CN102526762B - Application of ALC1 to preparation of leukaemia drug resistance reversing agent and as drug-resistant leukaemia diagnosing reagent - Google Patents

Abstract

The invention relates to the field of leukemia drug resistance, and discloses the application of ALC1 inhibitors in the preparation of leukemia drug resistance reversal agents, and the application of ALC1 gene in the preparation of drug-resistant leukemia diagnostic kits. Based on the determination of ALC1 gene expression in peripheral blood or bone marrow samples of patients, the high expression of the gene is related to the enhanced resistance of cells to chemotherapeutic drugs, and this characteristic can be used to screen sensitive chemotherapeutic agents. The present invention also includes the use of ALC1 gene in the preparation of drug-resistant leukemia reversal agent, inhibiting the expression of ALC1 gene can reverse the drug resistance of cells, improve the sensitivity of leukemia cells to drugs, and enhance the inhibitory effect of various chemotherapeutic drugs on cells and promote Therefore, ALC1 inhibitors can be used to prepare acute and chronic leukemia multi-drug resistance reversal agents, providing a new method for the treatment of drug-resistant leukemia.

Description

Application of ALC1 in the preparation of leukemia drug resistance reversal agent and as a diagnostic reagent for drug resistant leukemia

technical field

The invention relates to the field of leukemia drug resistance, in particular to the application of ALC1 gene in the diagnosis of drug-resistant leukemia, the screening of sensitive chemotherapeutic agents and the preparation of leukemia drug resistance reversal agent.

Background technique

Leukemia is a clonal disease of hematopoietic stem cells, which is characterized by uncontrolled proliferation and differentiation disorder of leukemia cells in clones, blocked apoptosis, and stagnation at different stages of cell development. The treatment of leukemia is mainly chemotherapy, targeted therapy and bone marrow transplantation. Bone marrow transplantation is limited by many factors such as donor source and patient age. Therefore, only a small number of patients can actually receive bone marrow transplantation, and most patients need chemotherapy. and targeted drug therapy. In the process of drug treatment of leukemia, combined chemotherapy has made great progress, and most patients can be relieved, but most of them end in chemotherapy failure. Leukemic cells will produce multidrug resistance, that is, cells become resistant to an antitumor drug. At the same time, cross-resistance to other anti-tumor drugs with different structures and different mechanisms of action occurs. This is the main reason for the failure and recurrence of leukemia chemotherapy, and it is a clinical problem that needs to be solved urgently. Currently, there are more and more types of drugs for the treatment of leukemia. Selecting the best chemotherapy regimen according to the molecular phenotype of drug resistance of leukemia cells and the characteristics of their response to drugs will directly improve the curative effect, and protect patients from the toxic and side effects of ineffective drugs. Survival, therefore, individualized drug susceptibility testing and resistance reversal therapy are critical.

Contents of the invention

The object of the present invention is to provide a kit for diagnosing drug-resistant leukemia and a leukemia drug-resistant reversal agent.

The first aspect of the present invention is to overcome the shortcoming that conventional drug treatment of leukemia patients cannot effectively predict their chemotherapeutic agent tolerance, and use peripheral blood or bone marrow samples of patients to conduct in vitro culture and drug effect measurement to determine the expression level of ALC1 gene before and after treatment. The change is used as an evaluation index of drug tolerance, and is combined with the growth characteristics of leukemia cells after drug treatment to screen out sensitive chemotherapy or targeted therapy drugs. The invention provides the application of the ALC1 gene as a diagnostic reagent for drug-resistant leukemia, and a kit for diagnosing drug-resistant leukemia, which includes: Oligo(dT) ₁₅ , reverse transcriptase (M-MLV), M-MLV RT enzyme reaction buffer, RNase inhibitor, PCR buffer, dNTPs, MgCl ₂ , specific primers, Taq enzyme, used for qualitative and quantitative detection of ALC1 gene. The specific primer sequences are: SEQ ID NO: 5: 5'-CCTCCTTCTATGATCATGTTG-3' and SEQ ID NO: 6: 5'-TGTTCTCATCCCAGGCCTTG-3'. The preferred kit also contains control substances, wherein the positive control is the ALC1 cDNA sample of normal cells, and the negative control is sterilized double distilled water without ALC1.

The primers designed by the above kit were used to carry out reverse transcription PCR to conduct qualitative and semi-quantitative analysis of the ALC1 gene.

The second aspect of the present invention is the use of ALC1 in the preparation of leukemia drug resistance reversal agent. The ALC1 gene inhibitor can reverse drug tolerance and improve the cell proliferation inhibitory and apoptosis-promoting effects of various drugs, so it can be used to prepare acute and chronic leukemia multi-drug resistance reversal agents. The ALC1 gene inhibitor can be antisense nucleic acid, RNA interference (RNAi) molecule (which can be siRNA, shRNA, ddRNAi construct or its transcription template, such as DNA encoding shRNA), ALC1 specific antibody, etc. . The ALC1 inhibitors and/or complexes of the invention may be administered by any suitable route. Preferably, the ALC1 gene inhibitor of the present invention is ALC1 siRNA. All kinds of commercial or synthetic ALC1 siRNAs can realize the present invention. Preferably, the sequence of the ALC1 siRNA is:

Sense strand SEQ ID NO: 1:5'-GGAUUCACCUACGCUCUUATT-3', antisense strand SEQ ID NO: 2:5'-UAAGAGCGUAGGUGAAUCCCT-3'; or:

Sense strand SEQ ID NO: 3:5'-GCAUCGUGAUCGUUCCAAUTT-3', antisense strand SEQ ID NO: 4:5'-AUUGGAACGAUCACGAUGCTG-3'.

The invention also provides a drug compound for treating drug-resistant leukemia, said compound comprising an ALC1 inhibitor, a chemotherapeutic agent, a drug-related excipient, a diluent or a carrier. The chemotherapeutic agent can be any commonly used clinical chemotherapeutic agent.

ALC1 (Amplified in Liver Cancer 1) is a new oncogene isolated and cloned in the chromosome 1q21 region of human liver cancer cells by chromosome cutting and screening hybridization technology. It has no expression in normal liver cells, but is highly amplified in liver cancer cells. It is closely related to the occurrence and development of liver cancer. The results of homology comparison and functional domain analysis show that the encoded protein contains conserved SNF2_N functional domain, helicase functional domain and Macro functional domain, and belongs to the SWI2/SNF2-related ATPase superfamily , associated with chromatin unwinding. ALC1 can quickly and transiently localize to DNA damage sites, relying on poly(ADP ribose) polymerase [poly(ADP ribose) polymerase, PARP] (a DNA-binding protease), selectively recognizes and binds to DNA gaps, and regulates chromatin Structure, abnormal expression of ALC1 will significantly increase the sensitivity of DNA to damage ^[2-3] .

In the experiment of studying the physiological function of ALC1 gene, we found that ALC1 is always expressed in hematopoietic tissue from normal embryonic development to adulthood, suggesting that it is related to hematopoietic function. There is a basic expression of ALC1 in the bone marrow of adult mice, which can be detected by peripheral blood to a weak ALC1 mRNA expression signal. In view of the important role of ALC1 in the occurrence of liver cancer, we are prompted to further explore the relationship between ALC1 and human hematological tumors. Abnormal amplification and expression of ALC1 is commonly found in the bone marrow and peripheral blood of various acute and chronic leukemia patients by RT-PCR detection, and the expression level of ALC1 in the bone marrow and peripheral blood of drug-resistant leukemia patients is higher than that of non-drug-resistant leukemia patients Leukemia patients; in vitro experiments show that the ALC1 expression level and cell proliferation ability of drug-resistant leukemia cell lines under the action of chemotherapy drugs are significantly higher than those of non-drug-resistant parental cell lines, knocking down the expression of ALC1 can reverse the drug resistance of leukemia cells , Improve the inhibitory effect of chemotherapy drugs on cell proliferation and the effect of promoting apoptosis. The role of abnormal expression of ALC1 in the formation of leukemia drug resistance and the related mechanism have not been reported so far. Invention discovered for the first time that ALC1 can be used as a marker molecule for drug-resistant leukemia.

The invention uses the RT-PCR method to detect the expression of ALC1 in the peripheral blood of normal people, patients with elevated leukocytes caused by infection, and patients with various acute and chronic leukemias, but the expression level of ALC1 in the peripheral blood of leukemia patients is significantly higher than that of patients with elevated infectious leukocytes and In normal people, the latter two are weakly expressed and there is no significant difference. The expression of ALC1 in the bone marrow of patients with acute and chronic leukemia was significantly higher than that of normal people.

In vitro experiments compared human chronic myeloid leukemia acute erythroleukemia cell line K562 with drug-resistant cell line K562/ADR (doxorubicin-resistant cell line, 1.0 μg/ml doxorubicin culture system), human acute myeloid leukemia cell line HL -60 and its multidrug-resistant cell line HL-60/ADR (doxorubicin-resistant cell line, 0.5 μg/ml doxorubicin culture system) ALC1 expression level, the results showed that the ALC1 expression of drug-resistant strains was significantly higher than that of non-resistant strains The difference in gene expression in the parental cell lines of the drug was more than 3 times, indicating that the ALC1 gene plays an important role in the drug resistance mechanism of different types of leukemia cells, so the abnormal expression of ALC1 can be used as a leukemia marker gene for clinical screening and drug resistance diagnosis.

K562/ADR cells and HL-60/ADR cells were subjected to ALC1 siRNA interference and chemotherapy drugs were used. MTT test results showed that the sensitivity of K562/ADR and HL-60/ADR cells to various chemotherapy drugs was significantly improved, indicating that the expression of ALC1 was inhibited. It can reverse the drug resistance of leukemia cells, and the ALC1 inhibitor can be used as a drug resistance reversal agent for clinical drug-resistant leukemia for adjuvant chemotherapy drugs.

Compared with the prior art, the present invention has the following beneficial effects:

The gene ALC1 detected by the drug-resistant leukemia diagnostic kit is a specific marker molecule for abnormal proliferation of hematopoietic cells. It can be directly detected in peripheral blood or bone marrow samples. The detection time period is short and the efficiency is high. Semi-quantitative analysis, easy to operate and low cost, easy to promote and use in clinic. The use of ALC1 to prepare a drug-resistant leukemia reversal agent has a remarkable effect, can significantly improve the sensitivity of drug-resistant leukemia cells to drugs, and is effective for all types of leukemia. It can be used as a broad-spectrum drug-resistant reversal agent, so it has broad clinical applications. Application prospects.

Description of drawings

Figure 1. Expression of ALC1 in human peripheral blood and bone marrow

a: ALC1 expression in human peripheral blood. N is normal human blood (n=10); F is blood of infected patients after general surgery (n=6); AML: acute myeloid leukemia (n=8); ALL: acute lymphoblastic leukemia (n=6); CML: chronic myelogenous leukemia (n=5); CLL: chronic lymphocytic leukemia (n=5). b: The relative value of ALC1 expression in human peripheral blood, showing a significant difference from patients with elevated infectious leukocytes (**P<0.01), and a significant difference from normal people (ΔP<0.01). There was no significant difference between patients with elevated infectious leukocytes and normal controls (P>0.05). c: ALC1 expression in bone marrow. The expression of ALC1 in bone marrow of patients with acute and chronic leukemia was significantly higher than that of normal people (**P<0.01).

Figure 2. ALC1 expression in acute and chronic leukemia drug-resistant cells and non-drug-resistant parental cells

a: ALC1 mRNA expression level of K562/ADR, K562, HL-60/ADR, HL-60 cells; b: relative value of ALC1 expression, the relative value of ALC1 mRNA in K562/ADR drug-resistant cells was 3.76 times higher than that of non-drug-resistant parental cells (P <0.05); the relative value of ALC1 mRNA in HL-60/ADR drug-resistant cells was 1.87 times higher than that of non-drug-resistant parental HL-60 cells (P<0.05).

Figure 3. The effect of ALC1 siRNA on the reversal of drug resistance in acute and chronic leukemia cells

a: HL-60/ADR and HL-60 cells, K562/ADR, K562 the effect of ADR alone or ALC1 siRNA combined with ADR on ALC1 expression; b: ALC1 siRNA reversal effect on drug resistance of each cell.

Figure 4. Detection of clinical peripheral blood samples by the ALC1 kit

a: Detection of ALC1 in peripheral blood samples. N is normal human blood (n=10); AML: acute myeloid leukemia (n=8); ALL: acute lymphoblastic leukemia (n=6); CML: chronic myeloid leukemia (n=5); CLL: chronic Lymphocytic leukemia (n=5); b: comparison of ALC1 levels in patients with acute and chronic leukemia before and after treatment.

Detailed ways

Example 1 Detection of ALC1 in peripheral blood and bone marrow samples

Peripheral blood and bone marrow were collected from normal people and patients with various types of acute and chronic leukemia (the bone marrow of normal people was obtained from bone marrow donors who matched). In order to compare the expression of ALC1 in the normal and abnormal proliferation of white blood cells, the experiment also selected infection after general surgery. Peripheral blood (total white blood cells > 10×109/L) was used as a control. Total RNA was extracted, total RNA was quantified, and cDNA was synthesized by reverse transcription according to the kit instructions. The cDNA product was stored at -20°C or used immediately. PCR primers were synthesized by Shanghai Sangong Company: ALC1 (800bp) Sence: 5′-CCTCCTTCTATGATCATGTTG-3′, Antisence: 5′-TGTTCTCATCCCAGGCCTTG-3′; GAPDH (310bp) Sence: 5′-GCCTCAAGATCAGCAAT-3′, Antisence: 5 '-AGGTCCACCACTGACACGTT-3'. The PCR reaction uses a 50 μl reaction system, denaturation at 95°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 60 seconds, extension at 72°C for 60 seconds, 28 cycles, 10 minutes at 72°C. 1% agarose gel electrophoresis, use the software Genesnap to take the gel electrophoresis band image, software Genetools for gray value analysis, and use the ratio of the target band to the internal reference band (ALC1/GAPDH) to represent the relative level of the target gene ALC1 mRNA (figure 1).

Conclusion: As shown in Figure 1, ALC1 was expressed in the peripheral blood of various populations, but the expression level of ALC1 in the peripheral blood of leukemia patients was significantly higher than that of patients with elevated infectious leukocytes and normal people (P<0.01), the peripheral blood of the latter two groups ALC1 was weakly expressed, and there was no significant difference in the expression intensity (P>0.05) (Fig. 1a, 1b). The expression of ALC1 in the bone marrow of patients with acute and chronic leukemia was significantly higher than that of normal people (P<0.01) (Fig. 1c, 1d). It shows that the abnormal expression of ALC1 is a symptom of various acute and chronic leukemias, representing the abnormal proliferation of bone marrow cells.

Example 2 Semi-quantitative analysis of ALC1 expression in acute and chronic leukemia drug-resistant cells and non-drug-resistant parental cells

Human chronic myeloid leukemia acute erythroleukemia cell line K562 and its drug-resistant cell line K562/ADR (doxorubicin-resistant cell line, 1.0μg/ml doxorubicin culture system maintains drug resistance), human acute myeloid leukemia cells Strain HL-60 and multidrug-resistant cell line HL-60/ADR (doxorubicin-resistant cell line, 0.5 μg/ml doxorubicin culture system to maintain drug resistance) in RPMI1640 culture medium containing 15% fetal bovine serum cultured in an incubator at 37°C, containing 5% CO ₂ and saturated humidity, passaged once every 2-3 days, and cultured for 4 passages before the experiment, and all experiments were performed on cells in the exponential growth phase. Extract total RNA from logarithmic growth phase cells (K562 and K562/ADR cells, HL-60 and HL-60/ADR cells) with RNA extraction kit (RNeasy mini columns from Qigen Company), quantify total RNA, synthesize cDNA by reverse transcription and PCR The method is the same as in Example 1, and the expression levels of ALC1 in two pairs of cells are compared.

Conclusion: The expression of ALC1 gene in drug-resistant cell lines was significantly higher than that in non-drug-resistant parental cell lines. Compared with K562 cells, the relative values of ALC1 mRNA in K562/ADR cells were 2.18±0.24 and 0.76±0.15( P<0.01), the relative value of ALC1 mRNA in the drug-resistant cell line was 3.76 times higher than that of the parental cell line (Fig. 2a, 2b). 0.13 and 0.66±0.1 (P<0.01), the relative value of ALC1 mRNA in the drug-resistant cell line was 1.87 times higher than that of the parental cell line (Figure 2a, 2b), suggesting that the enhanced expression of ALC1 can be used as a marker of drug resistance. For this characteristic, the qualitative and quantitative determination of ALC1 can make judgments on the curative effect and drug resistance characteristics of various acute and chronic leukemias.

Example 3 Reversal effect of ALC1 siRNA interference on drug resistance of leukemia cells

K562 cells and K562/ADR cells, HL-60 cells and HL-60/ADR cells were selected for pair verification.

(1) The half maximal inhibitory concentration (IC ₅₀ ) of doxorubicin (ADR) was detected by MTT assay: K562 cells and K562/ADR cells (1×10 ⁵ /ml), HL-60 cells and HL -60/ADR cells (2×10 ⁵ /ml) were seeded in 96-well culture plates, and each cell line was divided into 4 groups: control group (blank group without drug addition), drug group, ALC1 siRNA interference group, (siRNA+drug group) )Group. For siRNA interference, the adenoviral vector (ad-ALC1 siRNA) carrying the ALC1 siRNA fragment that has been successfully constructed is preferred. The cells in each group were added with Ad-mock (MOI 100, blank group, ADR group) and ad-mock diluted with serum-free RPMI-1640. -ALC1 siRNA (MOI 100, siRNA interference group, siRNA+drug group), the total amount of liquid added to each well is 0.1 ml. Gently shake the culture plate to make the liquid evenly cover the cells, suck out the culture medium after 2 hours, add 0.2ml RPMI-1640+10%FBS normal culture solution, add the corresponding concentration of drug (half inhibitory concentration) to the drug group and siRNA+drug group respectively After culturing in a 37°C, 5% _CO2 incubator for 72 hours, add 20 μl of MTT solution (5 mg/ml) to each well and continue to incubate for 4 hours, discard the supernatant, add 150 μl dimethyl sulfoxide, mix well, and measure with a microplate reader Absorbance value (A570) at 570nm wavelength, calculated cell growth inhibition rate, inhibition rate=[1-(administration group A570-blank control A570/control group A570-blank control A570)]100%. IC ₅₀ is the drug concentration at which cell growth is inhibited by 50%, using linear regression method. Calculate the half inhibitory concentration (IC ₅₀ ) of doxorubicin to K562/ADR cells before and after transfection, and calculate the drug resistance multiple and the drug resistance reversal multiple: drug resistance multiple = drug-resistant cell IC ₅₀ / parental cell IC ₅₀ , reversal multiple = IC ₅₀ of drug-resistant strain/IC ₅₀ of drug-resistant strain plus reversal agent. The experiment was repeated three times, and the data were analyzed by paired t-test and analysis of variance.

Conclusion: According to the IC ₅₀ value of HL-60/ADR and HL-60 cells, the drug resistance of HL-60/ADR to ADR is 20.4 times. The sensitivity of K562/ADR to ADR was significantly improved, and the drug resistance reversal factor was 2.55 times; the drug resistance factor of K562/ADR to ADR was 42 times based on the IC ₅₀ value of K562/ADR and K562 cells, and chemotherapy drugs were added after incubation with ALC1 siRNA When , the sensitivity of K562/ADR to chemotherapeutic drugs was significantly improved, and the drug resistance reversal factor was 3.25 times (Table 1, Figure 3). The results of the same experimental method showed that ALC1siRNA also had drug resistance reversing effects on other chemotherapeutic drugs (vincristine, vinblastine, homoharringtonine, daunorubicin) (IC ₅₀ value, drug resistance multiple and reversal effect). Multiples are shown in Table 2 and Table 3), so ALC1 can be used for the treatment of drug-resistant leukemia, and its inhibitor can be used for the preparation of drug-resistant leukemia reversal agent.

Table 1 IC ₅₀ values of drug-resistant and non-resistant parental cells under the action of ADR and ALC1 siRNA (μmol/L)

Table 2 HL-60/ADR resistance to different drugs and siRNA reversal times (mean±s)

Note: VCR: vincristine; VBL: vinblastine; HHT: homoharringtonine; DNR: daunorubicin. The doses of each drug were calculated by the MTT method to calculate the concentration obtained by _IC50 .

Table 3 K562/ADR cells' drug resistance to different drugs and siRNA reversed multiples (mean±s)

Note: VCR: vincristine; VBL: vinblastine; HHT: homoharringtonine; DNR: daunorubicin.

The doses of each drug were calculated by the MTT method to calculate the concentration obtained by _IC50 .

Example 4 Application of ALC1 kit in clinical testing:

(1) Extraction of peripheral blood or bone marrow RNA: Use 2.5ml BD company blood collection tubes (containing RNase inhibitors and erythrocyte lysate components), collect 2.5ml peripheral blood or bone marrow of the individual to be tested, and gently shake the blood collection tubes back and forth to make the blood Or the bone marrow and the liquid in the tube are fully mixed. Use Qiagen’s PAXgene Blood RNA Kit to quickly extract total RNA, take 99ul of DEPC water and add 1ul of RNA, measure the RNA concentration and quality with a spectrophotometer, and the OD260/OD280 ratio is between 1.6-1.8 , EB gel electrophoresis to detect the quality of RNA, and store at -70°C.

(2) ALC1-specific reverse transcription: 20 μl reverse transcription reaction system is

Reverse transcription reaction program: 50 minutes at 42°C, 15 minutes at 70°C.

(3) ALC1-specific PCR:

Perform PCR program: 94°C, 5min; 94°C denaturation for 30 seconds, 60°C annealing for 60 seconds, 72°C extension for 60 seconds, 25-30 cycles; 72°C, 10 minutes, 4°C, continuous.

ALC1-specific primers are:

Sence: 5′-CCTCCTTCTATGATCATGTT G-3’

Antisence: 5'-TGTTCTCATCCCAGGCCTTG-3';

Taking GAPDH as an internal reference gene, its sequence is:

Sence: 5′-GCCTCAAGATCAGCAAT-3′,

Antisence: 5′-AGGTCCACCACTGACACGTT-3′

(4) Analysis and determination of expression level: perform agarose gel electrophoresis, observe the results under ultraviolet light, use gel image processing software to measure the gray value, and calculate the relative value of mRNA expression. Calculation method: Relative value of ALC1 mRNA expression = ALC1 gray value/GAPDH gray value.

Conclusion: In the present invention, the average value of ALC1/GAPDH in peripheral blood of normal people is 0.2097, while the ALC1/GAPDH values in peripheral blood of patients with various types of leukemia are significantly higher than the average value of ALC1/GAPDH in normal people, so the super high level of ALC1/GAPDH value indicates that there is For the occurrence of hematological tumors (Figure 4a), the blood samples of the same patient were compared before and after chemotherapy, and the results showed that the ALC1/GAPDH value decreased in patients with clinical symptoms relief, while the ALC1/GAPDH value in patients without clinical symptom relief remained at the level before treatment. Therefore, the occurrence of drug resistance can be judged according to the peripheral blood or bone marrow ALC1/GAPDH value before and after treatment (Figure 4b).

SEQUENCE LISTING

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<120> Application of ALC1 in the preparation of leukemia drug resistance reversal agent and as a diagnostic reagent for drug resistant leukemia

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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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