CN102492656A - High-flux anti-tumor drug screening cell model based on STAT3 and NF-kB two-signal channel serving as target, as well as building and application of high-flux anti-tumor drug screening cell model - Google Patents

Abstract

The invention provides a high-flux anti-tumor drug screening cell model based on an STAT3 and NF-kB two-signal channel serving as a target. In the cell model, the specific binding sequence of STAT3 and that of NF-kB are bound to build a reporting system carrier; and the cell model is built by binding competent cells comprising both constitutive STAT3 and constitutive NF-kB. The cell model is used for screening and treating cell cancerization caused by continuous activization of STAT3 and/or NF-kB, and medicines generated by the combination and the mutual promotion of STAT3 and NF-kB, and has a higher efficiency than a single-signal medicine screening system. As the competent cells comprising both constitutive STAT3 and constitutive NF-kB are adopted, the reporting gene in the cell model is in a highly active state, no stimulus is required to be added additionally, the screening process is simplified, the screening cost is reduced, the stability, the high efficiency and the easiness in the use of a system are improved, and the cell model is more suitable for high-flux medicine screening.

Description

Based on STAT3 and NF-κ B dual signal path is screening anti-tumor medicine cell model and the structure and the application of target spot

Technical field

The invention belongs to biomedicine field, relate to a kind of screening anti-tumor medicine cell model, relate in particular to a kind of based on STAT3 and NF- κB dual signal path is the high-throughput screening anti-tumor medicine cell model of target spot; The present invention also relates to the construction process and the application of this screening anti-tumor medicine cell model simultaneously.

Background technology

In many tumour cells, look like especially in the cells such as colorectal carcinoma, mammary cancer, neuroglial cytoma, lung cancer, all have continuous activation STAT3 (signal transduction and activating transcription factor 3) and NF-kB (nf- κB) activity.NF- κB family mainly comprises following member, is specially RelA (p65), RelB, c-Rel, p50 and p52, usually NF- κB exists with the form of homology or heterodimer, and wherein the p65/p50 dimer is modal NF-in the Mammals κThe B form.Under the normal circumstances, NF- κThe B dimer is owing to combined its arrestin I κB (Inhibitor of NF- κB) and mainly be trapped in the tenuigenin, add under stimulation or the pathologic condition because I κB is caused NF-by excessive degraded κThe B activation is gone into nuclear and is exercised a series of functions.And STAT3 is as a member of STATs molecule family, receives that the stream signal cytokine stimulates or under the state of some Tyrosylprotein kinase continuous activation, 705 tyrosine of its C end are formed dimer by phosphorylation, and then goes into nuclear and regulate and transcribe.STAT3 and NF-kB can both combine special nucleotide sequence adjusting to transcribe (be generally at promoter region and strengthen transcribing of downstream gene) after going into nuclear; NF- κThe binding sequence of B is generally row 5 '-GGGRNNYYCC-3 ' (R represents purine, and Y represents pyrimidine, and N represents one of any four kinds of bases), and the binding sequence of STAT3 is 5 '-TT (C/A) CNNNAA-3 ' (N is one of any four kinds of bases).NF- κThe downstream gene that the activation of B and STAT3 is regulated and control often all with suppress apoptosis, promote cell proliferation relevant with transfer.A large amount of evidences show, active or its upstream and downstream signal that suppresses above-mentioned two kinds of transcription factors all will suppress existence, propagation and the migration of tumour cell (people: Science 1996 such as Wang greatly; People such as Wu: EMBO J 1996; People such as Wang: Nat Med 1999; People such as Ni: Cancer Res 2000; People such as Leong: Proc Natl Acad Sci 2003; People such as Barton: Mol Cancer Ther 2004; People such as Kortylewski: Nat Med 2005; People such as Xin: Cancer Res 2009).

At present more and more investigators recognize that with STAT3 and NF-kB be the feasibility that signal path carries out screening anti-tumor medicine; And begin the method for attempting being correlated with; But based on STAT3 and NF-kB signal path is that the sieve medicine system of target spot is because the STAT3 or the NF-kB specific activity of the clone of being selected for use itself are lower; During the screening specific inhibitor, often need add the cytokine irritation cell in advance, carry out screening compound again; The use of cytokine not only makes cost improve greatly, and every one step of increase of sieve medicine system will increase the unstable of system greatly; The stimulation that adds simultaneously can not simulate fully that tumour cell is actual to have higher STAT3 or an active state of NF-kB.In addition, nearest research shows, in numerous tumour cells, and STAT3 and NF- κThe B signal transducers also exist mutually combine, acting in conjunction promotes tumor-related gene to express and tumour takes place, the phenomenon of development (people: Cancer Res 2005 such as Yang J; People: Genes & Dev 2007 such as Yang J; People: Nat Cell Biol 2008 such as Torres J; People: Cancer Cell 2009 such as Lee H; People such as Yu H: Nat Rev Cancer 2009).

Summary of the invention

The objective of the invention is provides a kind of based on STAT3 and NF-to the problem that exists in the prior art κB dual signal path is the high-throughput screening anti-tumor medicine cell model of target spot.

Another object of the present invention provides a kind of based on STAT3 and NF- κB dual signal path is the construction process of the high-throughput screening anti-tumor medicine cell model of target spot.

A further object of the invention just provides a kind of based on STAT3 and NF- κB dual signal path is the application of screening anti-tumor medicine cell model in screening antineoplastic drugs of target spot.

(1) based on STAT3 and NF- κB dual signal path is the high-throughput screening anti-tumor medicine cell model of target spot

The present invention is based on STAT3 and NF- κB dual signal path is the screening anti-tumor medicine cell model of target spot, adopts STAT3 specificity binding sequence and NF- κThe reporting system carrier is set up in the combination of B specificity binding sequence; In conjunction with having composing type NF- κThe active cells of B and STAT3 is built into and stablizes easy-to-use high-throughput antitumor drug sieve medicine model.

STAT3 specificity binding sequence and NF- κThe combination of B specificity binding sequence, the reporting system carrier of foundation can both have significant response to the activation of above-mentioned two signal paths, can characterize STAT3 and/or NF-simultaneously κThe B activity change; Utilization has composing type NF- κThe medicaments sifting model that the active cells of B and STAT3 makes up; Make whole reporting system carrier reach the ideal active state; And on fluorescence level, significant variation can both be arranged when suppressing a signal path separately, finally make this system can be used for high-flux medicaments sifting delicately.

Said NF- κB specificity binding sequence is 5 '-GGGRNNYYCC-3 ', and wherein R is a purine, and Y is a pyrimidine, and N is a base.Preferred sequence is: 5 '-GGGAATTTCC-3 ' (is called for short κB).

The specificity binding sequence of said STAT3 is 5 '-TT (C/A) CNNNAA-3 ', and N is a base.Preferred sequence is: 5 '-TTCCCGTAA-3 ' is or/and 5 '-TTCCTGTAA-3 ' (general designation SIE).

Said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include STAT3 specificity binding sequence and NF- κB specificity binding sequence is as the response sequence of reporting system carrier, the reporting system carrier that is built into.

In the said reporting system carrier, STAT3 specificity binding sequence and NF- κB specificity binding sequence makes up by any way.In order to reach optimum screening effect, said STAT3 specificity binding sequence and NF- κThe array mode of B specificity binding sequence is: binding sequence comprises n STAT3 specificity binding sequence and m NF- κB specificity binding sequence, and n:m=1:1 ~ 16:1, two kinds of sequences can make up front and back, also can nestedly make up.

Said have a composing type NF- κThe active cells of B and STAT3 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.

(2) based on STAT3 and NF- κB dual signal path is the structure of the high-throughput screening anti-tumor medicine cell model of target spot

The present invention is based on STAT3 and NF- κB dual signal path is the structure of the screening anti-tumor medicine cell model of target spot, specifically comprises following process step:

(1) reporting system vector construction: utilize molecular biology method in containing the report carrier MCS of luciferase reporter gene, to insert and include STAT3 specificity binding sequence and NF- κB specificity binding sequence and 3 ' end contain the DNA fragment of TATA box sequence, as the response sequence of reporting system carrier, are built into the reporting system carrier.

Above-mentioned STAT3 specificity binding sequence and NF- κB specificity binding sequence includes n STAT3 specificity binding sequence and m NF- κB specificity binding sequence, and n:m=1:1 ~ 16:1.

Insertion sequence is positioned at the upper reaches of luciferase reporter gene, as STAT3 and/or NF- κDuring the B 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; Suppress STAT3 and/or NF- κWhen B was active, the level of translating with luciferase of transcribing of luciferase reporter gene all reduced, 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: the transfection of reporting system carrier is had composing type STAT3 and NF-simultaneously κBehind the B active cells, carry out the positive colony screening, choose STAT3 and NF-with tetracycline κThe agent of B signal suppressing has the clone of response, is based on STAT3 and NF- κB dual signal path is the screening anti-tumor medicine cell model of target spot.

Have composing type STAT3 and NF-at the same time κIn the active cell of B, the reporting system carrier can be activated well, when using STAT3 and/or NF- κFluorescence is significantly suppressed, and the cell model of structure also just need not under exogenous stimulation, to be used for delicately high-flux medicaments sifting.

(3) based on STAT3 and NF- κB dual signal path is the application of the high-throughput screening anti-tumor medicine cell model of target spot

The present invention is based on STAT3 and NF- κB dual signal path is that the screening anti-tumor medicine cell model of target spot is used for screening treatment STAT3 and/or NF- κThe cell carcinogenesis that the B continuous activation causes, STAT3 and NF- κB mutually combines, the medicine of the cell carcinogenesis that causes of mutually promoting.Concrete screening method is following:

(1) with said screening anti-tumor medicine cell model with 100 ml culture medium inoculateds in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40%;

(2) SCREENED COMPOUND 0.1 ml ~ 2ml is treated in adding in the substratum, continues to cultivate 24 hours;

(3) in 96 orifice plates, add 50 ml 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) eliminate the coup injury of compound pair cell model with the compound cytotoxicity experiment, the fluorescence inhibiting rate reaches still obtaining multiple sieve product more than 40%, is with STAT3 and/or NF- κThe B signal path is the tumor suppression medicine of target spot.

The present invention has the following advantages with respect to prior art: with STAT3 and NF- κB dual signal path is a target spot, adopts based on having composing type STAT3 and NF-simultaneously κThe active clone of B is strategy, and make reporting system this is in the height active state in cell, need not to use to add stimulator, 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 whole screening process from " cell cultures → activation STAT3 and/or NF-simultaneously κB → add compound → detection " be reduced to " cell cultures → add compound → detection ", shortened the cycle of screening, reduced operation steps, promoted stability, high efficiency and the ease for use of system, more be applicable to high-flux medicaments sifting.In addition, the present invention can screen treatment STAT3 and/or NF-simultaneously κThe cell carcinogenesis that the B continuous activation causes, STAT3 and NF- κB mutually combines, the medicine of the cell carcinogenesis that causes of mutually promoting, and with respect to single-signal medicament sifting motion system, tool is highland efficient more.

Description of drawings

Fig. 1 detects STAT3 and NF-in the cell involved in the present invention for using the Western-Blot method κThe active result of B.

Fig. 2 prevents test (EMSA) to detect the active result of STAT3 in the cell involved in the present invention for using gel electrophoresis.

Fig. 3 prevents test (EMSA) to detect NF-in the cell involved in the present invention for using gel electrophoresis κThe active result of B.

Fig. 4 is the ultimate principle figure of the used reporting system of the present invention.

Fig. 5 is for implementing row 1 used report carrier basic framework.

Fig. 6 is the constructed reporting system carrier S IE-of embodiment 1 κThe sequencing result figure of B-luc insertion sequence.

Fig. 7 utilizes the responding ability of two kinds of binding sequences in the 293T cell detection reporting system carrier for embodiment 1.

Fig. 8 starts transcriptional capability for embodiment 1 utilizes A549 cell detection reporting system carrier.

Fig. 9 is that two kinds of embodiment 1 screening anti-tumor medicine cell models surely change cell strain SKA and SKK to STAT3 or NF- κThe active response that raises of B.

Figure 10 is that two kinds of embodiment 1 screening anti-tumor medicine cell models surely change cell strain SKA and SKK to STAT3 or NF- κThe active response that reduces of B.

Figure 11 is screened in the natural product compound library by embodiment 1, and the specificity that can demonstrate,prove through document that filters out suppresses the inhibiting rate of compound.

Figure 12 for use the Western-Blot method detect institute sieve the active inhibition effect of its STAT3 behind unknown natural product h31 (10 μ M) the processing DU145 cell.

Figure 13 is the constructed reporting system carrier (SIE-of embodiment 2 κB)-the sequencing result figure of luc insertion sequence.

Figure 14 utilizes (SIE-in the 293T cell detection reporting system carrier for embodiment 2 κB)-luc is to STAT3 and/or NF- κB signal path activatory responding ability.

Figure 15 is that embodiment 2 screening anti-tumor medicine cell models are to STAT3 or NF- κThe active response that reduces of B.

Figure 16 is the constructed reporting system carrier S IE/ of embodiment 3 κThe sequencing result figure of B-luc insertion sequence.

Embodiment

Further specify through structure, construction process and the application of specific embodiment below screening anti-tumor medicine cell model of the present invention.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, comprises that common luciferase substrate and stable form luciferase substrate (Steady-Glo) are available from Promega company; Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF α) are available from Peprotech company; PD180970, 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; Carrier extracts and DNA product purification related kit, available from sky root biochemical corp; Synthetic and the order-checking of primer, Shanghai is given birth to the worker and is provided; DMEM (high sugar) and foetal calf serum are available from Hyclone (Thermo company); Transfection reagent GenEscort II is available from the intelligent basic biotech firm in Nanjing; The compound of screening is provided by national micromolecular compound resource center; Various antibody are available from Cell Signaling Technology company.

Embodiment 1

1, reporting system vector construction

By ultimate principle figure shown in Figure 4, artificial constructed response sequence wherein comprises STAT3 binding sequence and NF- κThe B binding sequence.The STAT3 binding sequence is called for short SIE, NF- κThe B binding sequence is called for short κB.The STAT3 binding sequence here is by each 8 alternately series connection of 5 '-TTCCCGTAA-3 ' and 5 '-TTCCTGTAA-3 '; NF- κThe B binding sequence is 5 '-GGGAATTTCC-3 '; Two kinds of binding sequences are cascaded, and the back adds general TATA box auxiliary sequencel 5 '-TATATAA-3 ', is inserted into general luciferase reporting carrier pBR322-luc-puro, obtains recombinant vectors called after SIE- κB-luc; As contrast, will comprise STAT3 binding sequence or NF-separately κThe nucleotide sequence of B binding sequence (all the other constitute the same) inserts on the luciferase reporting carrier, respectively called after SIE-luc with κB-luc.Here the ratio of two kinds of response element sequences is 16:1, and this ratio is used when making up cell model to have a composing type NF- κB and STAT3 active cells (referring to Fig. 1, Fig. 2, practical situation Fig. 3) and to the adjustment of the startup situation of reporter gene.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. 5).

During structure, insert earlier and contain NF- κThe sequence of B binding sequence and TATA box is inserted the STAT3 binding sequence then, finally obtains SIE- κThe whole insertion sequence of B-TATA box; Concrete steps are following: the amount test kit extracted during 1. luciferase reporting carrier pBR322-luc-puro carried with a day root high purity plasmid for a short time, got 10 μ g and did double digestion, 37 ℃ with Xho I and Hind III (each 3 μ l); 8 h; Agarose gel electrophoresis uses a day root DNA purifying and recovering test kit to reclaim, and obtains the product I; The complementary primer of two band toughness terminal (and can match with Xho I and Hind III restriction enzyme site) (has been comprised NF- κB binding sequence and TATA box sequence, line as follows):

。

Be diluted to 50 μ M solution, after respectively getting 15 μ l and mixing, 5 min in 95 ℃ of boiling water baths, naturally cooling in the water-bath obtains the product II; 2. with product I and II with amount of substance than 1:3, use the T4 dna ligase to connect, 16 ℃ of connections are spent the night; 3. transformed into escherichia coli competence DH5 α is applied to the agarose culture plate that contains amicillin resistance, and 37 ℃ are spent the night; 4. 5 of pickings are cloned in the LB liquid nutrient medium that contains penbritin and are cultured to logarithmic phase respectively; The upstream and downstream primer PCR is identified the product (contrasting not, the carrier of insertion sequence does not then have the specificity product) that can arrive 140bp; Get PCR and identify that two correct appearance are sent order-checking; The result shows equal successful connection, promptly inserts κThe success of B-TATA box sequence obtains control vector this moment κB-luc; 5. get the successful sample of above-mentioned order-checking κB-luc extracts plasmid, and double design contains the complementary primer of STAT3 binding sequence; With above-mentioned same mode respectively from Kpn I and Sac I; And insert the STAT3 binding sequence in Nhe I and two pairs of restriction enzyme sites of Xho I, during respectively the sample presentation order-checking identify, obtain complete SIE- κThe whole insertion sequence of B-TATA box, whole carrier called after SIE- κB-luc.6. another control vector SIE-luc has then dispensed NF-when inserting TATA box sequence κB binding sequence 5 '-GGGAATTTCC-3 ', all the other steps are with making up SIE- κThe process of B-luc.All carriers are final to be identified through order-checking, shows that insertion sequence is correct.So far, SIE- κThe complete insertion sequence of B-luc on the luciferase reporting carrier be (sequencing result is referring to Fig. 6) 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 outside when cytokine stimulates and detect the activation situation of corresponding signal path, thus when using this cell to detect two types of binding sequences and their combinations respectively as representative to signal activatory response condition (Fig. 7).Two types of binding sequences responded normally when the result was illustrated in the activation of corresponding signal path, wherein NF- κB uses TNF α to stimulate, and STAT3 uses IL-6 to activate.The 293T cell is grown to 50%-70% density in 12 orifice plates, the corresponding carrier of transfection κB-luc, SIE- κB-luc, SIE-luc (each 2 μ g/ hole), the usage ratio of carrier and transfection reagent is 1:2 (i.e. 2 μ g plasmids, 4 μ l transfection reagents; Respectively be dissolved in 50 μ l DMEM serum free mediums, be mixed into 100 μ l premixed liquids, room temperature leaves standstill 20 min; Add 400 μ l serum-free DMEM again, add 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; Use IL-6 (250 ng/ml) respectively, TNF α (50 ng/ml) handled 24 hours, inhaled and removed substratum; 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 (* * *, p<0.001).

3, the medicaments sifting model cell is selected

Prevent STAT3 and NF-in the experimental analysis cell through Western-Blot and EMSA gel κThe activity of B obtains the multiple NF-that is suitable for this report system carrier κB and STAT3 activity are all than the tumor cell line (Fig. 1 of higher (need not cytokine irritation cells such as extra use TNF α and IL-6); Fig. 2; Fig. 3), Fig. 1 detects STAT3 and NF-in the cell involved in the present invention for using the Western-Blot method κThe active result of B.The result shows that a series of tumor cell ratio normal cells that comprise A549 have higher STAT3 and NF- κB is active, and the phosphorylation sign with the 705th tyrosine of STAT3 (Tyr705) and the 536th Serine of p65 (Ser536) is labeled as " pTyr705-STAT3 " and " pSer536-p65 ", and phosphorylation level is high more, and activity is high more; Fig. 2 and Fig. 3 prevent test (EMSA) to detect NF-in the cell involved in the present invention for using gel electrophoresis κThe active result of B and STAT3.Show used several kinds of tumour cell activatory STAT3 and NF-among Fig. 1 κB has higher DNA binding ability than normal cell HME1.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

Select wherein a kind of NF- κB and STAT3 composing type activatory cell A549, the above-mentioned reporting system carrier of transfection (grows to 50% ~ 70% density with the A549 cell inoculation in 12 orifice plates, the corresponding carrier of transfection κB-luc (has only 1 NF- κThe control vector of B binding sequence referring to the step 1 of present embodiment, only characterizes NF- κThe B activity), SIE- κ(the reporting system carrier characterizes NF-to B-luc simultaneously κB and STAT3 are active), (control vector of having only the STAT3 binding sequence comprises 16 SIE sequences, referring to the step 1 of present embodiment to SIE-luc; Only characterize the STAT3 activity), each 2 μ g/ hole, 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 400 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 36 hours, inhale and remove substratum, add 200 μ l lysate lysing cell; Jog 10min gets 150 μ l in 96 orifice plates, adds the common Photinus pyralis LUC substrate of 20 μ l); Detect the actual fluorescence efficiency of SIE-μ B-luc, the result is illustrated in the A549 cell, SIE- κThe fluorescence efficiency of B-luc be not SIE-luc with κThe simple superposition of B-luc fluorescence (utilizing A549 cell detection reporting system carrier to start the transcriptional capability situation referring to Fig. 8) is found ability both stacks much larger than the back of reporting system carrier characterization signal: SIE- κB-luc is κAbout 14 times of B-luc, be about 6 times of SIE-luc, on theoretical value, in case suppress active about 83% (inhibition NF-that one of them signal path will reduce luciferase at least κBe 1-1/14 ≈ 93% behind the B, suppressing STAT3 is 1-1/6 ≈ 83%), after making up steady commentaries on classics cell strain, suppress separately can both significantly to indicate behind each signal path like this; Provide the A549 cell of similar number in the prerequisite test to grow to 50%-70% density in 12 orifice plates, the corresponding plasmid of transfection (2 μ g/ hole) is after 36 hours; Directly measure the activity of luciferase; Contrast and each 3 repetition of processing, and independent revision test 3 (* * *, p<0.001).

Use the stable cell model of A549 cell construction, and detect performance.When the A549 cell grows to 30%-40% density in 10 cm petridish, with SIE- κ(8 μ g carriers, 16 μ l transfection reagents respectively are dissolved in 500 μ l serum-free DMEM substratum to B-luc transfection A549 cell; And make the 1ml premixed liquid, add in the petridish behind the adding 3ml serum-free DMEM mixing behind 20 min), after 48 hours; With pancreatin cell dissociation and 1 is passed 3, add tetracycline simultaneously and carry out the positive colony screening, the starting point concentration of tetracycline is 5 μ g/ml; Reduce to 2.5 μ g/ml after 2 weeks; Behind the clone to be formed (about 3 weeks altogether), in 96 orifice plates, change 48 orifice plates after having grown again over to the trysinization mono-clonal; 6 orifice plates are until 10 cm petridish and frozen, during use final concentration be 2.5 μ g/ml tetracyclines continue kept for 2 weeks.First-selection is chosen 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, use STAT3 or NF-owing to be constitutive activation κThe suppressor factor of B signal path can both make the activity of luciferase reduce, and finally obtains the cell that reacts more satisfactory through screening, chooses wherein two strain SKA and SKK (SIE- κB A and SIE- κB B), result such as Fig. 9 and Figure 10, Fig. 9 are that two kinds of screening anti-tumor medicine cell models surely change cell strain SKA and SKK to STAT3 or NF- κThe active response that raises of B.Show that these two the steady cell strains that change are to STAT3 and NF- κThe further activation of the signal of B still can be reacted.Cell inoculation renews nutrient solution, and contains IL-6 (250 ng/ml) or TNF α (50 ng/ml) in 96 orifice plates (10,000/hole) growth 12 hours, continues to cultivate the activity of surveying luciferase after 24 hours (n=5 wherein; * *, p 0.001).Figure 10 is that two kinds of screening anti-tumor medicine cell models surely change cell strain SKA and SKK to STAT3 or NF- κThe active response that reduces of B.The suppressor factor TPCA-1 and the PD180970 that are selected for use are respectively known NF- κThe suppressor factor of B and STAT3 signal path, TPCA-1 acts on NF- κB upper reaches IKK2 finally reduces NF- κThe B activation, and PD180970 is the suppressor factor of Tyrosylprotein kinase Src, the STAT3 of bibliographical information A549 cell continuous activation is responsive to PD180970; TPCA-1 that is selected for use (1 μ M) and PD180970 (250 nM) concentration do not have growth-inhibiting (24 hours) through the MTT experiment confirm to the A549 cell.Through checking; Two kinds of known suppressor factor have all significantly suppressed the activity of luciferase under the concentration that does not influence the cell growth, show as goodly especially with SKA, handle through TPCA-1; Uciferase activity reduces 62.4%, handles uciferase activity through PD180970 and reduces 52.5%.Cell inoculation is in 96 orifice plates (10; 000/hole; 100 μ l) growth is 12 hours, changes the new nutrient solution that contains above-mentioned cytokine or suppressor factor, continues 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 from national micromolecular compound resource center; Choose 502 kinds of isolating monomeric compounds from sources such as natural phant; Based on above-mentioned SKK cell model, in 96 orifice plates, being cultured to cell degree of converging is 30% ~ 40% with 100 ml culture medium inoculateds; Use the compound (2 μ g compounds are used for 3 multiple holes, every hole 0.33 μ l) of 6.7 μ g/ml to handle cell, continue to cultivate 24 hours; 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; The completion primary dcreening operation obtains the maximum inhibition of 25 kinds of luciferases at effective inhibition compound more than 40%; Do repetition to these 25 kinds of compounds; Concentration is the same; Simultaneously do a cell growth inhibition test (MTT) eliminating the error that the direct toxicity of compound causes, and reject and repeat inconsistent compound for twice, finally obtain the fluorescence inhibiting rate and still effectively sieving 4 kinds of compounds again more than 40% with the concentration of equivalent.Through consulting document; Wherein the compound R adicicol of a kind of b22 of being numbered is the specific inhibitor (people such as Kwon: Biosci Biotechnol Biochem 1992) of STAT3 upper reaches Src molecule in the known A549 cell; Be numbered the compound R ottlerin of b122, report NF- κThe B signal has restraining effect (people such as Torricelli: Life Sci 2008), suppress effect referring to seeing Figure 11; Other two kinds of h31 and g13 can further investigate; Use wherein a kind of h31 that DU145 cell (having the active prostate cancer cell of composing type STAT3) is handled (10 μ M) and be respectively 2h; 6h; 24h, Western-Blot result find that this product has inhibition significantly (Figure 12 characterizes with STAT3 Tyr705 phosphorylation level) to the STAT3 activity among the prostate cancer cell DU145.

Embodiment 2

1, reporting system vector construction

1. with the SIE-among the embodiment 1 κThe amount test kit extracted during the B-luc carrier was carried with a day root high purity plasmid for a short time, got 10 μ g and did double digestion, 37 ℃ with Xho I and Bgl II (each 3 μ l); 8 h, agarose gel electrophoresis uses a day root DNA purifying and recovering test kit to reclaim; Obtain the product I, the product of this moment has excised κThe B sequence; With the complementary primer of two band toughness terminal (and can match with Xho I and Bgl II restriction enzyme site), SIE and two sequences that to have comprised two sequences be 5 '-TTCCCGTAA-3 ' are 5 '-GGGAATTTCC-3's ' κB sequence, line as follows)

5 '-TCGAGC GGAAATTCCCGTAAAC GGAAATTCCCGTAAAA-3 ', and

5’-GATCTT TTACGGGAATTTCCGT TTACGGGAATTTCCGC-3’

Be diluted to 50 μ M solution, after respectively getting 15 μ l and mixing, 5 min in 95 ℃ of boiling water baths, naturally cooling in the water-bath obtains the product II; 2. with product I and II with amount of substance than 1:3, use the T4 dna ligase to connect, 16 ℃ of connections are spent the night; 3. transformed into escherichia coli competence DH5 α is applied to the agarose culture plate that contains amicillin resistance, and 37 ℃ are spent the night; 4. 2 of pickings are cloned in the LB liquid nutrient medium that contains penbritin and are cultured to logarithmic phase respectively, and the upstream and downstream primer PCR is identified the product (SIE-in the control Example 1 that can arrive 280bp κThe B-luc carrier then is 260bp), get PCR and identify that two correct appearance are sent order-checking, the result shows equal successful connection.So far, new reporting system vector construction is accomplished, and the complete insertion sequence on the luciferase reporting carrier is (sequencing result is referring to Figure 13) 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, and background NF- κB and STAT3 activity are all lower, the examining report carrier (SIE-of system when being convenient to add outside the cytokine stimulation κB)-and luc is to corresponding signal path activatory response situation (Figure 14), and the reporting system carrier responded normally when the result was illustrated in the activation of corresponding signal path, and NF- κThe fluorescent value that reporting system can reach during the activation simultaneously of B and STAT3 signal path is about 2 times of individual signals path activation sum, like this for to have composing type NF- κWhen making up sieve medicine cell model in the active cell of B and STAT3, suppress separately can both significantly indicate behind each signal path, prerequisite is provided.NF-wherein κB uses TNF μ to stimulate, and STAT3 uses IL-6 to activate.The 293T cell is grown to 50%-70% density in 24 orifice plates, transfection reporting system carrier (SIE- κB)-and luc (1 μ g/ hole), the usage ratio of carrier and transfection reagent is 1:2 (i.e. 1 μ g plasmid, 2 μ l transfection reagents; Respectively be dissolved in 50 μ l DMEM serum free mediums, be mixed into 100 μ l premixed liquids, room temperature leaves standstill 20 min; Add 150 μ l serum-free DMEM again; Add behind the mixing in each hole of 24 orifice plates) change perfect medium (the DMEM substratum that promptly contains 10% foetal calf serum) into after 4 hours, after 12 hours, use IL-6 (250 ng/ml) and/or TNF μ (50 ng/ml) processing 24 hours respectively; Substratum is removed in suction; Add 150 μ l lysate lysing cell, jog 10min gets 120 μ l in 96 orifice plates; Add the common Photinus pyralis LUC substrate of 20 μ l, (statistical is with embodiment 1 to use fluorescence/chemiluminescent analyzer to measure the activity of (in the 1min) luciferase immediately; *, p<0.05; *, p<0.01; * *, p<0.001).

3, the medicaments sifting model cell is selected

Prevent STAT3 and NF-in the experimental analysis cell through Western-Blot and EMSA gel κThe activity of B is with embodiment 1.

4, medicaments sifting model makes up and performance test

Select wherein a kind of NF- κB and STAT3 composing type activatory cell DU145 make up stable cell model, and detect performance.When the DU145 cell grows to 30%-40% density in 10 cm petridish, with reporting system carrier (SIE- κB)-(8 μ g carriers, 16 μ l transfection reagents respectively are dissolved in 500 μ l serum-free DMEM substratum to luc transfection DU145 cell; And make the 1ml premixed liquid, add in the petridish behind the adding 3ml serum-free DMEM mixing behind 20 min), after 48 hours; Add tetracycline and carry out positive-selecting; The concentration of tetracycline is 4 μ g/ml, uses trysinization and 1 to pass 1 after 3 days, and the continuation working concentration is that the tetracycline of 4 μ g/ml was kept 10 days; Obtain positive cell group and go down to posterity with frozen, obtain required cell model by normal cell.

Through detecting STAT3 or the NF-of cell model to adding κThe activator IL-6 or the TNF alpha reaction of B signal path are not obvious, but to known STAT3 or NF- κThe suppressor factor of B signal path all has tangible reaction (Figure 15), and that need during drug screening is STAT3 and/or NF- κThe suppressor factor of B signal path is so this model remains the ideal cell model.Figure 14 for this cell model after use suppressing suppressor factor to STAT3 or NF- κThe active response that reduces of B.The suppressor factor TPCA-1 and the PD180970 that are selected for use are respectively known NF- κThe suppressor factor of B and STAT3 signal path, TPCA-1 acts on NF- κB upper reaches IKK2 finally reduces NF- κThe B activation, and PD180970 is the suppressor factor of Tyrosylprotein kinase Src, suppresses Src in the bibliographical information DU145 cell and can partly suppress the STAT3 activity; TPCA-1 that is selected for use (1 μ M) and PD180970 (250 nM) concentration do not have growth-inhibiting (24 hours, or growth inhibition ratio is less than 5%) through the MTT experiment confirm to the DU1745 cell.Through checking, two kinds of known suppressor factor have all significantly suppressed the activity of luciferase under the concentration that does not influence the cell growth (fluorometric assay mode and statistical are with embodiment 1; *, p < 0.01; * *, p 0.001).

5, model is used

Screening mode is with embodiment 1; Use 502 kinds of isolating monomeric compound storehouses from sources such as natural phant, through obtaining 3 kinds of compounds behind the multiple sieve, (embodiment 1 proof h31 can suppress the STAT3 activity to comprise embodiment 1 said g13 and h31; Referring to Figure 12), and another kind of compound c 22.

Embodiment 3

1, reporting system vector construction

Cut method of attachment with embodiment 1 identical primer annealing and enzyme, the complete insertion sequence that on luciferase reporting carrier pBR322-luc-puro MCS, inserts is following:

2, reporting system carrier property test

With embodiment 2; But the fluorescent value that reporting system can reach when STAT3 was with NF-kB signal path while activation when using the 293T cell detection is only big by about 37% than individual signals path activation sum; And the fluorescent value that the STAT3 activation increases is merely the NF-kB activation increases about 10% of fluorescent value, and the fluorescent value that can reduce when using same suppressor factor to suppress the STAT3 signal path like this is obvious not as the reporting system carrier among the embodiment 1 and 2.

3, the medicaments sifting model cell is selected

With embodiment 2.

4, medicaments sifting model makes up and performance test

With embodiment 2.

5, model is used

Screening method uses 502 kinds of isolating monomeric compound storehouses from sources such as natural phant with embodiment 1, through being inhibited rate at 2 kinds of compounds more than 40% behind the multiple sieve, is c22 and g13.

Claims (10)

1. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path, it is characterized in that:

Adopt the combination of STAT3 specificity binding sequence and NF-κ B specificity binding sequence, set up the reporting system carrier; In conjunction with the active cells that has composing type STAT3 and NF-κ B simultaneously, be built into and stablize easy-to-use high-throughput screening anti-tumor medicine model.

2. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path according to claim 1, it is characterized in that: said NF-κ B specificity binding sequence is 5 '-GGGRNNYYCC-3 ';

Wherein R is a purine, and Y is a pyrimidine, and N is a base.

3. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path according to claim 1, it is characterized in that: the specificity binding sequence of said STAT3 is 5 '-TT (C/A) CNNNAA-3 ', and N is a base.

4. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path according to claim 1; It is characterized in that: said reporting system carrier is in containing the report carrier MCS of luciferase reporter gene, to insert to include STAT3 specificity binding sequence and the NF-κ B specificity binding sequence response sequence as the reporting system carrier, the reporting system carrier that is built into.

5. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path according to claim 1; It is characterized in that: in the said reporting system carrier; The mode of the combination of STAT3 specificity binding sequence and NF-κ B specificity binding sequence is: binding sequence comprises n STAT3 specificity binding sequence and m NF-κ B specificity binding sequence, and n:m=1:1 ~ 16:1.

6. be the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path according to claim 1, it is characterized in that: the said active cells that has composing type NF-κ B and STAT3 simultaneously 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.

7. be the construction process of the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path 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 STAT3 specificity binding sequence and 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 STAT3 and NF-kB activity cell simultaneously; Carry out the positive colony screening with tetracycline; Choose the clone who STAT3 and the agent of NF-κ B signal suppressing is had response, being based on STAT3 and NF-κ B dual signal path is the screening anti-tumor medicine cell model of target spot.

8. of claim 7 is the construction process of the screening anti-tumor medicine cell model of target spot based on STAT3 and NF-κ B dual signal path; It is characterized in that: said STAT3 specificity binding sequence and NF-κ B specificity binding sequence include n STAT3 specificity binding sequence and m NF-κ B specificity binding sequence, and n:m=1:1 ~ 16:1.

9. be that the screening anti-tumor medicine cell model of target spot is used to screen cell carcinogenesis, STAT3 and the NF-κ B that treatment STAT3 and/or NF-κ B continuous activation cause mutually combine, the mutually promote medicine of the cell carcinogenesis that causes based on STAT3 and NF-κ B dual signal path according to claim 1.

10. of claim 9 is that the screening anti-tumor medicine cell model of target spot is being used to screen cell carcinogenesis, STAT3 and the NF-κ B that treatment STAT3 and/or NF-κ B continuous activation cause mutually combine, the mutually promote medicine of the cell carcinogenesis that causes based on STAT3 and NF-κ B dual signal path, and it is characterized in that: the method for said screening antineoplastic drugs comprises following process step:

(1) with said screening anti-tumor medicine 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 STAT3 and/or NF-κ B signal path is the tumor suppression medicine of target spot.

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