CN105838677A - Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method - Google Patents
Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method Download PDFInfo
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Abstract
The invention belongs to the field of cell engineering, in particular to a method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and an application of the method. According to the invention, fusion genes are subjected to gene recombination to an AAVS1 (Adeno-Associated Virus Integration Site 1) transposon through a plasmid, and the freezing/thawing step is introduced to the NK cell expansion step taking a modified K562 cell line as a basis in the intermediate stage of cell expansion; finally, the NK cells are expanded and saved. A K562-mbIL15-41BBL-mbIL21 cell line developed by the integration technology of site specific genes is used, and the activity, purity and cytotoxicity of the NK cells are high to realize high expansion efficiency of the NK cells to improve NK expansion. The method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells and the application of the method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells disclosed by the invention have great market prospects and economic values.
Description
Technical field
The invention belongs to cell engineering field, be specifically related to the side of a kind of efficient amplification freezen protective NK cell
Method and application thereof.
Background technology
People's NKT (NK) cell is by expressing CD56 and lacking natural the exempting from of CD3 (CD-CD56+)
The lymphocyte composition of epidemic disease system.NK cell has the function of the killing tumor cell of wide spectrum, it is possible to logical
Cross cytotoxicity particle such as perforin and granzyme, and interacted by such as death receptor, Fas-Fa
S part combines the mechanisms play antitumor actions such as the apoptosis-inducing ligand combination relevant with TNF.NK
Cell can also pass through membrane receptor FcR γ III (CD16) mediate antibody dependent cellular cytotoxicity (ADCC).
The targets identification of NK cell and activation by suppression and activate NK cell surface receptor and express target cell
On this balance of associated ligands play a role, it allows pathogen invasion and cancerates and make an immediate response.
The mechanism of the targets identification NK cell of this uniqueness is different from major histocompatibility complex (MHC) limit
The T cell dependent mechanism of property φt cell receptor (TCR) processed.Therefore, allogeneic NK cell is used
Treat cancer and may will not cause graft versus host disease(GVH disease) (GVHD).
In health volunteer, NK cell constitutes about the 5-20% of PMNC (PBMC).
In adoptive immunotherapy, heavy dose of NK cell context is from 5 × 10^6-5 × 10^7/kg body weight every dose
Amount, most 4 dosage.In prior art, it is possible to the side of Enrichment Amplification NK cell from substantial amounts of peripheral blood
Method, majority is by mean of using invasive leucocyte to separate and Large Magnetic separator, and this mode is very
Costliness, and patient is caused certain risk.Another kind of popular NK cell therapy is to use feeder cells to expand
Increase NK cell, such as the film binding molecule of K562 cell modification, such as interleukin (IL)-15 and 4-1B
B part (K562-mb15-41BBL).These feeder cells can be promptly from PMNC
Amplification NK cell, makes the averagely amplification 21.6 times at the 7th day of NK cell, within the 21st day, expands 277 times.
But, the Telomerase caused due to aging shortens, so continuation amplification can be limited.
The heavy dose of NK cell of acquisition typically requires and expands several weeks, and the NK cell of feedback must be fresh,
The coordination that cell is cultivated between multiple dose cell infusion has extremely challenging.Therefore, amplification
After the frozen of NK cell be highly desirable to very much.But, traditional NK cell frozen to cell
There are injurious effects, as reduced NK cell viability, cytotoxicity and NK vital to function
The expression of cell receptor.Generally NK cell cannot crack cancer cell, the N of recovery after frozen defrosting immediately
K cell needs to stimulate cytotoxicity (Denman, the Seny of overnight frozen with recovery NK cell with IL-2
ukov et al.,2012；Lapteva N et al.,2012).Therefore, in prior art, cell is deposited
NK cell survival after activity remains a problem, and recovery can not be continued above one week.Meanwhile,
The NK cells containing sequences of recovery expands further still to be failed to understand.Therefore, this area is highly desirable to a kind of new at present
Holding NK cytoactive and original function and there is the cryopreservation methods of well amplification property.
Summary of the invention
To this end, the technical problem to be solved is, thus a kind of holding NK cytoactive is proposed,
Function and and the cryopreservation methods of amplification property.
For solving above-mentioned technical problem, the invention discloses the side of a kind of efficient amplification freezen protective NK cell
Method, described method by fusion is carried out genetic recombination to AAVS1 transposons by plasmid, and
The cell amplification interstage introduces the NK cell expansion based on freezing/thawing step extremely modifies K562 clone
Increase step；Finally carry out amplification and the preservation of NK cell.
Preferably, described fusion is that human chromosome 19 encodes mbIL15,4-1BBL and mbIL21.
Preferably, the gene expression plasmid skeleton of described fusion is by pFastBac1.
Preferably, the promoter of described plasmid backbone is cytomegalovirus.
Preferably, the restructuring of described plasmid uses the homologous recombination technique of Zinc finger nuclease-mediation.
Preferably, described genetic recombination step particularly as follows:
K562 cell is carried out suspension process；
PFastBac-ZFN and PFB-fusion host's electricity is gone to cell；
The cell of electroporation is subsequently transferred to K562 growth medium cultivate.
After cultivation, carry out expanding unicellular, clone and carry out pcr gene parting, flow cytometry,
Prepare the cell stably expressing fusion.
Preferably, the amplification of described NK cell concretely comprises the following steps:
First taking PMNC, line density of going forward side by side gradient method analyzes and processes；
Then use serum-free stem cell growth culture medium that described NK cell is expanded, and use PBMC
With gamma-radiation K562 feeder cells co-incubation in NK cell growth medium；
NK cell amplification obtained is at the freezing culture medium and 1 containing SCGM culture medium and 10%FBS
In the cryovial of 0%DMSO frozen；
It is more highly preferred to, carries out again after cell is placed in frozen process thawing and washing；Finally by defrosting
NK cell carries out stimulating cultivation with the ratio of 1:1 with K562 feeder cells again.
The method that the invention also discloses described efficient amplification freezen protective NK cell preserves neck at cell
Application in territory.
The technique scheme of the present invention has the following advantages compared to existing technology, by using site-specific
Property gene integration technology exploitation K562-mbIL15-41BBL-mbIL21 clone, high by having
NK cytoactive, purity and cytotoxicity, it is achieved high NK cell amplification efficiency is to improve NK amplification.
Accompanying drawing explanation
In order to make present disclosure be more likely to be clearly understood, being embodied as below according to the present invention
Example also combines accompanying drawing, and the present invention is further detailed explanation, wherein
Fig. 1 is the fusion process signal to K562 cell of the site-specific integration in embodiment
Figure；
Fig. 2 is that flow cytometry analysis mbIL15,4-1BBL and mbIL21 in embodiment is modifying
K 562 cell in protein expression level；
Fig. 3 is NK cells expanded in embodiment；
Fig. 4 is to use the purity ratio of NK cell after K562mbil15-41BBL-mbil21 in embodiment
Relatively；
Fig. 5 is NK cell-stimulating acceptor, suppression acceptor and NK cell marking after amplification in embodiment
Expression.
Fig. 6 is the killing activity expanding the NK cell obtained in embodiment to K562 human leukemia cell line
Schematic diagram；
Fig. 7 is that in embodiment, in the presence of anti-CD20 human antibody, NK cell antagonism RaJi B-cell drenches
The ADCC effect schematic diagram of bar clone；
Fig. 8 be in embodiment EpCAM specific C AR modify initial NK cell to PCRC7 Colon and rectum
The killing activity schematic diagram of cancer cell；
Fig. 9 is that the initial NK cell that in embodiment, EpCAM specific C AR modifies is thin to MCF7 breast cancer
The killing activity of born of the same parents.
Detailed description of the invention
Embodiment 1 present embodiment discloses a kind of method of efficient amplification freezen protective NK cell, specifically
Step is as follows:
1, the cultivation of human archeocyte system
Wild type K562 and Raji clone is obtained from American type culture collection (ATCC).
And these cells are placed on containing 10% hyclone (GIBCO) RPMI-1640 culture medium (GIBCO)
In, and in putting into 37 DEG C, 5%CO2Incubator in cultivate.
2, the structure of plasmid
It is used for expressing ZF as plasmid backbone by pFastBac1 (Invitrogen, Carlsbad, CA)
N and the fusion of coding mbil15,4-1BBL and mbIL21, wherein promoter is cytomegalovirus
(CMV).Wherein, it is reported (Tay, Tan et al.2 before the concrete structure of pFastBac-ZFN
013)。
For the structure of pFB-fusion host, the DNA fragmentation of this coding fusion is first by AI
The construct of synthesis, according to the sequence gene synthesis of report in prior art, is cloned into p by T biotechnology
UC57 (ammonia benzyl resistance), is then expanded this fragment by PCR.
It addition, CMV promoter, internal ribosome entry site (IRES), neomycin resistance gene (newly)
Expand also by PCR with WCHV posttranscriptional regulatory element (WPRE).All these fragments
The seamless clone of GENEART and assembling kit (Invitrogen) is used to be cloned into the matter of a pUC19
Grain.Fragment excision will be merged subsequently, use Asc/ClaI I to be cloned into pFastBac1 key, wherein
Comprise the left homology arm of the 810bp belonging to AAVS1 transposons and the right homology arm of a 837bp
(Tay, Tan et al.2013's).
3, the fusion of site-specific integration is to K562 cell AAVS1 locus
By 2 × 106Individual K562 clone is resuspended in the Opti-MEM of 100 μ l respectively.Use NE
PA 21 electroporation and Bio-Rad company test tube are by each pFastBac-ZFN and PFB-of 3 μ g
Fusion host's electricity goes to cell.The cell of electroporation is subsequently transferred to K fresh in 6 orifice plates
562 growth mediums.After electroporation the 5th day, under 200 μ g/mL concentration, carry out 1 month by a definite date
G418 selects.Culture medium is changed for every 2 days.After the selection of 1 month, use cell sorter, BD FA
CSAria (BD Biosciences company) is by unicellular 96 orifice plates being sorted into.Expand unicellular and
Clone carries out pcr gene parting and flow cytometry, to confirm the fusion of this site-specific integration
The expression of gene and stable expression.Pcr gene sorting is with whole with AAVS1 site of identification of cell clone
Close.The genomic DNA of cell, usesBlood and Tissue kit (Qiagen, Xi Erdeng,
Germany) separate.Pcr gene parting is used for detecting the site-specific integration of OKSM box.KAP
A high-fidelity thermal starting premixing (KAPA Biosystem, Woburn, MA) is used together, forward primer:
ATATTGCTGAAGAGCTTGGCGGCGAATGGG and reverse primer: CGGGGATGCAGGGGAACGGGGCT
CAGTCTG.Amplified production is analyzed on 1% Ago-Gel.
4, the amplification of NK cell and frozen
Take 20 healthy donor's PMNCs, and use Ficoll-Paque PREMIUM
(GE Medical Group life science), carries out density gradient from the fresh buffycoat of PMNC
Method analyzes and processes.Use is supplemented with the IL-2 (Pep of 10%FBS (GIBCO) and 10 or 50IU/ml
RoTech, Rocky.Hill, NJ, USA) serum-free stem cell growth culture medium (the SCGM) (C of GMP
EllGro/CellGenix) NK cell is expanded, and raise with PBMC and gamma-radiation K562 thin
Born of the same parents (quantity is than 1:2) co-culture in NK cell growth medium.In T75 blake bottle 2 × 106
Individual PBMC and 4 × 106K562 feeder cells co-culture in the NK culture medium of 10ml.Every 2-3
It half amount is changed liquid and adds 100IU/mlIL-2.
After NK cell expands 7 days, the NK cell that amplification obtains is with 2 × 107Cell/mL is containing SCGM
At cryovial (NUNC) in the freezing culture medium+10%DMSO (Invitrogen) of culture medium+10%FBS
In frozen.Cell is placed in refrigerated container (Thermo Fisher Scientific, the Ro of freezing 1 DEG C
Chester, NY) in-80 DEG C of refrigerators frozen 24 hours, then shift and be stored in liquid nitrogen vapor storage tank.
Freezing NK cell thaws until being left a small amount of frozen matter, then at NK cell in 37 DEG C of water-baths
Growth medium washs.Every 7 days of defrosting NK cell again with K562 feeder cells with the ratio of 1:1
Stimulate.Frozen NK cell can at least expand 3 months.
5, flow cytometry analysis
Flow cytometer used is BD Accuri C6 flow cytometer (BD Biosciences, Fran
klin Lakes,NJ)。
The antibody of the APC mark used is purchased from U.S. sky Ni (Germany Bergisch Gladbach), BD
Biosciences company, or Beckman Coulter (Brea, CA).
6, cytotoxicity analysis
DescribedThe cytotoxic reagent box (Perkin Elmer) of cell is used for testing wild
Type K562 and the cytotoxicity of Raji cell.
NK cell and target cell under 1:1,1:5 and 1:10 ratio at 37 DEG C, 5%CO2Cultivation
Case co-cultures 2 hours.
7, experimental result
The fusion of site-specific integration is to K562 feeder cells
Utilize the Zinc finger nuclease (BV-ZFN) (Tay, Tan et al.2013) set up, two matter
Grain constructing technology: one coding ZFN, targeting and cracking AAVS1 transposons (pFastBac-ZFN) and
Other were used containing CMV promoter to drive the host mbil15,4-1BB of the expression of fusion coding
L, and mbil21 and neomycin gene.It is specifically shown in figure).In figure, the fusion of site-specific integration
Gene is to K562 cell.The structure of pFastBac-fusion host, AAVS1 to PPP1R12C base
Cause, shearing site BV-ZFN, and the AAVS1 modified is after homologous recombination.E1, E2 and E3: front
The extron of three PPP1R12C genes.Arrow represents PCR front and reverse starting (FP and RP)
3 ' the AAVS1 modified in conjunction with the 2.4kb fragment formed
This expression cassette is that the right homology arm of the left homology arm flank by a 810-bp and a 837-bp closes
In described AAVS1 transposons (pFastBac-mbIL21-host) (Fig. 1).K562 cell is by electricity
Two kinds of plasmids are realized site-specific integration by perforation.After electroporation, K562 cell is carried out G418 choosing
Select, after 5 days, carry out unicellular sorting.DNA is extracted from the genome of G418 patience single cell clone,
And be specific to the neomycin of No. 19 chromosomes by use and be present in described pFastBac-mbIL21-place
Key-gene (downstream of the 3' end of right homology arm) primer is to carrying out pcr gene parting.2.5-kb
The amplification of fragment shows that the homologous recombination success AAVS1 mediated by ZFN is modified.To prove successfully to repair
Changing and will cause the fusion stable protein expression clone, described clone carries out flow cytometer and divides
Analysis.One clone demonstrates that the high level expression of protein is chosen to be the feeder cells of NK cell amplification.
It is specifically shown in Fig. 2.
The amplification of frozen rear NK cell
Cell is harmful to, such as cell viability, Cytotoxic decline, N by the frozen of already known NK cell
The minimizing that K cell receptor is expressed.In order to overcome this problem, we have developed a kind of scheme, by fresh
The K562 cell that the NK cell separated obtains with us is cultivated short 7 days, then with the ratio of 1:2
The cell that frozen amplification obtains, then with ratio and the K562 co-culture of cells of 1:1 after recovery.With
After every 7 days, NK cell K562 feeder cells are stimulated again, the amplification of NK cell be up to 3 months.
It was found that this method can overcome the problem that cell viability caused by freezen protective declines.Such as Fig. 3 institute
Showing, the NK cell of freezen protective can be bred at least 35 days after recovery.Figure shows, NK cell
Average total amplification times be 78,880,152, scope 280,354 to 226,456,888 times.
Every a line represents a PBMC patient (n=3).NK cell K562mbil15-41BBL-mbil21 raises
Support confluent monolayer cells amplification PBMC:K 562 ratio and be 1:2 first 7 days, frozen after-80 DEG C.Frozen
NK cell was recovered after one week, again expanded after stimulating with K 562.
For determining the optimal culture condition of NK cell proliferation, evaluate PBMC to K562-mbIL15-4 respectively
The difference of 1BBL-mbIL21 ratio, 1:2,1:1.5 and 1:1.Observe, in dispensing initial p B
The reduction that MC stimulates may cause the reduction of NK cells expanded, and PBMC:K562 is the ratio of 1:2
Example produces the highest NK cells expanded.The increase of ratio also will keep higher NK cell purity, with
PBMC:K562 be 1:2 can cultivate 21 days (Fig. 4) after reach 94.9 ± 2.8% the highest
Purity.
Experimental example
1, the Phenotypic examination experiment of the NK cell of amplification
To one of the NK cell adhesion molecule that obtains of amplification and acceptor widely array test.Greatly
Part high expressed acceptor and adhesion molecule, have a few exceptions-activated receptor, NKp44, and Inhibitory receptor,
Surface C D158a, h, CD158i and CD158e1/e2 (Fig. 5).
Also compares simultaneously and cultivate the expression of adhesion molecule after the NK cell recovery of 7 days and with K562
-mbIL15-41BBL-mbIL21 again stimulate 1 week after adhesion molecule and the expression of acceptor.Suppression
Property acceptor, surface C D158a, h's and CD158e1/e2 and activated receptor, NKG2D and NKp44
Surface expression occur dramatically increasing.The expression of result display surface mark CD16 can be because of NK cell (Sa
Kamoto, Ishikawa et al.2015) freezen protective and lower.Consistent with these results
It is that significantly declining (up to occurs in the expression of surface markers CD16 of NK cell after frozen and recovery
86%).But, this problem obtains after using K562-mbIL15-41BBL-mbIL21 to stimulate 1 week again
To improving.CD16 surface expression shows and increases to average 90.6 ± 1.8%.
2, the lethal test of NK cells on cancer cells of amplification
The NK cell of amplification can directly kill the K562 Leukaemia that NK is sensitive, and average 16.1%
(scope from 0.9% to 31.2%), 69.8% (scope from 58.5% to 98.9%) and 86.3%
(from 74.9% to 98.7%), is respectively 1:1,5:1 and 10:1 (Fig. 6) with 1 effect target ratio.
Wherein NK cell and K562 cell co-culture 2 hours with the ratio of 1:1,5:1 and 10:1.
Every a line represent a PBMC healthy volunteer (n=6) but, Raji lymphoma cell is to NK cell
Less sensitive.By utilizing the ADCC function of these NK cells, at antibody CD20-hIgG1 (Fig. 7)
In the presence of can substantially observe the cytotoxicity to Raji cell.Therefore, the NK that amplification obtains is thin
The ADCC function of born of the same parents can be significantly expanded the spectrum of the killing to malignant cell.
Immunocyte Chimeric antigen receptor (CAR) can be improved their killing activity to cancer cell.
The cancer cell killing-efficiency of the NK cell of amplification is increased further, by testing anti-EpCA after freezen protective
M CAR-redirects whether NK cell shows the positive colorectal cancer of anti-EpCAM and breast cancer cell
Cytotoxicity.After the mRNA of electroporation coding anti-EpCAM, NK cell and the pCRC7 National People's Congress that will modify
Colon-cancer cell (Fig. 8) and MCF-7 breast cancer cell (Fig. 9) co-culture.Fig. 8, EpCAM is in 9
The initial NK cell that specific C AR modifies；CAR is the initial NK cell that comparison is modified；Wt is initial
NK cell.
Obtain following result: anti-EpCAM CARNK cell shows strong cellular cytoxicity activity, and can be at E:
During T ratio 10:1 100% kill tumour cell, the initial NK cell of this relatively unmodified and mGFP
The comparison NK cell that CAR modifies has more effective killing activity.
In view of the foregoing it is apparent that adoptive NK cell therapy is a kind of effective cancer treatment method,
Show high antitumor potentiality, there is in clinical testing extremely low graft versus host disease(GVH disease) (G simultaneously
VHD) risk.But, it is thus achieved that sufficient amount of NK cell is that NK is thin for the difficulty transplanted of adopting
One restriction of born of the same parents' therapy.The amplification in vitro of NK cell generally with complicated scheme and the cost phase of costliness
Association.By using the K562-mbIL15-41BBL-mbI of the integration technology exploitation of site-specific genetic
L21 clone, by having high NK cytoactive, purity and cytotoxicity realize high NK cell and expand
Increasing Efficiency expands scheme to improve NK.The reverse of random integration coding NK stimulation molecule dye K562 cell
The gene of record viral vectors is used for producing genetically modified K562 cell, and this may cause stimulation molecule
Expression different, therefore this will be that the clone of challenging duplication may express transgenic
Identical expression.AAVS1 swivel base is introduced by the integration problem of site-specific genetic by overcoming
Son.AAVS1 is positioned at protein phosphatase 1, regulates (inhibitor) subunit 12C (PPP1R12C) base
Within to No. 19 chromosomes (19q13.3-qter) of people.This site is used as specific integration gene
The non-pathogenic human parvovirus of seat AAV serotype 2 (AAV2).AAVS1 has transcriptional capability and includes
One unlimited chromatin Structure and comprise natural insulator, makes the gene integration to transgene silencing hinder
Power.Owing to there is the destruction of the transcriptional capability of the transgenosis box from PPP1R12C gene and insertion point
Not having known adverse effect to be maintained at different types of cell on the cell caused, AAVS1 is considered
Be one " safe port " be the one-tenth human genome of addition transgenosis.
The NK cell of amplification has more preferable efficiency after being frozen preservation, and in follow-up amplification procedure
There is the NK cell function of improvement.
At present, the impact expressed CD16 due to freezing, the NK cell recovered immediately cannot be used for ADCC
Antineoplaston.This therapy will become can by again stimulating with K562-mbIL15-41BBL-mbIl21
OK.NK cytotherapeutic treatment leukaemia relatively solid tumor is more successful at present.This is owing to this display suppresses signal
Impact with the solid tumor microenvironment factor of the lethal effect of NK cell.Therefore, the genetic modification of CAR
NK cell there is targeting solid tumor have drug-fast NK cell, open widely NK cell therapy
The possibility of various cancer types.Therefore, after the NK cell recovering frozen, anti-EpCAM CAR-resets
EpCAM positive colorectal cancer and breast cancer cell can be effectively killed to NK cell.
Obviously, above-described embodiment is only for clearly demonstrating example, and not to embodiment
Restriction.For those of ordinary skill in the field, can also do on the basis of the above description
Go out change or the variation of other multi-form.Here without also cannot all of embodiment be given exhaustive.
And the obvious change thus extended out or variation still in the invention protection domain it
In.
Claims (8)
1. the method for an efficient amplification freezen protective NK cell, it is characterised in that described method is passed through
Fusion is carried out genetic recombination to AAVS1 transposons by plasmid, and expands the interstage at cell
Introduce the NK cell expansion step based on freezing/thawing step extremely modifies K562 clone；Finally carry out
The amplification of NK cell and preservation.
2. the method for claim 1, it is characterised in that described fusion is human chromosome 1
9 coding mbIL15,4-1BBL and mbIL21.
3. method as claimed in claim 2, it is characterised in that the gene expression matter of described fusion
Grain skeleton is by pFastBac1.
4. method as claimed in claim 3, it is characterised in that the promoter of described plasmid backbone is
Cytomegalovirus.
5. method as claimed in claim 4, it is characterised in that the restructuring of described plasmid uses zinc to refer to core
The homologous recombination technique of acid enzyme-mediation.
6. method as claimed in claim 5, it is characterised in that the step of described genetic recombination particularly as follows:
K562 cell is carried out suspension process；
PFastBac-ZFN and PFB-fusion host's electricity is gone to cell；
The cell of electroporation is subsequently transferred to K562 growth medium cultivate.
After cultivation, carry out expanding unicellular, clone and carry out pcr gene parting, flow cytometry,
Prepare the cell stably expressing fusion.
7. method as claimed in claim 6, it is characterised in that the amplification of described NK cell specifically walks
Suddenly it is:
First taking PMNC, line density of going forward side by side gradient method analyzes and processes；
Then use serum-free stem cell growth culture medium that described NK cell is expanded, and use PBMC
With gamma-radiation K562 feeder cells co-incubation in NK cell growth medium；
NK cell amplification obtained is at the freezing culture medium and 1 containing SCGM culture medium and 10%FBS
In the cryovial of 0%DMSO frozen；
Carry out again after cell is placed in frozen process thawing and washing；Finally by thaw NK cell again with
K562 feeder cells carry out stimulating with the ratio of 1:1 to be cultivated.
8. the method for the efficient amplification freezen protective NK cell as described in any one of claim 1-7 is carefully
Application in born of the same parents' preservation field.
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| CN105838677B (en) | 2019-07-02 |
| CN110358736A (en) | 2019-10-22 |
| CN110358736B (en) | 2023-07-07 |
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