CN104711227A - Method for activation of Notch-1 signal channel on the basis of external co-culture - Google Patents
Method for activation of Notch-1 signal channel on the basis of external co-culture Download PDFInfo
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- CN104711227A CN104711227A CN201310686754.2A CN201310686754A CN104711227A CN 104711227 A CN104711227 A CN 104711227A CN 201310686754 A CN201310686754 A CN 201310686754A CN 104711227 A CN104711227 A CN 104711227A
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Abstract
The invention discloses a method for activation of Notch-1 signal channel on the basis of external co-culture, and belongs to the technical field of biological information. The method is as follows: respectively culturing breast cancer cells and stable expression Notch-1 ligand interstitial cells in DMEM (dulbecco's modified eagle medium) culture medium containing 10% by volume of calf serum, then cleaning with PBS (phosphate buffered solution), and collecting the stable expression Notch-1 ligand interstitial cells; culturing the interstitial cells in the DMEM (dulbecco's modified eagle medium) culture medium containing 10% by volume of calf serum, and adding the stable expression Notch-1 ligand interstitial cells into the breast cancer cells in the ratio of 1:1-2:1 for culturing to realize activation of the Notch-1 signal channel in the breast cancer cells. According to the method, the activation of the Notch-1 signal channel can be realized by simulation of interaction of Notch-1 in cancer cells in in-vivo environment and Jagged1 expressed by the interstitial cells, the defects that overexpression standard quantity and the efficiency transfection are difficult to determine, and part of cells are not transfected in the traditional methods can be overcome, and the situations that the Notch-1 signal channel cannot be fully and effectively activated by overexpression in the overexpression methods, and obtained biological results are not objective can be avoided.
Description
[technical field]
The invention belongs to bioinformatics technique field, particularly a kind of method activating Notch-1 signal path based on co culture system in vitro.
[background technology]
Notch-1 participates in the development of mammary cancer and an important oncogene of progress.Notch signal path plays a part very crucial in the transfer of cell proliferation, apoptosis, differentiation, invasion and attack, vasculogenesis, tumour and the process of breast carcinoma stem cell self.It is transmembrane protein receptor family, by activating its activity with ligand binding.Up to now, four Notch acceptors (Notch-1,2,3,4) and 5 ligands (Dll-1, Dll-3, Dll-4, Jagged-1 and Jagged-2) are had to be determined in this family.After Notch and ligand binding, need to be cut by two step enzymes to form final active Notch(Notch intracellular portion intracellular Notch ICN).The enzyme finally performing shearing is gamma-secretase (gamma secretase), use micromolecular inhibitor GSI(gamma-secretase inhibitor) activity of gamma secretase can be suppressed, stop last endonuclease reaction, and then suppress the signal transduction of Notch.The Notch-1 of activation transfers in nucleus, mainly through carrying out regulate gene expression in conjunction with transcription factor CBF-1.In mammary cancer, Notch-1 is very important oncogene and new drug target.But how Notch-1 regulates and controls, and cancer cells is survived, the mechanism of growth is unclear.
The inhibitor GSI of Notch has entered the clinical one/second phase research of mammary cancer, (referring to http://www.clinicaltrials.gov/ct2/results term=gamma-secretase++breast+cancer & Search=Search).Therefore, the medicine that the mechanism of carcinogenesis studying Notch-1 is Clinical practice target Notch-1 provides solid translational medicine basis.In the process of the mechanism of carcinogenesis of research Notch-1, the signal path effectively activating Notch-1 in vitro in culture environment becomes the indispensable instrument successfully disclosing mechanism of carcinogenesis.
[summary of the invention]
For overcoming the shortcoming and defect of above-mentioned prior art, the object of the present invention is to provide a kind of method activating Notch-1 signal path based on co culture system in vitro.The present invention, particular by by breast cancer cell and the inoblast co-cultivation expressing Jadde-1, realizes the object of the stable Notch-1 signal path activated in breast cancer cell by fibroblasts to secrete Jadde-1 and the contact of analogue body inner cell.
Object of the present invention is achieved through the following technical solutions: a kind of method activating Notch-1 signal path based on co culture system in vitro, comprises the steps:
(1) mesenchymal cell of breast cancer cell and stably express Notch-1 part is cultivated respectively in containing the DMEM substratum of volume fraction 10% calf serum;
(2) the two kinds of cells treating in step (1) grow into the density of 80% respectively, collect the mesenchymal cell of stably express Notch-1 part by PBS buffer solution for cleaning;
(3) cultivate the mesenchymal cell of the stably express Notch-1 part collected in resuspended step (2) in containing the DMEM substratum of volume fraction 10% calf serum, by 1:1-2:1, the mesenchymal cell of stably express Notch-1 part is joined in breast cancer cell; Co-cultivation 0.5 ~ 48h, realizes activating Notch-1 signal path in breast cancer cell.
In step (1):
Described breast cancer cell is preferably three cloudy breast carcinoma cell strain MDA-MB-231;
The mesenchymal cell of described stably express Notch-1 part is preferably the l cell LTK-JAG cell of expressing Jaddeg-1;
The preparation method of described LTK-JAG cell asks for an interview document: Lindsell C.E., Shawber C.J., Boulter J., Weinmaster G.Jagged:a mammalian ligand that activatesNotch1.Cell.1995; 80:909-917;
The condition optimization of described cultivation is in 37 DEG C, the CO of volume fraction 5%
2in cultivate.
PBS buffering described in step (2) clean and the inoblast of stably express Notch-1 part is preferably scraped by cell harvesting and is collected in 5ml PBS damping fluid by the step of collecting, and centrifugal 1000 × g, 5 minutes, abandons supernatant;
The condition optimization of the cultivation described in step (3) is in 37 DEG C, the CO of volume fraction 5%
2in cultivate.
Invention mechanism of the present invention is: activation mechanism in present method analogue body, uses and expresses the Jagged-1 l cell of Notch part and the method for breast cancer cell Dual culture, realizes the object activating the signal path of Notch-1 in vitro.Notch classical signal path as shown in Figure 1.After Notch discharges from cytolemma, NIC(intracellular Notch) be transferred to nucleus and carry out regulate gene expression in conjunction with transcription factor CBF-1.Under the state not having Notch, CBF-1 is the gene expression inhibition factor.It is by comprising SMRT(silencing mediator of retinoid and thyroid hormone receptors in promoter region in conjunction with Corepressors), nCOR(Nuclear corepressor) and histon deacetylase (HDAC) suppressor gene (HDAC1) expression.Notch in nucleus replaces these Corepressors and comprises acetylation of histone enzyme PCAF, GCN5 or p300 and MAML1 (mastermindlike1) in conjunction with co-activator.Notch target gene has HES (hairy enhancer of split genes) and HERP (HES-related repressor protein), HEY family, cyclin D, c-Myc and NF-к B transcription factor family.
The present invention has following advantage and beneficial effect relative to prior art:
The novel method based on breast cancer cell and the inoblast Dual culture of expressing Notch part Jagged-1 in the present invention activates tumour cell Notch-1 signal path in vitro, compared to the traditional method of process LAN, the method is better simulated the Jagged-1 that in environment in vivo, in cancer cells, Notch-1 and mesenchymal cell are expressed and is interacted to activate Notch-1 signal path, overcomes that the normal content of process LAN in traditional method is difficult to determine, the efficiency of transfection is difficult to determine, part cell does not have transfected defect at all; Avoid process LAN in process LAN method and cannot activate Notch-1 signal path comprehensively and effectively, the one sided situation of gained biological results.In the present invention the method for two kinds of co-culture of cells simulate transmembrane protein acceptor Notch-1 family under physiological condition in vivo by and ligand binding activate the characteristic of its activity, more biological activation Notch-1 signal path.
[accompanying drawing explanation]
Fig. 1 is the schematic diagram of Notch classical signal path.
Fig. 2 is the schematic diagram activating Notch signal path in embodiment 1
[embodiment]
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but protection scope of the present invention is not limited to this.
Three cloudy breast carcinoma cell strain MDA-MB-231 used in following examples derive from ATCC, LTK-PAR(l cell does not express Jaddeg-1) and LTK-JAG cell (l cell express Jaddeg-1) by Dr.G.Weinmaster (University of California at Los Angeles, LosAngeles, CA) build and provide.
Embodiment 1
(1) by three cloudy breast cancer cell MDA-MB-231, LTK-PAR(l cell does not express Jaddeg-1) or LTK-JAG cell (l cell express Jaddeg-1) cultivate in containing the DMEM substratum of volume fraction 10% calf serum respectively, the condition of cultivation is in 37 DEG C, the CO of volume fraction 5%
2middle cultivation;
(2) three cloudy breast cancer cell MDA-MB-231, LTK-PAR or LTK-JAG cells are gone down to posterity to the density of second day 80%, second day, wash LTK-PAR or LTK-JAG cell with PBS, scrape with cell harvesting and collect LTK-PAR or LTK-JAG cell to 5ml PBS, centrifugal 1000 × g, 5 minutes, abandon supernatant;
(3) with containing cultivating in the DMEM substratum of volume fraction 10% calf serum (in 37 DEG C, the CO of volume fraction 5%
2middle cultivation; ) middle LTK-PAR or the LTK-JAG cell collected of resuspended step (2), by 1:1, the mesenchymal cell of stably express Notch-1 part is joined in breast cancer cell (not needing digestion); Co-cultivation 24h, realizes activating Notch-1 signal path in breast cancer cell.After Dual culture 24h, use traditional method CBF-1 luciferase reporter gene to detect Notch-1 signal path active, detect and find compared with the cell of LTK-PAR Dual culture, improve 2-5 doubly with its Notch signal path activity of cell of LTK-JAG Dual culture.
Embodiment 2
(1) by three cloudy breast cancer cell MDA-MB-231, LTK-PAR(l cell does not express Jaddeg-1) or LTK-JAG cell (l cell express Jaddeg-1) cultivate in containing the DMEM substratum of volume fraction 10% calf serum respectively, the condition of cultivation is in 37 DEG C, the CO of volume fraction 5%
2middle cultivation;
(2) three cloudy breast cancer cell MDA-MB-231, LTK-PAR or LTK-JAG cells are gone down to posterity to the density of second day 80%, second day, wash LTK-PAR or LTK-JAG cell with PBS, scrape with cell harvesting and collect LTK-PAR or LTK-JAG cell to 5ml PBS, centrifugal 1000 × g, 5 minutes, abandon supernatant;
(3) with containing cultivating in the DMEM substratum of volume fraction 10% calf serum (in 37 DEG C, the CO of volume fraction 5%
2middle cultivation; ) middle LTK-PAR or the LTK-JAG cell collected of resuspended step (2), by 2:1, the mesenchymal cell of stably express Notch-1 part is joined in breast cancer cell (not needing digestion); Co-cultivation 48h, realizes activating Notch-1 signal path in breast cancer cell.After Dual culture 48h, use traditional method CBF-1 luciferase reporter gene to detect Notch-1 signal path active, detect and find compared with the cell of LTK-PAR Dual culture, improve 3-5 doubly with its Notch signal path activity of cell of LTK-JAG Dual culture.
Embodiment 3
(1) by three cloudy breast cancer cell MDA-MB-231, LTK-PAR(l cell does not express Jaddeg-1) or LTK-JAG cell (l cell express Jaddeg-1) cultivate in containing the DMEM substratum of volume fraction 10% calf serum respectively, the condition of cultivation is in 37 DEG C, the CO of volume fraction 5%
2middle cultivation;
(2) three cloudy breast cancer cell MDA-MB-231, LTK-PAR or LTK-JAG cells are gone down to posterity to the density of second day 80%, second day, wash LTK-PAR or LTK-JAG cell with PBS, scrape with cell harvesting and collect LTK-PAR or LTK-JAG cell to 5ml PBS, centrifugal 1000 × g, 5 minutes, abandon supernatant;
(3) with containing cultivating in the DMEM substratum of volume fraction 10% calf serum (in 37 DEG C, the CO of volume fraction 5%
2middle cultivation; ) middle LTK-PAR or the LTK-JAG cell collected of resuspended step (2), by 1.5:1, the mesenchymal cell of stably express Notch-1 part is joined in breast cancer cell (not needing digestion); Co-cultivation 0.5h, realizes activating Notch-1 signal path in breast cancer cell.After Dual culture 0.5h, use traditional method CBF-1 luciferase reporter gene to detect Notch-1 signal path active, detect and find compared with the cell of LTK-PAR Dual culture, improve 2-5 doubly with its Notch signal path activity of cell of LTK-JAG Dual culture.
The above the specific embodiment of the present invention, does not form limiting the scope of the present invention.Any various other done by technical conceive of the present invention change and distortion accordingly, all should be included in the protection domain of the claims in the present invention.
Claims (6)
1. activate a method for Notch-1 signal path based on co culture system in vitro, it is characterized in that comprising the steps:
(1) mesenchymal cell of breast cancer cell and stably express Notch-1 part is cultivated respectively in containing the DMEM substratum of volume fraction 10% calf serum;
(2) the two kinds of cells treating in step (1) grow into the density of 80% respectively, collect the mesenchymal cell of stably express Notch-1 part by PBS buffer solution for cleaning;
(3) in containing the DMEM substratum of volume fraction 10% calf serum, cultivate mesenchymal cell, by 1:1-2:1, the mesenchymal cell of stably express Notch-1 part is joined in breast cancer cell; Co-cultivation 0.5 ~ 48h, realizes activating Notch-1 signal path in breast cancer cell.
2. the method activating Notch-1 signal path based on co culture system in vitro according to claim 1, is characterized in that: the breast cancer cell described in step (1) is three cloudy breast carcinoma cell strain MDA-MB-231.
3. the method activating Notch-1 signal path based on co culture system in vitro according to claim 1, is characterized in that: the mesenchymal cell of the stably express Notch-1 part described in step (1) is the l cell LTK-JAG cell of expressing Jaddeg-1.
4. the method activating Notch-1 signal path based on co culture system in vitro according to claim 1, it is characterized in that: the PBS buffering described in step (2) is cleaned and the step of collecting is scraped by cell harvesting by the inoblast of stably express Notch-1 part to be collected in 5ml PBS damping fluid, centrifugal 1000 × g, 5 minutes, abandon supernatant.
5. the method activating Notch-1 signal path based on co culture system in vitro according to claim 1, is characterized in that: the temperature of the cultivation described in step (1) is 37 DEG C.
6. the method activating Notch-1 signal path based on co culture system in vitro according to claim 1, is characterized in that: the mesenchymal cell described in step (3) is the mesenchymal cell of the stably express Notch-1 part collected in resuspended step (2).
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| US20110027881A1 (en) * | 2009-07-31 | 2011-02-03 | St. Marianna University School Of Medicine | Production method of immune cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110027881A1 (en) * | 2009-07-31 | 2011-02-03 | St. Marianna University School Of Medicine | Production method of immune cells |
Non-Patent Citations (5)
| Title |
|---|
| HE MENG ET AL.: "Thrombospondin 2 Potentiates Notch3/Jagged1 Signaling", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| KANDIS V. BACKUS ET AL.: "Abstract LB-261: A novel notch-AKT-NF-KB axis in triple negative breast cancer cells", 《EXPERIMENTAL AND MOLECULAR THERAPEUTICS》 * |
| L HAO ET AL.: "Notch-1 activates estrogen receptor-α-dependent transcription via IKKα in breast cancer cells", 《ONCOGENE》 * |
| YIN PENG: "Notch-1 Activates Nf-Кb Activity in Cervical Cancer and Estrogen Receptor Negative Breast Cancer", 《LOYOLA UNIVERSITY CHICAGO LOYOLA ECOMMONS DISSERTATIONS》 * |
| YIN YUAN ET AL.: "Jagged1 contributes to the drug resistance of Jurkat cells in contact with human umbilical cord‑derived mesenchymal stem cells", 《ONCOLOGY LETTERS》 * |
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