CN105602904B - The new application of CD133 plasmid - Google Patents

The new application of CD133 plasmid Download PDF

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CN105602904B
CN105602904B CN201610092181.4A CN201610092181A CN105602904B CN 105602904 B CN105602904 B CN 105602904B CN 201610092181 A CN201610092181 A CN 201610092181A CN 105602904 B CN105602904 B CN 105602904B
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cik
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csc
plasmid
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CN105602904A (en
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孙雯雯
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TIANJIN 4TH CENTER HOSPITAL
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention provides the new application of CD133 plasmid, the CD133 plasmid can be effectively used for enhancing DC-CIK to the killing effect in vitro of breast carcinoma stem cell.

Description

The new application of CD133 plasmid
Technical field
The present invention relates to the new applications of CD133 plasmid.
Background technique
Breast cancer is the most common malignant tumour of women in world wide.A large amount of evidences show dry thin just because of breast cancer The presence of born of the same parents (Breast Cancer Stem Cell, BCSC) causes it to be easy transfer, recurs and resists to chemicotherapy.And it is close The adoptive cellular immunotherapy (adoptive cell immunotherapy, ACI) risen over year is that one kind is directed to chemicotherapy not The new treatment of susceptible neoplasms, clinical landscapes are wide, obtain more and more pay attention to.
The presence of CSC is to influence one of breast cancer treatment effect and the most important factor of prognosis, is clinically applied at present Chemicotherapy is typically only capable to eliminate normal breast cancer cell, and the remaining BCSC resisted to chemicotherapy remains to constantly be proliferated, and leads to cream Gland cancer relapse and metastasis, so a kind of method for finding effective killing CSC becomes the new direction of breast cancer treatment.ACI is A kind of novel therapies that the amplification in vitro progress tumour cell relying on self responsiveness immunocyte is effectively killed, are applied first In the treatment of hematological malignancies, after gradually extend to the entity tumor including breast cancer, breast cancer, lung cancer etc..The effect of ACI Answering cell mainly includes cytokine induced kill cell (CIKs), Tumor-infiltrating lymphocytes (lymphokine- Activated killer cells, LAKs), tumor infiltrating lymphocyte (tumor infiltrating lymphocytes, TILs), inosculating antibody cytotoxic T lymphocyte (cytotoxic T lymphocyte cells, CTLs) and proposed recently T cell (chimeric antigen receptor T cells, CAR-T) of original receptor etc..And it is most powerful as in vivo functionality The DC that offers of professional antigen be also the important component part of ACI.
CIK cell is quick with proliferation, kill tumor power by force, kills tumor spectrum extensively, to normal cell without lethal effect, to drug-resistant tumor The advantages that sensitive is the highest non-specific killing immune effector cell of activity being currently known.DC can mention tumour antigen In to immunocompetent cell, promote its Proliferative Activated, enhances the killing activity to tumour cell.Some researches show that load tumour The DC immunophenotype of stem cell antigen is mature earlier above, and by it with after CIK cell co-incubation, DC-CIK immunophenotype is further Mature and secrete cytokines level correspondinglys increase.
CD133 is the most important molecular labeling of breast carcinoma stem cell, it is a kind of single span membrane glycoprotein, by matching with a variety of The combination of body plays key effect in the behaviors such as cell adherence and migration.
Summary of the invention
Technical problem to be solved by the present invention lies in provide the new application of CD133 plasmid.
In order to solve the above technical problems, the technical scheme is that
CD133 plasmid is in enhancing DC-CIK to the application in terms of the killing effect in vitro of breast carcinoma stem cell.
Preferably, the application of above-mentioned CD133 plasmid, afterwards and CIK with CD133 plasmid impact DC (dendritic cell) (cytokine-induced killer) Coculture increases DC-CIK to the Cytotoxicity in vitro effect of breast carcinoma stem cell By force.
Preferably, the application of above-mentioned CD133 plasmid, the partial nucleotide sequence of the CD133 plasmid are SEQ ID NO:1 Shown sequence.
The beneficial effects of the present invention are:
CD133 plasmid has important application valence in terms of enhancing DC-CIK is to the killing effect in vitro of breast carcinoma stem cell Value.
Detailed description of the invention
The Dendritic Cells of Fig. 1 CSC-RNA induction；
The Dendritic Cells of Fig. 2 CSC- lysate antigen induction；
Fig. 3 helper plasmid and purpose plasmid co-transfection 293T for 24 hours with the green fluorescence intensity of 48h；
The slow virus of Fig. 4 concentration goes to infect 293T cell, and 12h changes liquid, the fluorescence intensity after 48.
Specific embodiment
In order to make those skilled in the art better understand technical solution of the present invention, With reference to embodiment Technical solution of the present invention is described in further detail.
Embodiment 1
One, the preparation of the full cell lysate of MCF-7 Breast Cancer Cell stem cell
It collects MCF-7 Breast Cancer Cell and its stem cells, adjust separately cell concentration to 1 × 107(MCF-7 cell And its stem cells: DC cell=10:1), it goes in cryopreservation tube, cryopreservation tube is put into liquid nitrogen 15min, is put immediately after taking-up Enter 15min in 37 DEG C of water-baths, 4 times repeatedly, after cracking, 4 DEG C of centrifugations, 10000g is centrifuged 10min, collects supernatant, 0.22um filter Film filtration sterilization, be stored in -80 DEG C it is spare.Measured with BCA method it is above-mentioned splitting object protein concentration, by BCA kit specification carry out Determination of protein concentration.
The extraction of MCF-7 Breast Cancer Cell stem cell total serum IgE
It is operated according to Qiagen Rneasy mini kit kit specification.Collect the MCF-7 cell of logarithmic phase growth And the MCF-7 stem cell of magnetic bead sorting, count no less than 1 × 107It is a, it moves in the 1.5ml EP pipe of no RNase, 600 μ is added L Buffer RLT blows and beats 5-8 times with 1ml asepsis injector repeatedly and cracks completely to cell, then, 600 μ l, 70% second is added Alcohol mixes, and 8 000g are centrifuged 15s, and abandoned stream wears liquid, 700 μ l RW1Buffer is added to collecting in column, 8 000g are centrifuged 15s washing Column is collected, the 20-30 DEG C of incubation 15min in collection column of the 80 μ l of Buffer RDD containing 10 μ l DNase is added and thoroughly disappears Change genomic DNA.350 μ l RW1 Buffer are added to collecting in column, 8 000g are centrifuged 15s washing and collect column, are then added 500 μ l RPE Buffer are to collecting in column, and 8 000g centrifugation 2min, which is thoroughly washed, collects column, and abandoned stream wears liquid.Column will be collected to be placed in In new 1.5ml EP pipe, deionized water of the 30-50 μ l without RNase is added, 8 000g of centrifugation are centrifuged 1min, and collection elutes RNA.The RNA concentration of 2000 spectrophotometer Detection and Extraction of Nanodrop.
The building of CD133 slow virus expression system
1.1.1.1 design of primers
With pCDNA3.1+CD133 (s16-s24) for template
Primer: CD133F:5-CGGAATTCATGGGTGCAAATGTGGA-3 ' (sequence shown in sequence table<400>2)
CD133R:5-ATAAGAATGCGGCCGCTCACAAGGGGTCGATAATGTAGC-3
(sequence shown in sequence table<400>3)
1.1.1.2PCR amplification
Reaction system: being sequentially added into following 7 kinds of ingredients, mixes:
Response procedures:
The recycling of PCR product glue, is connected with the overexpression control vector provided after EcoRI and NotI double digestion with us.
1.1.1.3 digestion
Segment digestion:
37 DEG C of digestion 2-3h.Electrophoresis gel extraction (digestion QIAquick Gel Extraction Kit, TaRaKa)
Carrier digestion:
37 DEG C of digestion 2-3h.Electrophoresis gel extraction (Ago-Gel DNA QIAquick Gel Extraction Kit, BPI)
1.1.1.4 connection, conversion
(1) connect: 1 μ l carrier, 3 μ l digestion recovery products, 1 μ l ligase (BPI), 5 μ l 2 × Rapid Buffer are mixed It is even, react at room temperature 30min.
(2) it converts: taking out 100 μ l competent cells (BL21) of -80 DEG C of preservations, be placed on ice slowly defrosting.It will impression State cell is added in connection product and mixes, and places 30min on ice.42 DEG C of heat shock 90s.After ice bath 2min, 800 μ l non-resistants are added LB culture medium.37 DEG C of culture 45min.5000rpm is centrifuged 3min, abandons most of supernatant, stays about 100-150 μ l, thallus is resuspended, Selection has the LB plate of corresponding resistant, coated plate.It dries, is inverted overnight incubation in 37 DEG C of incubators.Screening positive clone, plasmid It extracts, and is sequenced.
1.1.1.5 plasmid extracts (removing endotoxin)
E.coli with plasmid is inoculated in 200ml LB/ antibiotic culture solution, 37 DEG C of shaking table culture 12h；It takes The bacterium solution of 200ml, 5000g is centrifuged 10min and collects bacterium at room temperature.Abandon culture medium.10ml Solution I/ is added RNaseA mixed liquor, vortex oscillation make cell suspend completely.10ml Solution II is added toward being resuspended in mixed liquor, gently runs Mixed liquor can be placed at room temperature for 2min to improve yield after mixing 7-10 times.It avoids acutely mixing lysate and cracking reaction not It to be more than 5min.5ml ice bath Buffer N3 is added, and mildly overturns centrifuge tube 10 times and extremely forms white flock precipitate.It is ready to Filter.Column sleeve is measured in HiBind in collecting pipe, 3ml Buffer GPS is added into pillar, stands 3-5min.Room temperature 5000g is centrifuged 5min.Filtrate is gone, pillar is placed back in collecting pipe.10ml sterile water is added, 5000g is centrifuged 5min.It will split Solution liquid pour into preprepared syringe filter (syringe is opening up, below exit receive collector), stand 2min.It inserts Enter and touching piston flows into lysate in following collecting pipe.The ETR of 1/10 volume is added in filtering clear lysate, Solution becomes muddy after 7-10 times reverse, ice bath 20min.After 42 DEG C of water-bath 5min, 25 DEG C, 5000g is centrifuged 5min, ETR solution Blue layering will be formed in tube bottom.After centrifugation, if there are a large amount of suspending drops in solution, stands 10min and make drop naturally heavy Drop.Transfer supernatant moves in centrifuge tube, and 0.5 times of volume dehydrated alcohol (room temperature) is added, is stored at room temperature 2min after mixing.It will The a large amount of columns of HiBind are in 50ml collecting pipe.It shifts in 20ml mixed liquor to pillar, 5000g is centrifuged 5min at room temperature, goes Filtrate.The step is repeated, remaining filtered fluid is transferred to pillar, until all solution is all filtered out from pillar.Pillar weight New clothes recycle collector, and 10ml HB Buffer is added, and are centrifuged by above-mentioned condition, abandon filtrate.Pillar is reinstalled collecting pipe, is added Enter 15ml DNA Wash Buffer, be centrifuged by above-mentioned condition, abandons filtrate, be repeated once.Pillar is reinstalled collecting pipe, most Big speed (< 6000g) centrifugation 15min is to dry column matrix.Pillar on clean 50ml centrifuge tube, it is added 70 DEG C in advance On hot 3mlEndotoxin free Elution Buffer to column matrix (added amount depends on expected final product concentrations), 4000g speed centrifugation 5min is after standing 5min at room temperature with eluted dna.Take 10 μ l quantitative with trace dna quantitative instrument.
1.1.1.6 slow virus is packed
It is incubated overnight at 4 DEG C with gelatin (0.2%) coating 10cm dish.Pancreatin digests the 293T cell of culture early period, Uniform bed board waits for that cell fusion degree reaches the side 70%-90% and can be packed.It is careful to draw cell culture medium supernatant, it is added 6mL serum-free DMEM without double antibody, it is stand-by to be put into incubator.Transfection reagent and DNA compound are prepared, takes sterile no enzyme EP pipe 2, Wherein 1 is separately added into No. 1 helper plasmid 6.5 μ g, No. 2 helper plasmids 2.5 μ g, No. 3 helper plasmids 3.5 μ g, 15 μ of purpose plasmid 55 μ l transfection reagents are added in g (if purpose plasmid is changed to control plasmid by packaging control slow virus) another EP pipe (1mg/ml).Two EP are diluted to 500 μ l respectively using aseptic filtration PBS or DMED, 500 μ l of transfection reagent is drawn and is added 4 Softly piping and druming mixes (note: make sure to keep in mind to be vortexed, can not overturn addition sequence) in plasmid.Static placement 20min, quickly dropwise It is added in stand-by culture dish, 8 words are put into incubator after mixing culture dish.Fresh complete medium 12mL is changed after 6h.After changing liquid 48h collects supernatant, 1000g 10min centrifugation or 0.45 μm of sterile filters filtering.
1.1.1.7 PEG-8000 is concentrated in slow virus:
5 times of damp and hot extinction 30min of concentration PEG8000,121 DEG C of 30min；It is stored in 4 DEG C；It is filtered using 0.45 μm of filter slow Vial supernatant；Every filtered initial liquid of virus of 30ml, is added 5 times of concentration PEG-8000 and adds NaCl mother liquor 7.5ml；Often 30min mixing is primary, carries out 3-5 times altogether；4 DEG C stand overnight；4 DEG C, 4000g, it is centrifuged 20min；It inhales and abandons supernatant, stand pipe 2min siphons away residual liquid；Suitable (corresponding 100 μ l of 10ml) slow virus lysate (PBS) dissolution slow virus precipitating is added；It is dense Viral suspension after contracting is distributed into 50 every part of μ l, is stored in production tube, with being stored in -80 DEG C after broken dry ice quick-frozen.
1.1.1.8 the low detection of virus: virus titer measurement is carried out using GFP fluorecyte number
Titre unit: TU/ml refers to the biologically active virion number contained in every milliliter." TU " is The abbreviation of " transducing units ", Chinese are " transduced unit ", and expression can be infected and enter the virus in target cell Genome number.
By the good 293T cell dissociation of growth conditions, 6 orifice plates, the hole 2ml/ is added in the dilution ratio for passing six according to one.It puts Enter 37 DEG C, 5%CO2It is cultivated in incubator.Adding virus, convergence degree reaches 20%-50%, and it is added and dilutes virus, dilution ratio (1: 500).It is diluted with complete medium, every hole culture volume 1ml.3 blank wells are chosen simultaneously, carry out cell count.Additional training Nutrient solution sucks with virulent culture solution after 12h, adds the fresh complete culture solution of 2ml, in each hole in favor of cell Growth.Streaming surveys positive rate and calculates titre, vitellophag after 48h-72h, and PBS is cleaned twice, stream measuring GFP positive rate, MOI refers to infection multiplicity (a batch cell is by several virus infections)
Titre (TU/ml)=cell number × fluorescence percentage × MOI × viral dilution multiple
1.1.1.9DC preparation and identification
DC cell culture: the leucocyte 50ml of separating health people, adherent cell collecting, be added containing IL-41 000U/ml, It is cultivated in 1640 culture medium of 1 000U/ml of GM-CSF and 10% calf serum.It is impacted with different alluviums within the 5th day in culture DC, experimental group are that MCF-7 cell stem cell (CSC) constructs tumour-specific biomarker CD133 Transfected Recombinant Plasmid CD133- DC-CIK (MOI=80), MCF-7 cell stem cell CSC-RNA (RNA concentration is respectively 20ug) and CSC- lysate (CSC:DC Cell is 10:1) and MCF-7 cell MCF-7-RNA extract (RNA concentration is respectively 20ug), MCF-7 cell lysate (MCF-7 cell: DC cell is respectively 10:1)；Control group is the DC cell directly cultivated.The 6th day of culture, respectively to experiment TNF-α 10ng/ml is added in group and control group, is cultivated for 2-3d and obtains mature DC cell.
DC cellular identification: collecting the 0th, 5,8 day DC cell of culture respectively, and 200g/min is centrifuged 3min；Cell 2 is washed with PBS Time, it counts；PBS adjusts cell concentration to 1 × 105/ 100 μ l set blank tube and experiment tube, and every group sets 3 multiple pipes；Respectively plus Enter the anti-human CD80 antibody of mouse antibodies against human CD 40, mouse, mouse anti-human HLA-DR antibodies, the anti-human CD86 antibody of mouse, mixes well, 4 DEG C It is protected from light and is incubated for 30min；After incubation, cell is resuspended with 1-2ml PBS, 218g/min is centrifuged 3min, is repeated 1 times；Supernatant is abandoned, Cell is resuspended with 1ml PBS, mixes well, is placed in 4 DEG C of ice chests；Flow cytomery records result.
Table 1DC Immunophenotype analysis (n=3, %,)
*P<0.01
The preparation and identification of CIK cell
1.1.1.10CIK cell culture
Above-mentioned non-adherent cell is collected, is added in 10% calf serum, 1640 culture medium containing IFN-γ 50ng/m.24h Every 2-3 days IL-2500U/m, IL-1 α 5ng/ml, antibody (e.g., CD3) 50ng/ml half amounts are added afterwards and changes liquid.In the 8th day of culture By CIK cell in DC cell co-cultivation, every 2 days half amounts of co-cultivation process change liquid, and add IL-2, until 14 days CIK cells at It is ripe.
The analysis of table 2CIK Immunophenotyping (n=3, %,)
*P<0.01
Two, the sequence (DNA sequence dna) of CD133 plasmid, partial nucleotide sequence are sequence shown in SEQ ID NO:1:
ATGGGTGCAAATGTGGAAAAACTGATCTGTGAACCTTACACGAGCAAGGAATTATTCCGGGTTTTGGAT ACACCCTACTTACTAAATGAAGACTGGGAATACTATCTCTCTGGGAAGCTATTTAATAAATCAAAAATGAAGCTCAC TTTTGAACAAGTTTACAGTGACTGCAAAAAAAATAGAGGCACTTACGGCACTCTTCACCTGCAGAACAGCTTCAATA TCAGTGAACATCTCAACATTAATGAGCATACTGGAAGCATAAGCAGTGAATTGGAAAGTCTGAAGGTAAATCTTAAT ATCTTTCTGTTGGGTGCAGCAGGAAGAAAAAACCTTCAGGATTTTGCTGCTTGTGGAATAGACAGAATGAATTATGA CAGCTACTTGGCTCAGACTGGTAAATCCCCCGCAGGAGTGAATCTTTTATCATTTGCATATGATCTAGAAGCAAAAG CAAACAGTTTGCCCCCAGGAAATTTGAGGAACTCCCTGAAAAGAGATGCACAAACTATTAAAACAATTCACCAGCAA CGAGTCCTTCCTATAGAACAATCACTGAGCACTCTATACCAAAGCGTCAAGATACTTCAACGCACAGGGAATGGATT GTTGGAGAGAGTAACTAGGATTCTAGCTTCTCTGGATTTTGCTCAGAACTTCATCACAAACAATACTTCCTCTGTTA TTATTGAGGAAACTAAGAAGTATGGGAGAACAATAATAGGATATTTTGAACATTATCTGCAGTGGATCGAGTTCTCT ATCAGTGAGAAAGTGGCATCGTGCAAACCTGTGGCCACCGCTCTAGATACTGCTGTTGATGTCTTTCTGTGTAGCTA CATTATCGACCCCTTGTAA
Three, result
The form of DC cell
The 5-7 days DC cells are cultivated, volume increases, and attached cell occurs, quantity increases, pleomorphism cytosis.5th day It is impacted respectively with antigen prepared by this research, most cells aggregation is agglomerating after cultivating 5d, and cell space is larger, and shape is by When circle become irregular shape, cultivate that cell after 7d has the cytosis of dendron shape protrusion and protrusion becomes apparent.Electronic Speculum Lower that typical DC form is presented, bigger than lymphocyte volume, endochylema protrusion is obvious, grows in dendron sample, sees Fig. 1, Fig. 2.
CD133 slow-virus infection result
After CD133 slow-virus infection, the positive rate of pLenti-CMV-CD133-EF1-copGFP is 69.7% (Fig. 3,4), The titre range of pLenti-CMV-CD133-EF1-copGFP is 5.12 × 108TU/ml-15.37×108TU/ml。
Four, killing experiments in vitro of the difference DC-CIK cell system to MCF-7 Breast Cancer Cell and its stem cell
Experimental group is CD133-DC-CIK group, CSC-RNA-DC-CIK group, three groups of CSC- lysate-DC-CIK group, control Group is CIK and DC-CIK group, selects above-mentioned cell as effector cell, and MCF-7 Breast Cancer Cell and its stem cell are target cell, Killing experiments are carried out using lactic dehydrogenase (LDH) method for releasing.
LDH release: adjustment effector cell's concentration is 1 × 106/ mL, target cell concentration are 1 × 105/mL.It presses It is added in 96 orifice plates according to effect target ratio 10:1,20:1,40:1,80:1 ratio, under each effect target ratio, if 3 multiple holes.Laying effect simultaneously Cell Spontaneous release hole, target cell Spontaneous release hole, target cell maximum relief hole, culture solution ground control hole, lysate background Control wells, every group sets 3 holes again, mixes cell, and 4h is cultivated in 37 DEG C, 5% carbon dioxide incubator, collects supernatant, uses enzyme Mark instrument detects its absorbance (OD) value under 490nm wavelength.It calculates as follows:
Experimental data carries out statistical disposition using SPSS 18.0, in each group and between group identical index carry out paired t-test and Variance analysis, institute's measured data mean ± standard deviationIt indicates, P < 0.05 indicates that result is statistically significant.
Lethal effect of the different cells to MCF-7 Breast Cancer Cell
By CIK, DC-CIK, CSC133-DC-CIK, CSC-RNA-DC-CIK, CSC- lysate-DC-CIK, MCF-7- RNA-DC-CIK and MCF-7- lysate-DC-CIK is used as effector cell, and MCF-7 cell is target cell, is examined using LDH method for releasing It surveys, with the increase of effect target ratio, killing functions of immunocytes enhancing, when effect target ratio is 80:1, to the killing rate highest of MCF-7 cell. When effect target ratio is 10:1,20:1,40:1,80:1, CSC133-DC-CIK group, CSC-RNA-DC-CIK group, CSC- lysate-DC- The killing rate of CIK group is higher than CIK group and DC-CIK group, and difference is statistically significant (P < 0.05)；DC-CIK group is higher than CIK Group, difference are statistically significant (P < 0.05)；But CSC133-DC-CIK group, CSC-RNA-DC-CIK group, CSC- lysate-DC- Between CIK group, no significant difference (P > 0.05) is shown in Table 3.
Table 3LDH method detects different cells to the killing activity (%) of MCF-7 Breast Cancer Cell
Note: with CIK group ratio,*P<0.05；With DC-CIK group ratio,△P<0.05；
Lethal effect of the different cells to MCF-7 Breast Cancer Cell stem cell (CSC)
By CIK, DC-CIK, CSC133-DC-CIK, CSC-RNA-DC-CIK, CSC- lysate-DC-CIK, MCF-7- RNA-DC-CIK and MCF-7- lysate-DC-CIK is used as effector cell, and the CSC cell of MCF-7 cell is target cell, uses LDH method for releasing detection, with the increase of effect target ratio, killing functions of immunocytes enhancing, when effect target ratio is 80:1, to MCF-7 cell CSC The killing rate highest of cell.When effect target ratio is 10:1,20:1,40:1,80:1, CSC133-DC-CIK group, CSC-RNA-DC-CIK Group, the killing rate of CSC- lysate-DC-CIK group are higher than CIK group and DC-CIK group, and difference is statistically significant (P < 0.05)； DC-CIK group is higher than CIK group, and difference is statistically significant (P < 0.05)；But CSC133-DC-CIK group, CSC-RNA-DC-CIK Between group, CSC- lysate-DC-CIK group, no significant difference (P > 0.05) is shown in Table 4.
Table 4LDH method detects different cells to the killing activity (%) of CSC cell
Note: with CIK group ratio,*P<0.05；With DC-CIK group ratio,△P<0.05；
As it can be seen that constructing tumour-specific biomarker CD133 recombinant plasmid through MCF-7 Breast Cancer Cell stem cell (CSC) Transfection, RNA transfection DC cell or full cell cracking induce DC cell, co-culture with CIK cell, to MCF-7 Breast Cancer Cell and The lethal effect of its stem cell, as the result is shown CD133-DC-CIK group, CSC-RNA-DC-CIK group, CSC- lysate-DC-CIK The mature expression rate of the CIK immunophenotype (CD3, CD8, CD56) and DC immunophenotype (CD40, CD80, CD86, HLA-DR) of group is equal It is apparently higher than CIK group and DC-CIK group, difference is statistically significant (P < 0.05).But CD133-DC-CIK group, CSC-RNA- Between DC-CIK group, CSC- lysate-DC-CIK group, no significant difference (P > 0.05).With effect target ratio increase, CIK group, DC-CIK group, CSC-RNA-DC-CIK group, CSC- lysate-DC-CIK group, CD133-DC-CIK group are thin as effect Born of the same parents also gradually increase the lethal effect of MCF-7 cell and its stem cell, each to imitate in target ratio, CD133-DC-CIK group, CSC- RNA-DC-CIK group, the killing rate of CSC- lysate-DC-CIK group are higher than CIK group and DC-CIK group, and difference has statistics meaning Adopted (P < 0.05)；DC-CIK group is higher than CIK group, and difference is statistically significant (P < 0.05)；But CD133-DC-CIK group, CSC- Between RNA-DC-CIK group, CSC- lysate-DC-CIK group group, no significant difference (P > 0.05).
The above-mentioned detailed description that the new application of the CD133 plasmid is carried out referring to specific embodiment, be it is illustrative and It is not restrictive, several embodiments can be enumerated according to limited range, therefore in the case where not departing from present general inventive concept Change and modification, should belong within protection scope of the present invention.

Claims (2)

1.CD133 is in enhancing DC-CIK to the application in terms of the killing effect in vitro of breast carcinoma stem cell, the nucleosides of the CD133 Acid sequence is sequence shown in SEQ ID NO:1.
2. the application of CD133 according to claim 1, it is characterised in that: thin with the recombinant plasmid impact DC containing CD133 After born of the same parents, enhance DC-CIK to the Cytotoxicity in vitro effect of breast carcinoma stem cell mature DC cell and CIK cell Combined culture, The nucleotides sequence of the CD133 is classified as sequence shown in SEQ ID NO:1.
CN201610092181.4A 2015-08-24 2016-02-19 The new application of CD133 plasmid Expired - Fee Related CN105602904B (en)

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