, both treatments can cause injury of cranial nerve, lead
Cause serious complication.Targeted drug treatment is the emphasis studied at present, but up to the present, lacks effective drug therapy side
Method.In addition, NF2 gene is also the important base for leading to distribute the formation such as neurinoma (Sporadic schwannoma), meningioma
Cause.
During pathology and drug research, good neurinoma cell line can quickly, in depth analyze tumor cells
With the function of cell, and potential therapeutic compounds is screened, and establish the basis of neurinoma animal model.So
And the whole world only has 1 at present for cytology and experimental animal NF2 vestibular schwannomas cell line, is ground by U.S.'s House ear
Gene doctor Hung et al. creation.They obtain tumor tissues for 56 years old from one with NF2 patient, cultivate it is primary apply it is prosperous
Schwann Cells tumor, and the E6-E7 gene by importing human papilloma virus (HPV) successfully makes cell immortality, which is known as
HEI193[1].But the transfection of oncogene causes HEI193 cell line compared with primary cell, in characterization of molecules and cell
It changes in form, limits its effect [1] as people's vestibular schwannomas model.In addition, HEI193 cell line is derived from
56 years old NF2 patients, NF2 gene mutation are 15 exon shearing site regions, and coding albumen Merl in partial function is caused to lack
It loses, most NF2 patient mutations are nonsense mutation or large fragment deletion, form truncation albumen (truncat ing
protein).Therefore, which can not be entirely used for realizing all biological feature of NF2 neurinoma.
There are also some laboratories by improve primitive cell culture method, using vestibular schwannomas primary cultured cell into
The research of the external drug study of row, but it still has following problem:
1) primary cell slow growth grows to 90% in general 7~14 days and converges.
2) after primary cell reached for 5 generations, cellular morphology changes, S100 (schwann's cell characteristic expresses albumen) egg
White expression decline, part cell ageing, the expression of primary signal pathways key protein change.
3) per generation cell quantity is less, and tumorigenesis rate is lower in nude mice, is not able to satisfy the relevant zoopery of drug screening
Required cell quantity.
It there are also certain methods is passed through in the primary tumor cell in-situ inoculating to immunodeficient mouse body by vitro culture
A period of time tumour growth to certain volume takes tumour to carry out cell culture again, to establish stable cell line.
Using the method for above-mentioned transplantable tumor animal model, it is successfully established medulloblastoma different molecular sub-types of cells system.
This method can utmostly provide swollen needed for cell growth to avoid the prolonged in-vitro culture medium screening process of tumour cell
Tumor microenvironment.Transplanting oncocyte after screening is in terms of histopathology and related gene expression with primary cell ratio without obvious poor
It is different.It but is successfully to realize this method, primary cell growth conditions needed for needing to establish Transplanted tumor model are good and have certain cause
Ratio of outflow.Moreover, the orthotopic transplantation for some tumour cells, surgical procedure are more difficult.
Therefore, there are also to be developed for the prior art.
Summary of the invention
Place in view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of NF2-/-Vestibular nerve is applied prosperous
The method for building up and its cell of cell line, it is intended to solve NF2 in the prior art-/-Vestibular nerve schwann cell system can not be good
The problem of applied to experimental study.
In order to achieve the above object, this invention takes following technical schemes:
A kind of NF2-/-The method for building up of vestibular nerve schwann cell system.Wherein, this method comprises:
A, using 10% fetal calf serum of addition and the advance DMEM/F12 culture medium of growth factor to NF2-/-Before
Front yard nerve schwann cells tumor carries out originally culture；
B, it is seeded in the primary cell of step A acquisition in BALB/c immunodeficient mouse, establishes sciatic nerve transplantable tumor
Model；
C, after inoculated tumour 28-42 days, the transplantable tumor of mouse of the diameter of tumor greater than 1cm is taken
D, using the advance DMEM/F12 culture medium of 10% fetal calf serum and growth factor is added to taking in step C
The transplanting oncocyte obtained carries out originally culture；
E, after the transplantable tumor vitro growth rates are stablized, the advance DMEM/ that 10% fetal calf serum is added is used instead
F12 culture medium carries out continuous passage culture；
F, in continuous passage incubation, screening is obtained and primary cell strain cellular morphology 1 and molecular biology shape
Unanimously, and the NF2 that can infinitely pass on-/-Vestibular nerve schwann cell.
The method, wherein the step A is specifically included:
A1, take pretreatment after vestibular schwannomas tumor sample, be put into containing 1 collagen type enzyme of 156U/ml,
1 type dispase of 1.25U/ml, 10,000IU penicillin, 10,000IU streptomysin and 50mg/ml gentamicin DMEM high sugar
In culture medium, at 37 DEG C, 5%CO2Under conditions of digest the scheduled time；
A2, the DMEM mixing that the postdigestive cell sample of step A1 is added to 10% fetal calf serum that volume ratio is 1:1
After liquid terminates reaction, supernatant is removed in centrifugation；
A3, will be gone in step A2 the cell precipitation after supernatant be resuspended to be added to 10% fetal calf serum, 1% penicillin/
Streptomysin is dual anti-, the advance DMEM/ of 0.5 μM of forskolin, 10ng/ml β-rhNRG-1beta S177-Q237,2.5ul/ml insulin
In the full nutrient medium of F12；
A4, it the cell suspension after resuspension is laid on is coated in poly-D-lysine/laminin 35mm culture dish, set
37 DEG C, 5%CO2Culture.
When A5, the culture cell in A4 reach 80-90% fusion, secondary culture is carried out according to the ratio of 1:2, until obtaining
Obtain the primary cell of predetermined quantity.
The method, wherein the step B is specifically included:
B1, the BALB/c immunodeficient mouse for choosing several 4-8 week old simultaneously separate its sciatic nerve；
B2, the culture medium that the primary cell obtained in A containing step is injected in sciatic nerve；
B3, sewing-up cut and injection of antibiotics.
The method, wherein the step B2 is specifically included: the culture medium containing primary cell is injected into sciatic nerve
5 μ l, total cell concentration are 1 × 105/m, and injection time is 15~20s.
The method, wherein the step F is specifically included: it is thin in different stand densities to shoot every generation cell
Born of the same parents' photo, observation tumour cell whether bipolar morphology growth, cell dia, volume, and between per generation cellular morphology, size
Difference；
After vitro growth rates are stablized, the gene sequencing that cell DNA carries out NF2 gene is extracted；
By protein immunization fluorescence colour, the expression intensity of the S100 albumen of cell is detected；
At interval of 5 generations, intracellular protein is extracted, by western blot test, identifies vestibular schwannomas GAP-associated protein GAP
Expression；
Cell line tumor formation experiment is carried out using the BALB/c immunodeficient mouse of 4-8 week old, and draws corresponding cell line
Growth curve.
A kind of NF2-/-Vestibular nerve schwann cell system, wherein as above any method for building up obtains for application.
A kind of NF2-/-Vestibular nerve schwann cell system is preserved in Chinese microorganism strain with deposit number CGMCC 14310
Preservation administration committee common micro-organisms center.
The cell, wherein the Merl in protein function of cell lacks.
The cell, wherein cell tumorigenesis rate in the BALB/c immunodeficient mouse of 4-8 week old is 95% or more.
The cell, wherein cell after 3 generation of secondary culture the speed of growth stablize, can stablize 2 generation of secondary culture with
On.
The utility model has the advantages that a kind of NF2 provided by the invention-/-The method for building up and its cell of vestibular nerve schwann cell system, it is excellent
Change the primary culture method for vestibular nerve schwannoma, it is ensured that when using transplantable tumor method, primary cell
It is in good condition, tumorigenesis rate with higher.Transfection of the cell line without foreign gene is obtained by such method, is remained
NF2-/-The cellular morphology and molecular biological characteristics of vestibular nerve schwannoma.Its gene mutation mode is more typical, cell line
The speed of growth is consistent after 3 generations, can stablize and it is more than generation to be passaged to 20.In continuous passage incubation, schwann cell specific proteins
S100, NF2 vestibular schwannomas GAP-associated protein GAP express stabilization in Immune deficient mice body.Also, cell line can be in a short time
A large amount of proliferation, immunodeficient mouse tumorigenesis rate are 95%, meet the related experiments demand such as external drug screening.
Detailed description of the invention
Fig. 1 is the NF2 of the specific embodiment of the invention-/-The method stream of the method for building up of vestibular schwannomas schwann cell system
Cheng Tu.
Fig. 2 is cellular morphology schematic diagram of the transplanting oncocyte of the specific embodiment of the invention in succeeding generations.
Fig. 3 is the growth curve of the transplanting oncocyte of the specific embodiment of the invention.
Fig. 4 is that the transplantable tumor cell line of the specific embodiment of the invention and the sequencing of patient tumors sample NF2 gene compare and show
It is intended to.
Fig. 5 is the S100 protein immunization fluorescent staining result figure of the transplantable tumor of the specific embodiment of the invention.
Fig. 6 is the result figure of the western blot test of the specific embodiment of the invention.
Fig. 7 is the cell line growth curve of the specific embodiment of the invention.
Fig. 8 is the schematic diagram that the immunodeficient mouse tumor formation of the specific embodiment of the invention is tested.
Fig. 9 is the STR qualification result of the specific embodiment of the invention.
Figure 10 is the STR qualification result of the specific embodiment of the invention.
Specific embodiment
The present invention provides a kind of NF2-/-The method for building up and its cell of vestibular schwannomas schwann cell system are to make the present invention
Purpose, technical solution and effect it is clearer, clear, as follows in conjunction with drawings and embodiments further specifically to the present invention
It is bright.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not intended to limit the present invention.
Fig. 1 is a kind of NF2 of the specific embodiment of the invention-/-The method for building up of vestibular schwannomas schwann cell system.Such as figure
Shown in 1, this method comprises the following steps:
100, using 10% fetal calf serum of addition and the advance DMEM/F12 culture medium of growth factor to NF2-/-
Vestibular nerve schwann cells tumor carries out originally culture.
200, it is seeded in the primary cell of step A acquisition in BALB/c immunodeficient mouse, establishes sciatic nerve transplanting
Tumor model.
300, after inoculated tumour 28-42 days, the transplantable tumor of mouse of the diameter of tumor greater than 1cm is taken.
400, using 10% fetal calf serum of addition and the advance DMEM/F12 culture medium of growth factor to step 300
The transplanting oncocyte of middle acquirement carries out originally culture.
500, after the transplantable tumor vitro growth rates are stablized, the advance that 10% fetal calf serum is added is used instead
DMEM/F12 culture medium carries out continuous passage culture.
600, in continuous passage incubation, screening is obtained and primary cell strain cellular morphology and molecular biology shape
Unanimously, and the NF2 that can infinitely pass on-/-Vestibular nerve schwann cell.
Specifically, screening and cell detection method can specifically include it is following several:
1, cell photo of every generation cell in different stand densities is shot, whether bipolar morphology is raw for observation tumour cell
Length, cell dia, volume, and the difference between per generation cellular morphology, size.
2, after vitro growth rates are stablized, the gene sequencing that cell DNA carries out NF2 gene is extracted.
3, by protein immunization fluorescence colour, the expression intensity of the S100 albumen of cell is detected.
4, at interval of 5 generations, intracellular protein is extracted, by western blot test, identifies vestibular schwannomas GAP-associated protein GAP
Expression.
5, cell line tumor formation experiment is carried out using the BALB/c immunodeficient mouse of 4-8 week old, and draws corresponding cell
It is growth curve.
Below in conjunction with specific embodiment, the method for building up is described in detail, which is carried out based on the method for transplantable tumor,
Following 3 parts can be greatly classified into:
One, vestibular schwannomas schwannoma originally culture:
Firstly, vestibular schwannomas tumor sample is aseptically taken to set in DMEM culture medium, rinsed 3 times with PBS
Afterwards, the impurity such as clot, capsule change are removed.Tumour is cut into 1mm with eye scissors3After the fragment of size, it is put into containing 1 type glue of 156U/ml
Former protease, 1 type dispase of 1.25U/ml, 10,000IU penicillin/streptomycin be dual anti-and the DMEM of 50mg/ml gentamicin
In high glucose medium, at 37 DEG C, 5%CO2Under conditions of digest 18-20 hours.
Then, the DMEM mixed liquor for postdigestive cell sample being added to 10% fetal calf serum that volume ratio is 1:1 is whole
After only reacting, supernatant is removed in centrifugation.Cell precipitation after removing supernatant, which is resuspended to, is added to 10% fetal calf serum, 1% penicillin/chain
Mycin is dual anti-, the advance DMEM/F12 of 0.5 μM of forskolin, 10ng/ml β-rhNRG-1beta S177-Q237,2.5ul/ml insulin
In full nutrient medium.
Then, the cell suspension after resuspension is laid on and is coated with poly-D-lysine/laminin 35mm culture dish
In, 37 DEG C are set, 5%CO2Culture.Visible cell and tissue block adherent after 24 hours of incubation change liquid in every 2-3 days.
Finally, secondary culture is carried out according to the ratio of 1:2, until obtaining in advance when culture cell reaches 80-90% fusion
The primary cell of fixed number amount.Primary cell can be expanded to 600,000 in 7-14 days by sampling above-mentioned primary culture method.
Two, Immune deficient mice Transplanted tumor model is established:
Firstly, the BALB/c immunodeficient mouse of 5 4-8 week old is chosen, with 8% chloraldurate (0.4ml/100g) abdomen
Chamber injecting anesthetic nude mice, right side back femoral region preserved skin, disinfection, takes left lateral position.Determine that the right lateral thigh position of bone postpones, in femur
At caudal 0.3-0.5cm, the notch of parallel femur is done.With vessel forceps along white line direction blunt separation muscle, it is seen that 2 pieces of muscle it
Between sciatic nerve.
It is then possible to provoke sciatic nerve using Smooth forceps.After isolating sciatic nerve, micro syringe syringe needle is parallel
Neural inserting needle, the 5 μ l of culture medium containing primary cell is injected into sciatic nerve, and total cell concentration is 1 × 105A/m, injection are thin
Born of the same parents are maintained at 15~20s the time.
Finally, finished in operation, after sewing-up cut, the injection of antibiotics prevention infection into nude mice abdominal cavity, heat preservation to revival.
After inoculation, every 7 days observation tumour growth situations.In the present embodiment, after inoculated tumour about 28-42 days, 2
The right femur caudal visual tumors protrusion of nude mice, tumorigenesis rate is 40%.At about 28-35 days, longest diameter of tumor was put to death when being more than 1cm
Nude mice cuts skin along former operative incision, separates tumor sample into DMEM culture medium, and 4 DEG C of transports to laboratory carry out cell
Culture.
Three, transplantable tumor primitive cell culture:
The transplantable tumor sample that step 2 is obtained is rinsed 3 times with PBS, to remove the impurity such as clot.Firstly, will with eye scissors
Tumour is cut into 1mm3The fragment of size, be put into 20ml containing 1 collagen type enzyme of 156U/ml, 1 type dispase of 1.25U/ml,
In the dual anti-DMEM high glucose medium with 50mg/ml gentamicin of 10,000IU penicillin/streptomycins, 37 DEG C, 5%CO2Condition
12 hours of lower digestion.
After the completion of digestion, reaction is terminated by DMEM mixed liquor of the addition containing 10% fetal calf serum of volume 1:1. 800r/
Min centrifugation 5min removes supernatant.Then, cell precipitation is suspended in and is added to 10% fetal calf serum, 1% penicillin/streptomycin pair
Anti- (Gibco), 0.5 μM of forskol in (forskolin), 10ng/ml β-hereugl in (β-rhNRG-1beta S177-Q237) and
In the full nutrient medium of advance DMEM/F12 of 2.5ul/ml insulin.
It is coated in poly-D-lysine/laminin 100mm culture dish finally, the cell suspension after resuspension is laid on,
37 DEG C are set, 5%CO2Culture.
After culture 18-24 hours, it is seen that cell and tissue block adherent.Changed liquid every 2 days, when cell reach 80%~
1:2 is passed on when 90% fusion.After being passaged to for the 6th generation, it is changed to 10% fetal calf serum and 1% penicillin/streptomycin is dual anti-
Continue to cultivate in advance DMEM/F12 culture medium, to screen, establish NF2-/-Vestibular schwannomas schwann cell system.
Four, cellular identification:
Different methods can be used, in different judgement angles, whole evaluation or identification are carried out to cell, is determined
Its use that whether can satisfy experiment.
It can specifically include:
1), shoot the cell photo of every generation cell in different stand densities, observation tumour cell whether bipolar morphology
Growth, cell dia, volume, and the difference between more per generation cellular morphology, size.
2), after vitro growth rates are stablized, the gene sequencing that cell DNA carries out NF2 gene is extracted.
3), by protein immunization fluorescence colour, the expression intensity of the S100 albumen of cell is detected.
4), at interval of 5 generations, intracellular protein is extracted, by western blot test, identifies vestibular schwannomas GAP-associated protein GAP
Expression.
5) cell line tumor formation experiment, is carried out using the BALB/c immunodeficient mouse of 4-8 week old, and draws corresponding cell
It is growth curve.
In the present embodiment, specific cellular identification result is as follows:
1) as shown in Fig. 2, transplanting oncocyte is in succeeding generations, with bipolar morphology growth, cell dia is longer, volume compared with
It is small, and per generation cellular morphology, size are without substantially changeing.As shown in figure 3, being cell growth curve.It can be seen according to the curve
Out, 3 instead of after tumour cell the speed of growth stablize.
2), as shown in figure 4, transplantable tumor cell line and patient tumors sample NF2 gene 2 exon (EXON2) 169bp
There are C > T nonsense mutations for position.
3) as shown in figure 5, it is glimmering to transplantable tumor sample primary cell, 5 generations, 10 generations, 15 generations progress S100 protein immunization respectively
Light dyes as the result is shown: primary cell S100 expression is most strong, and 5-15 is consistent for cell S100 protein expression intensity.
4) Fig. 6 is extraction albumen progress of every 5 generation western blot test (western blot) as a result, can be used for identifying
The expression of vestibular schwannomas GAP-associated protein GAP.Wherein, the sample of swimming lane 1-10 respectively is: 1, primary tumor cell；
2-5 is respectively as follows: the NF2 using addition growth factor culture medium-/-Schwann cell system primary (2), the 5th generation (3), the 10th generation (4) with
And the 15th generation (5)；6-9 is respectively as follows: using the NF2 for not adding growth factor culture medium-/Vestibular schwannomas schwann cell system 1
Generation (6), the 5th generation (7), the 10th generation (8), the 15th generation (9)；10,NF2-/-The primary sample of vestibular schwannomas schwann cell system transplantable tumor
This.As shown in figure 5, transplantable tumor primary cell and tumor sample Merl in, PDGFR- β, YAP, Erk1/2, p-S6 and cell week
Phase protein expression degree is consistent, and in vitro culture is affected to correlative protein expression
It 5) as shown in Figure 7 and Figure 8, is the schematic diagram of cell line growth curve.In this experiment, 20 are selected 4-8 weeks
BALB/c immunodeficient mouse carried out cell line tumor formation experiment, every 7 days measurement tumour growth volumes.After 14 days, 19 it is naked
Mouse right sciatic nerves visual tumors growth, into logarithmic growth phase, tumor formation rate 95%.
It 6) as shown in Figure 9 and Figure 10, is cell line STR (short-movie section repetitive sequence) qualification result.By the way that the identification is tied
Fruit compares in DSMZ database, matching degree 72%.
In conclusion being established using method provided in an embodiment of the present invention thin compared with existing HEI193 cell line
Born of the same parents system has the advantage that
Firstly, the transfection without foreign gene, remains NF2-/-The cellular morphology and molecule of vestibular nerve schwannoma are raw
Object feature.Moreover, gene mutation mode is more typical, Merlin protein function is lost.In addition, the speed of growth after 3 generation of cell line
Unanimously, it can stablize and it is more than generation to be passaged to 20；Schwann cell specific proteins S100 stablizes expression, and NF2 vestibular schwannomas is related
Albumen expresses stabilization in Immune deficient mice body.And cell line can be largely proliferated in a short time, immunodeficient mouse tumorigenesis
Rate is 95%, meets the related experiments demand such as external drug screening.
The NF2 that the embodiment of the present invention is established-/-The biomaterial of vestibular schwannomas schwann cell system is preserved in the micro- life of China
Object culture presevation administration committee common micro-organisms center；
Preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；
Classification naming: people NF2-/Apply the strain of ten thousand Schwann Cells；
Preservation date: on 07 03rd, 2017
Deposit number: 14310.
It, can according to the technique and scheme of the present invention and this hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement all should belong to the guarantor of appended claims of the invention
Protect range.
Claims (10)
1. a kind of NF2-/-The method for building up of vestibular nerve schwann cell system characterized by comprising
A. the vestibular schwannomas sample of 2 type neurofibromatosis (NF2) patients is obtained during surgery, and separation obtains people NF2-/-
Primary schwann cells (human NF2-/-Schwann cell)。
B. using the advance DMEM/F12 culture medium that 10% fetal calf serum and growth factor is added, to people NF2-/-Vestibular
Neurinoma schwann cells carry out originally culture；
C. on more BALB/c immunodeficient mouse sciatic nerves, the primary schwann cells of step B acquisition are seeded in, establishes and sits
Bone nerve-grafting tumor model；
D. in the mouse after inoculated tumour 28-42 days, screening diameter of tumor is greater than the transplantable tumor of 1cm, carries out cell separation, screening
Schwann cells are identified out；
E. using the advance DMEM/F12 culture medium that 10% fetal calf serum and growth factor is added, to being obtained in step D
And the transplantable tumor schwann cells of identification are completed, re-start originally culture；
F. after the transplantable tumor vitro growth rates are stablized, the advance DMEM/F12 training that 10% fetal calf serum is added is used instead
It supports base and carries out continuous passage culture；
G. in continuous passage incubation, screening is obtained and people NF2-/-Vestibular schwannomas, transplantable tumor primary cell strain cell
Form and main biological property are consistent, and the people NF2 that can infinitely pass on-/-Vestibular nerve tumor schwann cell system.
H. it is implanted into again using this cell line on the sciatic nerve of BALB/c immunodeficient mouse and establishes Transplanted tumor model, success rate
95% or more, the biological study of NF2 neurinoma can be effectively used for.
2. the method according to claim 1, wherein the step A is specifically included:
A1, take pretreatment after vestibular schwannomas tumor sample, be put into containing 1 collagen type enzyme of 156U/ml,
The DMEM high sugar training of 1 type dispase of 1.25U/ml, 10,000IU penicillin, 10,000IU streptomysin and 50mg/ml gentamicin
It supports in base, at 37 DEG C, 5%CO2Under conditions of digest the scheduled time；
A2, the DMEM mixed liquor that the postdigestive cell sample of step A1 is added to 10% fetal calf serum that volume ratio is 1:1 are whole
After only reacting, supernatant is removed in centrifugation；
A3, the cell precipitation after supernatant will be gone to be resuspended in step A2 to be added to 10% fetal calf serum, 1% penicillin/strepto-
Plain dual anti-, 0.5 μM of forskolin, 10ng/ml β-rhNRG-1beta S177-Q237, the advance DMEM/F12 of 2.5ul/ml insulin are complete
In nutrient medium；
A4, it the cell suspension after resuspension is laid on is coated in poly-D-lysine/laminin 35mm culture dish, set 37
DEG C, 5%CO2Culture.
When A5, the culture cell in A4 reach 80-90% fusion, secondary culture is carried out according to the ratio of 1:2, until obtaining pre-
The primary cell of fixed number amount.
3. the method according to claim 1, wherein the step B is specifically included:
B1, the BALB/c immunodeficient mouse for choosing several 4-8 week old simultaneously separate its sciatic nerve；
B2, the culture medium that the primary cell obtained in A containing step is injected in sciatic nerve；
B3, sewing-up cut and injection of antibiotics.
4. according to the method described in claim 3, it is characterized in that, the step B2 is specifically included:
The 5 μ l of culture medium containing primary cell is injected into sciatic nerve, total cell concentration is 1 × 105/m, injection time 15
~20s.
5. the method according to claim 1, wherein the step F is specifically included:
Cell photo of every generation cell in different stand densities is shot, whether bipolar morphology grows, is thin observation tumour cell
Born of the same parents' diameter, volume, and the difference between per generation cellular morphology, size；
After vitro growth rates are stablized, the gene sequencing that cell DNA carries out NF2 gene is extracted；
By protein immunization fluorescence colour, the expression intensity of the S100 albumen of cell is detected；
At interval of 5 generations, intracellular protein is extracted, by western blot test, identifies the expression of vestibular schwannomas GAP-associated protein GAP
Situation；
Cell line tumor formation experiment is carried out using the BALB/c immunodeficient mouse of 4-8 week old, and draws corresponding cell line growth
Curve.
6. a kind of NF2-/-Vestibular nerve schwann cell system, which is characterized in that apply foundation a method as claimed in any one of claims 1 to 5
Method obtains.
7. a kind of NF2-/-Vestibular nerve schwann cell is preserved in Chinese microorganism strain preservation with deposit number CGMCC 14310
Administration committee's common micro-organisms center.
8. cell according to claim 7, which is characterized in that the Merlin protein function of cell lacks.
9. cell according to claim 7, which is characterized in that cell is in the BALB/c immunodeficient mouse of 4-8 week old
Tumorigenesis rate is 95% or more.
10. cell according to claim 7, which is characterized in that cell speed of growth after 3 generation of secondary culture is stablized, energy
It is more than generation to stablize secondary culture 2.
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| CN113151437A (en) * | 2021-02-01 | 2021-07-23 | 浙江理工大学 | Application of NF2 gene in preparing product for diagnosing abnormal development of skull |
| CN114807043A (en) * | 2022-03-24 | 2022-07-29 | 上海交通大学医学院附属第九人民医院 | Human-derived vestibular schlemma immortalized cell line and construction method thereof |
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| CN113151437A (en) * | 2021-02-01 | 2021-07-23 | 浙江理工大学 | Application of NF2 gene in preparing product for diagnosing abnormal development of skull |
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Effective date of registration: 20220711 Address after: 100070 No. 6, Tiantan Xili, Dongcheng District, Beijing Patentee after: BEIJING NEUROSURGICAL INSTITUTE Address before: No. 6, Temple of heaven, Dongcheng District, Beijing 100050 Patentee before: Zhao Fu |