CN104962518A - New application of CD44 plasmid - Google Patents
New application of CD44 plasmid Download PDFInfo
- Publication number
- CN104962518A CN104962518A CN201510426392.2A CN201510426392A CN104962518A CN 104962518 A CN104962518 A CN 104962518A CN 201510426392 A CN201510426392 A CN 201510426392A CN 104962518 A CN104962518 A CN 104962518A
- Authority
- CN
- China
- Prior art keywords
- cell
- plasmid
- cik
- vitro
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides new application of a CD44 plasmid. The CD44 plasmid can be effectively used for enhancing the in-vitro damaging effect of DC-CIK on mammary cancer stem cells.
Description
Technical field
The present invention relates to the novelty teabag of CD44 plasmid.
Background technology
Mammary cancer is the modal malignant tumour of women in world wide.A large amount of evidence shows to cause it easily to shift just because of the existence of breast carcinoma stem cell (Breast Cancer Stem Cell, BCSC), recurs and resists chemicotherapy.And the adoptive cellular immunotherapy of rising in recent years (adoptive cellimmunotherapy, ACI) is a kind of new therapy for the insensitive tumour of chemicotherapy, clinical landscapes is wide, obtains and more and more payes attention to.
The existence of CSC affects one of breast cancer treatment effect and the most important factor of prognosis, the chemicotherapy applied clinically at present can only eliminate normal breast cancer cells usually, and the remaining BCSC of chemicotherapy opposing still can constantly be bred, breast cancer relapse is caused to shift, so a kind of method finding effective CSC of killing becomes the new direction of breast cancer treatment.ACI is the novel therapies that a kind of amplification in vitro relying on autologous responsiveness immunocyte carries out tumour cell and effectively kills, and first it be applied to the treatment of hematological malignancies, after progressively extend to the noumenal tumour comprising mammary cancer, colorectal carcinoma, lung cancer etc.The effector cell of ACI mainly comprises cytokine induced kill cell (CIKs), Tumor-infiltrating lymphocytes (lymphokine-activated killer cells, LAKs), tumor infiltrating lymphocyte (tumorinfiltrating lymphocytes, TILs), cytotoxic T lymphocyte (cytotoxic Tlymphocyte cells, the T cell (chimericantigen receptor T cells, CAR-T) etc. of the Chimeric antigen receptor CTLs) and recently proposed.And be also the important integral part of ACI as the DC that the professional antigen that function in body is the most powerful is offered.
CIK cell have propagation fast, kill knurl power strong, kill knurl spectrum wide, to normal cell without lethal effect, to advantages such as drug-resistant tumor are responsive, be that the highest non-specific of known at present activity kills and wounds immune effector cell.Tumour antigen can be offered to immunologically competent cell by DC, promotes that it is Proliferative Activated, strengthens the killing activity to tumour cell.There are some researches show, the DC immunophenotype of load tumor stem cell antigen comparatively before ripe, and by after its and CIK cell co-cultivation, maturation and the corresponding raising of secrete cytokines level further of DC-CIK immunophenotype.
CD44 is the most important molecule marker of breast carcinoma stem cell, and it is a kind of single membrane span glycoprotein, by playing keying action with being combined in the behavior such as cell adhesion and migration of multiple ligands.
Summary of the invention
Technical problem to be solved by this invention is the novelty teabag providing CD44 plasmid.
For solving the problems of the technologies described above, technical scheme of the present invention is:
CD44 plasmid is strengthening DC-CIK to the application in the killing effect in vitro of breast carcinoma stem cell.
Preferably, the application of above-mentioned CD44 plasmid, impacts DC (dendritic cell) with CD44 plasmid and with CIK (cytokine-induced killer) Coculture, the Cytotoxicity in vitro effect of DC-CIK to breast carcinoma stem cell is strengthened afterwards.
Preferably, the application of above-mentioned CD44 plasmid, the sequence of described CD44 plasmid is sequence shown in sequence table <400>1.
The invention has the beneficial effects as follows:
CD44 plasmid has significant application value at enhancing DC-CIK in the killing effect in vitro of breast carcinoma stem cell.
Accompanying drawing explanation
Fig. 1 is that MDA-MB-231 passage is cultivated and microsphere cultivation figure, wherein, and A: the 3rd day MDA-MB-231 cellular form (× 200) cultivated by microballoon, B: the tenth day MDA-MB-231 cellular form (× 200) cultivated by microballoon;
Fig. 2 is the qualification result figure of goal gene and object plasmid, wherein, and CD44 (s1-s5) band (1:CD44 (s1-s5) after A:PCR; M:100bp marker); B:CD44 plasmid is fragment PCDNA3.1+-CD44 (s1-s5) (1: indigestible plasmid after double digestion; 2: the plasmid of digestion; M1:1kb marker; M2:100bp marker);
Fig. 3 is CD44 plasmid sequencing result figure;
Fig. 4 is immunoblotting (western blotting) the result figure of CD44 protein expression;
Fig. 5 is the 293T cell after transfection CD44 plasmid, wherein, and A: 293T cell (× 100) under microscope, B: 293T fluorocyte (× 100) under microscope.
Embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
1 materials and methods
1.1 main raw DMEM substratum, foetal calf serum, mycillin, lymphocyte separation medium are purchased from Tianjin Hao ocean biological products Science and Technology Ltd.; The B27 factor, recombinant human epidermal growth factor (EGF), Basic Fibroblast Growth Factor (bFGF) purchased from American PeproTech company; GM-CSF (GM-CSF), interleukin-(IL)-4, (IL)-2, tumour necrosis factor (TNF)-α, Interferon, rabbit (IFN)-γ, monoclonal antibody CD3 equal purchased from American Gibco company; RNA extracts test kit purchased from Qiagen company; RT-PCR kit is purchased from Takara company; Sepharose recovery, little upgrading grain, carry plasmid kit all purchased from Tian Gen biochemical technology company limited greatly.Breast carcinoma cell strain MDA-MB-231 is purchased from ATCC.
1.2 method
The preparation of cultured cell line substratum 1.2.1MDA-MB-231: DMEM 445ml, 10% foetal calf serum 50ml, dual anti-5ml.Frozen breast cancer cell MDA-MB-231 is put into after 37 DEG C of water-baths melt, directly join in super clean bench fill the full substratum of 10mlDMEM 25ml culturing bottle in, in 37 DEG C, 5%CO
2, saturated humidity incubator in cultivate, next day observation of cell growing state, and full dose changes liquid, and every 2-3d changes a not good liquor afterwards.
The preparation of microballoon culturing stem cells substratum 1.2.2MDA-MB-231: DMEM 98ml, EGF2 μ g (final concentration 20ng/mL), bFGF 1 μ g (final concentration 10ng/mL), 2% (volume fraction) B272ml.Treat that above-mentioned breast cancer cell grows to logarithmic phase, collecting cell, add the resuspended rear counting of 3ml stem cell media, with 5 × 10
3individual/ml density is seeded to 25cm
2in culturing bottle, add 10ml stem cell media, in 37 DEG C, 5%CO
2, saturated humidity incubator in cultivate, every 2-3d changes a not good liquor.
1.2.3PCDNA3.1
+the structure of-CD44 (s1-s5) plasmid is according to people CD44 (s1-s5) (accession number: NM-001001391.1) gene order in GeneBank, Oligo software design enzyme is utilized to cut primer, after sending company to synthesize, with the cDNA of the RNA reverse transcription of extracting in MDA-MB-231 cell generation for template carries out PCR, product and carrier PCDNA3.1
+connect after carrying out double digestion respectively, transform JM109 competent cell and increase, identify with restriction enzyme digestion and electrophoresis and order-checking respectively after extracting plasmid in a small amount, the bacterium liquid that result is correct carries out a large amount of extractions of plasmid after connecing large bottle, be stored in-20 DEG C for subsequent use.
1.2.4CD44 plasmid transfection 293T cell detection transfection efficiency is from the above-mentioned PCDNA3.1 built
+-CD44 (s1-s5) plasmid cuts object fragment, and subclone enters green fluorescence carrier pEGFP-C3, builds eukaryon expression plasmid pEGFP-C3-CD44 (s1-s5).Cellar culture 293T cell, logarithmic phase to be grown to, adjustment cell count is 5 × 10
5paving hole is carried out in individual/hole, multiple hole is set and adds respective amount without dual anti-substratum, after 24h, above-mentioned green fluorescence plasmid and transfection reagent are joined in index aperture according to the ratio of 1:4 (m/v), mix, absorb original substratum after 5h and change into containing dual anti-perfect medium, after 48h, collecting cell carries out Western blot detection transfection efficiency.
1.2.5DC be separated normal people's Cord Blood Mononuclear Cell with the preparation collection of CIK cell, after PBS cleaning, under normal condition, cultivate 4h.Adherent cell collecting and suspension cell respectively.The former becomes DC at induction: absorb original substratum, add the serum free medium cellar culture containing GM-CSF 600U/ml and IL-4 500U/ml, and every 3d half amount changes liquid, and supplies cytokine.Add TNF-α 500U/ml at the 6d cultivated, induction DC is ripe; The latter's induction becomes CIK: resuspended with the serum free medium containing IFN-γ 1000U/ml after centrifugal, adds CD350ng/ml monoclonal antibody and IL-2 300U/ml after 24h, and every 3d half amount changes liquid, and supplies IL-2, gathers in the crops CIK cell for subsequent use in 7d.
1.2.6DC it is 5 × 10 that antigen load adjusts DC number in the 5d of DC inducing culture
5paving hole is carried out in individual/hole, arranges multiple hole, add respective amount without after dual anti-substratum respectively by CD44 plasmid and PCDNA3.1
+empty carrier and transfection reagent join in respective markers hole according to the ratio of 1:4 (m/v), mix, and absorb original substratum and change into containing dual anti-perfect medium, collect experimental port cell for subsequent use after 48h after 5h.
1.2.7 above-mentioned load C D44 plasmid and unloaded DC and CIK cell join in the serum free medium containing IL-2 300U/ml in the number ratio of 1:10 by the DC of Antigen and CIK cell co-cultivation, every 3d half amount changes liquid, and supply IL-2, in 7d collection, also counting cells is for subsequent use.
1.2.8 cell in vitro fragmentation test is 2 groups of effector cells with the above-mentioned CD44-DC-CIK cell of Dual culture 7d and zero load-DC-CIK cell, the MDA-MB-231 cell ball of microsphere culture method enrichment is target cell, equalizing effect cell and target cell concentration, make effect target ratio be followed successively by 40:1,20:1,10:1,5:1 often organizes and establishes 3 multiple holes respectively.Application LDH method for releasing measures cell killing activity, detects the absorbance value (OD value) at 492nm wavelength place with microplate reader.
Calculate kill rate as follows: kill rate (%)=(experimental group OD value-effector cell spontaneous release OD value-target cell spontaneous release OD value)/(target cell maximum release OD value-target cell spontaneous release OD value) × 100%
1.3 statistical methods adopt SPSS 17.0 statistical software analytical data, and measurement data data are used
represent, compare employing two independent samples t test between mean, compare and adopt one-way analysis of variance method between many groups, P < 0.05 has statistical significance for difference.
2 results
Before and after 2.1 microballoons cultivations, MDA-MB-231 cellular form changes the front cell of Figure 1A, B visible microballoon cultivation is adherent growth, and cultivates rear balling-up suspension growth.
The PCR of 2.2CD44 (s1-s5), plasmid enzyme restriction and gene sequencing identify Fig. 2 show PCR after CD44 (s1-s5) stripe size to conform to theoretical value and plasmid still can cut out this size fragment (in figure arrow indication) after double digestion.As shown in Figure 3, the sequence of described CD44 plasmid is sequence shown in sequence table <400>1 to gene sequencing result.
After 2.3 transfection 293T cells, CD44 protein expression and transfection results Fig. 4 A and B show when internal reference Actin expression level is suitable, and transfection empty carrier group is expressed without CD44, and transfection CD44 plasmid group CD44 protein normal is expressed; Fig. 5 A and B is respectively 293T cell picture under transfection complete CD44 plasmid conventional microscopy and fluorescent microscope.
2.4 fragmentation effects detect with LDH method for releasing detect 5:1,10:1,20:1,40:1 tetra-kinds imitate target than time, CD44-DC-CIK cell and zero load-DC-CIK cell to target cell, i.e. the killing activity of breast carcinoma stem cell.
Percentage of cytotoxicity (%) respectively organized by table 1
Visible, CD44 plasmid effectively can strengthen the killing effect in vitro of DC-CIK to breast carcinoma stem cell.
Above-mentioned detailed description of the novelty teabag of this CD44 plasmid being carried out with reference to embodiment; illustrative instead of determinate; several embodiments can be listed according to institute's limited range; therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention.
Claims (3)
1.CD44 plasmid is strengthening DC-CIK to the application in the killing effect in vitro of breast carcinoma stem cell.
2. the application of CD44 plasmid according to claim 1, is characterized in that: with CIK cell Combined culture, the Cytotoxicity in vitro effect of DC-CIK to breast carcinoma stem cell is strengthened after impacting DC with CD44 plasmid.
3. the application of CD44 plasmid according to claim 2, is characterized in that: the sequence of described CD44 plasmid is sequence shown in sequence table <400>1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510426392.2A CN104962518A (en) | 2015-07-20 | 2015-07-20 | New application of CD44 plasmid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510426392.2A CN104962518A (en) | 2015-07-20 | 2015-07-20 | New application of CD44 plasmid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104962518A true CN104962518A (en) | 2015-10-07 |
Family
ID=54216616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510426392.2A Pending CN104962518A (en) | 2015-07-20 | 2015-07-20 | New application of CD44 plasmid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104962518A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567633A (en) * | 2015-10-13 | 2016-05-11 | 王秋凤 | Method for inducing DC-CIK cells through cryopreserved cord blood cells |
| CN106811442A (en) * | 2015-12-01 | 2017-06-09 | 上海市中医老年医学研究所 | The preparation of DC-CIK cells and its application in treatment ovarian cancer is prepared |
-
2015
- 2015-07-20 CN CN201510426392.2A patent/CN104962518A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| LARANJEIRA P等: "NM_001001391.1", 《GENBANK》 * |
| 孟冉冉等: "树突状细胞肿瘤疫苗抗肿瘤研究进展", 《中华肿瘤防治杂志》 * |
| 庞春淼: "负载干细胞抗原的DC联合CIK细胞对MCF-7/ADR细胞体内外作用研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567633A (en) * | 2015-10-13 | 2016-05-11 | 王秋凤 | Method for inducing DC-CIK cells through cryopreserved cord blood cells |
| CN106811442A (en) * | 2015-12-01 | 2017-06-09 | 上海市中医老年医学研究所 | The preparation of DC-CIK cells and its application in treatment ovarian cancer is prepared |
| CN106811442B (en) * | 2015-12-01 | 2020-12-29 | 上海市中医老年医学研究所 | Preparation of DC-CIK cells and application of DC-CIK cells in preparation of medicines for treating ovarian cancer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wei et al. | Adipose-derived stem cells promote tumor initiation and accelerate tumor growth by interleukin-6 production | |
| Wakita et al. | Tumor‐infiltrating IL‐17‐producing γδ T cells support the progression of tumor by promoting angiogenesis | |
| Kuo et al. | Lung cancer-derived galectin-1 mediates dendritic cell anergy through inhibitor of DNA binding 3/IL-10 signaling pathway | |
| CN108220239B (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN102321577B (en) | Preparation method of antitumor adoptive immune cells and prepared immune cells | |
| Lapteva et al. | Enhanced activation of human dendritic cells by inducible CD40 and Toll-like receptor-4 ligation | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN105176927A (en) | Preparation method of cytotoxicity-enhanced efficient target killing NK/CIK (Natural Killer)/( Cytokine Induced Killer) cells | |
| CN105062968A (en) | DC-CIK (Dendritic cell-Cytokine Induced Killer) cell culture reagent and culture method thereof | |
| CN105343894B (en) | The recombination mescenchymal stem cell of efficient secretion prostaglandin E2 (PGE2) and its application | |
| KR20210111746A (en) | Methods for isolating and propagating cells | |
| CN105316286B (en) | A kind of method of preparation and reorganization mescenchymal stem cell and its obtained recombination mescenchymal stem cell | |
| CN104815323A (en) | Dendrite cell tumor vaccine and preparation method thereof | |
| CN103710304A (en) | Method for inducing adipose-derived stem cells to differentiate for natural killer cell and purpose thereof | |
| CN105238753A (en) | Kit used for preparing CTL and application thereof | |
| CN110106150B (en) | Preparation method and application of synovial sarcoma cell line hSS-005R | |
| CN115558641A (en) | High-purity effector immune cell population, and culture method, reagent composition and application thereof | |
| CN103173411A (en) | Method and kit for preparing dendritic cells | |
| CN103740643B (en) | The external evoked cultural method of the restricted killer T cell of a kind of MHC | |
| CN104962518A (en) | New application of CD44 plasmid | |
| Kerkar et al. | Timing and intensity of exposure to interferon‐γ critically determines the function of monocyte‐derived dendritic cells | |
| CN103981144A (en) | Preparation method for autologous-serum antigen-sensitized DC-CIK cells | |
| Takacs et al. | Immunogenic dendritic cell generation from pluripotent stem cells by ectopic expression of Runx3 | |
| CN111826354B (en) | NK cell and application thereof in tumor treatment | |
| Samara et al. | A cytokine cocktail augments the efficacy of adoptive NK-92 cell therapy against mouse xenografts of human cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151007 |