CN105534995B - Tyrosine kinase inhibitor is preparing the application in treating chronic myelocytic leukemia drug to compound Hu-17 alone or in combination - Google Patents

Tyrosine kinase inhibitor is preparing the application in treating chronic myelocytic leukemia drug to compound Hu-17 alone or in combination Download PDF

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CN105534995B
CN105534995B CN201610101491.8A CN201610101491A CN105534995B CN 105534995 B CN105534995 B CN 105534995B CN 201610101491 A CN201610101491 A CN 201610101491A CN 105534995 B CN105534995 B CN 105534995B
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tyrosine kinase
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myelocytic leukemia
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杜艳芝
刘建胜
易杨华
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Renji Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The present invention discloses compound Hu 17, and tyrosine kinase inhibitor is preparing the application in treating chronic myelocytic leukemia drug alone or in combination.New drug and joint tyrosine kinase inhibitor (Imatinib etc.) the invention discloses treatment for chronic myelocytic leukemia treat the new therapeutic scheme of chronic myelocytic leukemia.New selection is provided for the patient of imatinib-resistant, it is different from previous tyrosine kinase inhibitor drug action mechanism, the transcription and translation that can inhibit pathogenic fusion BCR ABL is used in combination with first-line treatment drug Imatinib for it, so as to fundamentally cure the disease.

Description

Tyrosine kinase inhibitor is chronic in preparation treatment alone or in combination by compound Hu-17 Application in granulocytic leukemia drug
Technical field
The invention belongs to field of pharmaceutical biology, more particularly, it relates to compound Hu-17 junket ammonia alone or in combination Acid kinase inhibitor medicaments are preparing the application in treating chronic myelocytic leukemia drug.
Background technology
Chronic myelocytic leukemia (Chronic Myeloid leukemia, CML) is a kind of by myeloid cell increasing extremely Hematologic disease caused by life, ten thousand people of annual morbidity 1.0-1.5/10, child morbidity is extremely low, and morbidity the median age is at 50-60 Sui Between.Clinical manifestation symptom is lethargic sleep, perspiration, anaemia, weight loss during morbidity, and abnormal bleeding and spleen increase (Perrotti,D.,Jamieson,C.,Goldman,J.&Skorski,T.J Clin Invest 120,2254-2264).By No. 9 and No. 22 chromosome translocation t (9；22)(q34；Q11) abnormal chromosome formed is first Philadelphia being found dyeing The main reason for body and chronic myelocytic leukemia cause a disease.The fusion that chromosome translocation is formed is named as BCR-ABL Gene, the BCR-ABL nonreceptor tyrosine kinases of sustained activation are pathogenesis of chronic myeloid leukemia and the main reason for deteriorate (Melo,J.V.&Barnes,D.J.Leuk Res 26,713-720.).Gleevec (Sti571) is first special target The micromolecular inhibitor of BCR-ABL tyrosine kinase, the inducing leukemia cell when it can specifically inhibit BCR-ABL kinase activities Apoptosis becomes chronic myelocytic leukemia first-line treatment drug, and the initial patient of more than 80% chronic myelocytic leukemia is through Gleevec Treatment can reach complete cytogenetics sustained release, curative effect very significantly (Deininger, M., Buchdunger, E.＆Druker, B.J.Blood 105,2640-2653.)。
Although evident in efficacy for treating chronic myelocytic leukemia chronic phase patient, medicine treatment accelerated period and urgency Patient's curative effect of change phase is not good enough, and sustained release rate is less than 40%.Drug resistance recurrence in drug treatment is the problem of often meeting, because It is presently found to Gleevec drug resistance that the mutation impairing in BCR-ABL kinase activities area Sti571 is combined with BCR-ABL kinases The main reason for.In addition, BCR-ABL gene magnifications cause fusion overexpression that can also generate Gleevec drug resistance.Because BCR- ABL kinases, which is undergone mutation, causes patient's generation drug resistance recurrence that scientific research personnel is promoted to research and develop the suppression of second generation BCR-ABL tyrosine kinase Preparation, i.e., the Dasatinib occurred later (Dasatinib) and nilotinib (Nilonitib).Tyrosine kinase suppression of new generation The Gleevec drug resistance patient of 35%-63% can be made to reach Major cytogenetic sustained release, but both medicines after preparation (TKI) treatment Object to tyrosine kinase activity area T315I mutation treatment without remarkable result (Soverini, S.et al.Clin Cancer Res 12,7374-7379.).There are the mutation of kinase activity area T315 amino acid sites, the positions in about 20% TKI drug resistance patients Point mutation has directly blocked TKI drugs to enter the ATP-binding site of BCR-ABL kinases, and the BCR-ABL kinases containing T315I assigns The drug-resistant effect high to the first generation and second generation tyrosine kinase inhibitor drug (N Engl J Med 367,2075- 2088.).Thereafter, scientific research personnel develops new tyrosine kinase inhibitor Ponatinib (Zhou, T.et al.Chem again Biol Drug Des 77,1-11.), Ponatinib is the small molecule junket designed using computing technique and architecture basics as strategy Histidine kinase inhibitor is avoided that space steric effect caused by T315 site mutations, effective to inhibit what is be mutated containing T315I BCR-ABL kinase activities, clinical data show that the medicine has good therapeutic effect to containing T315I mutation patients.
The characteristics of science of heredity unstability having due to chronic myelocytic leukemia, new mutation will also be further It was found that mutation more stronger than T315I is likely to be filtered out by new TKI (Dasatinib, Nilotinib, Ponatinib) Come.The reason for drug resistance recurrence of CML, can be divided into two classes：First, the mechanism that BCR-ABL kinases relies on, sky is generated by being mutated Between steric effect, weaken the combination of BCR-ABL kinases and tyrosine kinase inhibitor (TKI) and generate drug resistance, capture this kind of resistance to The drug resistance that medicine can be generated by transforming the structure of TKI come reversal mutations；Second is that the drug resistance of BCR-ABL kinase activities is not depended on, I.e. cell may be survived by other approach, and this kind of drug resistance is likely to occur (Sawyers, C.L.Nat in CML rapid change period Med 15,1158-1161.).In recent years it is found that BCR-ABL in CML leukemic stem cells (being called leukaemia initiator cell) Although kinase activity can be inhibited by tyrosine kinase inhibitors such as STi571, TKI drugs fail to successfully inducing leukemia Stem cell apoptosis, therefore BCR-ABL tyrosine kinase is only targeted in therapeutic process can not to remove chronic myelocytic leukemia white Blood disease stem cell (Corbin, A.S.et al.J Clin Invest 121,396-409.), this leaves to be recurred after TKI treatments Hidden danger, this kind of drug resistance belong to the typically resistance mechanism independent of BCR-ABL kinases.Research and develop new treatment chronic granulocyte The drug of leukaemia can particularly be used to treat the drug of drug resistance and prevention recurrence, still there is very big researching value and city Field potentiality.
Tyrosine kinase inhibitor (Imatinib/Sti571, nilotinib, Dasatinib) is to chronic myelocytic leukemia The effect unobvious of middle leukemic stem cells；Chronic myelocytic leukemia drug resistance patients with recurrent is to the weight of tyrosine kinase inhibitor Multiple therapeutic effect is bad；Although being treated using first generation second generation kinase inhibitor can have preferably without the progression of disease phase, But the effect of being satisfied with is not obtained yet to the therapeutic effect of rapid change period patient.It is currently available that the effect of tyrosine kinase inhibitor Mechanism is targeting BCR-ABL fusion proteins, inhibits the activation of the tyrosine kinase, inhibits pathogenic protein so as to fulfill special target The effect of BCR-ABL, but the content of overall BCR-ABL is had no effect on, the BCR-ABL tyrosine kinase in leukemic stem cells Activity inhibit can not inducing leukemia stem cell apoptosis, therefore be the defects of such inhibitor cannot fully erased peripheral blood And abnormal leucocyte in marrow.
The content of the invention
First of the present invention is designed to provide compound Hu-17 in treatment chronic myelocytic leukemia drug is prepared Application.
Second object of the present invention is that provide compound Hu-17 is preparing with tyrosine kinase inhibitor drug combination Treat the application in chronic myelocytic leukemia drug.
To realize above first purpose, the present invention discloses following technical scheme：Compound Hu-17 is chronic in preparation treatment Application in granulocytic leukemia drug.
To realize above second purpose, the present invention discloses following technical scheme：Compound Hu-17 presses down with tyrosine kinase Agents medication is preparing the application in treating chronic myelocytic leukemia drug.
As a preferred embodiment, the tyrosine kinase inhibitor refers to Imatinib (Imatinib), nilotinib (Dasatinib) and Dasatinib (Nilotinib).
The compound Hu-17 molecular formula：C63H96N4O11, chemical structural formula is as follows：
The advantage of the invention is that：The invention discloses the new drug for the treatment of for chronic myelocytic leukemia and joint tyrosine-kinases Enzyme inhibitor (Imatinib etc.) treats the new therapeutic scheme of chronic myelocytic leukemia.It is provided for the patient of imatinib-resistant New selection, different from previous tyrosine kinase inhibitor drug action mechanism, it joins with first-line treatment drug Imatinib It closes using the transcription and translation that can inhibit pathogenic fusion BCR-ABL, so as to fundamentally cure the disease.
Description of the drawings
Fig. 1 compounds Hu-17 can significantly inhibit K562 and drug resistant K562-R cell viabilities and induce it that apoptosis occurs.A) The compound Hu-17 of various concentration (0.5,0.6,0.7,0.8,0.9,1 μM) handle respectively K562 and K562-R cells 48 it is small when, MTT colorimetric determination cell viabilities.B) K562 and K562-R cells through Hu-17 processing 48 it is small when after, at mtt assay detection compound The block diagram of cell viability after reason.C after) K562 and K562-R cells are when (1,1.5,2 μM) processing 24 of various concentration Hu-17 is small The ratio of flow cytomery apoptotic cell.D) K562 cells are in (1,1.5, the 2 μM) processing 24 of compound Hu-17 various concentrations Hour or 48 it is small when, the ratio of flow cytomery apoptotic cell.E) K562 cells are after 1 μM of Hu-17 is handled, cytometer The total number of cells of number plate method statistical disposition different time points, calculates the cell quantity of different time points compared with initial repopulating cell Several percentage.F) K562 and K562-R cells through various concentration Hu-17 processing 24 it is small when, it is thin with Rhodamine 123 dye marker Born of the same parents, flow cytomery Mitochondrial transmembrane potential change.In triplicate, histogram data is with average for each group experiment independence ± standard deviation (mean ± SD) shows that comparison among groups uses sided t method of inspection, and wherein * represents P<0.05, * * represents P< 0.01, * * * represent P<0.001, ns represents no difference of science of statistics.
The compound Hu-17 of Fig. 2 low dosages can significantly inhibit the multiplication of K562 and K562-R cells, cause cell that G1 occurs Block with the G2/M phases.A) K562 and K562-R cells through 1 μM of Hu-17 handle 24 or 48 it is small when, punched using saponin and contaminated through PI Material mark cell, flow cytomery cell cycle.B cell cycle point after) K562-R cells are when Hu-17 processing 48 is small Butut.In triplicate, histogram data is shown each group experiment independence with average ± standard deviation (mean ± SD), comparison among groups Using sided t method of inspection, wherein, * represents P<0.05, * * represents P<0.01, * * * represent P<0.001.
Fig. 3 Hu-17 induce K562 and K562-R apoptosis and the content of the pathogenic fusion protein BCR-ABL reduced It is related.1.5 μM of Hu-17 handle respectively K562 cells 12,24 and 48 it is small when, protein immunoblot (Western Blot) method Detect the protein content of apoptosis marker PARP and pro apoptotic protein Bak.B) protein immunoblot detection BCR-ABL, phosphorylation BCR-ABL and BCR-ABL downstream targets Stat5 and cycle GAP-associated protein GAP P27 content.
Significantly induction K562 Apoptosis is used in combination with Imatinib (Imatinib) in Fig. 4 compounds Hu-17, and is turning The horizontal expression for inhibiting pathogenic fusion protein BCR-ABL of record.A) the K562 cells Hu-17 through various concentration (1,1.5 μM) respectively With (0.5 μM) of Sti571 processing 24 it is small when after, the ratio of flow cytomery apoptotic cell.B) Real-time PCR methods Detection Hu-17 is used alone (1 μM) or the variation of BCR-ABL mRNA level in-sites is used in combination with Imatinib (0.5 μM).C) K562 cells through 0.5 μM of Sti571 (Imatinib) and 1 μM of Hu-17 handle alone or in combination 20 it is small when, collect cell protein And detect the protein level of BCR-ABL total proteins and the BCR-ABL of phosphorylation.In figure, S Sti571；Each group experiment is only Vertical histogram data is shown with average ± standard deviation (mean ± SD) in triplicate, and comparison among groups is examined using sided t Method, * * * represent P<0.001.
The marrow of energy significantly inducing chronic myelocytic leukemia patients is used in combination with Imatinib by Fig. 5 compounds Hu-17 Or the generation apoptosis of the mononuclearcell of derived from peripheral blood.A) relevant information of the diagrammatic representation CML patient of Specimen origin with And sample is through Hu-17 treated apoptosis testing results.The mononuclearcell of Bone Marrow of Patients or derived from peripheral blood is through 3 μM of Hu-17 Handled alone or in combination with 6 μM of Sti571 (Imatinib) 48 it is small when, collect cell, flow cytometer detection Apoptosis.Wherein Con Represent the control group of DMSO processing, group is used in combination for Hu-17 and Sti571 in Combination.B) figure B show wherein an example is suffered from Person respectively through DMSO, Sti571, Hu-17 and during small two medicine Combined Treatments 48 after, the ratio of the apoptotic cell of streaming technology detection Example.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.Experimental method used in following embodiments for example without Specified otherwise is conventional method.The materials, reagents and the like used in the following examples, unless otherwise specified, can be from business way Footpath obtains.It is to be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
The preparation of 1. compound Hu-17 of embodiment
The chemical structural formula of compound Hu-17 is as follows：
Molecular formula：C63H96N4O11, molecular weight：1084Da.
Phytolaccagenin (phytolaccagenin) is a kind of active ingredient therefrom isolated in pharmacist land, is business The aglycon part of land saponin(e first (Esculentoside A, EsA), and the phytolaccagenin in the present invention passes through sour water solution Phytolacca acinosa Saponin(e first and obtain, structure is as follows：
Phytolaccagenin (26.6mg, 0.05mmol) is dissolved in dry 2ml DMF/THF (1:3, v/v) in, N is added in, N- Dicyclohexylcarbodiimides (DCC, 20.7mg, 0.1mmol) and N- hydroxy benzo triazoles (HOBt, 13.5mg, 0.1mmol), be stirred at room temperature 3 it is small when.After the completion of reaction, dry, residue dichloromethane (20ml) dissolving, mistake are concentrated under reduced pressure into Filter, filtrate are concentrated to dryness, and add in 23mg sulfuric acid aminoguanidine and 5ml dimethyl sulfoxides, fill N2After seal, oil bath (90~95 DEG C) magnetic force Be stirred to react 8 it is small when.After reaction solution cooling plus 2 times of distilled water, filtering, precipitation are washed with distilled water 2 times, 50 DEG C of drying to get HU-17 crude products.Crude product is again through silica gel column layer chromatographic purifying, eluant, eluent：Dichloromethane：Methanol (9.2：0.8) Hu-17 sterlings, are obtained 3.5mg, white powder, yield：19.4%.285~7 DEG C of mp；Molecular formula：C63H96N4O11；Molecular weight：1084；ESI-MS high Resolution Mass Spectrometry：m/z 1107.6879[M+Na]+,1083.6892[M-H]-；Infrared spectrum (IR)：vmax cm-13427(OH), 2948(CH3,CH2), 1729 (C=O), 1577,1462,1213,1150,1051 (C-O-C).
Tyrosine kinase inhibitor is preparing the treatment white blood of chronic granulocyte to 2. compound Hu-17 of embodiment alone or in combination Application in medicine
Experiment material and method：
1 experiment material
1.1 experiment cell lines and patient's sample cell
K562 cells K562 used is purchased from ATCC in experimentation, and Imatinib (Sti571) is resistance to Medicine cell cycling inhibiting-R is provided by Melo doctors JV.The peripheral blood or marrow in patients with chronic myelocytic leukemia source are through lymph Cell separating liquid obtains peripheral blood mononuclear cells (PBMC) or bone marrow mononuclear cells (BMMC) using density-gradient centrifugation method.
1.2 experiment main agents and material
1.3 major experimental instrument and equipments
2 experimental methods
The preparation of 2.1 drugs
The preparation of cell compound Hu-17：The Hu-17 of certain mass is weighed, normal temperature condition is with DMSO (Sigma) solvent Dissolving is made into the packing of 20mM mother liquors aliquot and is placed in -20 DEG C of preservations.
2.2 cell culture
K562 chronic myeloid leukemia cells are incubated at trains liquid and 10% hyclone containing 90%RPMI-1640 (FBS) in culture solution, K562-R (abbreviation KR) is incubated at containing 10% hyclone, 1% glutamine, penicillin (100U/ Ml), in the 1640 culture medium of streptomysin (100ug/ml), k562-R cells are tieed up in the drug culture solution containing 1 μM of Sti571 Hold culture.
Cell used in experiment is placed in cultivating in 37 DEG C of incubators containing 5% carbon dioxide.According to cell density and Growth rate situation, once, the passage in 2~3 days of K562-R cells is once for the passage in two days of K562 cells.
The detection (mtt assay) of 2.3 cell viabilities
Chronic myeloid leukemia cell viability examination：8 × 10 are inoculated in 48 porocyte culture plates4Cells/ holes it is slender 500 μ l of born of the same parents' suspension sequentially add the drug of various concentration, when dosing 48 is small after, per hole add in 50 μ l 5mg/ml tetrazolium bromide it is molten Liquid, after 37 DEG C of incubators are incubated 3h, 2000rpm is centrifuged 5 minutes and is absorbed supernatant discarding solution, adds in 500 μ l DMSO solutions, Fully dissolving purple crystal is rocked on decolorization swinging table, 570nm absorbing wavelengths detection sample light absorption value in microplate reader, at each drug Reason group sets three wells, and final result is obtained by independent experiment three times.
The phosphatidylserine of 2.4 Apoptosis turns up detection
The detection of chronic myeloid leukemia cell (K562, K562-R) agent-feeding treatment Apoptosis：K562 and K562-R Cell is collected by centrifugation cell in exponential phase and is plated on 12 orifice plates, contains 2 × 10 per hole5A cell adds in compound Hu- 17 or Gleevec (Sti571) processing 24 or 48 it is small when after collect cell, with 2000rmp centrifugal speeds centrifuge 5 minutes, centrifugation knot Supernatant is removed after beam, 500 μ l PBS buffer solution is added in and is resuspended once, 2000rmp is centrifuged 5 minutes, supernatant is abandoned after centrifugation, often Pipe adds 500 μ l 1 × AnnexinV binding buffer repeated centrifugations and goes supernatant step, and finally often pipe adds in 200 μ l The AnnexinV antibody of 1 × AnnexinV binding buffer, 5 μ l PI and 2.5 μ l FITC marks, room temperature after mixing Mark is protected from light 15 minutes, most after being detected on flow cytometer.Experiment at least detects 10000 cells every time, and each group experiment is equal Independently in triplicate.
The detection of 2.5 mitochondrial transmembrane potentials
In Apoptosis generating process, since mitochondrial inner membrane permeability changes, mitochondrial transmembrane potentials can drop It is low.It can be used to detect mitochondrial membrane potential using the PI dyestuffs of lipophilic anion dye, rhodamine 123 and detection cell survival Variation to study drug-induced Apoptosis.Dosing the previous day is inoculated with 2 × 10 in 12 orifice plates5A cell, agent-feeding treatment 24 it is small when after collect each group cell, 2000rpm centrifuge 5 minutes, abandon supernatant, with 500 μ l PBS buffer solution be resuspended cell, then with It centrifuges within 2000rpm5 minutes, supernatant is abandoned after centrifugation, often pipe 100 μ l PBS of addition and 100 μ l Rhodamine 123 dyestuffs, 37 DEG C Be protected from light incubation 30 minutes, 2000rpm is centrifuged and 5 minutes and is abandoned supernatant after incubation, with 500 μ l PBS cleaning once and repeat from Heart processing, finally often pipe adds in 300 μ l PBS, the PI of 3 μ l 1mg/ml, mixing and in being detected on flow cytometer, tests every time 10000 cells are at least detected, each group experiment independence is in triplicate.
The detection of 2.6 cell cycles
2 × 10 are inoculated in 12 orifice plates in dosing the previous day5A cell, when agent-feeding treatment 24 or 48 is small after collect cell, Often pipe collects 2 × 105A cell, 2000rpm are centrifuged 5 minutes, and supernatant is discarded after centrifugation, are resuspended with 1ml PBS buffer solution For single cell suspension, then with 2000rpm5 minutes, supernatant is abandoned, above step is repeated, washs cell 3 times with PBS buffer solution, finally 500 μ l are added in containing 0.5% saponin, the PBS solution (saponin is used for Cell-transmission model) of 0.1mg/ml Rnase A, 50 μ g/ml PI, Streaming pipe is placed in 37 DEG C of incubators after mixing and is protected from light mark 10-15 minutes, up flow type instrument detects after mark.Examination every time At least 10000 cells of detection are tested, each group experiment independence is in triplicate.
The detection of 2.7 protein levels
2.7.1 the cracking and extraction of total protein of cell
Collect cell (5-10 × 10 of the different time points of agent-feeding treatment6) in 15ml centrifuge tubes, 4 DEG C of 2000rpm from The heart 5 minutes removes supernatant, and cell is resuspended in the PBS for adding in 1ml precoolings, and single cell suspension is transferred in 1.5ml EP pipes, 2000rpm is centrifuged 5 minutes, after removing supernatant, is added in right amount containing proteasome inhibitor and phosphorglase inhibitor RIPA lysates, vortex mixing and on ice be incubated 30 minutes, during which interval be vortexed 2 times, 12000rpm4 DEG C centrifuge 10 minutes, Supernatant is drawn into new 1.5ml EP pipes.
2.7.2 protein concentration quantifies
Protein quantification uses DC protein assay kits, and protein concentration is measured according to Lowry methods principle.Specific step Suddenly：96 orifice plates sequentially add 2 μ l protein samples, and (A ' reagents are by 1mlA reagents in kit and 20 μ lS reagents for 25 μ l A ' reagents Mixing is matched somebody with somebody), 200 μ l B reagents, after being stored at room temperature 15 minutes, microplate reader absorbing wavelength is arranged to 750nm detections.Protein standard Curve is made into 0.1 respectively by BSA protein standard substances, 0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8,2.0mg/ml The protein solution of various concentration gradient measures, and protein sample does three wells with standard items to ensure that data stabilization is reliable respectively.
And protein immunoblot 2.7.3SDS-PAGE
Suitable 5 × albumen loading buffer are added in the protein sample after quantifying, vortex mixing is placed on It is boiled in 100 DEG C of boiling water 10 minutes, well-done sample can directly carry out SDS-PAGE electrophoresis or be placed in -80 DEG C to preserve.First The SDS-PAGE glue of different acrylamide concentrations is equipped with, after glue completely solidifies (can 4 DEG C preserve overnight), adds in proper volume Protein sample (30 μ g), in electrophoresis in BIO-RAD protein electrophoresis instruments, voltage is arranged to 100V, and electrophoresis time is arranged to 120 points Clock；Continuing transferring film (NC films) experiment after electrophoresis, electric current 200mA is set, the time is 120 minutes, after transferring film, egg White sample will be migrated to NC films by PAGE glue；After treating transferring film experiment, NC films are placed on filter paper and are air-dried, with containing 5% When the TBST solution room temperature of skim milk or 5%BSA closing 1 is small；After the completion of closing, 4 DEG C of primary antibody is incubated overnight；1×TBST It washes 5 times, every time 5 minutes；When the secondary antibody incubation at room temperature 1 marked with horseradish peroxidase (HRP) is small；After secondary antibody is incubated, 1 × TBST is washed 5 times, every time 5 minutes；It is developed the color with ECL plus western blotting reagent reagents in darkroom and exposes X-ray Piece, by the X-ray after exposure with highly dense scanner scanning into clear data picture.
2.8 real time fluorescent quantitative polymerase chain reactions (Real time-PCR)
RNA is extracted：After agent-feeding treatment cell certain time point, 1ml Trizol are added in 6 orifice plates, place 5 points on ice Clock, pipette tips are firmly blown and beaten；Lysate is collected into 1.5ml EP pipes, adds in 0.2ml chloroforms, whirlpool shakes 15 seconds.15~30 It is incubated at DEG C 2~3 minutes, 4 DEG C of 12,000g are centrifuged 15 minutes, and after centrifugation, (top layer is colourless water sample layer to three layers of liquid point Containing RNA, interlayer white is DNA, and bottom is red, is protein), careful supernatant liquid of drawing is added in into new EP pipes The isopropanol of equivalent volumes, mixing are incubated at 15~30 DEG C 10~30 minutes, and 4 DEG C of 12,000g are centrifuged 10 minutes.Suck supernatant, EP pipes are placed in super-clean bench 5~10 minutes dry.The water dissolution of 20 μ l RNase-free is added in, dissolved RNA puts -80 DEG C refrigerator preserves.With the RNA mass of agarose gel electrophoresis method Detection and Extraction, RNA concentration is detected with NanoDrop instruments.It uses QIAGEN RNeasy kit purify the RNA of extraction.
RNA is purified：2 μ g of RNA after purification are taken, add in 1 μ l oligo dT, add sigma water to 10 μ l, 72 DEG C of denaturation 5 minutes, 4 DEG C of rapid RNAse for cooling down, adding 5 μ l, 10mM dNTP of MLV RT buffer, 1.25 μ l, 40u/ μ l The 1 μ l of MLV enzymes of 0.625 μ l, 200u/ μ l of inhibitor, the RNase-free water of 7.125 μ l, when 42 DEG C of reverse transcriptions 1 are small.
Real time PCR：Reaction system includes 1 μM of forward primer 1 μ l, 1 μM of 1 μ l, SYBR Master of reverse primer 5 μ l, cDNA templates of Mix 2 μ l, 1 μ l H2O, overall reaction system are 10 μ l.After the completion of 384 orifice plates sample-adding, 4 DEG C of 2000rmp centrifugations 10 minutes.After centrifugation, PCR reactions are carried out in pressing machine on acquiescence response procedures on ABI 7900HT real-time PCRs.
The detected gene of experiment and primer sequence are as follows：
2.9 statistical analysis
Experimental data uses the GraphPad Prism5 softwares of specialty to map and makes corresponding results of statistical analysis, and two Comparison among groups has no difference of science of statistics between using student t test (sided t inspection) detection group, compares use between multigroup One-way analysis of variance, ns represents no difference of science of statistics in figure, and * represents P<0.05, * * represents P<0.01, * * * represent P< 0.001。
Experimental result：
Legend 1 illustrates：Chronic myeloid leukemia cell K562 and imatinib-resistant cell K562-R are through various concentration Compound Hu-17 processing 48 it is small when, influences of the mtt assay detection compound Hu-17 to cell viability finds that it can substantially inhibit The growth (Figure 1A and 1B) of two plants of cells；It is difference dense through compound Hu-17 that we have detected K562 and drug resistant K562-R cells It is significant to find that Hu-17 induction K562 and K562-R apoptosis has for the situation of Apoptosis after when degree processing 24 is small Dose dependent (Fig. 1 C)；We have found that Hu-17 significantly (schemes the apoptosis-induced effect of drug resistant K562-R cells than K562 1C)；We have detected apoptosis-induced effect to K562 cells after Hu-17 processing different time points, it is found that it lures K562 Leading apoptosis effect does not have significant difference (Fig. 1 D) when 24 is small and when 48 is small.By cell count, relatively low-dose (1 μM) is found Hu-17 can significantly inhibit cell Proliferation (Fig. 1 E).In addition, the testing result of Mitochondrial transmembrane potential is found, Hu-17 processing K562 And after K562-R, mitochondrial membrane potential is reduced, and prompts Hu-17 can be by mitochondria pathway inducing cell apoptosis (figure 1F).It these results suggest that, Hu-17 is respectively provided with K562 and K562-R cells the effect for inhibiting cell growth and multiplication, can pass through The notable inducing leukemia apoptosis of mitochondria pathway of apoptosis, wherein, Hu-17 makees drug resistant K562-R cell killings With becoming apparent from.
Legend 2 illustrates：The Hu-17 of low dosage can significantly induce K562 and K562-R cells generation G1 phases and G2/M phases to hinder It is stagnant, inhibit cell Proliferation.Phenomena of apoptosis caused by 1 μM of Hu-17 processing K562 and K562-R cell (is not schemed significantly 1C), but the inhibitory action of cell proliferation is fairly obvious (Figure 1A and 1E), and then we have detected 1 μM of Hu-17 processing After cell, the period profile figure of cell, it has been found that the apparent G1 phases have occurred in K562 and K562-R cells and the G2/M phases block (Fig. 2A and 2B), the cell proportion of S phases is substantially reduced after Hu-17 processing, and the cell proportion of G1 phases and G2/M phases dramatically increase, and carry The multiplication of leukaemia can be significantly inhibited by showing the Hu-17 of our low dosages, which matches with Fig. 1 results.
Legend 3 illustrates：Compound Hu-17 can significantly induce K562 apoptosis, apoptosis indication molecule (marker) The PARP (leaved PARP) of shearing-type substantially increases when 24 is small after Hu-17 processing, and the content of pro apoptotic protein Bak also has Certain increase (Fig. 3 A).
Stat5 molecules play an important role in chronic myelocytic leukemia occurrence and development, have real in animal body Testing result confirms that Stat5 molecules have conclusive effect (Andrea to the maintenance of BCR-ABL-positive leukaemia Hoelbl,Christian Schuster,Veronika Sexl.EMBO Molecular Medicine(2010)2,98- 110)；As the target molecule of BCR-ABL fusion proteins, experiment in vitro also confirm that Stat5 to promote leukaemia multiplication and It resists Apoptosis and plays an important role (Malgorzata Nieborowska-Skorska, Tomasz Skorski.J.Exp.Med.189,1229–1242).When our data (Fig. 3 B) show that Hu-17 processing K562 cells 24 are small, Reducing occur in whole Bcr-Abl and the Bcr-Abl protein levels of phosphorylation, BCR-ABL downstream target proteins, that is, Stat5 albumen Phosphorylation level also occur reduce, illustrate BCR-ABL-Stat5 signal paths Hu-17 processing after be suppressed.In addition, P27 increases after Hu-17 processing in a period of time, and the increase of the albumen and leukaemia block related (figure the generation G1 phases 2A)。
It these results suggest that, apoptosis and Cycle Arrest and BCR- occurs after Hu-17 processing K562 and K562-R cells ABL-Stat5 accesses are suppressed related.Illustrate that Hu-17 can be antileukemie by inhibiting pathogenic protein BCR-ABL performances Effect, this result shows the clinical treatment potentiality of Hu-17.
Legend 4 illustrates：Compound Hu-17 combines with Imatinib can significantly induce K562 Apoptosis, while causes a disease and melt The expression of hop protein BCR-ABL is suppressed in transcriptional level.The compound Hu-17 and Imatinib of various concentration are independent or join Processing K562 cells are closed, the induced killer effect that the results show is used in combination to K562 cells becomes apparent from (Fig. 4 A).We are simultaneously Have detected that BCR-ABL protein is horizontal and variation of the mRNA level in-site after drug-treated, find drug combination histone it is horizontal and The BCR-ABL contents of mRNA level in-site substantially lower (Fig. 4 B and 4C), illustrate that drug combination group can substantially inhibit BCR-ABL genes Transcript and expression.
Legend 5 illustrates：Compound Hu-17 can significantly kill the marrow or peripheral blood mononuclear cells in CML patient source, The scavenging action being used in combination with Imatinib (Sti571) to leucocyte is stronger.
Treatment for chronic myelocytic leukemia, existing drug is (including Imatinib, Dasatinib, nilotinib Deng) activity of BCR-ABL tyrosine kinase can only be inhibited, the transcription and expression on BCR-ABL protein influence (figure any 4B, the BCR-ABL protein of phosphorylation is reduced, and whole BCR-ABL protein level is constant), noval chemical compound Hu-17 can induce K562 cells and drug resistant K562-R Apoptosis, become apparent from the lethal effect of K562-R；Secondly, Hu-17 and her horse The transcript and expression of Disease-causing gene BCR-ABL can be inhibited by being used in combination for Buddhist nun, and drug action differs markedly from what is listed at present Tyrosine kinase inhibitor.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (3)

1. compound Hu-17 is preparing the application in treating chronic myelocytic leukemia drug.
2. compound Hu-17 is with tyrosine kinase inhibitor drug combination in treatment chronic myelocytic leukemia drug is prepared Using.
3. application according to claim 2, which is characterized in that the tyrosine kinase inhibitor refers to Imatinib, Buddhist nun Replace Buddhist nun and Dasatinib in Lip river.
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