CN107488635A - Monoclonal cell and its production and use - Google Patents
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Abstract
The present invention relates to cell engineering field, monoclonal cell and its production and use is disclosed.The deposit number of the monoclonal cell is CCTCC NO：C2017118.The invention also discloses a kind of method for expressing aryl hydrocarbon receptor, this method includes：Above-mentioned cell is cultivated.In addition, study the application during aryl hydrocarbon receptor regulates and controls to inflammatory cytokine in vitro the invention also discloses above-mentioned cell.Present invention obtains stable expression AhR monoclonal cell strain, its cell quality is unified, the gene expression of cell keeps stable in succeeding generations, therefore is tested using the stable monoclonal cell strain for expressing AhR of the present invention, and the result obtained is more reliable and more stable.
Description
Technical field
The present invention relates to cell engineering field, and in particular to monoclonal cell and its production and use.
Background technology
Aryl hydrocarbon receptor (aryl hydrocarbon receptor, AhR) is a kind of transcription factor of ligand activation, no
Poisonous substance and foreign substances metabolism are only involved in, and regulates and controls organism immune response and biological rhythm.Recent study shows that AhR is being mediated
Outstanding role in terms of immunization inflammatory reaction, it would be highly desirable to further elucidate its mechanism.AhR can modulating T cell, B cell, dendron shape
The function of cell (dendritic cell, DC), NK and neutrophil leucocyte, equally reacted in Macrophage Inflamatory
Middle AhR also plays an important role, but due to shortage experimental model, its correlative study is deep not enough at present.Macrophage is due to being
Differentiated mature cell, transient transfection extremely inefficient and easily causes to hit to cell, can not carry out subsequent experimental.RAW264.7
Cell is mouse monokaryon macrophage leukaemia, the vehicles cells of generally acknowledged macrophage research classical at present.So structure
Platform will be provided for follow-up study by building stable high expression AhR RAW264.7 cell lines.
The content of the invention
The problem of the invention aims to overcome AhR existing for prior art to be difficult to stable high expression, there is provided one plant
Monoclonal cell and its production and use.
After no matter plasmid transfection or slow-virus infection import target gene at present, obtained by drug resistant gene or fluorescent screening
To cell line be actually multiple cell clones combination, different cell clones have different gene integration positions and expression
Amount.This cell line, due to the difference in different cell clone proliferation speed, causes cloning in vivo group after multiple passage
Proportional change, so as to result in the change of destination gene expression amount.Because the cell of mass expressing external gene is in propagation
General not dominant, so usually occurring after removing screening medicine, exogenous gene expression amount declines；In the cell with fluorescence labels
In, showing as luciferase expression intensity gradually reduces.The present invention is successfully constructed steady by way of slow-virus infection resistance screening
Surely AhR RAW264.7 monoclonal cell strains are expressed.Therefore, to achieve these goals, one aspect of the present invention provides one plant
Monoclonal cell, the deposit number of the monoclonal cell is CCTCC NO：C2017118.
Second aspect of the present invention provides a kind of method for expressing aryl hydrocarbon receptor, and this method includes：Above-mentioned cell is entered
Row culture.
Third aspect present invention provides above-mentioned cell and studies aryl hydrocarbon receptor in vitro in inflammatory cytokine regulation and control
Application.
Fourth aspect present invention provides the method for preparing monoclonal cell, and this method includes：
(1) plasmid for carrying aryl hydrocarbon receptor encoding gene is subjected to slow virus packaging；
(2) use and pack successful slow-virus transfection cell；
(3) cell in the culture medium of purine-containing mycin after culture transfection, so as to screen to obtain monoclonal cell.
The present invention transfects 293T cells and collected on purified virus by building slow virus recombinant plasmid vector after packaging
Clearly, screening obtains stable expression AhR monoclonal cell strain after infecting RAW264.7 cells, and carries out gene and protein level mirror
It is fixed, it is thin that the macrophage that AhR induces for lipopolysaccharides (Lipopolysaccharides, LPS) is further inquired into cell is surely turned
The influence of born of the same parents' inflammatory Cytokines Expression, laid the foundation for AhR biological functions in follow-up further research macrophage.
More specifically, the cell quality of the present invention is unified, the gene expression holding of cell is stable in succeeding generations.Therefore profit
Tested with the stable expression AhR of present invention monoclonal cell strain, the result obtained is more reliable and more stable.
Brief description of the drawings
Fig. 1 is expression of results figure of the fluorescence microscope green fluorescent protein (GFP) on RAW264.7 cells, its
In, Figure 1A and Fig. 1 C are the observation result under white light, Figure 1B and Fig. 1 D are the observation result under green fluorescence；
Fig. 2 is the result figure that FCM analysis green fluorescent protein (GFP) is expressed on RAW264.7 cells；
Fig. 3 is the AhR gene level expressions of results of RT-PCR detection stable cell strains；
Fig. 4 is the AhR protein expression results of Western Blot detection stable cell strains；
Fig. 5 is the AhR protein expression results of Western Blot detection stable cell strain difference algebraically；
Fig. 6 is the influence result figure for being overexpressed the Raw cellular inflammation factors mRNA expression that AhR is stimulated LPS；
Fig. 7 is the influence result figure for being overexpressed the Raw cellular inflammations factor protein expression that AhR is stimulated LPS.
Biological deposits
The monoclonal cell of the present invention, China typical culture collection center (is deposited on July 26th, 2017
Location：Luojiashan, Wuchang, Wuhan City, Hubei Province, Wuhan University, postcode：430072) (depositary institution is abbreviated as CCTCC), protect
It is CCTCC NO to hide numbering：C2017118, the culture title of preservation and dated diagnostic characteristics：
It is overexpressed AhR RAW264.7 cells RAW/AhR.
Embodiment
The end points of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For number range, between the endpoint value of each scope, respectively
It can be combined with each other between the endpoint value of individual scope and single point value, and individually between point value and obtain one or more
New number range, these number ranges should be considered as specific open herein.
The deposit number of monoclonal cell (can stablize the cell for expressing AhR) provided by the invention is CCTCC NO：
C2017118.As it was previously stated, the monoclonal cell character of the present invention is unified, the gene expression holding of cell is steady in succeeding generations
It is fixed, therefore (experiment that such as aryl hydrocarbon receptor regulates and controls to inflammatory cytokine) is tested using it, the result obtained is more
It is reliable and stable.
Expression AhR provided by the invention method includes：The monoclonal cell of the present invention is cultivated.
According to a kind of special embodiment of the present invention, the culture is carried out in the presence of lipopolysaccharides, to inquire into AhR pairs
The regulating and controlling effect of free macrophage.Dosage and condition of culture to lipopolysaccharides do not require particularly, can be conventional choosing
Select.Under preferable case, the dosage of lipopolysaccharides is 1-10 μ g/mL culture mediums.
According to the present invention, in the case of the part without using AhR, be advantageous to know the AhR of no ligand activation to inflammatory
Macrophage has negativity regulating and controlling effect.Therefore, according to the preferred embodiment of the present invention, AhR's matches somebody with somebody in the cultivating system
The μ g/mL culture mediums of the content of body≤1 (or≤0.1 μ g/mL culture mediums, or≤0.01 μ g/mL culture mediums).It is it is highly preferred that described
Culture is in part (such as Polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAH) (PAHs), benzanthracene (BZA), plant part turmeric without AhR
Plain (Curcumin), micromolecular compound naphthoflavene (ANF)) in the presence of carry out.By the monoclonal cell table for making the present invention
Up to AhR, namely the monoclonal cell by (in the presence of lipopolysaccharides) culture present invention, verify the AhR without ligand activation
There is negativity regulating and controlling effect to free macrophage, proinflammatory cytokine IL-6 and IL-1 β expression can be lowered, raise IL-
10 expression, laid a good foundation for AhR immunoregulation functions in follow-up further research macrophage.
Study the application during AhR regulates and controls to inflammatory cytokine in vitro present invention also offers above-mentioned monoclonal cell.
For example, it can be realized by cultivating the monoclonal cell of the present invention.
The method provided by the invention for preparing monoclonal cell, it is characterised in that this method includes：
(1) plasmid for carrying AhR encoding genes is subjected to slow virus packaging；
(2) use and pack successful slow-virus transfection cell；
(3) cell in the culture medium of purine-containing mycin after culture transfection, so as to screen to obtain monoclonal cell.
In step (1), the plasmid for carrying AhR encoding genes is preferably between BamHI and AgeI restriction enzyme sites
GV358 carriers inserted with aryl hydrocarbon receptor encoding gene.The method for obtaining the plasmid can be conventional method, for example,
Performing PCR amplification (the primer used such as SEQ ID NO can be entered to AhR genes (such as NM_013464) in cDNA library：1 and 2
It is shown), amplified production and GV358 carriers are subjected to digestion with BamHI, AgeI respectively, are connected into after purifying recovery with T4 ligases
GV358 carriers, conversion, screening, so as to obtain described (restructuring) plasmid.In order to ensure plasmid carries AhR encoding genes, may be used also
Further to be identified (as being sequenced).
In step (1), the slow virus is preferably HIV slow virus (human immunodeficiency virus).
In step (2), the cell is preferably RAW264.7 cells.
Wherein, the method for transfection can be conventional method, and the method screened to the cell after transfection can be according to
Conventional mode is carried out, for example, when containing fluorescin (such as GFP) encoding gene in plasmid, except by purine-containing mycin
, can also be in the expression of observe observe under fluorescence microscope, according to the expression of fluorescin beyond culture medium
The stable infection cell of screening.
In the present invention, methods described can also include further identifying the monoclonal cell that screening obtains, example
Such as, total serum IgE and total protein are collected, so as to determine the expression of AhR genes.
The present invention will be described in detail by way of examples below.
Embodiment 1
The present embodiment is used for the acquisition for illustrating the cell line of the present invention.
(1) experiment material：RAW264.7 cell lines and HEK293T cell lines are purchased from Chinese Academy of Sciences's American Type Culture Collection
Committee's cell bank is preserved by Chongqing Field Surgery Inst. of The Third Military Medical Univ.；GV358 carriers, viral infectious agent
ENi.S. it is purchased from Shanghai JiKai Gene Chemical Technology Co., Ltd；BamHI, AgeI restriction enzyme, the purchase of T4DNA ligases
From U.S. NEB；Ago-Gel DNA QIAquick Gel Extraction Kits, plasmid be small to be carried middle amount kit and is purchased from Beijing Tiangeng；Puromycin is purchased from
Give birth to work in Shanghai；Mouse AhR (NM_013464) full length sequence reported according to ncbi database, designs primer P1/P2, and forward direction is drawn
Thing is 5'-GAGGATCCCCGGGTACCGGTCGCCACCATGAGCAGCGGCGCCAACATC ACC-3'(SEQ ID NO：1),
Reverse primer is 5'-TCCTTGTAGTCCATACCACTCTGCACCTTGCTTAGGAATGC-3'(SEQ ID NO：2)；PCR reflects
Determine primer P3/P4, forward primer is 5'-CGAAGTACGTCGCTGCCCTTC-3'(SEQ ID NO：3), reverse primer 5'-
CCTTATAGTCCTTATCATCGTC-3'(SEQ ID NO：4)；LPS is purchased from sigma companies of the U.S.；Mouse IL-6, IL-1 β,
IL-10 enzyme-linked immunosorbent assay kits are purchased from Wuhan Boster Biological Technology Co., Ltd.；Hyclone, RPMI 1640 are cultivated
Base (cell culture use), tryptic digestive juice, dual anti-it is purchased from Gibco companies of the U.S.；Total serum IgE Rapid extraction kit is purchased from north
The Tyke of capital hundred；Reverse transcription and qPCR kits are purchased from upper Hypon life biotech firm；AhR antibody is purchased from U.S. Enzo；Flag、β-
Actin antibody is purchased from U.S. CST；Flow cytometer is Hangzhou Essen Biology ACEANovoCyte products.
(2) plasmid construction：Enter performing PCR amplification to AhR genes with primer P1/P2 in cDNA library；Product is through 1% agar
After sugar is gel purified, uses BamHI, AgeI digestion respectively together with slow virus GV358 carriers, connected after purifying recovery with T4
Connect enzyme and be connected into GV358 carriers, conversion, screening recombinant plasmid.After double digestion is identified and confirmation (using primer P3/P4) is sequenced,
Extract recombinant plasmid, purifying dissolving, -20 DEG C of preservations.
(3) prepared by slow virus packaging：It is limited that the plasmid for carrying target gene AhR is sent to the lucky triumphant chemical gene technology in Shanghai
Company carries out HIV slow virus packagings, and carries out titer determination.Mainly comprise the following steps：Take the logarithm growth period culture HEK293T it is thin
Born of the same parents, use LipofectamineTM2000 transfection reagents transfect recombinant plasmid.The expression intensity of observe observe, collect cell
Culture supernatant, further after processing, obtain packing successful Ubi-AhR-3FLAG-SV40-EGFP-IRES-puromycin slow
Virus, it is 2E+8TU/ml with ELISA method detection virus titer, freezes in -80 DEG C of refrigerators.
(4) stable infection cell screening：By the RAW264.7 cells of exponential phase with 1 × 105It is individual to be inoculated in six orifice plates
In, viral dose is determined according to its infection multiplicity, the virus liquid that nutrient solution adds ENi.S. dilutions is changed before next day infection, after
Fresh medium is changed after continuous culture 12h, is screened after transfecting 72h with 5 μ g/ml puromycins, without the thin of infection after 7d
Born of the same parents are all dead, obtain the polyclonal cells strain for being overexpressed AhR, and visible GFP is expressed under fluorescence microscope, and pick out stable table
The monoclonal cell strain reached, persistently cultivated with 2 μ g/ml puromycins.
Treat the normal RAW264.7 cells of infection and carry out dose-response analysis, the results showed that the suitable screening of puromycin
Concentration is 5 μ g/ml.After virus infected cell 48h, it is glimmering that fluorescence microscope visible part is infected cell expression GFP greens
Photoprotein (Figure 1A -1B).Screened after infection 72h with 5 μ g/ml puromycins, be then discovered that part cell occur becomes rugula
Contracting, adherent loosely floating is dead, and liquid screening is changed by 7d, the cell being uninfected by due to all dead not with resistant gene,
The polyclonal cells strain for being overexpressed AhR is obtained, the more stable monoclonal cell strain of property is then picked out, is named as
RAW264.7 is overexpressed AhR cells, and strain number is D2 strains, and symbol is RAW/AhR cells and freezes conservation, namely has obtained this hair
Bright cell line, or the steady RAW264.7 cells for turning AhR.
Test case 1
This test case is used for the ability for the cell line expression AhR for illustrating the present invention.
In test case, analyzed using the statistical softwares of SPSS 13.0, measurement data is with mean ± standard deviation (x ± s)
Represent, between each group the comparison of data use variance analysis.P<0.05 represents that difference is statistically significant.Similarly hereinafter.
Take the logarithm the normal RAW264.7 cells in growth period, empty carrier transfectional cell, the steady RAW264.7 cells for turning AhR
6 orifice plates are inoculated in respectively, cultivate 12h, cell total rna and total protein are collected respectively after PBS washings.Cell add 1mL lysates/
Hole, with the Tyke RP1202 kits extraction total serum IgE of Beijing hundred, determine RNA concentration and purity；By TaKaRa RR047A kits
Operation, takes 1g RNA reverse transcriptions into cDNA；Operated by TaKaRa RR820A kits, carry out qPCR quantitative analyses, primer sequence
Row are shown in Table 1.According to obtained Ct values, using 2-△CtMethod calculates gene expression amount respectively.Cell adds protein lysate and carried always
Albumen, after protein quantification, albumen loading sample is prepared, by Western Blot methods electrophoresis, transferring film, the closing of 5% skimmed milk power, incubated
Educate primary antibody (AhR, Flag, β-actin) and secondary antibody, ECL developments, gel imaging system exposure.
The visible RAW/AhR cells of fluorescence microscope all express GFP green fluorescent proteins (Fig. 1 C-1D).Streaming is thin
Born of the same parents' instrument detects, and each cell expresses GFP green fluorescences (Fig. 2).Turn using the checking of qRT-PCR and Western Blot methods is steady
Whether AhR gene and protein level raise in cell, it is found that RAW/AhR surely turns group (85.68 ± 9.79) relative to RAW just
Normal group (control group, 1.00 ± 0.08) and RAW/Vector idle running group (1.58 ± 0.21), AhR mRNA level in-site substantially raise
(P<0.05, Fig. 3).Further detecting confirms, RAW/AhR surely turns a group AhR expression and significantly rises and express Flag tag fusion eggs
In vain, remaining two groups of low expression AhR albumen and Flag albumen (Fig. 4) is not expressed.
In addition, obtained RAW/AhR cells carry out to AhR protein level point again after repeatedly passing on (the 30th generation)
Analysis, it is found that its expression is not decayed, illustrates the cell line of the present invention and can stablize expression AhR (referring to Fig. 5).
Test case 2
Application of the cell line that this test case is used for illustrating the present invention in research AhR regulates and controls to inflammatory cytokine.
The idle running group cell in growth period, the steady group RAW264.7 cells that turn take the logarithm respectively with 2 × 106Individual/hole is inoculated in 6 holes
Plate, it is divided into idle running Control groups, idle running LPS groups, surely turns Control groups, surely turns LPS groups, after handling 4h with LPS (10 μ g/mL)
Cell total rna is collected, cell conditioned medium is collected after handling 12h.Carrying out qRT-PCR experiments according to the method described above, (primer sequence is shown in Table
1) IL-6, IL-1 β, IL-10 gene expression amount, are detected, the expression of cell conditioned medium secretory protein is detected using ELISA method
Amount.
Table 1
As described above, in order to verify influences of the AhR for RAW264.7 cellular immune functions, stimulate structure scorching using LPS
Disease cell model, detect inflammatory cytokine mRNA respectively and secrete the situation of change of supernatant.RAW264.7 cells are stimulated in LPS
After 4h, idle running LPS groups and the steady LPS groups that turn are relative to Control groups, intracellular IL-6, IL-1 β and IL-10 mRNA expression
Level significantly increases.Relative to idle running LPS groups, it is lower surely to turn proinflammatory cytokine IL-6, the IL-1 β expression of LPS groups,
Anti-inflammatory cytokines IL-10 expression it is higher (Fig. 6, wherein, A-C respectively illustrates IL-6, IL-1 β and IL-10 mRNA tables
Up to level).Equally, IL-6, IL-1 β and IL-10 protein expression level raise in cell conditioned medium is detected after LPS stimulates 12h,
Be overexpressed AhR after lower LPS stimulate RAW264.7 cells expression IL-6, IL-1 β, raise IL-10 expression (Fig. 7, wherein,
A-C respectively illustrates IL-6, IL-1 β and IL-10 protein expression level).Result above shows the macrophage that AhR is stimulated LPS
Cellular inflammation reaction has negativity regulating and controlling effect.
In summary, the present invention is screened after infection RAW264.7 cells and obtained by building the AhR high slow virus carriers expressed
Expression AhR monoclonal cell strain must be stablized, and carry out gene and protein level identification, then verify the AhR of no ligand activation
There is negativity regulating and controlling effect to free macrophage, lower proinflammatory cytokine IL-6, IL-1 β expression, raise IL-10 table
Reach.The Macrophage Model of the stable high expression AhR genes of structure, tentatively illustrates it and regulates and controls phenomenon to inflammatory cytokine, will
Laid the foundation for AhR immunoregulation functions in follow-up further research macrophage.
The preferred embodiment of the present invention described in detail above, still, the present invention is not limited thereto.In the skill of the present invention
In art concept, technical scheme can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, and these simple variants and combination should equally be considered as content disclosed in this invention, belong to
Protection scope of the present invention.
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Sequence table
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<213> Artificial Sequence
<400> 13
agccatgtac gtagccatcc 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 14
ctctcagctg tggtggtgaa 20
Claims (9)
1. one plant of monoclonal cell, it is characterised in that the deposit number of the monoclonal cell is CCTCC NO：C2017118.
A kind of 2. method for expressing aryl hydrocarbon receptor, it is characterised in that this method includes：Cell described in claim 1 is entered
Row culture.
3. according to the method for claim 2, wherein, the μ g/ of the content of the part of aryl hydrocarbon receptor in the cultivating system≤1
ML culture mediums.
4. according to the method for claim 2, wherein, the culture is carried out in the presence of the part without aryl hydrocarbon receptor.
5. the cell described in claim 1 studies the application during aryl hydrocarbon receptor regulates and controls to inflammatory cytokine in vitro.
A kind of 6. method for preparing monoclonal cell, it is characterised in that this method includes：
(1) plasmid for carrying aryl hydrocarbon receptor encoding gene is subjected to slow virus packaging；
(2) use and pack successful slow-virus transfection cell；
(3) cell in the culture medium of purine-containing mycin after culture transfection, so as to screen to obtain monoclonal cell.
7. the method according to claim 11, wherein, in step (1), the matter for carrying aryl hydrocarbon receptor encoding gene
Grain is the GV358 carriers inserted with aryl hydrocarbon receptor encoding gene between BamHI and AgeI restriction enzyme sites.
8. according to the method for claim 6, wherein, in step (1), the slow virus is HIV slow virus.
9. according to the method for claim 6, wherein, in step (2), the cell is RAW264.7 cells.
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| CN114350617A (en) * | 2022-01-24 | 2022-04-15 | 安徽医科大学第一附属医院 | Application of Raw264.7 cell line for stably expressing different human ApoE genotypes and preparation method thereof |
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