CN103520705B - Application of CD226 extracellular domain protein in inhibition of tumor cell proliferation - Google Patents
Application of CD226 extracellular domain protein in inhibition of tumor cell proliferation Download PDFInfo
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Abstract
The invention discloses an effect of inhibiting tumor of CD226 extracellular domain protein. The CD226 extracellular domain protein is a protein consisting of an amino acid sequence shown by SEQ ID NO:1. The protein disclosed by the invention can be combined with CD112 and CD155, can be used for effectively closing binding sites between CD226 and target cells and inhibiting the cellular poison activity of NK cells, can be used as an inhibitor of the NK cells for treating autoimmune diseases on the one hand, and can generate an inhibitory action on proliferation of tumor cells after being combined with CD112 or CD155 molecules on the surfaces of the tumor cells on the other hand. Therefore, the protein can be used for inhibiting the proliferation of tumors in a tumor patient with a low immunologic function. Moreover, because the protein is a human body natural protein, an immunologic reaction of an organism is rarely caused. The invention relates to application of the CD226 extracellular domain protein in preparation of medicaments for inhibiting tumor cell proliferation.
Description
Technical field
The present invention mainly belongs to tumor immunology field, mainly participates in preparation of the albumen of inhibition tumor cell growth and uses thereof.Specifically, the present invention relates to the purposes of CD226 Extracellular domain protein inhibition tumor cell propagation.
Background technology
Natural killer cell (Natural Killer, NK cell) plays very important effect in the natural immunity, mainly comprises killing tumor cell, kills and wounds the cell of viral infection, kills and wounds born of the same parents' endophyte and kill and wound fungus.NK cell is mainly through two kinds of direct functionatings of mode; One is by secrete cytokines, produces inhibitory action to target cell.Two is by the identification between NK cell surface receptor and part, kills and wounds after making NK cell activation to target cell.
NK cells play function is mainly realized by the various Activating receptor of NK cell surface and Inhibitory receptor.CD226 molecule is the Activating receptor of NK cell surface, has two immunoglobulin like domain in extracellular, belongs to immunoglobulin molecules superfamily with its part CD155 and CD112 molecule, is I type transmembrane protein.
In numerous Activating receptor, CD226 molecule has Activating receptor and adhesion molecule dual identity, both the combination of mediate NK cell and target cell, in born of the same parents, reactivity signal (Jun Shirakawa is transmitted again by signal path, Yinan Wang et, al.International Immunology, Vol.18, No.6, pp.951-957).In many tumor cells, the part CD155 of CD226 molecule and CD112 molecule all can up-regulated expressions (Sloan KE, Eustace BK, Stewart JK, et al.BMCCancer, 2004,4 (1): 73-78.).This is considered to be conducive to immunocyte the killing and wounding tumor such as NK cell.But this lethal effect but and not obvious in tumor patient.Tumor cell is blocked by the combination of CD155 and CD112 molecule to the CD226 molecule of the lymphocytic cell surfaces such as NK cell, T cell of secreting solubility, thus escape immune system identifies it and removes (Baury B, Masson D, et al.Biochem Biophys Res Commun.2003Sep12; 309 (1): 175-82.).
Summary of the invention
The object of the present invention is to provide a kind of can combination with tumor cell surface CD155/CD112 molecule thus the CD226 Extracellular domain protein that grows of inhibition tumor cell.
CD226 Extracellular domain protein provided by the invention, the albumen be made up of the aminoacid sequence shown in SEQ ID NO:1 (we are referred to as again soluble CD226, are called for short sCD226).
This Extracellular domain protein is the part that can be combined with CD155 and CD112 molecular specificity, directly can synthesize with artificial synthesis, also the encoding gene of albumen can be merged or be inserted into expression vector respectively, then transfection host cell, produces by gene engineering method.
Above-mentioned tumor refers to the tumor of CD155 or the CD112 molecule positive in various people source, such as, but not limited to, malignant adenoma, the former leukemia of chronic marrow etc.
The invention provides the medicine that active component is the inhibition tumor cell propagation of above-mentioned CD226 Extracellular domain protein.
Said medicine can comprise one or more pharmaceutically acceptable carriers.
Above-mentioned carrier can be diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier and/or lubricant.
Said medicine all can prepare following dosage form according to the conventional method of pharmaceutical field: injection and freeze-dried powder.
The solubility CD155 of tumor cells expression and CD112 molecule effectively can block NK cell and carry out lethal effect to it, thus make tumor cell escape immune surveillance.Experiment confirm: CD226 Extracellular domain protein of the present invention can in and solubility CD155 and CD112 molecule, and combine with CD155 and the CD112 molecule of tumor cell surface simultaneously, thus the growth of tumor cell is had an impact, reduce the speed of growth of tumor cell.
The present invention with CD226 molecule for reference template makes, can the growth of inhibition tumor cell, simultaneously less to immune impact cell.Because protein sequence of the present invention almost derives from the albumen of self, therefore seldom cause the immunoreation of body.Use tumor cell to carry out proliferation test, this albumen can realize the effect similar with CD155 or CD112 monoclonal antibody, it also avoid the side effect of antibody molecule simultaneously.Albumen of the present invention can adopt existing conventional method to prepare, technology maturation.These are all the Advantageous Effects of the present invention relative to prior art.
In sum, the invention provides the following:
1, the application of CD226 Extracellular domain protein in the medicine of breeding for the preparation of inhibition tumor cell, the aminoacid sequence of wherein said CD226 Extracellular domain protein is SEQ ID NO:1.
2, the application according to the 1st, wherein said tumor cell is the tumor cell of CD112 or the CD155 positive.
3, the application according to the 2nd, wherein said tumor cell is selected from Hela cell or K562 cell.
4, the application according to the 2nd, the tumor cell of wherein said CD112 or the CD155 positive is selected from malignant adenoma or the former leukemic cell of chronic marrow.
5, a medicine for inhibition tumor cell propagation, its active component is the CD226 Extracellular domain protein shown in SEQ ID NO:1.
6, the medicine according to the 5th, wherein said tumor cell is the tumor cell of CD112 or the CD155 positive.
7, the medicine according to the 6th, wherein said tumor cell is selected from Hela cell or K562 cell.
8, the medicine according to the 6th, the tumor cell of wherein said CD112 or the CD155 positive is selected from malignant adenoma or the former leukemic cell of chronic marrow.
Accompanying drawing is sketched
Fig. 1, the structure flow chart of RT-PCR expression vector pET32a6His-MBP-sCD226.
A) Tertiary structure predictions of CD226 albumen.The Smart instrument of Expasy is used to carry out Tertiary structure predictions to CD226 albumen;
B) secondary structure prediction of CD226 albumen.The Psipred instrument of Expasy is used to carry out secondary structure prediction to CD226 albumen;
C) the structure flow process of CD226 RT-PCR expression vector.
Fig. 2, CD226 extracellular domain recombinant protein is expressed, Isolation and characterization.
A) the recombiant protein 6His-MBP-sCD226 of ni-sepharose purification prokaryotic expression;
B) Superdex-200 molecular sieve purification 6His-MBP-sCD226;
C) SDS page detects TEV enzyme action 6His-MBP-sCD226;
D) SDS page detects ni-sepharose purification the destination protein of tape label and Superdex-200 is not further purified destination protein;
E) aminoacid sequence of destination protein that obtains of Mass Spectrometric Identification purification, the peptide section detected represents with runic and underscore.
Fig. 3, sCD226 protein active identify, sCD226 albumen can with the CD112 molecule of CD112 monoclonal antibody competition binding tumor cell surface.
The depression effect that Fig. 4, sCD226 albumen grows CD112 or CD155 positive tumor cell.
A) Phenotypic examination of Hela cell, K562 cell and CHO-K1 cell.By the CD226 ligand expression situation of several cell line of flow cytometer detection;
B) sCD226 albumen suppresses K562 and Hela cell proliferation.Add BSA respectively, sCD226, sCD226+aCD226mAb and sCD226+IgG albumen and K562 and Hela cell hatch the cell number of 3 days respectively;
C) sCD226 albumen does not affect CHO-K1 cell proliferation.Add BSA and sCD226 albumen and the CHO-K1 cell incubation cell number of 3 days respectively.
The time effect of Fig. 5, sCD226 albumen inhibition tumor cell growth.
A) CFSE after-dyeing K 562 breeds detection.Under not adding any albumen situation, K562 through CFSE dyeing after, the fluorescence intensity of CFSE in different time points born of the same parents;
B) sCD226 albumen is on the impact of K562 cell proliferation.The comparison of CFSE fluorescence intensity and blank in born of the same parents in the experimental group after 24h, 48h and 72h is hatched after adding equivalent sCD226 and BSA.
The dosage effect of Fig. 6, sCD226 inhibition tumor cell growth.
The migration of Fig. 7, sCD226 inhibition tumor cell.
A) scratch experiment detects sCD226 to the inhibitory action of its ligand positive tumor cell migration.Experimental system adopts the culture medium (K562 RPMI-1640 culture medium, CHO-K1 DMEM culture medium) containing 2% new-born calf serum.When albumen and cell hatch 0h and 36h altogether, Taking Pictures recording respectively organizes the width of cut respectively;
B) Hela cell migration distance calculates and each group migration distance statistics;
C) CHO-K1 cell migration distance calculates and each group migration distance statistics.
Fig. 8, sCD226 can affect Rac/Cdc42 and Cyclin D signal path in tumor cell.
A) Western blot detection Hela cell and corresponding protein hatch total expression of rear Rac/Cdc42 and the expression of Phospho-Rac1/cdc42 (Ser71);
B) to the statistics of band gray value in A figure.Statistical data derives from three experiments;
C) Western blot detection Hela cell and corresponding protein hatch total expression of rear Cyclin D and the expression of Phospho-Cyclin D1 (Thr286);
D) to the statistics of band gray value in C figure.Statistical data derives from three experiments.
Sequence explanation
The aminoacid sequence of SEQ ID NO:1 CD226 Extracellular domain protein
SEQ ID NO:2 sCD226 protein sequence is at the coding nucleotide sequence of expression in escherichia coli
The aminoacid sequence of SEQ ID NO:3 TEV enzyme
The coding nucleotide sequence in escherichia expression system of SEQ ID NO:4 TEV enzyme
Detailed description of the invention
Come by the following examples to illustrate the present invention further.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
In following embodiment, if no special instructions, conventional method is.
Embodiment instrument is as follows: pipettor purchased from French Gilson company, centrifuge purchased from German Hettich company, clean work station purchased from Jinan City, Shandong Province air purifying and sterilizing instrument factory, CO
2incubator purchased from American Thermo Fisher company, flow cytometer purchased from American Becton, Dickinson and Company, Superdex-200 molecular sieve and Ni-NTA purification column purchased from American General Electric company.
The synthesis of embodiment 1, albumen of the present invention and Function detection thereof
One, the expression and purification of albumen
1, the design of expression vector
1) fragment sequence of albumen is selected
The three dimensional structure of CD226 is not yet resolved, the Smart instrument of Expasy website is used to carry out structure prediction, obtain may there be two immunoglobulin like domain outside CD226 molecule born of the same parents, its cross-film district may between 243 amino acids residues to 250 amino acids residues.Adopt the secondary structure prediction instrument Psipred of Expasy afterwards, prediction draws after the 249th amino acids residue it is an alpha helical structure, and supposition is the beginning in cross-film district, and therefore, we select to intercept extracellular fragment at the 248th residue place.
2) vector construction and label are selected
With the cDNA of human peripheral PBMC for template, with primer (forward primer: 5 '-CGCGGATCCGAAGAGGTGCTTTGGCATAC-3 ' and reverse primer: 5 '-CCGCTCGAGTTAAACTCTAGTCTTTGGTC-3 '), the DNA sequences encoding of the fragment of CD226 molecule 1 9aa to 248aa is increased, be structured in respectively with 6His, 6His-GST, on 6His-Trx and 6His-MBP fusion tag pET32a carrier (pET32a carrier purchased from American Novagen company), after abduction delivering splits bacterium in a small amount, detect the expression of CD226 soluble protein in supernatant, find to only have with 6His-MBP tag fusion after can obtain the protein expression of solubility.Carrier used holds 6Xhis label and N to hold the pET32a carrier of MBP label with N.
3) enzyme action of fusion rotein and the purification of destination protein
Supernatant after prokaryotic expression, decomposition under high pressure antibacterial adopts Ni-NTA purification column (purchased from American General Electric company) to carry out purification, obtain the 6His-MBP-sCD226 fusion rotein fragment (Fig. 2 A) of purity about 95%, use Superdex-200 sieve chromatography (purchased from American GeneralElectric company) to be further purified (Fig. 2 B) again, obtain sample peak single on chromatography collection of illustrative plates.(can buy in market, the article No. produced as Suo Laibao bio tech ltd, Shanghai is P2060-1000 to adopt TEV enzyme (marmor erodens Tobacco etch virus protease); The TEV proenzyme nuclear expression system that also can be built by this laboratory is expressed and is obtained) (TEV protein sequence and nucleotide sequence are shown in SEQ ID NO:3 and SEQ ID NO:4), be cloned in expression vector pEGX4T2, soluble g ST-6His-TEV fusion rotein is gone out by IPTG abduction delivering in e. coli bl21 DE3 expression strain, this albumen produces self enzyme action owing to there is TEV restriction enzyme site between GST and 6His afterwards, thus removes GST label.Purify with Ni-NTA the 6His-TEV soluble protein enzyme that high pressure break bacterium supernatant obtains purity about 95% afterwards and enzyme action is carried out to MBP fusion tag, to obtain single CD226 extracellular protein.Add the TEV enzyme of the fusion rotein half quality will carrying out enzyme action, 4 DEG C of hold over night carry out enzyme action to fusion rotein, and fusion rotein is digested is 6His-MBP label protein and the destination protein not with any label (Fig. 2 C).Carry out Solid phase with Ni-NTA purification column to purify, by with His label and the 6His-MBP label protein produced after not digested fusion rotein, enzyme action and all removing with the TEV enzyme of His label.Protein solution, again through the purification of Superdex-200 sieve chromatography, obtains the destination protein fragment (Fig. 2 D) that purity is about 95%.CD226 ectodomain fragment (ectodomain) is accredited as with two-dimensional liquid chromatography multi-stage ms combined instrument (2D1C-MS) (purchased from American Thermo Fisher company), be called for short CD226ECD protein fragments, also known as sCD226 albumen (Fig. 2 E) in the present invention.
Two, the Function detection of albumen
1, sCD226 albumen and anti-CD112 monoclonal antibody competition binding test cell line
Experiment material: K562 cell (the former leukaemia of chronic marrow) purchased from American Type Tissue Collection (ATCC), numbering CCL-243
tMmice Anti-Human CD112 (R2.525) the purchased from American Becton of PE fluorescein coupling, Dickinson and Company (Cat#:337410), RPMI-1640 culture medium purchased from American Thermo Fisher company, new-born calf serum is purchased from Shanghai Excell Biology Product Co., Ltd., and BSA albumen is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Experimental apparatus: low-temperature and high-speed centrifuge purchased from American Sigma-Aldrich company, model SIGMA1-15K, tabletop refrigerated centrifuge purchased from German Hettich company, model Rotanta46R, flow cytometer purchased from American Becton, Dickinson and Company.
The setting of experimental group: blank group, isotype antibody controls group and antibody assay group be (comprising adding different protein contents: 20 μ g, 40 μ g, the sCD226 experimental group of 80 μ g and 160 μ g and different protein contents: 20 μ g, 40 μ g, the experiment contrast group of the BSA albumen of 80 μ g and 160 μ g).
1) with the centrifugal 5min of low-temperature and high-speed centrifuge 200g, the K562 cell of fresh cultured in 30ml culture fluid is collected;
2) with 1ml PBS (pH=7.2) solution re-suspended cell;
3) with the centrifugal 5min of tabletop refrigerated centrifuge 200g, collecting cell;
4) PBS (pH=7.2) repeated washing is used once;
5) counting cells, adds 2 × 10 in each experimental group
5individual cell;
6) add mice serum, 4 DEG C of lucifuge 30min close;
7) add 2 μ g CD112 monoclonal antibody and corresponding albumen in each group, 4 DEG C of lucifuge 30min are hatched;
8) the centrifugal 5min of tabletop refrigerated centrifuge 200g, abandons supernatant;
9) cell 1ml PBS (pH=7.2) is resuspended, and then the centrifugal 5min of continuation tabletop refrigerated centrifuge 200g washs cell;
10) repeat step 9, washing once;
11) washing is often organized cell after terminating and is proceeded to flow cytomery pipe with 300 μ l PBS (pH=7.2) are resuspended, detects with flow cytometer.
Interpretation of result finds, along with the increase of sCD226 albumen addition, the fluorescent antibody that K562 cell combines reduces gradually, unrelated protein BSA adds the amount not affecting the fluorescent antibody that K562 cell surface combines, and points out the sCD226 obtained to have the ability (Fig. 3) be combined with CD112 molecule.
Three, albumen inhibition tumor cell proliferation test
Experiment material: the sterile filters purchased from American Millipore company of 0.22 μm, IMDM culture medium, RPMI-1640 culture medium and DMEM culture medium equal purchased from American Thermo Fisher company, new-born calf serum is purchased from Shanghai Excell Biology Product Co., Ltd., 24 porocyte culture plate purchased from American Costar companies, the equal purchased from American Type Tissue Collection (ATCC) of K562 cell, CHO-K1 cell and Hela cell, numbering is respectively CCL-243
tM, CCL-61
tMand CCL-2
tM, above-mentioned three kinds of cells are relevant with malignant adenoma to chronic granulocytic leukemia, Chinese hamster ovary cancer respectively.BSA albumen is purchased from Sangon Biotech (Shanghai) Co., Ltd..The anti-CD226 of mice (DX11) antibody (aCD226mAb) of purification and the mouse IgG 1 Isotype antibody purchased from American Becton of purification, Dickinson and Company, article No. is respectively 559787 and 555749.
The setting of experimental group: each cell arranges five experimental grouies respectively: blank group only adds PBS solution, the every hole of experiment contrast group adds the BSA of 50 μ g, the every hole of sCD226 group adds the sCD226 albumen of 50 μ g, sCD226+ anti-CD226 antibody (aCD226mAb) organizes the blocking antibody DX11 (aCD226mAb) also adding the CD226 of 50 μ g while every hole adds 50 μ gsCD226 albumen, and the every hole of sCD226+IgG group also adds the mouse IgG antibody of 50 μ g as Isotype control while the sCD226 albumen adding 50 μ g.Often organize 3 multiple holes.
1) sCD226 albumen and BSA albumen use the sterile filters filtration sterilization of 0.22 μm respectively;
2) collecting cell, the centrifugal 5min of 200g, abandons supernatant;
3) according to the requirement of ATCC cell culture, add different culture medium respectively, Hela RPMI-1640 complete medium (containing 10% new-born calf serum), CHO-K1 DMEM complete medium (containing 10% new-born calf serum), K562 IMDM complete medium (containing 10% new-born calf serum), carry out cell counting;
4) cell concentration to 10 is adjusted
4/ ml, adds 24 orifice plates, every hole 1ml;
5) add corresponding protein solution respectively, blank group adds equal-volume PBS (pH=7.2);
6) culture plate is placed on 5%CO
2, in 37 DEG C of incubators, cultivate 72h;
7) collect the cell suspension in each culture hole respectively, carry out cell counting;
8) each test repetition 3 times.
SCD226 albumen is for the propagation inhibited (Fig. 4 A) of Hela cell and K562 cell.And CHO-K1 cell surface does not have CD112 or CD155 molecule, breed unaffected (Fig. 4 B).Unrelated protein BSA adds the propagation also not affecting tumor cell, tumor cell number can be made significantly to reduce (table 1) after sCD226 albumen adds 3 days.
The cell count data of each experimental group of table 1.
"-" expression is tested
Four, the time relationship of albumen inhibition tumor cell propagation
Experiment material: CFSE dyestuff (0.5mM/L) purchased from Bio Basic Inc, K562 cell purchased from American Type Tissue Collection (ATCC) numbering CCL-243
tM, IMDM culture medium purchased from American Thermo Fisher company, new-born calf serum purchased from Shanghai Excell Biology Product Co., Ltd., 24 porocyte culture plate purchased from American Costar companies.BSA albumen is purchased from Sangon Biotech (Shanghai) Co., Ltd.
The setting of experimental group: for a kind of cell, arranges three experimental grouies: blank group only adds PBS solution respectively, and the every hole of experiment contrast group adds the BSA of 50 μ g, and the every hole of sCD226 group adds 50 μ g destination proteins.Often group arranges 9 multiple holes, the multiple holes of each detection 3.
1) get the K562 cell of fresh cultured, the centrifugal 5min of 200g, abandons supernatant;
2) count with 5ml IMDM complete medium (containing 10% new-born calf serum) re-suspended cell;
3) cell concentration is adjusted to be 10 with 37 DEG C of PBS containing 0.1%BSA
6/ ml;
4) add the CFSE dyestuff of 2 times of volumes, make its final concentration be 10 μMs/L;
5) 10min is hatched for 37 DEG C;
6) 4 DEG C, the centrifugal 5min of 200g, abandons supernatant;
7) cell is washed twice with 1ml IMDM complete medium;
8) counting cells adjust cell concentration to 10
4/ ml;
9) add in 24 orifice plates, every hole 1ml;
10) add corresponding protein, be placed on 5%CO
2, cultivate in 37 DEG C of incubators;
11) respectively at 24h, 48h, 72h time point, each experimental group gets the CFSE fluorescence intensity in 3 multiple hole flow cytometer detection born of the same parents.
Because CFSE can give daughter cell uniformly in cell division, so the fluorescence intensity that cell often divides CFSE in born of the same parents all can decline, therefore fissional number of times can be judged according to the fluorescence intensity of CFSE in born of the same parents, like this in same time, Growth of Cells divides group often soon, and in born of the same parents, the fluorescence intensity of CFSE is just lower.Experimental result is consistent with cell counts, cell division slow after adding sCD226 albumen, and in born of the same parents, comparatively blank group is strong for CFSE fluorescence intensity, and the fluorescence intensity of BSA group cell compared with blank group is identical.And As time goes on, incubation time is longer, the inhibitory action of on cell proliferation also larger (Fig. 5 A and 5B).
Five, the dose relationship of albumen inhibition tumor cell propagation.
Experiment material: K562 cell purchased from American Type Tissue Collection (ATCC) numbering CCL-243
tM, BSA albumen purchased from Sangon Biotech (Shanghai) Co., Ltd., sCD226 albumen according to the method prokaryotic expression of embodiment 1 and purification, IMDM culture medium purchased from American ThermoFisher company.New-born calf serum, purchased from Shanghai Excell Biology Product Co., Ltd.
The setting of experimental group: three experimental grouies are set: blank group, BSA group and sCD226 group.Often organize 12 porocytes.Blank group only adds PBS solution, and BSA group and sCD226 group are divided into four dose gradients respectively, each gradient 3 porocyte.Four dose gradients of BSA group and sCD226 group are as follows: the Cell sap of every hole 1ml adds BSA or sCD226 albumen respectively, makes its final concentration be respectively 10 μ g/ml, 20 μ g/ml, 40 μ g/ml and 80 μ g/ml.
1) get the K562 cell of fresh cultured, the centrifugal 5min of 200g, abandons supernatant;
2) with 5ml IMDM complete medium (containing 10% new-born calf serum) re-suspended cell;
3) counting cells adjust cell concentration to 10
4/ ml;
4) cell suspension is added, every hole 1ml;
5) albumen of corresponding dosage is added respectively;
6) 5%CO is put
2, cultivate in 37 DEG C of incubators;
7) culture plate is taken out after 72h, with the cultivation night in hole by resuspended for cell rear suction EP pipe;
8) cell number in each hole is counted.
Add several experimental grouies of sCD226 albumen, along with the increase of sCD226 dosage, cell number reduces gradually, adds between the experimental group of BSA and blank group and does not then have significant difference.Therefore, sCD226 albumen suppresses the propagation of K562 cell to be have dose dependent (Fig. 6).
Six, albumen suppresses the migration of solid tumor cell.
Experiment material: CHO-K1 cell and the equal purchased from American Type Tissue Collection (ATCC) of Hela cell, numbering is respectively CCL-61
tMand CCL-2
tM, BSA albumen is purchased from Sangon Biotech (Shanghai) Co., Ltd., and sCD226 albumen is according to the method prokaryotic expression of embodiment 1 and purification, and RPMI-1640 cultivates and DMEM base purchased from American Thermo Fisher company.The anti-CD226 of mice (DX11) the antibody purchased from American Becton of purification, Dickinson and Company, article No. is 559787.24 porocyte culture plate purchased from American Costar companies.New-born calf serum, purchased from Shanghai Excell Biology Product Co., Ltd..
The setting of experimental group: Hela and CHO-K1 two kinds of cells arrange four experimental grouies respectively: blank group, BSA group, sCD226 group and CD226+antibody group.Often organize 3 multiple porocytes.Blank group only adds PBS solution, and BSA group and sCD226 group add 40 μ g BSA and 40 μ g sCD226 albumen respectively, and CD226+antibody group adds 40 μ g sCD226 albumen and 10 μ gDX11 antibody.
1) get Hela cell and the CHO-K1 cell of fresh cultured, the centrifugal 5min of 200g, abandons supernatant;
2) contain the complete medium re-suspended cell of 10% new-born calf serum with corresponding and be adjusted to 5 × 10
6the higher concentration of/ml;
3) in 24 orifice plates, cell suspension is added, every hole 1ml;
4) hatch and within two hours, treat that cell is completely adherent for 37 DEG C;
5) cut is carried out with 20 μ l pipettor gun heads in every hole;
6) inhale the culture fluid abandoned in each hole, wash some suspension cells removed cut and cause for three times by the corresponding culture medium containing 2% low serum;
7) every hole adds 1ml containing the corresponding culture medium of 2% low serum and corresponding albumen;
8) Taking Pictures recording each hole cut picture is as the result of 0h, then culture plate is placed on 37 DEG C of cell culture incubators and cultivates.
9) again cut is taken pictures after 36h.
10) respectively organize scratch width according to image measurement and calculate and add up the migration distance of cell.
In order to get rid of the impact of cell proliferation, we adopt the low blood serum medium of 2% to cultivate in an experiment.According to formulae discovery cell migration distance below:
Cell migration distance=(width of the width-36h cut of 0h cut) ÷ 2.
Finally find according to statistical result, after adding sCD226 albumen, the migration distance of Hela cell significantly declines, but if add blocking-up row antibody DX11 (antibody) of CD226 simultaneously, then can block the effect of sCD226 albumen and cell migration ability is recovered.Simultaneously for the CHO-K1 cell results of CD226 part feminine gender, add sCD226 albumen and impact be there is no on its transfer ability.Also demonstrate that sCD226 albumen can suppress the migration of solid tumor cell like this.(Fig. 7).
Seven, sCD226 suppresses the phosphorylation of Cyclin D and Rac/Cdc42 by ligand signal path.
Experiment material: Hela cell purchased from American Type Tissue Collection (ATCC), numbering CCL-2
tM, BSA albumen purchased from Sangon Biotech (Shanghai) Co., Ltd., sCD226 albumen according to the method prokaryotic expression of embodiment 1 and purification, RPMI-1640 culture medium and DMEM culture medium purchased from American Thermo Fisher company.Rac/Cdc42 antibody, Phospho-Rac1/cdc42 (Ser71) antibody, Cyclin D1 (92G2) antibody and Phospho-Cyclin D1 (Thr286)
antibody equal purchased from American Cell Signaling Technology, article No. is respectively 4651S, 2461P, 2978P and 3300P.24 porocyte culture plate purchased from American Costar companies.β-actin antibody purchased from Wuhan doctor's moral company, article No. BM0627.New-born calf serum, purchased from Shanghai Excell Biology Product Co., Ltd..The reagent of various solution needed for preparation cell pyrolysis liquid: Tirs (traditional Chinese medicines, 30188336), NaCl (traditional Chinese medicines, 10019318), Na
3vO
4(Sigma, S-6508), EDTA (traditional Chinese medicines, 10009719KP), EGTA (the raw work in Shanghai, E0732-5g), NP-40 (the raw work in Shanghai, 9016-45-9), PMSF (the raw work in Shanghai, PB0425), NaF (SCRC, 10019618).BSA (Sigma, A9647) standard solution (1mg/ml), Coomassie brilliant G-250 dyeing liquor (Bio-Rad, 500-0006), histiocyte cracking supernatant.OneDrop OD-1000 spectrophotometer (the adopted Science and Technology Ltd. in Nanjing five).Prestained protein marker (Fermentas, SM1811); Reagent: Acryl/Bis Solution (29:1) 40% (w/v) (the raw work in Shanghai needed for preparation SDS-polyacrylamide gel, SD6013), APS (the raw work in Shanghai, 7727-54-0), 4 × Tris-HCl (pH6.8) (raw work in Shanghai, SD6022), 4 × Tris-HCl (pH8.8) (raw work in Shanghai, SD6021), TEMED (Bio Basic Inc, 110-18-9), pvdf membrane (0.45 μm) (Millipore, IPVH00010); Methanol (Shanghai Ling Feng, 67-56-1); MouseAnti-β Actin mAb (Boster, BM0627); Stat3 antibody (Cell Signaling, 9132); Goat anti-rabbit igg-HRP (Boster, BA1054); Sheep anti-mouse igg-HRP (Boster, BA1050); The half-dried transfer groove of Supersignal West Pico Chemiluminescent (Pierce, 34080), U.S. Bio-rad Bole Trans-Blot SD; DYY-6C electrophresis apparatus; GE Molecular Imager LAS4000mini
The setting of experimental group: three experimental grouies are set: blank group, BSA group and sCD226 group.Often organize 3 porocytes.Blank group only adds PBS solution, and BSA group and sCD226 group add 40 μ g BSA and 40 μ g sCD226 albumen respectively.
1) get the Hela cell of fresh cultured, the centrifugal 5min of 200g, abandons supernatant;
2) use PBS solution washed cell three times, the centrifugal 5min of each 200g, abandons supernatant;
3) with PBS solution adjustment cell concentration to 10
5/ ml, gets 3ml respectively and is sub-packed in 3 EP pipes, and adds equal-volume albumen buffer (Control group), 40 μ g BSA and 40 μ gsCD226 albumen respectively;
4) 1 hour is hatched for 37 DEG C;
5) the centrifugal 5min of 200g abandons supernatant, washes three times with PBS;
6) prepare cell pyrolysis liquid 2ml according to the following formulation, prepare rearmounted ice bath.
7) often organize cell and add 50 μ l lysate mixings, place 30min cell lysis on ice;
8) 16000g, 4 DEG C of centrifugal 10min, get supernatant;
9) get 1ml Coomassie brilliant G-250 dyeing liquor and add the mixing of 4ml tri-distilled water, as working solution;
10) get 5 EP pipes BSA tri-distilled water is diluted to concentration is respectively 0mg/ml, each 10 μ l of standard substance of 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml;
11), after protein sample tri-distilled water being diluted 100 times, 10 μ l are taken out;
12) respectively to the working solution mixing adding 200 μ l in 6 pipe standards product and 10 μ l testing samples, room temperature leaves standstill 5min;
13) protein concentration of lysate is measured by the spectrophotometric Bradford method of OneDrop OD-1000;
14) 2 × SDS-PAGE sample-loading buffer 20 μ l histiocyte lysate capable supernatants and 20 μ l having been added DTT mixes, boiling water bath 8min;
15) electrophoresis loading, often group adds 150 μ g albumen, carries out SDS-PAGE electrophoresis;
16) electrophoresis terminates the position that destination protein is determined in the rear position according to Marker, the pvdf membrane of the suitable size of clip and filter paper;
17) 18V, 30 ~ 40min transferring film after half-dried transferring film membrane-transferring device is installed.
18) the TBST solution of film with the skim milk of 5% is closed after terminating by transferring film, room temperature shaker 1h;
19) close end, put into the TBST solution of 5% skim milk containing corresponding antibodies after being taken out by film, be placed on 4 DEG C of incubator overnight and hatch.The dilution proportion of destination protein antibody 1:1000 to specifications, internal reference antibody is 1:500 dilution proportion to specifications;
20) after primary antibodie hatches end, film TBST solution is washed three times, then resist hatch to dilute containing the TBST of 5% skim milk corresponding two.Two anti-dilution ratios are 1:5000 to specifications;
21) two anti-room temperature shaker hatch 50min, terminate rear TBST solution and film is washed three times.
22) add HRP substrate, in imager, carry out development take pictures.
Found that, after adding sCD226 albumen, sCD226 albumen can, by its part CD112 and CD155 to transmission of signal in cell Nectin path, make the phosphorylation of Rac/Cdc42 strengthen (Fig. 8 A and Fig. 8 B).There are some researches show that the enhancing of Rac/Cdc42 phosphorylation is the back of its ubiquitination degraded.The degraded of Rac/Cdc42 can cause cell migration ability to decline.Therefore sCD226 albumen inhibits the migration of tumor cell by phosphorylation-degradation Rac/Cdc42.In addition, we find that sCD226 also can make the phosphorylation of Cyclin D strengthen (Fig. 8 C and Fig. 8 D).There are some researches show that the phosphorylation of Cyclin D is equally also the back of its ubiquitination degraded.The degraded of CyclinD can make cellular retention in the G0 phase.Therefore, the time lengthening that sCD226 albumen makes cell stop in the G0 phase by phosphorylation-degradation Cyclin D, thus inhibit the propagation of tumor cell.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, do not deviating from by under the condition of the spirit and scope of the present invention as defined in the claims, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
Claims (6)
- The application of 1.CD226 Extracellular domain protein in the medicine of the tumor cell proliferation for the preparation of suppression CD112 or the CD155 positive, the aminoacid sequence of wherein said CD226 Extracellular domain protein is SEQ ID NO:1.
- 2. application according to claim 1, wherein said tumor cell is selected from Hela cell or K562 cell.
- 3. application according to claim 1, the tumor cell of wherein said CD112 or the CD155 positive is selected from malignant adenoma or the former leukemic cell of chronic marrow.
- 4. suppress a medicine for the tumor cell proliferation of CD112 or the CD155 positive, its active component is the CD226 Extracellular domain protein shown in SEQ ID NO:1.
- 5. medicine according to claim 4, wherein said tumor cell is selected from Hela cell or K562 cell.
- 6. medicine according to claim 4, the tumor cell of wherein said CD112 or the CD155 positive is selected from malignant adenoma or the former leukemic cell of chronic marrow.
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| WO2005107799A1 (en) * | 2004-05-06 | 2005-11-17 | Eisai R & D Management Co., Ltd. | REMEDY FOR CANCER CONTAINING ANTI-Necl-5 ANTIBODY AS THE ACTIVE INGREDIENT |
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Non-Patent Citations (4)
| Title |
|---|
| CD226分子在NK细胞杀伤肿瘤中的作用及其分子机制;荆琳等;《细胞与分子免疫学杂志》;20120818;第28卷(第8期);879-881 * |
| CD226及其配体CD112/CD155分子相互作用机制的研究;庄然;《中国博士学位论文全文数据库 医药卫生科技辑》;20080415(第04期);E059-42 * |
| Increased levels of soluble CD226 in sera accompanied by decreased membrane CD226 expression on peripheral blood mononuclear cells from cancer patients;Xu ZW et al.;《BMC immunology》;20090602;第10卷(第34期);摘要，结果和讨论，图1-2 * |
| NK细胞活化受体CD226的表达及功能研究;葛葵葵;《中国博士学位论文全文数据库 医药卫生科技辑》;20090915(第09期);E059-31 * |
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