HUMAN T-LYMPHOBLASTIC LEUKEMIA/LYMPHOMA CELL STRAIN AND USE THEREOF

Abstract

The present application discloses a human T-lymphoblastic leukemia/lymphoma cell strain named as ZYXY-T1, and its construction method and use thereof. It was conserved in China Center for Type Culture Collection (Wuhan, China) on Jan. 20, 2021, and the preservation number was CCTCC NO: C202143. The present application is obtained by separating mononuclear cells from peripheral blood of one ETP-ALL patient, and culturing the cells in vitro for continuous natural passage. The strain has the typical surface antigen expression characteristics of ETP-ALL, that is, it does not express CD1α, CD5 or CD8, and highly expresses a stem cell marker CD34, and has good proliferation ability in vitro and tumorigenesis ability in vivo; it can be used as a cell material to study the occurrence and development mechanism of ETP-ALL, and can also be used to screen and evaluate ETP-ALL drugs to guide clinical medication.

Claims

1. A human T-lymphoblastic leukemia/lymphoma cell strain, wherein the cell strain is named as a human T-lymphoblastic leukemia/lymphoma cell ZYXY-T1 and had been conserved in China Center for Type Culture Collection on Jan. 20, 2021, with a preservation number of CCTCC NO: C202143.

2. The human T-lymphoblastic leukemia/lymphoma cell strain according to claim 1, wherein the cell strain is a suspension cell, which conforms to typical ETP-ALL surface antigen expression characteristics: CD1a, CD8 and CD5 are not expressed, while a stem line marker CD34 is expressed.

3. A progeny cell of the human T-lymphoblastic leukemia/lymphoma cell ZYXY-T1 according to claim 1.

4. Use of the human T-lymphoblastic leukemia/lymphoma cell strain according to claim 1, which is selected from any one or more of the following: a. study on molecular characteristics and therapeutic mechanism of ETP-ALL; b. preparing a tumor cell model or preparing a tumor animal model; c. in vitro screening, in vitro evaluation or preparation of tumor therapeutic drugs; d. developing tumor drug targets; e. preparing tumor diagnosis products; f. in vitro screening of tumor biological therapy drugs/reagents; and g. in vitro development and detection of tumor-related bioengineering products.

5. The use according to claim 4, wherein an animal for preparing a tumor animal model in b is an immunodeficient mouse.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0019] The present application will be further explained with reference to the following examples and drawings.

[0020] FIG. 1 shows the results of microscopic observation and Wright-Giemsa staining of the human T-lymphoblastic leukemia/lymphoma cell strain;

[0021] FIG. 2 is the cell growth curve of the human T-lymphoblastic leukemia/lymphoma cell strain at different cell densities;

[0022] FIG. 3 shows the surface antigen expression result of the human T-lymphoblastic leukemia/lymphoma cell strain;

[0023] FIG. 4 shows the in vivo tumorigenicity of the human T-lymphoblastic leukemia/lymphoma cell strain, where a is the expression of CD45 in mouse peripheral blood, b is the expression of CD45 in mouse bone marrow, and c is the survival time of mice.

DESCRIPTION OF EMBODIMENTS

[0024] The following examples will further illustrate the present application, but the present application is not limited thereto. The experimental methods without specific conditions in the following examples are usually in accordance with conventional conditions.

Example 1 Preparation of the ZYXY-T1 Cell Strain

[0025] Primary cell culture: 6 ml of a peripheral blood sample (male) was taken from a patient with ETP-ALL diagnosed by the First Affiliated Hospital of Medical College of Zhejiang University, and leukemia mononuclear cells were immediately isolated; in the biosafety cabinet, 6 ml of the peripheral blood sample was dropped into a 15 ml sterile centrifuge tube into which 6 ml of a lymphocyte separation solution was added in advance, and the mixture was centrifuged at 2000 rpm for 20 minutes; after centrifugation, the mononuclear cell layer was put into a new 15 ml sterile centrifuge tube, 5 ml of sterile 1×PBS resuspended cells was added, and the mixture was centrifuged at 2000 rpm for 5 minutes; after discarding the supernatant, a sterile red blood cell lysate was added to lyse the cells at room temperature for 5 minutes, and then the mixture was centrifuged at 2000 rpm for 5 minutes; the supernatant was discarded 5 ml of an IMDM complete medium (IMDM 90%+fetal bovine serum 10%) was added to resuspend the cells, and the mixture was centrifuged at 1500 rpm for 5 minutes; the supernatant was discarded, 5 ml of the IMDM complete medium was added to resuspend the cells; a cell counting plate counted the cells, 1*10.sup.8 cells were added into a 25 cm culture flask, then the IMDM culture medium was added to 6 ml, the cells were mixed, and the culture flask was put into a constant temperature and humidity incubator at 37° C. to culture the cells; after 1 week, the IMDM culture medium was replaced with a new IMDM culture medium to remove the cell debris, and the culture continued; the culture medium was changed once a week.

[0026] Cell subculture: after 1-2 weeks of culture, the cells underwent apoptosis; the remaining cells without apoptosis were in a state of non-proliferation and non-death, and the culture medium was changed every week; when the cells were cultured for 2 months, they began to proliferate and grew in suspension; at this time, the cell culture medium was changed every 48-72 hours and the passage began; up to now, the cells have been passaged for more than 50 generations and is an immortalized cell strain.

[0027] According to the present application, cells grow in a suspension state, single cells grow, cells are round or oval, and the cell growth rate is stable. The cell stain was named ZYXY-T1, and was conserved in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on Jan. 20, 2021, with the preservation number of CCTCC NO: C202143.

Example 2 Biological Characteristics and Use of the Human T-Lymphoblastic Leukemia/Lymphoma Cell Strain

[0028] According to the present application, the cell ZYXY-T1 was cultured in an IMDM culture medium containing 10% fetal bovine serum, so that it could grow stably in vitro and be passaged stably; microscopically, the cells were suspended and grow individually, round or oval; wright-Giemsa staining showed that the cells were acute leukemia protocells with big hyperchromatic nuclei; by flow analysis, it was found that this cell strain has the typical surface antigen characteristics of ETP-ALL, and it is the first ETP-ALL cell strain established internationally; the cell strain can be used to study the pathogenesis of ETP-ALL, screen and/or evaluate/prepare tumor therapeutic drugs; developing tumor drug targets; preparing tumor diagnosis products; screening tumor biotherapy drugs/reagents; develop tumor-related bioengineering products, which are specifically as follows:

[0029] Morphological Observation

[0030] The cultured ZYXY-T1 cell strain was observed under an inverted microscope and photographed. As shown in FIG. 1a, the cells grew in a single suspension, and the cells were round or oval. 1*10.sup.6 cultured cells were put in a 1.5 ml EP tube and centrifuged at 1500 rpm for 5 minutes; the supernatant was discarded, 10 ul of a culture medium was added to suspend the cells, and then the slides were pushed; after the cell smear was dried, the cell smear was stained with a Gareth-Giemsa staining solution for 5 minutes, then rinsed and dried; the morphology of the cells was observed under the inverted microscope. As shown in FIG. 1b, the cells showed big hyperchromatic nuclei with a mononuclear state, and there were obvious spinous processes on the surface of the cells.

[0031] Observation of Proliferation Ability In Vitro

[0032] The cultured ZYXY-T1 cell strain was plated on a 96-well plate at the concentration of 1, 2, 4*10.sup.5/ml, and each well was plated with 100 P. L; at 0, 24, 48, 72 and 96 hours, 20 ul of a cell proliferation reagent MTS was added respectively; after 4 hours, the absorbance value of the 96-well plate was measured with a microplate reader, and the proliferation curves of cells at different plating concentrations were drawn by graphpad software, as shown in FIG. 2; the cells of the cell strain had good proliferation ability in vitro and showed malignant growth.

[0033] Flow Cytometry Surface Antigen Examination

[0034] The cultured cells (1*10.sup.6) were divided into 5 parts, packed in 5 clean and sterile EP tubes, and centrifuged at 1500 rpm; after 5 min, the supernatant was discarded and the cells were washed with 1×PBS; the cells were centrifuged at 1500 rpm, the supernatant was discarded after 5 min, 100 ul of 1×PBS resuspended cells were added into each EP tube, the first tube without antibody, the second tube with a CD1α antibody, the second tube with a CD8 antibody, the third tube with a CD5 antibody, and the fourth tube with a CD34 antibody, each antibody added with 10 ul; the cells were incubated at room temperature for 30 min, then 1 ml of 1×PBS was added, and the tube was centrifuged at 1500 rpm for 5 min; the supernatant was discarded, and 300 ul of 1×PBS was added into each tube to resuspend the cells; the expressions of CD1a, CD8, CD5 and CD34 were measured by a flow cytometry. As shown in FIG. 3, this cell strain does not express CD1a, CD5 and CD8 antigens, but highly expresses CD34, which accords with the definition of ETP-ALL and is a typical surface antigen expression characteristic of ETP-ALL.

[0035] Observation on the Tumorigenic Ability of Cells In Vivo

[0036] The cultured ZYXY-T1 cell strain was injected into the immunocompromised NCG mice of 6-8 weeks old through the tail vein at the amount of 5*10.sup.6/mouse. On the 18th and 25th day after the injection of cells and when the mice were dying, 100 ul of peripheral blood of the mice was taken, and 2 ml of an erythrocyte lysate was added to lyse the peripheral blood of the mice at room temperature for 5 min, and then the mixture was centrifuged at 2000 rpm for 5 min; the supernatant was removed, and the cells were resuspended with 1 ml of 1×PBS, and were centrifuged at 2000 rpm for 5 min; after one repetition, the cells were equally divided into two parts, 300 ul of 1×PBS was added into one part, and 300 ul of 1×PBS+anti-human CD45 antibody was added into the other part; after incubation in the dark at room temperature for 30 min, 1 ml of 1×PBS was added, and then the mixture was centrifuged at 2000 rpm for 5 min; the supernatant was discarded, and 300 ul of 1×PBS was added to each tube to resuspend the cells; then, the expression of CD45 was measured by a flow cytometry; the mice were killed when they were dying, the bone marrow cells of the mice were taken and resuspended with 1×PBS, then centrifuged at 2000 rpm for 5 min, the supernatant was removed, and the cells were resuspended with 1 ml of 1×PBS and centrifuged at 200 rpm for 5 min; after one repetition, the cells were divided into two equal parts, 300 ul of 1×PBS was added into one part, and 300 ul of 1×PBS+anti-human CD45 antibody was added into the other part; after incubation in the dark at room temperature for 30 min, 1 ml of 1×PBS was added, and then the mixture was centrifuged at 2000 rpm for 5 min; the supernatant was discarded, and 300 ul of 1×PBS was added to each tube to resuspend the cells; then, the expression of CD45 was measured by a flow cytometry; finally, the survival time of mice was recorded; the results are shown in FIG. 4; after the mice were injected with cells, the expression of CD45 in peripheral blood gradually increased with time (FIG. 4a); after the mice were injected with cells, they showed a good bone marrow homing ability (FIG. 4b), and all the mice finally got sick and died, with a median survival time of 34 days (FIG. 4c). It shows that this cell strain has good tumor ability in vivo, and it is a good cell material for constructing T-lymphoblastic leukemia/lymphoma animal model. It can be used as a cell material to study the development mechanism of ETP-ALL and individualized treatment in vitro. It can also be used to screen and evaluate ETP-ALL drugs in vivo and in vitro, and guide clinical medication.

[0037] STR Identification Of cells

[0038] The cultured cell strains and the newly isolated cells of the patients were all sent to Shanghai Yihe Biology for genotyping of STR and Amelogenin loci. The results indicated that the cell strain was unique and unmatched with the existing cell strains in the world, and it was completely matched with the genotype of the STR and Amelogenin loci of the newly isolated cells from patients, that is, there was no cross-contamination during the correct culture of the cell source. The genotyping of the STR and Amelogenin loci in cells is shown in Table 1.

TABLE-US-00001 TABLE 1 Genotyping results of STR and Amelogenin loci of cells STR information of cells for inspection Name of cells for inspection: ZYXY-T1 Loci Allele1 Allele2 Allele3 D5S818 10 13 D13S317 9 9 D7S820 12 13 D16S539 8 11 VWA 14 15 TH01 6 7 AMEL X Y TPOX 8 11 CSF1PO 12 12 D12S391 19 21 FGA 23 24 D2S1338 23 23 D21S11 30 31 D18S51 14 19 D8S1179 13 14 D3S1358 14 17 D6S1043 13 13 PENTAE 15 19 D19S433 13 13.2 PENTAD 10 11 D1S1656 17.3 18.3

[0039] The above examples are used to explain, but not to limit the present application. Any modifications and changes made to the present application within the scope of protection of the spirit and claims of the present application fall within the scope of protection of the present application.