CN102492657A - Drug screening cell model using NF-kappa B as target and building and applications thereof - Google Patents
Drug screening cell model using NF-kappa B as target and building and applications thereof Download PDFInfo
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Abstract
The invention provides a drug screening cell model using nuclear factor (NF)-kappa B as the target, belonging to the biomedical field. The model is built on the basis of the active cells of constitutive NF-kappa B by using a reporting system carrier containing a NF-kappa B specific combination sequence and is used to screen drugs for curing the tumors and immunity diseases which are caused by the activation of the constitutive NF-kappa B. As the active cells of the constitutive NF-kappa B are adopted, the reporter gene in the modal cell is in a highly activated state and no external irritant is required; the obtained stable cell model can be directly used in the screening of compounds, thus the screening cost is greatly reduced, the entire screening flow is simplified, the screening cycle is shortened, the operating steps are reduced, the stability, high efficiency and usability of the system are increased; and the cell model is more suitable for high throughput drug screening. The original entire screening flow comprises the following steps: culturing cells, activating NF-kappa B, adding compounds and detecting. The simplified screening flow comprises the following steps: culturing cells, adding compounds and detecting.
Description
Technical field
The invention belongs to biomedicine field, relating to a kind of is the drug screening cell model of target spot with NF-κ B, relates in particular to a kind of to have the drug screening cell model that composing type NF-kB activity cell is the basis; The present invention also relates to the construction process and the application of this drug screening cell model simultaneously.
Background technology
Nuclear Factor-Kappa B (NF-κ B; Nuclear factor kappa-light-chain-enhancer of activated B cells) be important transcription factor; Mainly comprise RelA (p65), RelB, c-Rel, p50 and p52; Usually NF-κ B exists with the form of homology or heterodimer, and wherein the p65/p50 dimer is modal NF-κ B form.Participated in multiple physiological response under the normal circumstances NF-κ B, as with cell adhesion, differentiation, apoptosis, degradation of extracellular matrix, inflammatory cell chemotactic, immuno-stimulating or the like gene.Multiple factor can activate NF-κ B and cause downstream reaction, comprises the various kinds of cell factor (like TNF (Tumor Necrosis Factor) alpha and interleukin-IL-1 β etc.), stress stimulation, LPS (LPS), virus or other exogenous nucleic acid, radical or the like.In the path of classics; Major part is trapped in the tenuigenin NF-κ B dimer owing to combined its arrestin I κ B (Inhibitor of NF-κ B); Add stimulate or pathologic condition under (such as above-mentioned induction factor) because I κ B is caused NF-κ B activation to go into to examine by excessive degraded, exercise a series of functions; In recent years research simultaneously show NF-κ B dimer such as p65/p50 can with some other protein bound process in be activated functionating (like STAT3).
NF-κ B is one of relatively thorough and more generally acknowledged key molecule of in disease, playing an important role of a few research so far.The NF-kB activity of abnormal activation common and tumour generation, development and transfer, disease-relateds such as dysimmunity such as sacroiliitis, autoimmunization, asthma, some heart trouble.Discover that in the tumour of many types, as mammary cancer, colorectal carcinoma, prostate cancer, malignant lymphatic oncocyte or the like all has the NF-kB activity of continuous activation.Research shows, in this type tumour cell, suppresses processes such as survival that the NF-kB activity will effectively reduce tumour cell, propagation, reduces its grade malignancy effectively; Therefore, active the becoming naturally that suppresses NF-κ B slowed down and treats one of important aspect of disease of immune system (people: Science 1996 such as Wang; People such as Wu: EMBO J 1996; People such as Wang: Nat Med 1999, people such as Yamamoto: J Biol Chem 1999; People such as Yamamoto: Curr Mol Med 2001; People such as Yamamoto: J Clin Invest 2001).
For these reasons, all the time investigators to attempt to seek specificity good, the NF-kB inhibitor that spinoff is low, clinical finally to be used for, alleviate and the huge associated patient of treatment number.Yet the ideal product is few even to this day, largely because of there not being cover screening method efficiently and easily.Though practice has appearred and has been used in the high-throughput screening method based on reporting system; And obtained certain effect, but still some unsurmountable shortcomings are arranged, (activate NF-κ B with activator earlier such as the needs exogenous stimulation; Remove to screen suppressor factor again); It is many so not only to screen step, and the screening cost is also higher, especially will increase cost greatly as the use of activator such as cytokine IL-1 β and TNF α; Even picture LPS Class Activation agent use cost is lower, but, limited its use because itself toxicity is bigger; In every one step of increase of the system of screening simultaneously, can make the unstable of reporting system increase (extra operation will inevitably bring extra uncertain factor).
Summary of the invention
The objective of the invention is to the problem that exists in the prior art, providing a kind of is the drug screening cell model of target spot with NF-κ B.
Another object of the present invention provides
OnePlanting with NF-κ B is the construction process of the drug screening cell model of target spot.
A further object of the invention, just providing a kind of is the application of drug screening cell model in drug screening of target spot with NF-κ B.
(1) with NF-κ B is the drug screening cell model drug screening cell model of target spot
The present invention is the drug screening cell model of target spot with NF-κ B; Be to utilize the reporting system carrier that contains NF-κ B specificity binding sequence; Active cells to have composing type NF-kB is the basis, and being built into stable is target spot high-flux medicaments sifting cell model with NF-κ B signal.
The specificity binding sequence of said NF-κ B is 5 '-GGGRNNYYCC-3 '; Wherein R is a purine, and Y is a pyrimidine, and N is a base.
Said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include the response sequence of NF-κ B specificity binding sequence as the reporting system carrier, the reporting system carrier that is built into.
Said have a composing type NF-kB activity cell; It is one type of cell with continuous activation NF-kB activity; Comprise DU145; Prostate cancer cell, HepG2, human liver cancer cell, H1299 human lung carcinoma cell, A549 human lung carcinoma cell, HCT119 human colon cancer cell, Hela human cervical carcinoma cell, MCF-7 human breast cancer cell, MDA-MB-468 human breast cancer cell or MDA-MB-231 human breast cancer cell or the like.
(2) with NF-κ B be the structure of the drug screening cell model of target spot
The present invention is that the structure of the drug screening cell model of target spot comprises following process step with NF-κ B:
(1) reporting system vector construction: utilize molecular biology method in containing the report carrier MCS of luciferase reporter gene, to insert to include NF-κ B specificity binding sequence and 3 ' end to contain the DNA fragment of TATA box sequence; As the response sequence of reporting system carrier, be built into the reporting system carrier.
Insertion sequence is positioned at the upper reaches of luciferase reporter gene; When the NF-kB activation; Just can strengthen transcribing and the luciferase translation of luciferase reporter gene, thereby strengthen the activity of luciferase, can detect stronger fluorescence when adding the luciferase substrate; When suppressing the NF-kB activity, the level of translating with luciferase of transcribing of luciferase reporter gene all reduces, and also just suppresses the activity of luciferase in the cell, and fluorescence intensity also just weakens when adding the luciferase substrate.
(2) structure of cell model: after the transfection of reporting system carrier had composing type NF-kB activity cell; Carry out the positive colony screening with tetracycline; Choose the agent of known NF-κ B signal suppressing (like TPCA-1; Known NF-κ such as Bay11-7082 B stream signal suppressor factor) clone of response is arranged, being with NF-κ B is the drug screening cell model of target spot.
In cell with composing type NF-kB activity; The reporting system carrier can be activated well; When using NF-κ B signal pathway inhibitor, fluorescence is significantly suppressed, the cell model of structure also just need not under exogenous stimulation, to be used for delicately high-flux medicaments sifting.
(3) with NF-κ B be the application of the drug screening cell model of target spot
The present invention is that the drug screening cell model of target spot is used to screen the tumour that caused by the activation of NF-κ B composing type and the medicine of Immunological diseases with NF-κ B.The method of concrete screening of medicaments is following:
(1) with said drug screening cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%;
(2) SCREENED COMPOUND 0.1 μ l ~ 2 μ l are treated in adding in substratum, continue to cultivate 24 hours;
(3) in 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate, obtain the primary dcreening operation product more than 40% when the fluorescence inhibiting rate reaches;
(4) with the coup injury of compound cytotoxicity experiment elimination compound pair cell model, the fluorescence inhibiting rate reaches still and is obtaining multiple sieve product more than 40%, and being with NF-κ B is the inhibition medicine of target spot.
The present invention has the following advantages with respect to prior art: with NF-κ B signal path is target spot; Employing is strategy based on the clone with composing type NF-kB activity (promptly continuing the NF-kB activity); Make reporting system this is in the height active state in cell, need not to use to add stimulation, and the stabilized cell model that obtains can directly be used for the screening of compound; Not only greatly reduce the cost of screening; Also make simultaneously whole screening process be reduced to cell cultures → add compound → detection, shortened the cycle of screening, reduced operation steps from cell cultures → activations NF-κ B → add compound → detection; Promote stability, high efficiency and the ease for use of system, more be applicable to high-flux medicaments sifting.
Description of drawings
Fig. 1 detects the active result of NF-κ B in the cell involved in the present invention for using the Western-Blot method.
Fig. 2 prevents test (EMSA) to detect the active result of NF-κ B in the cell involved in the present invention for using gel electrophoresis.
Fig. 3 is used report carrier basic framework.
Fig. 4 is the sequencing result figure of constructed reporting system carrier κ B-luc insertion sequence.
Fig. 5 is for utilizing 293T cell detection reporting system carrier κ B – luc responding ability.
The response that Fig. 6 raises to the NF-kB activity for antiradiation drug screening cell model.
Fig. 7 changes for using the Western-Blot method to detect the NF-kB activity of DU145 cell after known suppressor factor TPCA-1 handles.
The response that Fig. 8 reduces the NF-kB activity for antiradiation drug screening cell model.
Fig. 9 is the inhibition effect of the resulting compound in process multiple sieve back to NF-κ B signal.
Figure 10 is 8215 and 11017 the compound that uses the Western-Blot method to detect the to be sieved inhibition ability to the NF-kB activation.
Embodiment
Be example explanation the present invention with NF-κ B with the DU145 cell below be that structure, construction process and the application of the drug screening cell model of target spot is described further.
The instrument and the reagent that are adopted in the experiment are following:
Instrument: PerkinElmer Victor3 fluorescence/chemiluminescent analyzer.
Reagent
:Photinus pyralis LUC is measured test kit available from Promega company; TNF α and IL-1 β are available from Peprotech company; TPCA-1 and tetracycline are available from Sigma-Aldrich company; 96 orifice plates that are used to sieve medicine are available from Corning company; Restriction enzyme, T4 dna ligase are available from Fermentas company; Synthetic and the order-checking of sequence, Shanghai is given birth to the worker and is provided; DMEM (high sugar) and foetal calf serum are available from Hyclone (Thermo company), and transfection reagent GenEscort II is biological available from the intelligent base in Nanjing; The compound of screening is provided by national micromolecular compound resource center; Various antibody are available from Cell Signaling Technology company.
1, reporting system vector construction
The dna fragmentation that includes NF-κ B specificity binding sequence (two 5 '-GGGACTTTCC-3 ' and two 5 '-GGGAATTTCC-3 ') and the auxiliary preface 5 ' of general TATA box-TATATAA-3 ' with synthetic; Restriction enzyme site with KpnI and HindIII is inserted into general luciferase reporting carrier pBR322-luc-puro, the reporting system carrier called after κ B-luc that obtains.Luciferase reporting carrier pBR322-luc-puro is by inserting North America Photinus pyralis LUC (Firefly Luciferase between pBR322 carrier Hind III and the Sal I; Referring to people such as J R de Wet: reporter gene and tetracycline gene (Puromycin Mol. Cell. Biol. 1987); Referring to people: Gene.1989 such as Lacalle RA; GenBank:M25346.1) gained; And luciferase reporter gene gets into sequence (IRES) with the tetracycline screening-gene through internal ribosome and links to each other, and it is poly A tailing signal that there is the AATAAA sequence in tetracycline gene terminator codon downstream.The pBR322-luc-puro MCS is between former EcoR I and Hind III (referring to Fig. 3).
So far, the complete insertion sequence on pBR322-luc-puro luciferase reporting carrier (sequencing result is seen Fig. 4) as follows:
2, reporting system carrier property test
Because the 293T cell is relatively more responsive to the stimulation of the various foreign cell factors, be convenient to add the activation situation that detects the corresponding signal path when cytokine stimulates outside, so use this cell as representing examining report system carrier whether to respond normally.(see figure 5) shows as a result: used reporting system carrier responds normally when the activation of NF-κ B signal path, and NF-κ B uses TNF α to activate.The 293T cell grows to 50%-70% density in 12 orifice plates, transfection κ B-luc (2 μ g/ hole), and the usage ratio of carrier and transfection reagent is 1:2 (i.e. 2 μ g plasmids; 4 μ l transfection reagents respectively are dissolved in 50 μ l DMEM serum free mediums, are mixed into 100 μ l premixed liquids; Room temperature leaves standstill 20 min, adds 150 μ l serum-free DMEM again, adds behind the mixing in each hole of 12 orifice plates) change perfect medium (the DMEM substratum that promptly contains 10% foetal calf serum) into after 4 hours; After 12 hours, handled 24 hours, inhale and remove substratum with TNF α (50 ng/ml); Add 200 μ l lysate lysing cell, jog 10min gets 150 μ l in 96 orifice plates; Add the common Photinus pyralis LUC substrate of 20 μ l, use fluorescence/chemiluminescent analyzer to measure the activity of (in the 1min) luciferase immediately, contrast and each 3 repetition of processing; And independent revision test 3 times (* * *, p 0.001).
3, the medicaments sifting model cell is selected
The activity of preventing NF-κ B in the experimental analysis cell through Western-Blot and EMSA gel; Obtain multiple being suitable for and (see Fig. 1 with the tumor cell line of the NF-kB activity high (need not extra use cytokine irritation cell) of this report system carrier; Fig. 2), Fig. 1 detects the active result of NF-κ B in the cell involved in the present invention for using the Western-Blot method.The result shows that a series of tumor cell ratio normal cells that comprise DU145 have higher NF-kB activity, and the phosphorylation sign with the 536th Serine of p65 (Ser536) is labeled as " pSer536-p65 ", and phosphorylation level is high more, and activity is high more.Fig. 2 prevents test (EMSA) to detect NF-kB activity result in the cell involved in the present invention for using gel electrophoresis.Show that used several kinds of tumour cell activatory NF-κ B have higher DNA binding ability than normal cell HME1 among Fig. 1.Wherein, HME1, people's mammary gland epidermic cell (normal control cell); DU145, prostate cancer cell; HepG2, human liver cancer cell; H1299 and A549 are human lung carcinoma cell, HCT119, human colon cancer cell; Hela, human cervical carcinoma cell; MCF-7, MDA-MB-468 and MDA-MB-231 are human breast cancer cell.
4, medicaments sifting model makes up and performance test
After 48 hours, 1 passes 3 with κ B-luc transfection DU145 cell (10cm petridish), and the adding tetracycline carries out the positive colony screening; Starting point concentration is 5 μ g/ml, reduces to 2.5 μ g/ml after 2 weeks, behind the clone to be formed (about 3 weeks altogether); With the trysinization mono-clonal in 96 orifice plates; Change 48 orifice plates after having grown again over to, 6 orifice plates are until 10cm petridish and frozen, during to use concentration be that the tetracycline of 2.5 μ g/ml continued to keep 2 weeks.At first choose luciferase male clone; Whether detect these clones again responds normal; Even because the composing type activation, signal path generally can also externally add cytokine and react to some extent, so the uciferase activity of selected positive colony should increase when cytokine stimulates to some extent; And owing to be constitutive activation, the suppressor factor of NF-κ B signal path should make the activity of luciferase reduce, and finally obtains the cell strain that reacts more satisfactory, called after 145 VIII through screening.The suppressor factor TPCA-1 that is selected for use is the suppressor factor of known NF-κ B signal path, acts on NF-κ B upper reaches IKK2, finally reduces NF-κ B activation and (analyzes referring to Fig. 7 Western-Blot.The DU145 passage is in 10 cm petridish; During to 60% ~ 70% degree of converging; Add 1 μ M or 2 μ M TPCA-1 and handle 24h; Add DMSO simultaneously as contrast), the TPCA-1 that selects for use (1 μ M) concentration does not have obvious growth-inhibiting (24 hours) through the MTT experiment confirm to the DU145 cell.Fig. 6 shows that 145 VIII fluorescence when external source TNF α (50 ng/ml) or IL-1 β (50 ng/ml) stimulation also has 2 ~ 3 times of liftings.Fig. 8 shows through TPCA-1 and handles, and clones 145 VIII uciferase activities and reduces 49.3% (* * *, p < 0.001).Cell inoculation is in 96 orifice plates (10; 000/>hole, 100 μ l) grew 12 hours, change and contain above-mentioned cytokine TNF alpha (50 ng/ml) or IL-1 β (50 ng/ml); Or the new nutrient solution of suppressor factor TPCA-1 (1 μ M); Continue to cultivate after 24 hours, add 50 μ l stable form luciferase substrates (Steady-Glo), lucifuge uses fluorescence/chemiluminescent analyzer to measure the activity of luciferase after reacting 10 min.
5, model is used
Suppressor factor to be screened is chosen 1440 kinds of natural products from national micromolecular compound resource center.
With the drug screening cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%; Add and treat SCREENED COMPOUND 0.25 μ l (25 μ M), continue to cultivate 24 h, in 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate.Obtain 28 kinds and inhibition and inhibiting rate are arranged at the compound more than 40%; The screening that reduces by half once more of these 28 kinds of compound concentrations; And eliminate the coup injury of compound pair cell model here with compound cytotoxicity experiment (testing with MTT); Obtain still drug screening cell model uciferase activity inhibiting rate being had 2 kinds at the compound more than 40%, be the multiple sieve product that the ability specificity suppresses NF-κ B, two kinds of compounds (Fig. 9) of 8215 and 11017 when being respectively numbering.
This is numbered 8215 and 11017 the compound fluorescence that NF-κ B reporting system is produced in can suppressing the DU145 cell through the Western-Blot analysis verification; NF-kB activity to human breast cancer cell MDA-MB-231 continuous activation has extraordinary inhibition effect (referring to Figure 10); The activity level of NF-κ B p65 has obtained suppressing effectively; Prove that this medicaments sifting model not only has good feasibility and safety, good versatility (compound that is sieved can suppress the activity of STAT3 under multiple situation) is arranged simultaneously.Wherein, DU145 cell or MDA-MB-231 go down to posterity in 10 cm petridish, during to 60% degree of converging, add and to be numbered 8215 and 11017 compound (10 μ M) and to handle 2h, the while with DMSO as contrast.
Above-mentioned experiment showed, through being that the drug screening cell model of target spot sieves the compound that obtains again really for being the inhibition medicine of target spot with NF-κ B with NF-κ B, thus the validity of this drug screening cell model proved.
A large amount of experiments are illustrated in the multiple cell with composing type NF-kB activity, and to be that the drug screening cell model of target spot can both be obtained convenient, screening effect fast and efficiently with NF-κ B in the present invention.
Claims (7)
1. with NF-κ B the drug screening cell model of target spot; It is characterized in that: utilize the reporting system carrier that contains NF-κ B specificity binding sequence; Active cells to have composing type NF-kB is the basis, and being built into stable is target spot high-flux medicaments sifting cell model with NF-κ B signal.
2. be the drug screening cell model of target spot with NF-κ B according to claim 1, it is characterized in that: the specificity binding sequence of said NF-κ B is 5 '-GGGRNNYYCC-3 '; Wherein R is a purine, and Y is a pyrimidine, and N is a base.
3. be the drug screening cell model of target spot according to claim 1 with NF-κ B; It is characterized in that: said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include the response sequence of NF-κ B specificity binding sequence as the reporting system carrier, the reporting system carrier that is built into.
4. be the drug screening cell model of target spot according to claim 1 with NF-κ B; It is characterized in that: said active cells with composing type NF-κ B is DU145; Prostate cancer cell, HepG2, human liver cancer cell, H1299 human lung carcinoma cell, A549 human lung carcinoma cell, HCT119 human colon cancer cell, Hela human cervical carcinoma cell, MCF-7 human breast cancer cell, MDA-MB-468 human breast cancer cell or MDA-MB-231 human breast cancer cell.
5. be the structure of the drug screening cell model of target spot with NF-κ B according to claim 1, comprise following process step:
(1) reporting system vector construction: utilize molecular biology method in containing the report carrier MCS of luciferase reporter gene, to insert to include NF-κ B specificity binding sequence and 3 ' end to contain the DNA fragment of TATA box sequence; As the response sequence of reporting system carrier, be built into the reporting system carrier;
(2) structure of cell model: after the transfection of reporting system carrier had composing type NF-kB activity cell; Carry out the positive colony screening with tetracycline; Choosing to NF-κ B signal suppressing agent has the clone of response, and being with NF-κ B is the drug screening cell model of target spot.
6. be that the drug screening cell model of target spot is used to screen the tumour that caused by the activation of NF-κ B composing type and the medicine of Immunological diseases with NF-κ B according to claim 1.
7. of claim 6 is that the drug screening cell model of target spot is used to screen the tumour that caused by the activation of NF-κ B composing type and the medicine of Immunological diseases with NF-κ B, and it is characterized in that: the method for said drug screening comprises following process step:
(1) with said drug screening cell model with 100 μ l culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%;
(2) SCREENED COMPOUND 0.1 μ l ~ 2 μ l are treated in adding in substratum, continue to cultivate 24 hours;
(3) in above-mentioned 96 orifice plates, add 50 μ l stable form luciferase substrates, use fluorescence/chemiluminescent analyzer to measure fluorescence intensity and analysis of fluorescence inhibiting rate, obtain the primary dcreening operation product more than 40% when the fluorescence inhibiting rate reaches;
(4) with the coup injury of compound cytotoxicity experiment elimination compound pair cell model, the fluorescence inhibiting rate reaches still and is obtaining multiple sieve product more than 40%, and being with NF-kB is the inhibition medicine of target spot.
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| CN114437202A (en) * | 2022-01-18 | 2022-05-06 | 四川大学华西医院 | Androgen response element and application thereof in detecting environmental androgen |
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