Combined medicine for treating leukemia

文档序号：158912 发布日期：2021-10-29 浏览：17次中文

阅读说明：本技术 一种治疗白血病的联合用药物 (Combined medicine for treating leukemia ) 是由刘广洋刘拥军李欣张晨亮米一徐立强苗丽于 2021-08-06 设计创作，主要内容包括：本发明提供了一种治疗白血病的联合用药物,包括TRAIL基因修饰的脐带间充质干细胞和地塞米松,属于医药技术领域。本发明成功构建了TRAIL-MSCs,具有分泌sTRAIL的功能,MSCs特异性地向肿瘤部位迁移并在局部分泌sTRAIL,靶向发挥促肿瘤凋亡作用,具有更为强大的抗肿瘤作用。同时本发明采用地塞米松联合TRAIL-MSCs对急性B淋巴细胞白血病作为联合用药物,发现其能够明显地抑制急性B淋巴细胞白血病细胞的增殖,促进急性B淋巴细胞白血病细胞的凋亡,且具有较好的靶向性,为开发针对复发难治的ALL新的有效治疗方法提供了参考。(The invention provides a combined medicine for treating leukemia, which comprises an umbilical cord mesenchymal stem cell modified by TRAIL gene and dexamethasone, and belongs to the technical field of medicines. The invention successfully constructs TRAIL-MSCs, has the function of secreting sTRAIL, specifically migrates MSCs to tumor parts and locally secretes sTRAIL, and has the function of promoting tumor apoptosis in a targeted manner, thereby having stronger anti-tumor effect. Meanwhile, the dexamethasone and TRAIL-MSCs are used as combined medicines for acute B lymphocyte leukemia, and the dexamethasone and TRAIL-MSCs can obviously inhibit the proliferation of acute B lymphocyte leukemia cells and promote the apoptosis of the acute B lymphocyte leukemia cells, have better targeting property, and provide reference for developing a new effective treatment method for ALL difficult to treat due to relapse.)

1. An application of combination of an umbilical cord mesenchymal stem cell modified by a TRAIL gene and dexamethasone in preparing a medicament for treating leukemia.

2. Use according to claim 1, characterized in that: the concentration of the dexamethasone is 12.5-50 mu mol/L; the ratio of the TRAIL gene modified umbilical cord mesenchymal stem cells to the tumor cells is 1:1-1: 5.

3. Use according to claim 2, characterized in that: the concentration of the dexamethasone is 25 mu mol/L; the ratio of the TRAIL gene modified umbilical cord mesenchymal stem cells to the tumor cells is 1: 2.

4. Use according to claim 3, characterized in that: the leukemia is acute lymphocytic leukemia, acute myelocytic leukemia, acute monocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia, eosinophilic leukemia, basophilic leukemia, tissue basophilic leukemia or hairy cell leukemia.

5. Use according to claim 4, characterized in that: the leukemia is acute B lymphocyte leukemia.

6. A combination drug for treating leukemia, which is characterized in that: the combined medicine comprises umbilical cord mesenchymal stem cells TRAIL-MSCs modified by TRAIL genes and dexamethasone DEX.

7. The combination of claim 6, wherein: the concentration of the dexamethasone is 12.5-50 mu mol/L; the ratio of the number of TRAIL-MSCs to the number of tumor cells is 1:1-1: 5.

8. The combination according to claim 7, wherein: the combined medicine also comprises a pharmaceutically acceptable carrier, wherein the carrier is any one or more of a sustained-release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

9. The combination according to claim 8, wherein: the combined medicine is any one of an external preparation, an oral preparation or an injection preparation.

10. The combination of claim 9, wherein: the external preparation is a spray or an aerosol; the oral preparation is any one of granules, capsules, tablets or capsule agents; the injection preparation adopts intradermal, subcutaneous, intramuscular, local or intravenous injection as the administration mode.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a combined medicine for treating leukemia.

Background

Leukemia is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues in large quantities due to mechanisms such as uncontrolled proliferation, differentiation disorder, apoptosis blockage, etc., infiltrate other non-hematopoietic tissues and organs, and inhibit normal hematopoietic function. Different degrees of anemia, bleeding, infectious fever, and swelling of the liver, spleen, lymph nodes and bone pain are seen clinically. Acute and chronic leukemia can be classified according to the differentiation degree and natural disease course of leukemia. The differentiation of acute leukemia cells is arrested in the early stage, mainly primitive and early juvenile cells, and the disease develops rapidly and the course of the disease lasts for months. Chronic leukemia cells are well differentiated, mainly immature or mature cells, slowly develop and have a disease course of several years. The cells were classified by the diseased cell series, including myeloid, mono-, erythro-, megakaryoid and lymphoid T and B cell lines. Clinically, leukemia is often classified into lymphocytic leukemia, myelocytic leukemia, mixed cell leukemia, and the like. Acute B-lymphoblastic leukemia (B-ALL) is a common hematological malignancy, accounting for 70-80% of Acute Lymphoblastic Leukemia (ALL) patients in children and adults. At present, the Complete Remission (CR) rate of B-ALL can reach 70-90%, the disease-free survival rate (DFS) in 3-5 years can reach 30-60%, and the long-term DFS rate of children ALL can reach more than 85%. However, 30-60% of patients have refractory or relapsed disease, which is one of the main causes of death in B-ALL patients.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein, the carboxyl terminal of which is located outside the cell membrane and contains a receptor binding domain. After membrane type TRAIL is cleaved by specific matrix metalloprotease, its extracellular region forms soluble molecule (sTRAIL). The TRAIL protein monomer molecule has high oligomerization tendency, and can form dimer with molecular weight of 48KDa and trimer with molecular weight of 66 KDa. The configuration stability and aggregation form of the protein have close relationship with the tumor killing activity, receptor affinity, stability and the like of the protein, and when TRAIL forms a tripolymer structure in space, the anti-tumor activity of the TRAIL is optimal and the stability is highest. The current studies targeting TRAIL receptors mainly include 2 that utilize TRAIL as a recombinant ligand to function and develop humanized monoclonal antibodies against DR4, DR 5. However, the method has the limitation, and preclinical researches find that the half-life period of the recombinant human sTRAIL in vivo is short, namely 30-60 min; the half-life of the DR4 monoclonal antibody Mapatumumab was extended (14-21d), but potential toxic side effects could increase. The patent CN110669145A successfully constructs a genetic engineering stem cell of high expression soluble tripolymer TRAIL fusion protein, and researches the treatment effect of the stem cell on malignant tumors.

Glucocorticoids, which cause apoptosis and cell cycle G1 arrest and inhibit growth and proliferation of lymphocytes, are an indispensable chemotherapeutic agent in the treatment of ALL. Dexamethasone (DEX) is a typical glucocorticoid, acts by combining with a glucocorticoid receptor, has a strong killing effect on lymphocyte, is one of clinically common prescription drugs, and is widely applied to the treatment of leukemia/lymphoma. However, the long-term and large-dose use of DEX in the treatment process not only easily causes adverse reactions such as hypertension and osteoporosis, but also has poor curative effect and easily causes drug resistance, thereby causing poor prognosis.

Patent CN111658636B discloses that andrographolide and dexamethasone are combined to be used for preparing a compound medicine for resisting acute lymphoblastic leukemia, the treatment effect is obvious, the toxic and side effects are small, and drug resistance is not easy to generate. Patent CN108315302B discloses a glucocorticoid-resistant cell line of human acute B-lymphocyte leukemia, which is prepared by the following steps: firstly culturing a human acute B lymphocyte leukemia cell strain NALM-6 under a hypoxia condition, then adding 0.25 mu M dexamethasone at one time, continuing the hypoxia culture, and finally performing amplification culture under an normoxic condition, thereby obtaining a human acute B lymphocyte leukemia glucocorticoid resistant cell strain NALM-6/HDR, wherein the cell can be used for treating glucocorticoid resistant acute B lymphocyte leukemia.

Currently, there is no clinically effective treatment for B-ALL that is refractory to relapse, and even when allogeneic hematopoietic stem cell transplantation is performed, the DFS rate is still unsatisfactory, so that a new effective treatment strategy for B-ALL is urgently needed to be found.

Disclosure of Invention

Aiming at the defects, the invention provides a combined medicine for treating leukemia. The combined medicine comprises the umbilical cord mesenchymal stem cells (TRAIL-MSCs) modified by TRAIL genes and Dexamethasone (DEX), can obviously inhibit the proliferation of acute B lymphocyte leukemia cells, promotes the apoptosis of the acute B lymphocyte leukemia cells, has better targeting property, and provides a new thought and a new method for the targeted therapy of tumors.

In order to achieve the above object, the technical solution of the present invention is as follows:

in one aspect, the invention provides an application of a combination of TRAIL gene modified umbilical cord mesenchymal stem cells (TRAIL-MSCs) and Dexamethasone (DEX) DEX in preparing a medicament for treating leukemia.

Specifically, the concentration of the dexamethasone is 12.5-50 mu mol/L, and preferably 25 mu mol/L.

Specifically, the ratio of the cell number of the TRAIL-MSCs to the tumor cells is 1:1-1:5, and preferably 1: 2.

Specifically, the leukemia includes, but is not limited to, acute lymphocytic leukemia, acute myelocytic leukemia, acute monocytic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia, eosinophilic leukemia, basophilic leukemia, histiophilic leukemia or hairy cell leukemia, preferably acute B lymphocytic leukemia.

In another aspect, the invention provides a combined medicine for treating leukemia, wherein the combined medicine comprises the TRAIL gene modified umbilical cord mesenchymal stem cells TRAIL-MSCs and dexamethasone DEX.

Specifically, the concentration of the dexamethasone is 12.5-50 mu mol/L, and preferably 25 mu mol/L.

Specifically, the ratio of the cell number of the TRAIL-MSCs to the tumor cells is 1:1-1:5, and preferably 1: 2.

Specifically, the combined medicine further comprises a pharmaceutically acceptable carrier, wherein the carrier is any one or a mixture of more than two of a slow release agent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.

Specifically, the combined medicament is any one of an external preparation, an oral preparation or an injection preparation.

Further specifically, the external preparation is a spray or an aerosol.

More specifically, the oral preparation is any one of granules, capsules, tablets or vesicular agents.

More specifically, the injection preparation adopts intradermal, subcutaneous, intramuscular, topical or intravenous injection as the administration mode.

Compared with the prior art, the invention has the advantages that:

(1) the invention successfully constructs TRAIL-MSCs, has the function of secreting sTRAIL, specifically migrates MSCs to tumor parts and locally secretes sTRAIL, and has the function of promoting tumor apoptosis in a targeted manner, thereby having stronger anti-tumor effect. Meanwhile, the expression of the TRAIL-MSCs surface marker after gene modification and the basic biological characteristics of osteogenesis, adipogenic differentiation and the like are not changed.

(2) The invention adopts dexamethasone combined with TRAIL-MSCs to treat acute B lymphocyte leukemia as a combined medicament, finds that the dexamethasone combined medicament can obviously inhibit the proliferation of acute B lymphocyte leukemia cells and promote the apoptosis of the acute B lymphocyte leukemia cells, has better targeting property, and provides reference for developing a new effective treatment method for ALL difficult to treat relapse.

Drawings

FIG. 1 is a graph showing the results of detecting the proliferation inhibitory effect of DEX on Nalm-6 cells.

FIG. 2 is a graph showing the result of detecting the relative expression level of DEX to mRNA levels of Nalm-6 cell TRAIL receptors DR4 and DR 5.

FIG. 3 is a graph showing the results of the detection of the proliferation inhibition effect of TRAIL-MSCs in combination with DEX on Nalm-6 cells.

FIG. 4 is a diagram showing the result of cell state detection of Nalm-6 by the combined action of TRAIL-MSCs and DEX.

FIG. 5 is a graph showing the results of detecting the effect of DEX in combination with TRAIL-MSCs on Nalm-6 apoptosis.

FIG. 6 is a graph showing the results of the detection of mRNA levels of Nalm-6 cell TRAIL receptors DR4 and DR5 relative to the expression level.

FIG. 7 is a graph showing the results of protein expression detection of Nalm-6 cell TRAIL receptors DR4 and DR 5.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

The examples, where no specific techniques or conditions are indicated, are carried out according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, ed. by Huang Pe, et al) or according to the instructions of the product.

The invention adopts SPSS 19.0 statistical software to measure the data useExpressing, using one-way analysis of varianceAnd t-test for statistical analysis.

1. Reagents or materials for the experiments

(1) Acute B-lymphocytic leukemia cell Nalm-6 was purchased from the institute of basic medicine of Chinese academy of medical sciences;

(2) RPMI 1640 culture medium, DMEM/F-12 culture medium, fetal bovine serum, etc. were purchased from Thermo Fisher, USA;

(3) the 4 th generation lentiviral vector was purchased from AddGene, USA;

(4) RNA extraction, reverse transcription kit and SYBR Green qPCR kit purchased from Thermo Fisher company of USA;

(5) DR4, DR5, beta-actin primers were synthesized by Jinzhi Biotechnology, Inc., Suzhou;

(6) the CCK-8 kit and the Annexin V-FITC/PI apoptosis kit are purchased from Donglian chemical technology Co., Ltd;

(7) RIPA and PMSF were purchased from CST Inc.;

(8) loadingbuffer is available from Zhongke Ruita BioLimited;

(9) marker available from Bio-Rad;

(10) DR4, DR5, GAPDH, Goat Anti-RabbitIgG H & L (HRP) were purchased from Abcam corporation.

Example 1 preparation and detection of TRAIL Gene-modified MSCs

Human umbilical cord tissue was derived from a healthy fetus born by caesarean section in a gynecology hospital, and umbilical cord collection was approved by the hospital ethics committee and signed with a donor informed consent. The preparation of human umbilical cord mesenchymal stem cells (hUC-MSCs), the establishment of seed banks and working banks, quality detection and the like are all finished in a GMP workshop and a quality control center of Beijing Beibei Biotechnology Ltd. The preparation method of the TRAIL-MSCs comprises the following steps: the UC-MSCs are transfected by the SPD-TRAIL gene through a lentiviral vector, so that the UC-MSCs can express dTRAIL protein (see the research on the SPD-TRAIL gene modified mesenchymal stem cells and the lung adenocarcinoma killing cells thereof in Miyi, Wanglihua, Lexin, and the like in China medicinal biotechnology 2020,15(2): 157-162).

Example 2 inhibition of Nalm-6 cell proliferation by DEX

Nalm-6 cells at 1X 10⁴Inoculating to 96-well plate, standing at 37 deg.C and 5% CO₂Culturing in an incubator. The test solution is divided into a control group (without adding medicine) and DEX 6.25, 12.50, 25.00, 50.00 and 100.00 mu mol/L groups, and each group is provided with 6 multiple holes. After 24h of culture, detecting the proliferation inhibition of DEX on Nalm-6 cells by adopting a CCK8 kit, detecting the absorbance (A) value of the cells at 450nm, and calculating the cell proliferation inhibition rate.

Inhibition of proliferation rate of 1-A_DEX/A_Control

As shown in FIG. 1, DEX showed a concentration-dependent proliferation inhibitory effect on Nalm-6 cells, and the median inhibitory concentration (IC50) was 25 to 30. mu. mol/L.

Example 3 Effect of DEX on the DR4, DR5 of Nalm-6 cells

Nalm-6 cells were arranged in a 3X 10 format⁵Inoculating to 6-well plate at 37 deg.C and 5% CO₂Culturing in an incubator. The test pieces were divided into control group (without drug) and DEX 6.25, 12.50, 25.00, 50.00, 100.00. mu. mol/L groups, each group was provided with 2 multiple wells. After 24h of culture, the real-time fluorescence quantitative RCR (qRT-PCR) method detects the influence of different concentrations of DEX on the change of DR4 and DR5 of Nalm-6 cells on the mRNA level.

The Trizol method is used for extracting Nalm-6 cell total RNA, reverse transcription is carried out to prepare cDNA, operation is carried out according to ABI instructions, and the primer sequence is shown in the following table 1. Each sample was repeated 3 times, and the qRT-PCR reaction procedure was as follows: at 95 ℃ 20s 1 cycle, at 95 ℃ 3s, at 60 ℃ 3s 40 cycles. By using 2^-△△CtThe method analyzes the data.

TABLE 1 detection primers

As shown in fig. 2, when the concentration of DEX was 6.25, 12.5, 25, 50 μmol/L, the expression amounts of DR4 and DR5 increased with the increase in the concentration of DEX, wherein the concentration of 12.5 to 100 μmol/L was significantly different from the control group (P < 0.05 or P < 0.01); on the other hand, the expression level of DR4 and DR5mRNA was reduced at a DEX concentration of 100. mu. mol/L as compared with 25-50. mu. mol/L. Further comparison shows that the level of DR4 and DR5 expressed by Nalm-6 cells is highest when the concentration of DEX is 25-50 mu mol/L.

In combination with examples 2-3, the concentration of DEX was chosen to be 25. mu. mol/L for optimal treatment of Nalm-6 cells for subsequent experiments in combination with TRAIL-MSCs.

Example 4 inhibition of Nalm-6 cell proliferation by DEX in combination with TRAIL-MSCs

Nalm-6 cells were divided into a control group, a UC-MSCs group, a TRAIL-MSCs group, a DEX + UC-MSCs group, and a DEX + TRAIL-MSCs group. Nalm-6 cells were as per 6.25X 10⁴After 6 hours, the control group, UC-MSCs group and TRAIL-MSCs group were replaced with normal medium, and the DEX group, DEX + UC-MSCs group and DEX + TRAIL-MSCs group were replaced with medium containing DEX at a concentration of 25. mu. mol/L, and the culture was continued for 24 hours. Mixing 3.13X 10⁴UC-MSCs/well were inoculated into the Transwell upper chambers of UC-MSCs and DEX + UC-MSCs groups, respectively, and 3.13X 10⁴The TRAIL-MSCs of each hole are inoculated in the upper chamber of a Transwell of TRAIL-MSCs and DEX + TRAIL-MSCs groups and are co-cultured with Nalm-6 cells for 48h, the upper chamber is removed after the co-culture is finished, the Nalm-6 cells in the chamber are taken down and inoculated in a 96-well plate, CCK-8 detects the absorbance (A) value of the cells at 450nm, and the proliferation inhibition rate of each group of cells is calculated.

As shown in FIG. 3, UC-MSCs and TRAIL-MSCs can inhibit proliferation of Nalm-6, and have significant difference (P < 0.01) compared with the control group, but the inhibition rates are below 30%; DEX can inhibit the proliferation of Nalm-6 cells, the inhibition rate is 43%, and the difference is significant compared with a control group (P < 0.01); the proliferation inhibition effect of DEX combined TRAIL-MSCs on Nalm-6 cells is strongest, the inhibition rate reaches 85%, and the difference is obvious compared with a control group and a DEX group (P < 0.01). Meanwhile, DEX can improve the proliferation inhibition effect of UC-MSCs and TRAIL-MSCs on cells Nalm-6, and has obvious difference (P < 0.01) compared with UC-MSCs and TRAIL-MSCs.

Example 5 Effect of DEX in combination with TRAIL-MSCs on apoptosis of tumor cells Nalm-6

Nalm-6 cells were arranged in a 3X 10 format⁵Perwell in 6-well Transwell plate, the procedure and grouping were as described in example 4, 1.5X 10 after 24h DEX drug action⁵The UC-MSCs and TRAIL-MSCs per well are respectively inoculated in the upper chamber of the Transwell of the UC-MSCs/DEX + UCMSCs group and the TRAIL-MSCs/DEX + TRAIL-MSCs group and are connected withNalm-6 cells were co-cultured. After the cell culture is carried out for 48h, the cell is removed from the chamber, Nalm-6 cells in the chamber are taken out, the cells are observed and photographed under a microscope, and the apoptosis condition of the cells is detected by a flow cytometer.

After Nalm-6 cells are treated in different modes, the cell morphology and the cell density are observed by different times (40X and 100X) of a microscope respectively, and the results are shown in figure 4, wherein the Nalm-6 cells have certain killing effect on tumor cells in different modes, and the killing effect of the combination of DEX and TRAIL-MSCs is most obvious.

After Nalm-6 cells are treated in different modes for 48 hours, Nalm-6 cells are dyed by fluorescent dyes Annexin-V and PI respectively, and the apoptosis conditions of different treatment groups are detected by a flow cytometer, and the result is shown in figure 5, TRAIL-MSCs can promote Nalm-6 cell apoptosis, and the apoptosis rate reaches 10%; when DEX acts alone, Nalm-6 cell apoptosis can be promoted, the apoptosis rate reaches 15%, and the difference with a control group is significant (P < 0.05, 0.01); and after the DEX combined with TRAIL-MSCs acts on Nalm-6 cells for 48 hours, the apoptosis rate is increased to 36%, and the obvious difference (P < 0.01) is compared with that of the DEX and TRAIL-MSCs which are used alone.

Example 6 Effect of DEX in combination with TRAIL-MSCs on expression of DR4, DR5 in Nalm-6 cells

Cell inoculation and group administration As described in example 4, after 24h of co-culture, the chamber was removed, and Nalm-6 cells were removed, and changes in mRNA and protein levels of DR4 and DR5 in Nalm-6 cells by DEX-combined TRAIL-MSCs were detected by real-time fluorescence quantitative RCR (qRT-PCR) and Western blotting, respectively.

(1) qRT-PCR method: the detection method was the same as in example 3.

As a result, as shown in FIG. 6, the expression amounts of the mRNAs DR4 and DR5 in the TRAIL-MSCs group were significantly decreased (P < 0.01) as compared with the control group; DEX can promote expression of DR4 and DR5, and has significant difference compared with control group (P < 0.01); compared with the single use of DEX, the combined action of DEX and TRAIL-MSCs has the advantages that the expressions of DR4 and DR5 are obviously reduced (P < 0.01).

(2) Western blotting method: the cell is cracked for 20min at 0 ℃ by the mixed solution of RIPA and PMSF, the total protein is extracted, the protein concentration of each sample is detected by the BCA protein detection kit, the samples are adjusted to be uniform concentration, 5 Xloading buffer solution is added, and the denaturation is carried out for 5min at 100 ℃. Samples (30. mu.g) were separated by 10% SDS-PAGE and transferred to PVDF membrane, blocked with 5% skimmed milk powder in TBST for 1h, and the primary antibody incubated overnight. After washing 3 times with TBST, the secondary antibody was incubated at room temperature for 1h, washed 3 times with TBST, and the band was observed with ECL luminescence detection reagent. Image J software quantitatively analyzes the images.

The results are shown in FIG. 7, after TRAIL-MSCs act on Nalm-6 cells for 24h, DR4 and DR5 protein expression is slightly reduced compared with a control group, but no significant difference exists; DEX can promote the expression of DR4 and DR5 proteins of Nalm-6 cells, and has obvious difference compared with a control group (P < 0.01); compared with the effect of DEX alone, the effects of DEX combined with TRAIL-MSCs are obviously reduced in the expression of DR4 and DR5 (P < 0.01).

The results show that DEX can obviously improve the expression of DR4 and DR5 of tumor cells, the promotion effect of DEX on DR5 expression is better than that of DR4, and DEX is proved to enhance the sensitivity of the tumor cells to TRAIL by improving TRAIL apoptosis receptors on the surface of Nalm-6; it is noted that the combined action of DEX and TRAIL-MSC on Nalm-6 significantly reduced the expression of DR4 and DR5 compared to the use of DEX alone, because the combined action of DEX and TRAIL-MSC more easily killed TRAIL-sensitive tumor cells (these cells highly express DR4/5), while leaving DR4/5 low-expression tumor cells, which indicates that the combined action of DEX and TRAIL-MSC has higher targeting on tumor cells.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Beijing Beibei Biotech Co., Ltd

<120> a combined medicine for treating leukemia

<130> 20210804

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA