CN105467119A - Method for predicting sensitivity of colorectal cancer cells to p38MAPK kinase inhibitor - Google Patents

Abstract

A method for predicting sensitivity of colorectal cancer cells to a p38MAPK kinase inhibitor comprises steps as follows: the PP2AC expression level of the colorectal cancer cells is detected and H-score is measured and calculated with an immunohistochemical or immunofluorescence technique; if the value of H-score is smaller than 100, the colorectal cancer cells are judged to be sensitive to the p38MAPK kinase inhibitor; if the value of H-score is larger than 200, the colorectal cancer cells are judged to be resistant to the p38MAPK kinase inhibitor. The PP2AC expression level of the colorectal cancer cells is detected and H-score is measured and calculated with the immunohistochemical or immunofluorescence technique of a general laboratory, whether the colorectal cancer cells are sensitive or resistant to the p38MAPK kinase inhibitor can be accurately judged according to the value of H-score, and the gap of a means for predicting the sensitivity of the colorectal cancer cells to the p38MAPK kinase inhibitor is filled.

Description

A kind of forecast colorectal cancer cell is to the method for p38MAPK inhibitors of kinases susceptibility

Technical field

The present invention relates to the sensitivity testing method of a kind of cell to certain drug, particularly relate to a kind of forecast colorectal cancer cell to the method for p38MAPK inhibitors of kinases susceptibility.

Background technology

Along with the raising of people's living standard and the change of dietary structure, colorectal cancer is at the incidence of disease of China in increasing trend, and the mechanism that development occurs due to colorectal cancer is still not clear, therefore lacks effectively preventing target spot and means.Especially Advanced Colorectal Cancer, its treatment means can only based on chemotherapy, curative effect and patient's prognosis all poor.Targeted therapy is on cellular and molecular level, corresponding medicine is designed for clear and definite carcinogenic site (protein molecular in tumour cell or genetic fragment), medicine enters can specifically in conjunction with carcinogenic site in body, make tumor cell specific dead, and do not involve normal tissue cell, represent the new direction of oncotherapy.

P38MAPK (p38 MAPK) phosphorylation comprises a large amount of substrate (MK2 of transcription factor and kinases, HSP27 etc.), play a significant role in the transcribing of cell, albumen synthesis, signal transduction and cell surface receptor expression etc., widely studied by the target spot as neoplasm targeted therapy, part p38MAPK inhibitors of kinases (p38in) enters early studies in man.Preclinical study shows: in the treatment of colorectal cancer, and p38in can improve the susceptibility of part colorectal cancer to 5 FU 5 fluorouracil, the resistance of reduction Irinotecan.But, also there is Partial tumors to p38in resistance.

Tumour be multifactor, multi-step, the gene mutation of the heterogeneity of cell, the complicacy of cell-signaling pathways and high probability in addition, prior art can not reach the object of precisely treatment.Therefore, the method for a kind of forecast colorectal cancer to p38in susceptibility is found to seem particularly important.Have not yet to see bibliographical information and have the measurable colorectal cancer of method to the susceptibility of p38in.

PP2A is the serine/threonine protein phosphatase extensively existed in most eukaryotes, the heterotrimer be made up of catalytic subunit C subunit (PP2AC), Structural subunits A subunit (PP2AA) and adjustment subunit B subunit (PP2AB).There is between PP2AC species the conservative property of height, made the protein dephosphorylation of phosphorylation activation by reversible phosphorylation, participate in numerous biological event in body.

Summary of the invention

The present invention be solve the aforementioned problems in the prior proposition a kind of forecast colorectal cancer cell to the method for p38MAPK inhibitors of kinases susceptibility, effectively filled up the technological gap of forecast colorectal cancer cell to the susceptibility means of p38in.

Applicant is surprised to find that by large quantifier elimination and experiment, and PP2AC is active by participating in regulation and control P38/ERK/TSC2/mTOR signal path, determines that colorectal cancer is to the susceptibility of p38in; The colorectal cancer cells of the low expression of PP2AC is responsive to p38in, and the colorectal cancer cells of PP2AC high expressed is to p38in resistance.Application SABC combined immunization fluorescent technique (IHC/IF) or detect the PP2AC expression of Colorectal Carcinoma individually, measurable colorectal cancer is responsive or resistance to p38in.

In order to solve the problems of the technologies described above, the technical measures that the present invention takes are:

Forecast colorectal cancer cell, to a method for p38MAPK inhibitors of kinases susceptibility, comprises the PP2AC expression of application SABC or immunofluorescence technique detection colorectal cancer cells, and calculates the step of H-score; Wherein, if H-score numerical value is less than 100, then judge that colorectal cancer cells is responsive to p38MAPK inhibitors of kinases, if H-score numerical value is greater than 200, then judge that colorectal cancer cells is to p38MAPK inhibitors of kinases resistance.

In order to optimize technique scheme, the technical measures that the present invention takes also comprise:

Preferably, SABC operation is specially:

Step one, section dewaxing is to water:

Dimethylbenzene I, II dewaxing 5-10min, fully sloughs paraffin; Graded ethanol takes off dimethylbenzene: washing, is then inserted in antigen repairing box by slice, thin piece, with distillation washing 2 times;

Step 2, deactivating endogenous peroxydase:

The section of step one gained being added 3% hydrogen peroxide makes liquid level covering organize 10min, with distillation washing 2 times, and each 3min;

Step 3, antigen retrieval:

The section of step 2 gained is placed in citrate buffer solution microwave heating reparation, first with distillation washing 2 times after naturally cooling to room temperature, then washes 3 times with PBS, each 5min;

Step 4, close with NIS, hatch:

Tissue moisture is around exhausted by step 3 gained section thieving paper as far as possible, around tissue, draws a circle with SABC pen, then add serum and cover tissue, put into wet box incubated at room 10-30min, bottom wet box, add a small amount of water;

Step 5, primary antibodie is hatched:

Discard serum, drip primary antibodie 4 DEG C of night incubation, after second day rewarming 45min, with PBS, section is washed 3 times, each 5min;

Step 6, two anti-hatch:

Drip two anti-incubated at room 30min, PBS and wash 3 each 5min;

Step 7, chromogenic reaction:

Add DAB colour developing 5-10min, immerse pure water color development stopping;

Step 8, redye:

Haematoxylin dyeing, then uses water to rinse, and 1% hydrochloride alcohol differentiated tissue color reddens to take out immediately and puts into water, water rinses and returns indigo plant, gradient alcohol dehydration, each two minutes of I, II dimethylbenzene dealcoholization, dry or dry up, dripping a small amount of neutral resins and get a cover glass and carry out mounting;

Step 9, H-score calculates.

Preferably, immunofluorescence operation is specially:

Step one, paraffin section cut by embedded sample microtome, dries sheet 2 hours through 60 DEG C, and dimethylbenzene dewaxes, graded ethanol rehydration;

Step 2, antigen retrieval, uses and repairs liquid, microwave steam 2 minutes, and 98 degree of water-baths 25 minutes, cool 25 minutes;

Step 3, PBS washes 3 times, each 5min;

Step 4, uses 10% sheep blood serum to close 60min;

Step 5, primary antibodie 4 DEG C of overnight incubation;

Step 6, two resist 25 DEG C hatches 1h;

Step 7, mounting and detection, drip the mountant mounting that includes DAPI;

Step 8, H-score calculates.

Method of the present invention can also be combine to utilize SABC and immunofluorescence technique forecast colorectal cancer cell to p38MAPK inhibitors of kinases susceptibility, and namely the H-score numerical value of binding immunoassay group and immunofluorescence judges.Wherein, H-score numerical value can be determined by conventional algorithm, also can pass through Aperio systems (Vista, CA, USA) calculates.

The present invention adopts technique scheme, compared with prior art, there is following technique effect: applicant has found that a kind of Accurate Prediction colorectal cancer cells is to the method for p38MAPK inhibitors of kinases susceptibility in a creative way, the SABC all possessed by common laboratory or immunofluorescence technique, detect the PP2AC expression of colorectal cancer cells and calculate H-score, by H-score numerical value, can judge that colorectal cancer cells is to p38MAPK inhibitors of kinases sensitivity or resistance exactly, Forecasting Methodology is simple and convenient, fill up the blank of colorectal cancer cells to p38MAPK inhibitors of kinases sensitivity prediction means.

Accompanying drawing explanation

Fig. 1 is Westernblot experimental result in embodiment one.PP2AA, PP2AB and PP2AC of showing are in the expression situation of different colorectal carcinoma cell line.

Fig. 2 is SRB experimental result in embodiment two.The proliferation of colorectal cancer cells showing different PP2AC expression is different to the susceptibility of p38in.Upper figure is the impact of SB202190 on Cell Growth in Human Colorectal Cancer; Figure below is the impact of LY2228820 and BIRB796 on Cell Growth in Human Colorectal Cancer.

Fig. 3 is embodiment three middle plateform colony formation result.The colorectal cancer cells clonality showing different PP2AC expression is different to the susceptibility of p38in.Left figure is the impact of SB202190 on colorectal cancer cells Clone formation.Histogram is that plate clone forms the quantitative of experimental result.

Fig. 4 is embodiment four kinds of soft-fractrue rock mass experimental results.The colorectal cancer cells soft-fractrue rock mass ability showing different PP2AC expression is different to the susceptibility of p38in.Left figure is the impact of SB202190 on colorectal cancer cells soft-fractrue rock mass ability.Histogram is the quantitative of soft-fractrue rock mass experimental result.

Fig. 5 is colorectal cancer cells transplanted tumor in nude mice drug sensitive test result in embodiment five.The colorectal cancer cells transplanted tumor in nude mice showing different PP2AC expression is different to the susceptibility of p38in.The low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in resistance.

Fig. 6 is SABC testing result in embodiment six.Colorectal cancer cells nude mice model tumor tissue P-S6, Ki67 of showing different PP2AC expression are different to the susceptibility of p38in.

Fig. 7 is the Westernblot testing result of embodiment seven.Show the on-targeteffect of p38in at colorectal cancer cells transplantable tumor tissue.VC: control group; SB20:SB202190 processed group.

Fig. 8 is the result of the application SABC detection organization chip of embodiment eight.Show PP2AC and there is comparatively Big mutation rate at the expression of human large intestine cancer.Left figure is the colorectal cancer photo of different PP2AC expression, and brown is PP2AC albumen; Right figure is PP2AC expression quantitative (H-score).

Fig. 9 is the result (HE dyeing) of the 40 routine PDX model pathology detection that embodiment nine is set up.

Figure 10 is the result that embodiment ten is applied SABC combined immunization fluorescent technique (IHC/IF) and detected.

Figure 11 applies the primary Transplanted tumor model of human large intestine cancer to carry out drug sensitive test in embodiment 11, detect the PDX of different PP2AC expression to the result of the susceptibility of p38in (SB202190).VC: control group; SB20:SB202190 processed group.Data represent means standard deviation.Scatter diagram is that T/C value is quantitative, PP2AC low expression group vs.PP2AC high expressed group, P<0.0001.

Figure 12 applies SABC (IHC) to detect P-S6, P-MK2, Ki67 of PDX to the result of the susceptibility of p38in in embodiment 12.The low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in insensitive (or resistance).VC: control group; SB20:SB202190 processed group.

Figure 13 applies the primary Transplanted tumor model of human large intestine cancer to carry out drug sensitive test in embodiment 13, detect the PDX of different PP2AC expression to the result of the susceptibility of p38in (LY2228820).VC: control group; LY:LY2228820 processed group.Data represent means standard deviation.

Embodiment

The invention provides a kind of forecast colorectal cancer cell to the method for p38MAPK inhibitors of kinases susceptibility, comprise the PP2AC expression of application SABC or immunofluorescence technique detection colorectal cancer cells, and calculate the step of H-score; Wherein, if H-score numerical value is less than 100, then judge that colorectal cancer cells is responsive to p38MAPK inhibitors of kinases, if H-score numerical value is greater than 200, then judge that colorectal cancer cells is to p38MAPK inhibitors of kinases resistance.

Carry out detailed and concrete introduction below by specific embodiment to the present invention, to make better to understand the present invention, but following embodiment does not limit the scope of the invention.

In each embodiment following of the present invention, Human Large Intestine Carcinoma Cells system CACO-2, HCT116, RKO, SW1116, SW480 and SW620 are all purchased from ATCC, RPMI1640 nutrient culture media purchased from American Sigma company; Animal used as test is BALB/c nude mice, male, 4 week age, inbred strais, purchased from zoopery base, Chinese Academy of Sciences Songjiang (SCXK (Shanghai) 2003-0003), raise in Medical College, Shanghai Communication Univ. IVC Animal Lab. (SYXK (Shanghai) 2003-0007).

Embodiment one. Western blot colorectal cancer cells PP2AC protein level

Antibody used (CellSignalingTechnology): PP2AA (#2041), PP2AB (#2290), PP2AC (#2059), Tubulin (#3873).Concrete operations are as follows:

1. prepare HEPES electrophoretic buffer: HEPES23.8g, Tris12.1g, SDS1.0g, add deionized water dissolving, is settled to 1000ml.

2. prepare Tris-glycocoll electrotransfer damping fluid (PH8.3): Tris3.03g, glycocoll 14.4g, methyl alcohol 200ml, adds deionized water dissolving, is settled to 1000ml.

3. prepare confining liquid: calf serum and TBS solution (Tris12.1g, NaCl9g, deionized water is settled to 1000ml, regulates pH to 7.5) are made into the confining liquid containing 10% calf serum with 1: 9 ratio.

4. colorectal carcinoma cell line CACO-2, HCT116, RKO, SW1116, SW480 and SW620 are with RIPA cracking process extracting total protein of cell.Mixed with 5 × loadingbuffer by protein sample quantitatively, in 100 DEG C of heat denatured 3-5min, simple centrifugal rear order adds (10%SDS-PAGE) in well.With 150V electrophoresis until bromophenol blue arrives bottom separation gel.

5. take out gel, wherein one piece for coomassie brilliant blue staining to observe application of sample amount and the electrophoresis situation of sample, another block for transferring film, balances 10-15min in glycocoll electrotransfer damping fluid.Cut out size and gel phase with 6 filter paper and 1 pvdf membrane.Pvdf membrane leaching, with methyl alcohol pre-service 15s, is soaked in Tris-glycocoll electrotransfer damping fluid jointly with filter paper.The one side of gel contacted with filter membrane, be sandwiched between 6 filter paper, be neatly stacked in electrotransfer device, pvdf membrane is near anode side.Add Tris-glycocoll electrotransfer damping fluid, on ice with the strength of current Constant Electric Current transferase 12 ~ 3h of 0.65mA/cm2.

6.PVDF film in confining liquid 4 DEG C spend the night.Discard confining liquid, TTBS (999mlTBS, Tween-201ml) vibration washes film 5 minutes, incubates primary antibodie (incubated at room 2-3h.TTBS concussion washes film 3 times, each 15min.The two anti-incubated at room 1-2h marked with horseradish peroxidase (HRP).TTBS concussion washes film 3 times, each 15min.

7. carry out chemiluminescence with ECL kit, by A, B luminescent solution equal proportion dilution mixture in darkroom.Film deionized water rinsing a little, filter paper pastes angle and blots, and anti-subsides method is overlying on A, B mixed liquor, is placed in preservative film internal fixtion in film magazine, covers film rapidly, closes glue box, exposes according to finding fluorescence intensity.Demarcate Marker, analyze.

As shown in Figure 1, in Human Large Intestine Carcinoma Cells system, PP2AC expression has notable difference, and PP2AA and PP2AB expression difference is not obvious.In CACO-2, RKO and SW480, the low expression of PP2AC; In HCT116, SW1116 and SW620, PP2AC high expressed.

Embodiment two. Sulforhodamine B (SulforhodamineB, SRB) colourimetry

Get one bottle, the cell just growing up to intact monolayer, collecting cell after Trypsin Induced, even with transfer pipet piping and druming, get two cell suspension Trypan Blues, in counted under microscope number of viable cells (dead cell number must not more than 5%), with complete culture solution adjustment cell number to 1 × 10 ⁵individual cells/ml.

In 96 porocyte culture plates, every hole adds 100 μ l cell suspensions, culture plate is placed in CO2 incubator to cultivate 24 hours, in every hole, the p38in (SB202190 of variable concentrations is added after taking out culture plate, LY2228820, BIRB796) complete culture solution, each concentration establishes 5 parallel holes, separately establishes the capable cell of A to add not pastille complete culture solution and does normal control hole (C).

After dosing completes, nutrient solution in sucking-off blank well, deionization washes 5 times.Culture plate vibrates mixing on microwell plate oscillator, is placed in CO2 incubator and continues to cultivate 24-72 hour.

Taking-up culture plate, the trichloroacetic acid (TCA) adding 50% of 50 μ l precoolings in every hole, nutrient solution surface is gently fixed, and leaves standstill after 5 minutes, then culture plate is moved to 4 DEG C of placements 1 hour.

Outwell immobile liquid, every hole deionized water washes 5 times, dries, air oxygen detrition.Every hole adds 100 μ lSRB liquid, places 10 minutes in room temperature, does not wash 5 times with 1% acetic acid, air oxygen detrition with the SRB of protein bound.In conjunction with SRB with 150 μ l10mmol/L non-buffered Tris alkali lye (pH10.5) vibration dissolve.

Measure the light absorption of each hole in BIO-TEK microplate reader, mensuration wavelength is 540nm.The impact of p38in on cell proliferation is calculated according to each hole OD value.Often organize repetition measurement 5 hole, average after revision test three times.

As shown in Figure 2, p38in (SB202190, LY2228820, BIRB796) growth (CACO-2 of the low expression colorectal cancer cells of PP2AC can obviously be suppressed, RKO, SW480), to the growth unrestraint effect of PP2AC high expressed colorectal cancer cells, or present facilitation (HCT116, SW1116 and SW620) on the contrary.Namely the low expression colorectal cancer cells of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer cells is to p38in insensitive (or resistance).

Embodiment three. plate clone forms experiment

Complete culture solution dilution single cell suspension, is seeded to 12 porocyte culture plates with 200/hole, after SB202190 process, cultivates 1-2 week, with the dyeing of subunit indigo plant, observes Clone formation and counts.Often group establishes 3 multiple holes, averages after revision test three times.

As shown in Figure 3, p38in significantly can suppress the clonality (CACO-2 of the low expression colorectal cancer cells of PP2AC, RKO, SW480), to the Clone formation unrestraint effect of PP2AC high expressed colorectal cancer cells, or present facilitation (HCT116, SW1116 and SW620) on the contrary.Namely the low expression colorectal cancer cells of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer cells is to p38in insensitive (or resistance).Histogram is that plate clone forms the quantitative of experimental result.Data represent means standard deviation.

Embodiment four. soft-fractrue rock mass is tested

To take the logarithm cell in growth period, blow and beat gently with 0.25% Trypsin Induced, make it to become unicellular, make viable count, with RPMI1640 nutrient solution adjustment cell density to the 1 × 106 cell/L containing 20% hyclone.Experimentally require to make gradient multiple dilutions.Prepare the LMP agar liquid glucose of 1.4% and 0.7% two concentration with distilled water respectively, after autoclaving, maintain in 40 DEG C and do not solidify.In 1:1 ratio mixing 1.4% agarose and 2 × RPMI1640 nutrient culture media (containing 2 × microbiotic and 20% calf serum), inject culture plate, cooled and solidified, puts in CO2 incubator for subsequent use as bottom-layer agar.In agarose and 2 × RPMI1640 nutrient culture media of 1:1 ratio mixing 0.7%, then add the cell suspension of 0.2mL, fully mix, inject and be covered with 1.4% agarose bottom culture plate, form two agar layer.After top-layer agar solidifies, insert in 37 DEG C of 5%CO2 incubators and cultivate 10 ~ 14 days.SB202190 is dissolved with DMSO, and is diluted to desired concn with RPMI1640 nutrient culture media, be added on agar surface, change liquid every other day.Under plate is placed on inverted microscope, observation of cell clone number, calculates cloning efficiency.

As shown in Figure 4, p38in obviously can suppress the soft-fractrue rock mass ability (CACO-2 of the low expression colorectal cancer cells of PP2AC, RKO, SW480), to the soft-fractrue rock mass unrestraint effect of PP2AC high expressed colorectal cancer cells, or present facilitation (HCT116, SW1116 and SW620) on the contrary.Namely the low expression colorectal cancer cells of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer cells is to p38in insensitive (or resistance).Histogram is the quantitative of soft-fractrue rock mass experimental result.Data represent means standard deviation.

Embodiment five. the drug sensitive test of Human Large Intestine Carcinoma Cells Nude Mouse Model

1. cultivate Human Large Intestine Carcinoma Cells strain RKO, SW480 and HCT116, SW620, with 0.25% trypsin digestion and cell, collected by centrifugation, washes twice with PBS, suspends as single cell suspension, and density is 5 × 10 ⁶-1 × 10 ⁷cells/ml.Be inoculated in armpit on the left of nude mice with micro syringe subcutaneous, 100 μ l/ only.After inoculation, 1w forms macroscopic cancerous node, starts drug sensitive test.

2. drug treating:

SB202190 effect group: SB202190 is dissolved in DMSO+ physiological saline, 5mg/kg, 0.1ml/, once a day, lumbar injection;

Control group: DMSO is dissolved in physiological saline, 0.1ml/, once a day, lumbar injection;

3. what application vernier caliper was tested in administration measures longest diameter of tumor (a) and minimum diameter (b), according to formula on the 1st, 3,5,7,9,11 day calculate gross tumor volume.Data with represent (n=6).Experimental endpoints puts to death animal used as test, and draw materials, take pictures, liquid nitrogen cryopreservation, formalin is fixed.Carry out the subsequent analysis such as IHC.

As shown in Figure 5, p38in (SB202190) obviously can suppress the growth (RKO of PP2AC low expression colorectal cancer cells transplantable tumor, SW480), to the growth unrestraint effect of PP2AC high expressed colorectal cancer cells transplantable tumor, present facilitation (HCT116, SW620) on the contrary.Namely the low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in resistance.Data represent means standard deviation.* P<0.01vs. control group.

Embodiment six. P-S6, Ki67 that Immunohistochemical Method analyzes colorectal cancer cells transplantable tumor tissue express

The zoopery of embodiment five, to experimental endpoints, put to death animal used as test, tumor material obtaining, formalin is fixed, and further row SABC detects.Antibody used: P-S6 (S235/236) (#4858), Ki67 (#9449).Concrete operations are as follows:

1. section dewaxing is to water: dimethylbenzene I, II dewaxing 5-10min, fully sloughs paraffin; Graded ethanol takes off dimethylbenzene: 100% → 100% → 95% → 95% → 80% (each one minute); Wash from the beginning, then slice, thin piece is inserted in antigen repairing box, outwell for 2 times with distillation washing.

2. deactivating endogenous peroxydase: add 3% hydrogen peroxide and make liquid level covering organize 10min, with distillation washing 2 times, each 3min.

3. antigen retrieval: put microwave heating reparation in the 0.01M citrate buffer solution (pH6.0) filled it up with, high fiery 7min, low fiery 10min, first with distillation washing 2 times after naturally cooling to room temperature, then washes 3 times with PBS, each 5min.

4. close with NIS, hatch: section is taken out, with thieving paper, tissue moisture is around exhausted as far as possible, around tissue, a circle is drawn with SABC pen, then add serum and cover tissue, put into wet box incubated at room 10-30min, bottom wet box, add a small amount of water.

5. discard serum, drip primary antibodie 4 DEG C of night incubation, after second day rewarming 45min, with PBS, section is washed 3 times, each 5min.

6. drip two anti-incubated at room 30min, PBS and wash 3 each 5min.

7. chromogenic reaction, adds DAB colour developing 5-10min (controlling under microscope, aobvious brown), immerses tap water color development stopping

8. redye, haematoxylin dyeing (dye core), tap water, 1% hydrochloride alcohol differentiated tissue color reddens to take out immediately and puts into tap water, and tap water returns indigo plant, gradient alcohol dehydration: 95% I → 95% II (each 1min) → 100% I → 100% II (each 2min), I, each two minutes of II dimethylbenzene dealcoholization, dries or dries up, and drips a small amount of neutral resins and gets a cover glass and carry out mounting, finally labelled, analysis result under microscope.

P-S6 represents mTOR signal path activity (mTOR signal path increased activity will promote tumor cell proliferation), and Ki67 represents tumor cell proliferation ability.As shown in Figure 6, p38in (SB202190) obviously can suppress mTOR signal path activity and the cell proliferation (RKO of PP2AC low expression colorectal cancer cells transplantable tumor, SW480), active and the cell proliferation unrestraint effect to the mTOR signal path of PP2AC high expressed colorectal cancer cells transplantable tumor, present facilitation (HCT116, SW620) on the contrary.Namely the low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in resistance.

Embodiment seven. Western blot p38in is at the on-targeteffect of colorectal cancer cells transplantable tumor tissue

Antibody used: P-MK2 (T334) (#3007), MK2 (#3042), P-Hsp27 (S82) (#9709), Hsp27 (#2402).Concrete operations are with embodiment one.

MK2 and Hsp27 is the kinase whose substrate of p38MAPK.As shown in Figure 7, each sample source is individual in different transplantable tumor.VC: control group; SB20:SB202190 processed group.P38in is at responsive colorectal cancer cells transplantable tumor tissue (RKO, and resistance colorectal cancer cells transplantable tumor tissue (HCT116 SW480), SW620) suppress the phosphorylation of MK2 and Hsp27 albumen all completely, illustrate that p38MAPK signal path activity is suppressed completely by p38in; Colorectal cancer has nothing to do to the susceptibility of p38in and the kinase whose substrate phosphorylation level of p38MAPK.

Embodiment eight. application IHC and tissue array technology detect the expression of PP2AC at human large intestine cancer

Antibody used: PP2AC (#2059), dilutability 1:300.Collector's colorectal neoplasm operation sample 75 example, builds organization chip.Application IHC detects PP2AC expression, and concrete operations are with embodiment six.Pass through Aperio systems (Vista, CA, USA) calculates H-score.H-score is greater than 200, is PP2AC high expressed; H-score is less than 100, is the low expression of PP2AC.

As shown in Figure 8, PP2AC is significantly different at the expression of the colorectal cancer of Different Individual, and H-score is distributed as 0-300.Wherein 22 examples are PP2AC high expressed (H-score>200,29.33%), and 14 examples are the low expression of PP2AC (H-score<100,18.67%).(left figure is the colorectal cancer photo of different PP2AC expression, and brown is PP2AC albumen; Right figure is that PP2AC expression is quantitative).

Embodiment nine. the foundation of the primary Transplanted tumor model (Patientderivedxenograft, PDX) of human large intestine cancer

1. the collection of patient's tumor specimen: collect tumor tissues in tumour in vitro latter 30 minutes, Specimen origin is in biopsy and tumor radical cure specimens from pri.Fresh tumor tissues is soaked in the RPMI1640 of aseptic serum-free.

2. with sterilization tissue shear, tumor tissues is cut into 3 × 3 × 3mm, and cleans 3 times with above nutrient culture media.

3. the nude mice side incision of skin of anaesthetizing lower 5 week age is about 5mm, and isolates a packed space, organizes 2-3 block to plant in subcutaneous, use tissue adhesive's binding skin by what prune without downright bad tumour fritter.

4. detect tumor size weekly, when growth of transplanted human is to 1500mm ³(the 0th generation, P0), takes out transplantable tumor, with sterilization tissue shear, tumor tissues is cut into 3 × 3 × 3mm, and cleans 3 times with above nutrient culture media.Repeat above operation, carry out PDX amplification.

5.HE dyeing confirmation transplantable tumor pathological characters.

PDX is and clinical animal model for tumour the most close, is also the current the most approved animal model can simulating clinical tumor test in the world.As shown in Figure 9, successfully set up PDX model 40 example, confirm through HE dyeing, it remains the malignant phenotype of the primary colorectal cancer of people, and other pathological characteristicses, comprises atypia and the mesenchyma stroma of tumors of tumor tissues.

Embodiment ten. application SABC combined immunization fluorescent technique (IHC/IF) detects the PP2AC expression of PDX

Antibody used: primary antibodie PP2AC (#2059), dilutability 1:50.SABC operation is with embodiment six.Immunofluorescence concrete operations are as follows:

1. paraffin section cut by embedded sample microtome, dries sheet 2 hours through 60 DEG C, and dimethylbenzene dewaxes, graded ethanol rehydration.

2. antigen retrieval (98 degree of water-baths 25 minutes, cool 25 minutes for the reparation liquid of ph=6, microwave steam 2 minutes)

3.PBS washes 3 times, each 5min.

4. close.10% sheep blood serum is used to close 60min.

5. primary antibodie is hatched 4 DEG C and is spent the night.

6. two anti-hatch room temperature lucifuge 1h.

7. mounting and detection.Drip mountant one (include DAPI and redye nucleus), mounting, fluorescence microscopy is also taken pictures.

8. calculate H-score value.

As shown in Figure 10, it is sepia that SABC (IHC) detects PP2AC positive expression, and immunofluorescence (IF) detects PP2AC positive expression for red.According to H-score, application IHC/IF screens acquisition 9 routine PP2AC high expressed PDX (case numbering 2,3,8,11,19 in 40 routine PDX, 28,30,31,40) and 9 routine PP2AC low expression PDX (case numbering 1,12,13,14,17,25,26,33,34).Prediction case 1,12,13,14,17,25,26,33,34 pairs of mapk kinase inhibitor are responsive; Prediction case 2,3,8,11,19,28,30,31,40 pairs of mapk kinase inhibitor resistances.

Embodiment 11. the primary Transplanted tumor model (Patientderivedxenograft, PDX) of application human large intestine cancer carries out SB202190 drug sensitive test, verifies method of the present invention

1. the 9 routine PP2AC high expressed PDX (case numbering 2,3,8,11,19,28,30,31,40) and 9 routine PP2AC low expression PDX (case numbering 1,12,13,14,17,25,26,33,34) that increase carry out drug sensitive test to P2-P4.

2.PDX grows to 100-200mm ³time, be divided into 2 groups at random, often organize 6.SB202190 effect group: SB202190 is dissolved in DMSO+ physiological saline, 5mg/kg, 0.1ml/, once a day, lumbar injection; Control group: DMSO is dissolved in physiological saline, 0.1ml/, once a day, lumbar injection; 15-21 days altogether.

3. vernier caliper measures longest diameter of tumor (a) and minimum diameter (b), according to formula in every 3 days calculate gross tumor volume.To experimental endpoints, calculate T/C value.T/C value is that experimental group measures the difference (i.e. △ T) measuring gross tumor volume with first time for the last time, measures the difference (i.e. △ C) measuring gross tumor volume with first time than upper control group for the last time.T/C=0, complete growth inhibition; T/C<0 tumor regression; T/C=100, without growth inhibition; T/C>100 growth promoting effects.Experimenter is to patient's tumor specimen case number and corresponding PP2AC expression all unknown (blind).

As shown in figure 11, p38in (SB202190) obviously suppresses PP2AC low expression PDX growth, and makes part PP2AC low expression PDX tumor regression; P38in (SB202190) can not suppress PP2AC high expressed PDX to grow, and promotes part PP2AC high expressed PDX tumor growth on the contrary.Namely the low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in insensitive (or resistance), consistent with the result that the inventive method is predicted.VC: control group; SB20:SB202190 processed group.Data represent means standard deviation.Scatter diagram is that T/C value is quantitative, PP2AC low expression group vs.PP2AC high expressed group, P<0.0001.

Embodiment 12. application SABC (IHC) detects the P-S6 of PDX, P-MK2, Ki67 expression

Antibody used: Ki67 (#9449), P-MK2 (T334) (#3007), P-S6 (S235/236) (#4858).PDX drug sensitive experiment to terminal, put to death animal used as test, tumor material obtaining, fix by formalin, and further row SABC detects.SABC concrete operations are with embodiment six.

It is active that P-MK2 represents P38MAPK signal path, and P-S6 represents mTOR signal path activity (mTOR signal path increased activity will promote tumor cell proliferation), and Ki67 represents tumor cell proliferation ability.As shown in figure 12, p38in (SB202190) suppresses the P38MAPK signal path of the low expression of PP2AC PDX and PP2AC high expressed PDX active completely; P38in (SB202190) obviously can suppress mTOR signal path activity and the cell proliferation of PP2AC low expression PDX, the active and cell proliferation unrestraint effect to the mTOR signal path of PP2AC high expressed PDX, or presents facilitation.Namely the low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in insensitive (or resistance).

Embodiment 13. the primary Transplanted tumor model (Patientderivedxenograft, PDX) of application human large intestine cancer carries out LY2228820 drug sensitive test, verifies method of the present invention

The routine PP2AC of random amplification 2 low expression PDX (case numbering 13,33) and 2 routine PP2AC high expressed PDX (case numbering 3,40), to P2-P4, carries out LY2228820 drug sensitive test, verifies method of the present invention.Concrete operations are with embodiment 11.

LY2228820 is the p38MAPK inhibitors of kinases having entered early studies in man.As shown in figure 13, p38in (LY2228820) obviously suppresses PP2AC low expression PDX growth; P38in (LY2228820) can not suppress PP2AC high expressed PDX to grow (promoting tumor growth on the contrary in case 40).Namely the low expression colorectal cancer of PP2AC is responsive to p38in, and PP2AC high expressed colorectal cancer is to p38in insensitive (or resistance), consistent with the result that the inventive method is predicted.VC: control group; LY222:LY2228820 processed group, 10mg/kg/day.Data represent means standard deviation.

By the above embodiments and confirmatory experiment, illustrate Forecasting Methodology of the present invention can Accurate Prediction colorectal cancer cells to p38MAPK inhibitors of kinases susceptibility, the SABC all possessed by common laboratory or immunofluorescence technique, detect the PP2AC expression of colorectal cancer cells and calculate H-score, by H-score numerical value, can judge that colorectal cancer cells is to p38MAPK inhibitors of kinases sensitivity or resistance exactly, Forecasting Methodology is simple and convenient, has filled up the blank of colorectal cancer cells to p38MAPK inhibitors of kinases sensitivity prediction means.

Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.

Claims (3)

1. forecast colorectal cancer cell is to a method for p38MAPK inhibitors of kinases susceptibility, it is characterized in that, comprises the PP2AC expression of application SABC or immunofluorescence technique detection colorectal cancer cells, and calculates the step of H-score; Wherein, if H-score numerical value is less than 100, then judge that colorectal cancer cells is responsive to p38MAPK inhibitors of kinases, if H-score numerical value is greater than 200, then judge that colorectal cancer cells is to p38MAPK inhibitors of kinases resistance.

2. method according to claim 1, is characterized in that, SABC operation is specially:

Step one, section dewaxing is to water:

Dimethylbenzene I, II dewaxing 5-10min, fully sloughs paraffin; Graded ethanol takes off dimethylbenzene: washing, is then inserted in antigen repairing box by slice, thin piece, with distillation washing 2 times;

Step 2, deactivating endogenous peroxydase:

The section of step one gained being added 3% hydrogen peroxide makes liquid level covering organize 10min, with distillation washing 2 times, and each 3min;

Step 3, antigen retrieval:

The section of step 2 gained is placed in citrate buffer solution microwave heating reparation, first with distillation washing 2 times after naturally cooling to room temperature, then washes 3 times with PBS, each 5min;

Step 4, close with NIS, hatch:

Tissue moisture is around exhausted by step 3 gained section thieving paper as far as possible, around tissue, draws a circle with SABC pen, then add serum and cover tissue, put into wet box incubated at room 10-30min, bottom wet box, add a small amount of water;

Step 5, primary antibodie is hatched:

Discard serum, drip primary antibodie 4 DEG C of night incubation, after second day rewarming 45min, with PBS, section is washed 3 times, each 5min;

Step 6, two anti-hatch:

Drip two anti-incubated at room 30min, PBS and wash 3 each 5min;

Step 7, chromogenic reaction:

Add DAB colour developing 5-10min, immerse pure water color development stopping;

Step 8, redye:

Haematoxylin dyeing, then uses water to rinse, and 1% hydrochloride alcohol differentiated tissue color reddens to take out immediately and puts into water, water rinses and returns indigo plant, gradient alcohol dehydration, each two minutes of I, II dimethylbenzene dealcoholization, dry or dry up, dripping a small amount of neutral resins and get a cover glass and carry out mounting;

Step 9, H-score calculates.

3. method according to claim 1, is characterized in that, immunofluorescence operation is specially:

Step one, paraffin section cut by embedded sample microtome, dries sheet 2 hours through 60 DEG C, and dimethylbenzene dewaxes, graded ethanol rehydration;

Step 2, antigen retrieval, uses and repairs liquid, microwave steam 2 minutes, and 98 degree of water-baths 25 minutes, cool 25 minutes;

Step 3, PBS washes 3 times, each 5min;

Step 4, uses 10% sheep blood serum to close 60min;

Step 5, primary antibodie 4 DEG C of overnight incubation;

Step 6, two resist 25 DEG C hatches 1h;

Step 7, mounting and detection, drip the mountant mounting that includes DAPI;

Step 8, H-score calculates.