CN108588028A

CN108588028A - A kind of CIC cell models and preparation method thereof of targeting CDKN2A

Info

Publication number: CN108588028A
Application number: CN201810385867.1A
Authority: CN
Inventors: 孙强; 梁剑青; 牛祖彪; 黄红艳
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2018-09-28
Anticipated expiration: 2038-04-26
Also published as: CN108588028B

Abstract

The invention discloses a kind of cell in cell (CIC) cell models and preparation method thereof of targeting CDKN2A.The method provided by the present invention for preparing cell in cell relevant cell models is that cell in cell relevant cell models are prepared by regulating and controlling the expression of CDKN2A genes in target cell.Inhibit the expression of CDKN2A genes in target cell that can prepare induction cell in cell cell models；Make to be overexpressed CDKN2A genes in target cell and can prepare to inhibit cell in cell cell models.Utilize this method for promoting CIC structure to be formed by intervening CDKN2A gene expressions provided by the present invention.Complete tumour cell is expressed for CDKN2A, the purpose that the intracellular for promoting CIC to mediate is dead, realizes kill tumour cell, inhibits tumour growth is expected to by this method.

Description

A kind of CIC cell models and preparation method thereof of targeting CDKN2A

Technical field

The invention belongs to biotechnologies, are related to a kind of cell-in-cell (CIC) cell model of targeting CDKN2A And preparation method thereof.

Background technology

Cell-in-cell (CIC) structure refers to that one or more living cells are located at another cell interior and are formed by solely Special cell is nested spline structure.CIC phenomenons are widely present in each species, including unicellular lower eukaryote Amoeba, raw after arriving In biological caenorhabditis elegan and mammlian system.It is different according to the cell category for forming CIC structure, homogeneity CIC can be divided into, such as The CIC structure being formed between the identical tumour in source or epithelial cell；With heterogeneous CIC, be formed in the different cell in source it Between CIC structure.

Currently, tumour, which is homogeneity CIC structure biological significance, studies field the most active.The research of clinical tissue sample Report, CIC phenomenons are common in mankind tumor tissue, such as breast cancer, colon cancer, cervical carcinoma, prostate cancer, liver cancer, and fresh It sees in normal structure.Since the formation of homogeneity CIC structure can mediate internal cell death, there is scholar to think homogeneity CIC Substantially a kind of cell death mechanism can remove the cell of the cell or vicious transformation that are detached from extracellular matrix, it is seen that tumour The formation of iuntercellular CIC structure has to kill tumour cell, limits tumour growth.

There is the cell model for lacking to be regulated and controled by specific gene in existing CIC researchs, it is difficult in vitro study system is obtained, The defects of can not achieve targeting specific gene.

Invention content

The present inventor has found that tumor suppressor gene CDKN2A expresses reduction in the cell can promote CIC structure for the first time Formation, and then mediate internal cell death, realize the purpose for killing cell.The model is not only suitable for non-tumor cell system and also fits For tumor cell line.

In a first aspect, a kind of claimed method preparing cell-in-cell relevant cell models.

The method provided by the present invention for preparing cell-in-cell relevant cell models is by regulating and controlling in target cell The expression of CDKN2A genes is prepared cell-in-cell relevant cell models.

Wherein, the target cell both can be tumour cell, or non-tumor cell.The target cell can be people source Cell.

Further, the method can be following method A or method B：

Method A：The method for preparing induction cell-in-cell cell models, it may include following steps：Inhibit the target thin The expression of CDKN2A genes in born of the same parents.

Method B：The method is to prepare the method for inhibiting cell-in-cell cell models, it may include following steps：Make Overexpression CDKN2A genes in the target cell.

Further, in the method A, the target cell can be the cell of CDKN2A gene effective expressions (as led to The expression of CDKN2A gene coded proteins can be detected by crossing Western blot).In the method B, the target cell can be The cell of CDKN2A gene expressions or afunction (such as can not detect CDKN2A gene coded proteins by Western blot Expression).

The present invention specific implementation mode in, in the method A, the target cell be specially HEK293 cells (just Ordinary person's embryonic kidney cells).In the method B, the target cell is specially MCF10A cells (non-transformed galactophore epithelial cell system) Or MCF7 cells (breast cancer cell line).

Further, in the method A, " expression for inhibiting CDKN2A genes in the target cell " can pass through Any technological means that can realize this purpose is realized, such as passes through sequence specific nuclease (such as CRISPR/Cas9 nucleases) Specific cleavage is carried out to CDKN2A genes, to reduce its expression in the target cell；Or inhibited by compound The protein function of CDKN2A gene expressions, such as CDKN2A inhibitor.

In the specific implementation mode of the present invention, in the method A, inhibit the table of CDKN2A genes in the target cell Up to what is realized particular by CRISPR/Cas9 technologies.Specifically to meet 5 '-GN in CDKN2A gene orders₁₉- NGG-3 ' sequences The segment of row queueing discipline is target sequence；N indicates any one of A, G, C and T, N₁₉Indicate 19 continuous deoxyribose cores Glycosides.If can not be found in target position point range and meet 5 '-GN₁₉The sequence of-NGG-3 ' queueing disciplines, then find 5 '-N₂₀- NGG-3 ', And add G, i.e. 5 '-G-N before sgRNA₂₀-NGG-3’.Correspondingly, two complementary sgRNA corresponding to the target sequence Oligo is specially SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 or SEQ ID No.5 With SEQ ID No.6.

Further, in the method B, it is by such as lower section to make overexpression CDKN2A genes in the target cell What formula was realized：Make at least one in four transcripts of overexpression CDKN2A genes in the target cell, the CDKN2A bases Four transcripts of cause are p16INK4a, p16 γ, p12 and p14ARF.

In the specific implementation mode of the present invention, make the transcript of overexpression CDKN2A genes in the target cell P16INK4a is particular by the recombination table for importing the encoding gene containing the transcript p16INK4a into the target cell It is realized up to carrier；Make the transcript p16 γ of overexpression CDKN2A genes in the target cell particular by the target The recombinant expression carrier realization of the encoding gene containing the transcript p16 γ is imported in cell；Make mistake in the target cell The transcript p12 of amount expression CDKN2A genes is particular by volume of the importing containing the transcript p12 into the target cell What the recombinant expression carrier of code gene was realized；Keep the transcript p14ARF of overexpression CDKN2A genes in the target cell specific It is to be realized by importing the recombinant expression carrier of the encoding gene containing the transcript p14ARF into the target cell.

Wherein, the recombinant expression carrier is the structure using retroviral vector pQCXIP-EGFP-N1 as skeleton CDKN2A over-express vectors.Four transcripts of the CDKN2A genes are respectively inserted into the retroviral vector Between the EcoRI and MfeI of pQCXIP-EGFP-N1,4 recombinant expression carriers are obtained.

In the present invention, the nucleotide sequence such as GenBank of the CDKN2A genes：The of NC_000009.12 (complement, 2018-3-26) shown in 21967752-21995043.The encoding gene of the transcript p16INK4a Nucleotide sequence is specific as shown in SEQ ID No.7.The nucleotide sequence of the encoding gene of the transcript p16 γ is specifically such as Shown in SEQ ID No.8.The nucleotide sequence of the encoding gene of the transcript p12 is specific as shown in SEQ ID No.9.It is described The nucleotide sequence of the encoding gene of transcript p14ARF is specific as shown in SEQ ID No.10.

In the present invention, the type of the cell-in-cell be specially homogeneity cell-in-cell (i.e. allogenic cell it Between be formed by cell-in-cell structures).

Second aspect, the claimed cell model being prepared by method previously.

The third aspect, the claimed cell model it is following it is any in application：

(A1) determinand (such as specific compound, extract, small molecule, polypeptide, albumen are evaluated by the cell model Matter, gene, therapeutic cells etc.) influence to disease process such as tumour growths；

(A2) by determinand (such as specific compound, extract, small molecule, polypeptide, protein, gene, therapeutic cells Deng) cell model is targeted to evaluate the biological safety of the determinand；

(A3) tumor therapeutic agent is prepared using the cell model；As for establishing new disease by cell-in-cell Sick (such as tumour) treatment means, including be used to treat cell, nucleic acid, protein and small molecule etc.；

(A4) new animal model is established by or by the cell model.

It is demonstrated experimentally that using normal human embryonic kidney cells HEK293 (cell effective expression CDKN2A), using CRISPR/ Cas9 systems strike the expression of low endogenous CDKN2A by designing sgRNA, CIC structure can be promoted to be formed.It is expressed using CDKN2A The non-transformed galactophore epithelial cell system MCF10A and breast cancer cell MCF7 of defect transfect weight by retroviral vector New expression CDKN2A, which can get, inhibits CIC cell models.Using it is provided by the present invention it is this pass through intervene CDKN2A gene tables Up to the method for promoting CIC structure to be formed.Effective tumour cell is expressed for CDKN2A, is expected to that CIC is promoted to be situated between by this method The intracellular led is dead, realizes the purpose killed tumour cell, inhibit tumour growth.

Description of the drawings

Fig. 1 is the expression that Western blot detect CDKN2A in each cell line.

Fig. 2 is the immunofluorescence schematic diagram of cell-in-cell structures.

Fig. 3 strikes low CDKN2A genes for targeting and cell-in-cell structures is promoted to be formed.A：Western blot detections three The CDKN2A-sgRNA's of a difference target spot strikes poor efficiency；B：Cell-in-cell formation rates.**p<0.01；C：pCS- The sequencing result of CDKN2A-sgRNA expression plasmids.

Fig. 4 is to be overexpressed the different transcripts of CDKN2A tetra- cell-in-cell structures is inhibited to be formed.A：Non-transformed mammary gland Epithelial cell line MCF10A；B：Breast cancer cell MCF7.**p<0.01.

Specific implementation mode

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

HEK293 cells：ATCC cell banks, #CRL-1573.

Cas9 work system plasmid precut pCS (puro)：Bioengineering Research Institute doctor Xi Yongyi gives, and is purchased from north Hundred Olympic Competition figure company of capital.

Retroviral vector pQCXIP-EGFP-N1：It is recorded in " Wang M, Ning X, Chen A, Huang H, Ni C,Zhou C,et al.Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression.Scientific reports.2015；5: 12223 " one texts, the public can obtain from applicant, can only be used to repeat present invention experiment use.

PCMV-VSV-G plasmids：Addgene, #8454.

Gag/pol plasmids：Addgene, #14887.

293FT cells：Northern Na Shengwuxibaoku, BNCC339263.

MCF10A cells：It is recorded in " Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al.Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression.Scientific reports.2015；5:12223 " one texts, The public can obtain from applicant, can only be used to repeat present invention experiment use.

MCF7 cells：It is recorded in " Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al.Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression.Scientific reports.2015；5:12223 " one texts, The public can obtain from applicant, can only be used to repeat present invention experiment use.

Embodiment 1, the foundation for inducing CIC cell models

Target cell in the present embodiment is HEK293 cells, and Western blot can detect CDKN2A in HEK293 cells The expression (Fig. 1) of gene coded protein, specific detection method is referring to Westernblot in the present embodiment step 16.

One, HEK293/CDKN2A-cas9 strikes the foundation of low cell line

1, design sgRNA oligo

According to PAM sequence principles are added after target sequence, with CDKN2A genes (GenBank：The of NC_000009.12 Shown in 21967752-21995043, complement, 2018-3-26) it is target sequence, 3 CDKN2A-sgRNA of design are such as Under：

sgRNA-1：5’-caccGCACCGAATAGTTACGGTCGG-3’(SEQ ID No.1)；

5’-aaacCCGACCGTAACTATTCGGTGC-3’(SEQ ID No.2)。

sgRNA-2：5’-caccGACCGTAACTATTCGGTGCGT-3’(SEQ ID No.3)；

5’-aaacACGCACCGAATAGTTACGGTC-3’(SEQ ID No.4)。

sgRNA-3：5’-caccGTGGGCCATCGCGATGTCGCA-3’(SEQ ID No.5)；

5’-aaacTGCGACATCGCGATGGCCCAC-3’(SEQ ID No.6)。

2, the plasmid of structure expression CDKN2A-sgRNA

After primer synthesis, by sgRNA oligo ddH₂O dissolves, final concentration of 100 μM, two complementary sgRNA Oligo respectively takes 15 μ L mixing, is put into boiling water bath after boiling 5min and waits for that it is naturally cooling to room temperature and anneals completion；Then pass through I digestion cas9 work system plasmid precut pCS (puro) of Bbs, make its linearisation, by digestion carrier and the sgRNA of annealing into Row connection obtains the plasmid pCS-CDKN2A-sgRNA of expression CDKN2A-sgRNA, and confirms that structure is correct through sequence verification.

3, cell is spread

First by HEK293 cells with every hole 1 × 10⁶The even density of a cell is laid on uses collagen (collagen I) in advance Six orifice plates of embedding, polishing culture medium DMEM (containing 10%FBS, % indicates volumn concentration) are put into incubator culture to 2mL Transfection experiment is carried out after 16h.

4, liposome transfection system

A liquid：The plasmid pCS-CDKN2A-sgRNA of 0.4 μ g mesh and 100 μ L Opti-MEM are mixed well, are stored at room temperature 5min；B liquid：Transfection reagent Lipo 2000 is gently overturned into mixing, 2.5 μ L Lipo 2000 is taken to be diluted to 100 μ L Opti- In MEM, mixing is gently overturned, 5min is stored at room temperature；C liquid：A liquid is mixed with B liquid, gently mixing, is stored at room temperature 30min；It will 800 μ L Opti-MEM are mixed with the C liquid of 200 μ L obtains transfection cocktail D.

5, transfectional cell

The 1mL D liquid mixed is lightly added along orifice plate inner wall for the former culture medium for discarding HEK293.It is abandoned after culture 5-6h Transfection liquid is changed to the normal incubation medium containing 10% (volumn concentration) FBS and continues to cultivate.

6, pressurization screening

Cell uses 1 μ g/m puromycins of final concentration pressurization screening 5 days for 24 hours in transfection, obtains HEK293/CDKN2A- Cas9 strikes low cell line.And poor efficiency is struck by the CDKN2A-sgRNA of Western blot three different target spots of detection.

Western blot detecting steps are as follows：Aim cell albumen is extracted, is placed in cell pyrolysis liquid is added on ice 30min, during which being shaken 2~3 times using vortex oscillator makes cell fully crack, and 4 DEG C of 12,000r/min centrifugations 20min are collected Supernatant simultaneously carries out protein quantification.The albumen of extraction is through 95 DEG C of denaturation 5min, using 15% SDS-PAGE glue, 10 μ g of applied sample amount, 100V transferring films 40min, 5%BSA room temperature closes 1h, and sanction film is carried out according to molecular weight of albumen size, adds primary antibody anti-E- respectively Cadherin antibody (BD；#610182), anti-β-Tubulin antibody (CWBIO；#CW0256) and anti-p16INK4a antibody (BOSTER；#BM1592), in 4 DEG C of overnight incubations, TBST cleans 3 times × 10min, the two of horseradish peroxidase-labeled is added Anti- incubation at room temperature 1h, TBST clean 3 times × 10min, finally by chemoluminescence method testing goal band.To ensure applied sample amount one It causes, is compareed using Tubulin bands as internal reference.

Two, Cell-in-cell forms experiment

1, prepare soft agar

The soft agar solution that 0.5% (0.5g/100mL) is prepared using the PBS buffer solution of preheating, rapidly with the amount in the holes 1mL/ It is added into six orifice plate of cell culture, gently shaking orifice plate keeps soft agar solution evenly laid out in board bottom.In room temperature horizontal rest A few hours wait for its solidification.

2, cell suspension cultures

Using pancreatin by cell dissociation at single cell suspension, 2 × 10 are counted with every hole⁵A cell is laid on the soft agar of solidification On, by culture medium polishing to the holes 2mL/, the culture 13h that suspends is placed in cell incubator.

3, cell rejection tablet

It suspends and cell can be observed after culture 13h under microscope suspends agglomerating phenomenon, cell suspension is gently sucked out, is set 800rpm centrifuges 4min in centrifuge tube, abandons supernatant, is resuspended using 1mL PBS, and blowing and beating 10-20 times with rifle makes it at unicellular outstanding Liquid is taken rapidly 200 μ L to be added into rejection tablet machine and is got rid of cell to glass slide with 500rpm rotating speed rejection tablets 4min.

4, film-making

The slice, thin piece got rid of fixes 10-15min using 4% paraformaldehyde in room temperature, and PBS is washed 2 times, each 5min, gently will Water wiped clean on slide, 0.2%Triton X-100/PBS permeable membranes 4min, PBS clean 2 times × 5min；Use 5% ox blood Pure albumen room temperature closes 1h；Primary antibody anti-E-cadherin antibody (1 is added after closing：400 dilutions；BD；# 610182), in 4 DEG C of overnight incubations of wet box；3 times × 10min is cleaned with PBS within second day；Add fluorescein-labeled secondary antibody (1:500 Dilution), it is incubated at room temperature 1h in dry box；PBS cleans 3 times × 10min, and mounting preservation, turntable laser are carried out with mountant containing DAPI Laser Scanning Confocal Microscope (Perkin Elmer) is observed and is photographed to record.

5, Cell-in-cell is counted

Fluorescence microscopy microscopic observation cellular morphology and cell-in-cell formational situations, each slice, thin piece randomly select four The even visual field, each visual field at least count 200 cells (not including the cell pierced).Mark nucleus identification thin according to DAPI Born of the same parents, to be denoted as 1 cell-in-cell structure by the fully wrapped around structure of external cellular.

Average value ± the SD of cell number/total cell number × 100 of cell-in-cell (%)=formation cell-in-cell.

Cell-in-cell structures are as shown in Fig. 2, blue-fluorescence indicates that nucleus, red fluorescence mark epicyte protein E- Cadherin, for distinguishing cell and intercellular boundary.Statistical result shows to strike low CDKN2A genes by sgRNA, can show It writes and cell cell-in-cell structures is promoted to form (Fig. 3).

Embodiment 2, the foundation for inhibiting CIC cell models

Target cell in the present embodiment is MCF10A cells and MCF7 cells, and Western blot can not detect MCF10A The expression (Fig. 1) of CDKN2A gene coded proteins in cell and MCF7 cells, specific detection method is referring to the present embodiment step 16 Middle Western blot.

One, the foundation of MCF10A/CDKN2A overexpressing cells system

Using retroviral vector pQCXIP-EGFP-N1 as skeleton, CDKN2A over-express vectors, including its four are built Different transcript p16INK4a, p16 γ, p12 and p14ARF.The nucleotide sequence of the encoding gene of transcript p16INK4a is specific As shown in SEQ ID No.7.The nucleotide sequence of the encoding gene of transcript p16 γ is specific as shown in SEQ ID No.8.Transcription The nucleotide sequence of the encoding gene of this p12 is specific as shown in SEQ ID No.9.The nucleosides of the encoding gene of transcript p14ARF Acid sequence is specific as shown in SEQ ID No.10.It will be reversed by double digestion using restriction endonuclease EcoRI and MfeI Viral vectors pQCXIP-EGFP-N1 linearisations are recorded, then (both ends add phase when artificial synthesized by the encoding gene of each transcript The restriction enzyme site answered) it is inserted respectively into retroviral vector pQCXIP-EGFP-N1, it obtains corresponding to four transcripts Four recombinant retroviral vectors, and confirm that structure is correct through sequence verification.

Then, destination carrier is imported MCF10A cells, specific experiment by the method packed and infected by retrovirus Steps are as follows：First by Viral packaging cell 293FT with every hole 1 × 10⁶A even density is laid on uses collagen (collagen in advance I) six orifice plates embedded, polishing culture medium DMEM (containing 10%FBS, % indicates volumn concentration) are put into incubator training to 2mL Transfection experiment is carried out after supporting 16h；By the carrier of 0.4 μ g mesh, (four recombinations built above reverse retrovirus packaging system Record viral vectors), 0.2 μ g pCMV-VSV-G plasmids, 0.25 μ ggag/pol plasmids and lipofectamine Lipo 2000 It constitutes；By viral packaging system infection cell make its production virus, after transfection for 24 hours, 48h, 72h collect viral supernatants, often It is secondary collect viral supernatants after add the fresh normal incubation mediums of 2mL；Viral supernatants 2000rpm is centrifuged into 5min, collects supernatant, point It fills and freezes spare in -80 DEG C.

Before infection cell, target cell MCF10A is first pressed 1 × 10⁵/ hole is inoculated in six orifice plates, waits for that 8~12h cells are adherent complete Quan Hou, the complete medium for taking above-mentioned viral supernatants 1mL fresh with 500 μ l mix, and 2 μ l polybrenes Polybrene (storages are added Deposit 10 μ g/mL of concentration), infection cell in six orifice plates is added after mixing, infects being changed to 2mL fresh cultures after 6h and continue to train It supports；Cell is screened 5 days using the pressurization of final concentration of 2 μ g/mL puromycins for 24 hours after infection, complete with blank control group cell It is criterion that dead and positive controls GFP expression rates, which are 100%, obtains the cell strain for stablizing expression CDKN2A.

MCF7/CDKN2A overexpressing cells system method for building up is same as above, and puromycin screening concentration is 2 μ g/ml.

Two, Cell-in-cell forms experiment

Specific steps are with 1 step 2 of embodiment, in addition to cell suspension time is 6h.Epithelial cell MCF10A or tumour cell Again CDKN2A is expressed in MCF7 can significantly inhibit cell-in-cell structures formation (Fig. 4).

<110>Military medical research institute of PLA Academy of Military Sciences

<120>A kind of CIC cell models and preparation method thereof of targeting CDKN2A

<130> GNCLN180921

<160> 10

<170> PatentIn version 3.5

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attctccgga aaaaggggag gcttcctggg gagttttcag aaggggtttg taatcacaga 300

cctcctcctg gcgacgccct gggggcttgg gaagccaagg aagaggaatg aggagccacg 360

cgcgtacaga tctctcgaat gctgagaaga tctgaagggg ggaacatatt tgtattagat 420

ggaagtcatg atgatgggca gcgcccgagt ggcggagctg ctgctgctcc acggcgcgga 480

gcccaactgc gccgaccccg ccactctcac ccgacccgtg cacgacgctg cccgggaggg 540

cttcctggac acgctggtgg tgctgcaccg ggccggggcg cggctggacg tgcgcgatgc 600

ctggggccgt ctgcccgtgg acctggctga ggagctgggc catcgcgatg tcgcacggta 660

cctgcgcgcg gctgcggggg gcaccagagg cagtaaccat gcccgcatag atgccgcgga 720

aggtccctca gacatccccg attgaaagaa ccagagaggc tctgagaaac ctcgggaaac 780

ttagatcatc agtcaccgaa ggtcctacag ggccacaact gcccccgcca caacccaccc 840

cgctttcgta gttttcattt agaaaataga gcttttaaaa atgtcctgcc ttttaacgta 900

gatatatgcc ttcccccact accgtaaatg tccatttata tcatttttta tatattctta 960

taaaaatgta aaaaagaaaa acaccgcttc tgccttttca ctgtgttgga gttttctgga 1020

gtgagcactc acgccctaag cgcacattca tgtgggcatt tcttgcgagc ctcgcagcct 1080

ccggaagctg tcgacttcat gacaagcatt ttgtgaacta gggaagctca ggggggttac 1140

tggcttctct tgagtcacac tgctagcaaa tggcagaacc aaagctcaaa taaaaataaa 1200

ataattttca ttcattcact caaaaaaaaa aaaaa 1235

<210> 10

<211> 1164

<212> DNA

<213> Artificial sequence

<400> 10

cgctcaggga aggcgggtgc gcgcctgcgg ggcggagatg ggcagggggc ggtgcgtggg 60

tcccagtctg cagttaaggg ggcaggagtg gcgctgctca cctctggtgc caaagggcgg 120

cgcagcggct gccgagctcg gccctggagg cggcgagaac atggtgcgca ggttcttggt 180

gaccctccgg attcggcgcg cgtgcggccc gccgcgagtg agggttttcg tggttcacat 240

cccgcggctc acgggggagt gggcagcgcc aggggcgccc gccgctgtgg ccctcgtgct 300

gatgctactg aggagccagc gtctagggca gcagccgctt cctagaagac caggtcatga 360

tgatgggcag cgcccgagtg gcggagctgc tgctgctcca cggcgcggag cccaactgcg 420

ccgaccccgc cactctcacc cgacccgtgc acgacgctgc ccgggagggc ttcctggaca 480

cgctggtggt gctgcaccgg gccggggcgc ggctggacgt gcgcgatgcc tggggccgtc 540

tgcccgtgga cctggctgag gagctgggcc atcgcgatgt cgcacggtac ctgcgcgcgg 600

ctgcgggggg caccagaggc agtaaccatg cccgcataga tgccgcggaa ggtccctcag 660

acatccccga ttgaaagaac cagagaggct ctgagaaacc tcgggaaact tagatcatca 720

gtcaccgaag gtcctacagg gccacaactg cccccgccac aacccacccc gctttcgtag 780

ttttcattta gaaaatagag cttttaaaaa tgtcctgcct tttaacgtag atatatgcct 840

tcccccacta ccgtaaatgt ccatttatat cattttttat atattcttat aaaaatgtaa 900

aaaagaaaaa caccgcttct gccttttcac tgtgttggag ttttctggag tgagcactca 960

cgccctaagc gcacattcat gtgggcattt cttgcgagcc tcgcagcctc cggaagctgt 1020

cgacttcatg acaagcattt tgtgaactag ggaagctcag gggggttact ggcttctctt 1080

gagtcacact gctagcaaat ggcagaacca aagctcaaat aaaaataaaa taattttcat 1140

tcattcactc aaaaaaaaaa aaaa 1164

Claims

1. a kind of method preparing cell-in-cell relevant cell models is the table by regulating and controlling CDKN2A genes in target cell It reaches cell-in-cell relevant cell models are prepared.

2. according to the method described in claim 1, it is characterized in that：The target cell is tumour cell or non-tumor cell.

3. method according to claim 1 or 2, it is characterised in that：The method is following method A or method B：

Method A：The method for preparing induction cell-in-cell cell models, includes the following steps：Inhibit in the target cell The expression of CDKN2A genes；

Method B：The method for inhibiting cell-in-cell cell models is prepared, is included the following steps：Make excessive in the target cell Express CDKN2A genes.

4. according to the method described in claim 3, it is characterized in that：In the method A, the target cell is CDKN2A genes The cell of effective expression；

In the method B, the target cell is the cell of CDKN2A gene expressions or afunction.

5. according to the method described in claim 4, it is characterized in that：In the method A, the target cell is that HEK293 is thin Born of the same parents；

In the method B, the target cell is MCF10A cells or MCF7 cells.

6. according to any method in claim 3-5, it is characterised in that：In the method A, inhibit the target cell The expression of middle CDKN2A genes is realized by CRISPR/Cas9 technologies；

In the method B, overexpression CDKN2A genes in the target cell is made to realize in the following way：Make described At least one in four transcripts of overexpression CDKN2A genes in target cell, four transcripts of the CDKN2A genes are P16INK4a, p16 γ, p12 and p14ARF.

7. according to the method described in claim 6, it is characterized in that：Make turn of overexpression CDKN2A genes in the target cell It is the recombination table by importing the encoding gene containing the transcript p16INK4a into the target cell to record this p16INK4a It is realized up to carrier；

Making the transcript p16 γ of overexpression CDKN2A genes in the target cell is contained by being imported into the target cell What the recombinant expression carrier of the encoding gene of the transcript p16 γ was realized；

It is by being imported into the target cell containing to make the transcript p12 of overexpression CDKN2A genes in the target cell State the recombinant expression carrier realization of the encoding gene of transcript p12；

Making the transcript p14ARF of overexpression CDKN2A genes in the target cell is contained by being imported into the target cell There is the recombinant expression carrier of the encoding gene of the transcript p14ARF to realize.

8. according to any method in claim 1-7, it is characterised in that：The type of the cell-in-cell is homogeneity cell-in-cell。

9. the cell model being prepared by any the method in claim 1-8.

10. cell model described in claim 9 it is following it is any in application：

(A1) influence of the determinand to tumor-related illness process is evaluated by the cell model；

(A2) determinand is targeted into the cell model to evaluate the biological safety of the determinand；

(A3) tumor therapeutic agent is prepared using the cell model；

(A4) new animal model is established by or by the cell model.