CN102526762B - Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent - Google Patents
Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent Download PDFInfo
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Abstract
The invention relates to the field of leukaemia drug resistance and discloses application of an ALC1 inhibitor to preparation of a leukaemia drug resistance reversing agent and application of an ALC1 gene to preparation of a drug-resistant leukaemia diagnosing reagent. The expression of the ALC1 gene in a peripheral blood sample or a bone marrow sample of a patient is detected; and based on a characteristic that the high expression of the gene is related to the increase of the resistance of a cell to chemotherapeutic medicines, sensitive chemotherapeutic agents can be screened. The invention further discloses an application of the ALC1 gene to preparation of the leukaemia drug resistance reversing agent. By inhibiting the expression of the ALC1 gene, the drug resistance of the cell can be reversed, the sensitivity of a leukemia cell to the medicine can be improved, and the cell inhibiting effects and the apoptosis promoting effects of various chemotherapeutic medicines can be improved, so that the ALC1 inhibitor can be applied to the preparation of acute and chronic leukaemia multidrug resistance reversing agents and a new method for treating the drug-resistant leukaemia is provided.
Description
Technical field
The present invention relates to the drug-resistant leukemia field, be specifically related to the purposes of screening and the preparation drug-resistant leukemia sexual inversion agent of the leukemic diagnosis of ALC1 gene pairs drug resistance, sensitivity chemotherapeutics.
Background technology
Leukemia is hematopoietic stem cell clone property disease, it is characterized by the leukaemia born of the same parents' proliferation out of control dysdifferentiation among the clone, and apoptosis is obstructed, and is stuck in cytocerastic different phase.Leukemic treatment mainly is chemotherapy, targeted therapy and bone marrow transplantation, wherein bone marrow transplantation is subjected to the multifactorial restrictions such as donor source, patient age, so the actual patient that can accept bone marrow transplantation only is minority, most of patient need carry out chemotherapy and targeted drug treatment.In leukemic Drug therapy process, combined chemotherapy has obtained considerable progress, overwhelming majority patient can be alleviated, yet mostly come to an end with the chemotherapy failure, the leukaemia can produce multidrug resistance, when namely cell drug resistance occurs to a kind of antitumor drug, to other configurations, the different antineoplastic agent deposits yields crossing drug resistant phenomenon of mechanism of action, this is the main cause of leukemia chemotherapy failure and recurrence, is problem demanding prompt solution clinically.The leukemic medicament categories of current treatment is on the increase, select best chemotherapy regimen according to leukaemia's drug resistance molecular phenotype and to the response feature of medicine, to directly improve curative effect, and make the patient avoid the toxic and side effects of invalid medicine, improve the sufferer survival rate, therefore, the detection of individuation drug susceptibility and reversal of drug resistance treatment are very crucial.
Summary of the invention
The object of the present invention is to provide a kind ofly to can be used for diagnosing the leukemic test kit of drug resistance, and drug-resistant leukemia sexual inversion agent.
First aspect of the present invention is to overcome the shortcoming that leukaemic's routine medication can not effectively be predicted its chemotherapeutics tolerance, utilize peripheral blood in patients or sample of bone marrow, carry out In vitro culture and drug effect effect measuring, expression with ALC1 gene before and after the medication changes as the Drug tolerance evaluation index, and leukaemia's growth characteristics filter out sensitivity chemotherapy or target therapeutic agent after the bound drug processing.Invention provides the ALC1 gene in the application aspect drug resistance leukemia diagnosis reagent, and the leukemic test kit of a kind of diagnosis drug resistance, and this test kit comprises: Oligo (dT)
15, reverse transcription (M-MLV), M-MLV RT enzyme reaction buffer solution, RNase inhibitor, PCR buffer, dNTPs, MgCl
2, Auele Specific Primer, Taq enzyme, in order to carry out the qualitative and detection by quantitative of ALC1 gene.Described specific primer sequence is: SEQ ID NO:5:5 '-CCTCCTTCTATGATCATGTTG-3 ' and SEQ ID NO:6:5 '-TGTTCTCATCCCAGGCCTTG-3 '.Also contain reference substance in the preferred test kit, wherein positive control is Normocellular ALC1 cDNA sample, and negative control is the sterilization distilled water without ALC1.
Utilize the designed primer of mentioned reagent box to carry out reverse transcriptional PCR, the ALC1 gene is carried out qualitative and semi-quantitative analysis.
Second aspect of the present invention is that ALC1 is in the purposes of preparation drug-resistant leukemia inversion agent.Described ALC1 gene inhibitor reversible Drug tolerance, cell inhibitory effect effect and the apoptosis-promoting effect of raising various kinds of drug are so can be used for preparing the leukemia multidrug-resistance reversal agent.This ALC1 gene inhibitor can be to regulate antisensenucleic acids, RNA that the ALC1 encoding gene expresses to disturb (RNAi) molecule (can be that siRNA, shRNA, ddRNAi construct or its are transcribed template, such as the DNA of coding shRNA), ALC1 specific antibody etc.ALC1 inhibitor of the present invention and/or complex can pass through any suitable administration.Preferably, ALC1 gene inhibitor of the present invention is ALC1 siRNA.All kinds of commercial or synthetic ALC1 siRNA all can realize the present invention.Preferably, the sequence of this ALC1 siRNA is:
Positive-sense strand SEQ ID NO:1:5 '-GGAUUCACCUACGCUCUUATT-3 ', antisense strand SEQ ID NO:2:5 '-UAAGAGCGUAGGUGAAUCCCT-3 '; Perhaps:
Positive-sense strand SEQ ID NO:3:5 '-GCAUCGUGAUCGUUCCAAUTT-3 ', antisense strand SEQ ID NO:4:5 '-AUUGGAACGAUCACGAUGCTG-3 '.
Invention provides a kind of leukemic medicinal composition of drug resistance that is used for the treatment of, described this complex to comprise ALC1 inhibitor, chemotherapeutics, the relevant excipient of medicine, diluent or carrier simultaneously.Described chemotherapeutics can be any clinical Common Chemotherapy agent.
ALC1 (Amplified in Liver Cancer 1) is that we use the chromosome cutting and screen the new oncogene that hybridization technique is distinguished from and cloned at the chromosome 1q21 of human liver cancer cell, nothing is expressed in normal liver cell, but hepatoma carcinoma cell camber amplification, closely related with generation, the development of hepatocarcinoma, homology comparison and functional domain analysis result show that the albumen of its coding contains conservative SNF2_N functional domain, unwindase functional domain and Macro functional domain, belong to the relevant ATP enzyme superfamily of SWI2/SNF2, unwind relevant with chromatin.ALC1 can be positioned the DNA damage position fast and momently, rely on poly-adenosine diphosphate ribose polymerase [poly (ADP ribose) polymerase, PARP] (a kind of DBP enzyme), selectivity identification and in conjunction with the DNA breach, the regulation and control chromatin Structure, the unconventionality expression of ALC1 will significantly improve the sensitivity of DNA to damage
[2-3]
We find from embryo's normal development to adult period in the experiment of research ALC1 gene physiological action, ALC1 expresses in hemopoietic tissue all the time, point out it relevant with hemopoietic function, the basal expression of ALC1 is arranged in the adult mice bone marrow, can detect faint ALC1 mrna expression signal by peripheral blood.In view of the important function of ALC1 in hepatocarcinoma occurs, impel us further to probe into the relation of ALC1 and human blood tumor.By being detected, all kinds of leukemia Bone Marrow of Patients and peripheral blood RT-PCR find the unusual amplification of ubiquity ALC1 and express that the expression that wherein produces ALC1 in its bone marrow of leukaemic of drug resistance and the peripheral blood is higher than non-drug resistance leukaemic; The experiment in vitro result show the drug resistance leukemia cell line under the chemotherapeutics effect its ALC1 expression, ability of cell proliferation apparently higher than the strain of non-drug resistance parental cell, strike the expression reversible leukaemia's who subtracts ALC1 drug resistance, improve inhibitory action and the apoptosis-promoting effect of chemotherapeutics on cell proliferation.The unconventionality expression of ALC1 there is no report so far at effect and the related mechanism of drug-resistant leukemia formation.Invention finds that first ALC1 can be used as drug resistance leukemia marker molecule.
Invention utilization RT-PCR method detects the normal person, infection causes leukocyte rising patient and all kinds of leukemia peripheral blood in patients all has ALC1 to express, but mutations in leukemia patients by peripheral blood ALC1 expression is significantly higher than infectious leukocyte rising patient and normal person, and both are faint expression and no significant difference afterwards.All kinds of leukemia Bone Marrow of Patients ALC1 express apparently higher than Normal Human Bone Marrow.
Experiment in vitro is human chronic myelogenous leukemia Di Guglielmo syndrome cell strain K562 and drug-resistant cell strain K562/ADR (Adriamycin resistant cell strain relatively, 1.0 μ g/ml amycin cultivating system), people's Acute Myeloid Leukemia Cells Contributing strain HL-60 and multidrug resistance cell strain HL-60/ADR (Adriamycin resistant cell strain thereof, 0.5 μ g/ml amycin cultivating system) ALC1 expression, the result shows that persister ALC1 expresses apparently higher than the strain of non-drug resistance parental cell, gene expression amount differs and reaches more than 3 times, show that the ALC1 gene plays a significant role in different classes of leukaemia's drug resistance mechanism, be used for Clinical screening and drug resistance diagnosis so the ALC1 unconventionality expression can be used as the leukemia marker gene.
K562/ADR cell and HL-60/ADR cell are carried out using chemotherapeutics after ALC1 siRNA disturbs, MTT testing result demonstration K562/ADR and HL-60/ADR cell significantly improve the sensitivity of various chemotherapeutic, show and suppress the drug resistance that ALC1 expresses the reversible leukaemia, the ALC1 inhibitor can be used as the auxiliary treatment that the leukemic reversal agent of drug resistance of clinical drug-resistant carries out chemotherapeutics.
Compared with prior art, the present invention has following beneficial effect:
The Gene A LC1 that drug resistance leukemia diagnosis test kit detects is the specificity marker molecule of hematopoietic cell abnormality proliferation, available peripheral blood or sample of bone marrow direct-detection, its cycle detection time is short, efficient is high, can carry out accurately qualitative and semi-quantitative analysis to the ALC1 transcript, easy and simple to handle and cost is lower, be easy to use at clinical expansion.It is remarkable that application ALC1 prepares the action effect of drug resistance leukemia inversion agent, can obviously improve the drug resistance leukaemia to the sensitivity of medicine, all effective to each quasi-leukemia, can be used as the reversal agent of drug resistance of wide spectrum, so wide potential applicability in clinical practice is arranged.
Description of drawings
The expression of Fig. 1 .ALC1 in human peripheral and bone marrow
A: human peripheral ALC1 expresses.N is normal human blood (n=10); F is general surgery postoperative infection blood samples of patients (n=6); AML: acute myeloid leukaemia (n=8); ALL: acute lymphoblastic leukemia (n=6); CML: chronic myelocytic leukemia (n=5); CLL: chronic lymphocytic leukemia (n=5).B: human peripheral ALC1 expresses relative value, and showing with infectious leukocyte rising patient difference has significant (* * P＜0.01), and comparing difference with the normal person has significant (Δ P＜0.01).Infectious leukocyte rising patient compares no significant difference (P＞0.05) with the normal person.C: ALC1 expresses in the bone marrow.All kinds of leukemia Bone Marrow of Patients ALC1 express apparently higher than Normal Human Bone Marrow (* * P＜0.01).
Fig. 2. leukemia mdr cell and non-drug resistance parental cell ALC1 express
A:K562/ADR, K562, HL-60/ADR, HL-60 cell ALC1mRNA expression; B:ALC1 expresses relative value, and K562/ADR mdr cell ALC1 mRNA relative value is higher than 3.76 times of non-drug resistance parental cells (P＜0.05); HL-60/ADR mdr cell ALC1 mRNA relative value is higher than 1.87 times in non-drug resistance parent HL-60 cell (P＜0.05).
Fig. 3 .ALC1 siRNA is to the effect of leukemia mdr cell drug resistance inversion
A:HL-60/ADR and HL-60 cell, K562/ADR, K562 give merely ADR or ALC1 siRNA unites the impact that ADR expresses ALC1; B:ALC1 siRNA is to the reversing effect of each cells resistance.
Fig. 4 .ALC1 test kit is to the detection of clinical peripheral blood sample
A: peripheral blood sample ALC1 detects.N is normal human blood (n=10); AML: acute myeloid leukaemia (n=8); ALL: acute lymphoblastic leukemia (n=6); CML: chronic myelocytic leukemia (n=5); CLL: chronic lymphocytic leukemia (n=5); B: the ALC1 level relatively before and after the leukemia patient medication.
The specific embodiment
The detection of ALC1 in embodiment 1 peripheral blood and the sample of bone marrow
Get normal person and all kinds of leukemia peripheral blood in patients and bone marrow (Normal Human Bone Marrow is taken from the bone marrow donations and joined type person), for the ALC1 that compares in the normal propagation of leukocyte and the abnormality proliferation situation expresses, (leukocyte sum＞10 * 109/L) compare the experiment alternative with general surgery postoperative infection peripheral blood in patients, extract total RNA, total RNA is quantitative, the synthetic cDNA of reverse transcription etc. all undertaken by the test kit description, and cDNA product-20 ℃ is preserved or namely used.The PCR primer is given birth to worker company by Shanghai and is synthesized: ALC1 (800bp) Sence:5 '-CCTCCTTCTATGATCATGTTG-3 ', Antisence:5 '-TGTTCTCATCCCAGGCCTTG-3 '; GAPDH (310bp) Sence:5 '-GCCTCAAGATCAGCAAT-3 ', Antisence:5 '-AGGTCCACCACTG ACACGTT-3 '.50 μ l reaction systems are adopted in PCR reaction, 95 ℃ of degeneration 5 minutes, 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds, 28 circulations, 72 ℃ 10 minutes.1% agarose gel electrophoresis, take the gel electrophoresis strip image with software Genesnap, software Genetools carries out the gray value analysis, represents genes of interest ALC1 mRNA relative level (Fig. 1) with the ratio (ALC1/GAPDH) of purpose band and internal reference band.
Conclusion: as shown in Figure 1, all kinds of crowd's peripheral bloods all have ALC1 to express, but mutations in leukemia patients by peripheral blood ALC1 expression is significantly higher than infectious leukocyte rising patient and normal person (P＜0.01), rear two kinds of crowd's peripheral blood ALC1 all are faint expression, its expression intensity no significant difference (P＞0.05) (Fig. 1 a, 1b).ALC1 expresses apparently higher than Normal Human Bone Marrow (P＜0.01) (Fig. 1 c, 1d) in all kinds of leukemia Bone Marrow of Patients.Illustrate that the ALC1 unconventionality expression is a kind of sign of all kinds of leukemias, represents the abnormality proliferation of medullary cell.
The semi-quantitative analysis that embodiment 2 acute and chronic leukemia mdr cells and non-drug resistance parental cell ALC1 express
The acute red white blood of human chronic myelogenous leukemia becomes cell strain K562 and its drug-resistant cell strain K562/ADR (Adriamycin resistant cell strain, 1.0 μ g/ml amycin cultivating system is kept drug resistance), people's Acute Myeloid Leukemia Cells Contributing strain HL-60 and multidrug resistance cell strain HL-60/ADR (Adriamycin resistant cell strain, 0.5 μ g/ml amycin cultivating system is kept drug resistance) in containing the RPMI1640 culture fluid of 15% hyclone, in 37 ℃, contain 5%CO
2And cultivate in the incubator of saturated humidity, went down to posterity 1 time in 2-3 days, reagent removal cultivated for 4 generations before experiment, and all are tested equal fetching and count the trophophase cell and carry out.With RNA extract test kit (the RNeasy mini columns of Qigen company) extract the exponential phase cell (K562 and K562/ADR cell, HL-60 and HL-60/ADR cell } total RNA, the methods such as total RNA is quantitative, the synthetic cDNA of reverse transcription and PCR are identical with embodiment 1, the expression of two couples of cell ALC1 of comparison.
Conclusion: the ALC1 gene is expressed in drug-resistant cell strain apparently higher than the strain of non-drug resistance parental cell, gray scale scanning the analysis showed that the K562/ADR cell compares with the K562 cell, ALC1 mRNA relative value is respectively 2.18 ± 0.24 and 0.76 ± 0.15 (P＜0.01), the drug-resistant cell strain cell is than high 3.76 times of (Fig. 2 a of parental cell strain ALC1 mRNA relative value, 2b), the HL-60/ADR cell is compared with the HL-60 cell, ALC1 mRNA relative value is respectively 1.24 ± 0.13 and 0.66 ± 0.1 (P＜0.01), the drug-resistant cell strain cell increases by 1.87 times of (Fig. 2 a than parental cell strain ALC1 mRNA relative value, 2b), prompting ALC1 expresses enhancing can be as producing chemical sproof sign, characteristic is carried out qualitative to ALC1 accordingly, quantitative assay can be to all kinds of urgency, the curative effect of chronic leukemia, the features such as resistant characterization judge.
Embodiment 3 ALC1 siRNA disturb the reverse effect to leukemia cell drug toleration
Select K562 cell and K562/ADR cell, HL-60 cell and HL-60/ADR cell to carry out pair verification.
(1) the MTT detection method detects the half-inhibition concentration (IC of amycin (ADR)
50): the K562 cell of the trophophase of taking the logarithm and K562/ADR cell (1 * 10
5/ ml), HL-60 cell and HL-60/ADR cell (2 * 10
5/ ml) being inoculated in 96 well culture plates, every strain cell is all established 4 groups: matched group (the not blank group of dosing), medicine group, ALC1 siRNA interference group, (siRNA+ medicine) group.The adenovirus vector that carries ALC1 siRNA fragment (ad-ALC1 siRNA) that siRNA disturbs preferred success to make up, each is organized cell and adds respectively Ad-mock with serum-free RPMI-1640 dilution (MOI 100, blank group, ADR group) and ad-ALC1 siRNA (MOI 100, siRNA interference group, siRNA+ medicine group), the total amount of liquid that every hole adds is 0.1ml.Rock gently culture plate, make uniform liquid cover cell, sucking-off culture fluid behind the 2h, the normal culture fluid of adding 0.2ml RPMI-1640+10%FBS, medicine group and siRNA+ drug component do not add the medicine (half-inhibition concentration) of respective concentration, in 37 ℃, 5%CO
2Every hole adds MTT solution (5mg/ml) 20 μ l continuation cultivation 4h after cultivating 72h in the incubator, abandon supernatant, add 150 μ l dimethyl sulfoxide, measure the absorbance (A570) of 570nm wavelength behind the mixing in microplate reader, calculate inhibitory rate of cell growth, suppression ratio=[1-(administration group A570-blank A570/ control group A 570-blank A570)] 100%.IC
50Drug level when reaching 50% for cell growth inhibiting adopts return law of the straight line.Calculate amycin to the half-inhibition concentration (IC before and after the K562/ADR cell transfecting
50), and calculate drug resistance multiple and drug resistance reversal fold: drug resistance multiple=mdr cell IC
50/ parental cell IC
50, reversal index=persister IC
50/ persister adds inversion agent IC
50Repeated experiments 3 times, the data paired t-test and variance analysis.
Conclusion: by the IC of HL-60/ADR and HL-60 cell
50Being worth HL-60/ADR is 20.4 times to the drug resistance multiple of ADR, and when adding ADR again after ALC1 siRNA is hatched, HL-60/ADR significantly improves the sensitivity of ADR, and its reversal of drug resistance multiple is 2.55 times; IC by K562/ADR and K562 cell
50Being worth K562/ADR is 42 times to the drug resistance multiple of ADR, and when adding chemotherapeutics again after ALC1 siRNA is hatched, K562/ADR significantly improves the sensitivity of chemotherapeutics, the reversal of drug resistance multiple be 3.25 times (table 1, Fig. 3).Identical experimental technique testing result shows that ALC1 siRNA has drug resistance inversion effect (IC equally to other Treated with Chemotherapeutic Drugs (vincristine, vincaleucoblastine, homoharringtonine, daunorubicin)
50Value, drug resistance multiple and reversal index see Table 2, table 3), so ALC1 can be used for the drug resistance leukemia treating, its inhibitor can be used for the preparation of drug resistance leukemia inversion agent.
The lower drug resistance of table 1 ADR and ALC1 siRNA effect and non-drug resistance parental cell IC
50Value (μ mol/L)
Table 2 HL-60/ADR is to the drug resistance multiple of different pharmaceutical and the reversal index under the siRNA effect (mean ± s)
Annotate: VCR: vincristine; VBL: vincaleucoblastine; HHT: homoharringtonine; DNR: daunorubicin.The used dosage of each medicine all is to calculate IC by mtt assay
50Gained concentration.
Table 3 K562/ADR cell is to the drug resistance multiple of different pharmaceutical and the reversal index under the siRNA effect (mean ± s)
Annotate: VCR: vincristine; VBL: vincaleucoblastine; HHT: homoharringtonine; DNR: daunorubicin.
The used dosage of each medicine all is to calculate IC by mtt assay
50Gained concentration.
Embodiment 4 application of ALC1 test kit in Clinical detection:
(1) extracting of peripheral blood or bone marrow RNA: adopt the BD 2.5ml of company blood taking tube (containing RNA enzyme inhibitor and erythrocyte splitting composition), gather to be measured peripheral body or bone marrow 2.5ml, gently shake back and forth blood taking tube, make the abundant mixing of blood or bone marrow and liquid in pipe.Adopt Qiagen company's blood rna extraction agent box (PAXgene Blood RNA Kit) total RNA of rapid extraction, the DEPC water of getting 99ul adds the RNA of 1ul, spectrophotometer instrumentation RNA concentration and quality, OD260/OD280 ratio is between 1.6-1.8, the EB gel electrophoresis detects RNA quality ,-70 ℃ of preservations.
(2) ALC1 specificity reverse transcription: reverse transcription 20 μ l reaction systems are
The reverse transcription reaction program: 42 ℃ 50 minutes, 70 ℃ 15 minutes.
(3) ALC1 specific PCR:
Carry out the PCR program: 94 ℃, 5min; 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds, the 25-30 circulation; 72 ℃, 10 minutes, 4 ℃, continue.
The ALC1 Auele Specific Primer is:
Sence：5′-CCTCCTTCTATGATCATGTT G-3’
Antisence：5′-TGTTCTCATCCCAGGCCTTG-3′；
As reference gene, its sequence is with GAPDH:
Sence：5′-GCCTCAAGATCAGCAAT-3′，
Antisence：5′-AGGTCCACCACTGACACGTT-3′
(4) the expression analysis is measured; Row agarose gel electrophoresis, observed result under the uviol lamp adopts the gel images process software to measure gray value, calculates the relative value of mrna expression.Computational methods: ALC1 mrna expression relative value=ALC1 gray value/GAPDH gray value.
Conclusion: normal person's peripheral blood ALC1/GAPDH meansigma methods is 0.2097 among the present invention, and all kinds of mutations in leukemia patients by peripheral blood ALC1/GAPDH values all obviously surpass normal person ALC1/GAPDH meansigma methods, (Fig. 4 a) so super high-caliber ALC1/GAPDH value indicates the generation of neoplastic hematologic disorder, same patient is compared with blood sample before and after the chemotherapeutic, the result shows that clinical symptom remission patient ALC1/GAPDH value descends, and clinical symptoms not its ALC1/GAPDH value of reduction of patient remain on level before the medication, so can judge whether to produce drug resistance (Fig. 4 b) according to peripheral blood before and after the medication or bone marrow ALC1/GAPDH value.
SEQUENCE LISTING
＜110〉Guangzhou Medical College
＜120〉ALC1 is in preparation drug-resistant leukemia inversion agent and as the application aspect the drug resistance leukemia diagnosis reagent
<130>
<160> 6
<170> PatentIn version 3.2
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<211> 21
<212> DNA
＜213〉artificial sequence
<400> 1
ggauucaccu acgcucuuat t 21
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 2
uaagagcgua ggugaauccc t 21
<210> 3
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 3
gcaucgugau cguuccaaut t 21
<210> 4
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 4
auuggaacga ucacgaugct g 21
<210> 5
<211> 21
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＜213〉artificial sequence
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Claims (8)
1.ALC1 the application of inhibitor aspect preparation drug-resistant leukemia sexual inversion agent.
2. application as claimed in claim 1 is characterized in that described ALC1 inhibitor comprises antisensenucleic acids, rnai molecule or the ALC1 specific antibody of regulating the expression of ALC1 encoding gene.
3. application as claimed in claim 2 is characterized in that described rnai molecule is siRNA, shRNA or ddRNAi.
4. application as claimed in claim 3 is characterized in that described siRNA sequence is: positive-sense strand SEQ ID NO:1, antisense strand SEQ ID NO:2; Perhaps: positive-sense strand SEQ ID NO:3, antisense strand SEQ ID NO:4.
5. one kind is used for the treatment of the leukemic medicinal composition of drug resistance, and described complex comprises ALC1 inhibitor, chemotherapeutics, medicine relevant excipient, diluent and carrier.
6.ALC1 the application of gene aspect preparation drug resistance leukemia diagnosis reagent.
7. a drug resistance leukemia diagnosis test kit is characterized in that comprising: reverse transcriptase (M-MLV), M-MLV RT enzyme reaction buffer solution, RNase inhibitor, PCR buffer, dNTPs, MgCl2, Auele Specific Primer and Taq enzyme; Described specific primer sequence is: SEQ ID NO:5 and SEQ ID NO:6.
8. claim 1 or 6 described application is characterized in that described leukemia is all kinds of leukemias.
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| C06 | Publication | ||
| PB01 | Publication | ||
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