HUMAN PRIMARY MYELOFIBROSIS CELL STRAIN AND USE THEREOF

Abstract

Providing a human primary myelofibrosis (PMF) cell strain, a construction method therefor and use thereof. The PMF cell strain is named as ZYXY-M2, and is deposited in China Center for Type Culture Collection (Wuhan University, Wuhan, China), with a preservation number of CCTCC NO: C202145. The present application is obtained by extracting and separating mononuclear cells from peripheral blood of a PMF patient and culturing in vitro for continuous natural passage. The leukemia cell strain is negative for JAK2, CALR, MPL mutation, positive for ASXL1, TP53, IKZF1, IDH1, FLT3 and TET1. The cell strain has good proliferation ability in vitro, and can be used as a cell material to study the pathogenesis of PMF and individualized treatment in vitro. Meanwhile, it can also be used to screen and evaluate drugs for in vitro and in vivo research of human PMF and guide clinical medication.

Claims

1. A human primary myelofibrosis cell strain, wherein the cell stain is named as a human myelofibrosis cell ZYXY-M2, which was deposited in China Center for Type Culture Collection on Jan. 20, 2021, with a preservation number of CCTCC NO: C202145.

2. The human primary myelofibrosis cell strain according to claim 1, wherein a cell morphology is primitive red blood cell, CD34 and CD11b are not expressed on a cell surface, but CD71 is expressed, and cells of the cell strain grow in suspension or with weak wall adherence.

3. The human primary myelofibrosis cell strain according to claim 1, wherein the cells are negative for JAK2 mutation, negative for CALR mutation, negative for MPL mutation, positive for ASXL1 mutation, positive for TP53 mutation, positive for IKZF1 mutation, positive for IDH1 mutation, positive for TET1 mutation and positive for FLT3 mutation.

4. A progeny cell of the human primary myelofibrosis cell strain according to any one of claim 1.

5. Use of the human primary myelofibrosis cell strain according to any one of claim 1 in any one or more selected from the following: a. study on molecular characteristic and therapeutic mechanism of bone marrow fibrosis; b. preparation of a tumor cell model or preparation of a tumor animal model; c. in vitro screening and/or in vitro evaluation of a prepared tumor therapeutic drug; d. development of a tumor drug target; e. preparation of a tumor diagnosis product; f. in vitro screening of a tumor biological therapy drug/reagent; and g. in vitro development and detection of a tumor-related bioengineering product.

6. The use according to claim 5, wherein an animal for preparing the tumor animal model is an immunodeficiency mouse or a C57BL6 mouse.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] The present application will be further explained with reference to the following examples and drawings.

[0023] FIG. 1 shows the results of staining of the human primary myelofibrosis cell strain Wright-Giemsa;

[0024] FIG. 2 is the expression chart of cell surface antigen of the human primary myelofibrosis cell strain;

[0025] FIG. 3 is a graph of cell growth of the human primary myelofibrosis cell strain at different cell densities;

[0026] FIG. 4 is the Circos diagram of genome variation of the human primary myelofibrosis cell strain.

DESCRIPTION OF EMBODIMENTS

[0027] The present application will be illustrated with reference to the following examples, but are not limited thereto. The experimental methods without specific conditions in the following examples are usually in accordance with conventional conditions.

Example 1 Preparation of a ZYXY-M2 Cell Strain

[0028] Primary cell culture: leukemia mononuclear cells were immediately isolated from the fresh high white blood cell isolated specimen (male, 62 years old, PMF, white blood cell 178.1*10.sup.9/L) obtained from the First Affiliated Hospital of Zhejiang University Medical College. In the bio-safety cabinet, 6 ml of a separation liquid was added dropwise into a 15 ml sterile centrifuge tube with 6 ml of lymphocyte separation liquid added in advance, and the mixture was centrifuged at 2000 revolutions for 20 minutes. After centrifugation, the mononuclear cell layer was added into a new 15 ml sterile centrifuge tube, and 5 ml of sterile 1×PBS resuspended cells was added, and the mixture was centrifuged at 2000 revolutions 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, then the mixture was centrifuged at 2000 revolutions for 5 minutes. After discarding the supernatant, 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 revolutions for 5 minutes. After discarding the supernatant, 5 ml of the IMDM complete medium was added to resuspend the cells. A cell counting plate was used to count cells, and 1*10.sup.8 cells were added into a 25 cm culture bottle, and then the IMDM medium was added to 6 ml of mixed cells, and the mixture was put into an incubator at 37 degrees Celsius with constant temperature and humidity to culture the cells. After 1 week, the medium was replaced by a new IMDM medium to remove the cell debris, and the culture continued. The culture medium was changed once a week.

[0029] Cell subculture: Cell apoptosis occurred after 2 weeks of culture. The remaining non-apoptotic cells proliferated very slowly, and the culture medium was changed every week. When the cells were cultured for 2.5 months, they began to proliferate and grew in suspension. At this time, the cell culture medium was changed every 72 hours and the passage began. Up to now, the passage of cells has exceeded 50 generations, showing an immortalized cell strain.

[0030] According to the present application, the cells grow in a suspended state or are weakly adherent to the wall (without trypsin digestion), and grow in groups, and the cells are round or oval, and the growth rate of the cells is stable. The cells are named ZYXY-M2, and the cells are preserved in China Typical Culture Collection Center (address: Wuhan, Wuhan University, China, zip code 430072) on Jan. 20, 2021, with a preservation number of CCTCC NO: C202145.

Example 2: Biological Characteristics and Application of a Human Acute Myeloid Leukemia Cell Line

[0031] According to the present application, an IMDM culture medium containing 10% fetal bovine serum was adopted to culture the cell strain, so that the cell strain could grow stably in vitro and be passaged stably. Microscopically, the cells were suspended or weakly adherent to the wall, growing individually or in groups, round or oval. According to Wright-Giemsa staining, the cells were mainly primitive red blood cells, with a large size, dark blue cytoplasm, a large number of vacuoles in some parts, a lightly stained area around the nucleus, fine granular nuclear chromatin and obvious nucleoli. The cell strain showed no expression of CD34 and CD11b, but high expression of CD71, as tested by flow cytometry. The whole exon sequencing of the cell strain showed that the cell strain negative mutations of JAK2, CALR and MPL, poor prognosis of PMF or positive mutations of TP53, ASXL1 and TKZF1 of genes related to leukemia transformation. In addition, it showed positive mutations of FLT3 and TET1 genes related to acute myeloid leukemia. The cell strain can be used for preparing tumor cell models or preparing tumor animal models; screening and/or evaluating/preparing tumor therapeutic drugs; developing tumor drug targets; preparing tumor diagnosis products; screening tumor biotherapy drugs/reagents; developing tumor-related bioengineering products, which are specifically as follows:

[0032] Morphological Observation

[0033] The cultured ZYXY-M2 cell strain was placed under an inverted microscope to observe that the cells grew in a clustered suspension or weakly adhered to the wall, and the cells were round or oval. 1*10.sup.6 cultured cells were added in a 1.5 ml EP tube, and were centrifuged at 1500 revolutions for 5 minutes. The supernatant was discarded, and 10 ul of a culture medium was added to suspend the cells, followed by smearing on a slide; 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 cell morphology was observed under the inverted microscope. As shown in FIG. 1, the cells were mainly primitive red blood cells, with a large size, dark blue cytoplasm, a large number of vacuoles in some parts, a lightly stained area around the nucleus, fine granular nuclear chromatin and obvious nucleoli.

[0034] Cell Surface Antigen Detection

[0035] 1*10.sup.6 of the cultured cells were taken and divided into three clean and sterile EP tubes, and centrifuged at 1500 revolutions. After 5 min, the supernatant was discarded and the cells were washed with 1×PBS. After centrifugation at 1500 revolutions, the supernatant was discarded after 5 min, and then 100 ul of 1×PBS resuspended cells was added into each EP tube, the first tube without antibody, the second tube with a FITC-labeled anti-human CD34 antibody and a PE-labeled anti-human CD11b antibody, the third tube with a FITC-labeled anti-human CD71 antibody, with each antibody being 10 ul; the mixture was incubated at room temperature for 30 min, 1 ml of 1×PBS was added, and the mixture was centrifuged at 1500 revolutions for 5 min. The supernatant was discarded, and 300 ul of 1×PBS was added into each tube to resuspend the cells, and then the expression of CD34, CD11b and CD71 was measured by an up-flow analyzer. As shown in FIG. 2, this cell strain does not express CD34 and CD11b antigens, but highly expresses the erythroid marker CD71(73.25%), which is consistent with the morphology of proerythroblasts.

[0036] Observation of Proliferation Ability In Vitro

[0037] The cultured ZYXY-M2 cell strain was plated on the 96-well plate at the concentrations of 1, 2, 4*10.sup.5/ml, with 100 ul per well. At 1, 24, 48, 72 and 96 hours, 20 ul MTS was added, respectively. After 4 hours, the absorbance of the 96-well plate was measured by an enzyme-labeled instrument, and the proliferation curves of the cells at different plating concentrations were drawn as shown in FIG. 3. The cells of the cell line have good proliferation ability in vitro, showing malignant growth.

[0038] Whole Exon Sequencing of Cells

[0039] After counting the cultured cells, a cell culture fluid containing 5*10.sup.6 cells were centrifuged at 1500 revolutions for 5 min, the supernatant was removed leaving the cell mass; 1×PBS was added to resuspend the cells, followed by centrifugation at 1500 revolutions for 5 min, and the supernatant was removed, leaving the cell mass. The genomic DNA was extracted from the samples, and the DNA samples qualified for electrophoresis were randomly broken into fragments of 150 bp-220 bp by Covaris. The library was built and captured by an Agilent Sureselect Human All Exon V6 kit. The DNA fragments were repaired at the end, added with a ployA tail, followed by steps such as sequencing adapter, purification, capturing by magnetic beads and PCR amplification, and finally the library was built. After the library was tested to be qualified, double-ended sequencing was carried out by a sequencer. After the sequencing machine got the raw sequencing data (Raw data), it entered the bioinformatics analysis process, which was divided into two stages: 1, quality evaluation of sequencing data: whether the sequencing of the database had reached the standard was evaluated mainly by the statistics of sequencing error rate, data volume, comparison rate, coverage and the like, and follow-up analysis was carried out if the standard was met.

[0040] 2, Variation information analysis: high-quality sequencing sequences were compared to the reference genome, and the variation information in the sample was detected, and the detected variation information was analyzed and interpreted. The genomic variation Circos is shown in FIG. 4. results of SNP and InDel mutation locus detection and analysis in ZYXY-M2 cell genome and mutation information closely related to hematological malignancies are summarized in Table 1. It can be known from the table that the cells of the cell strain showed negative mutations of JAK2, CALR and MPL, poor prognosis of PMF or positive mutations of TP53, ASXL1 and TKZF1 of genes related to leukemia transformation. In addition, it showed positive mutations of FLT3 and TET1 genes related to acute myeloid leukemia. It is suggested that the cells have certain value in the study of PMF transformation, including the study of the therapeutic mechanism or pathogenesis of PMF transformation into leukemia and the screening of targeted drugs for the above mutations.

TABLE-US-00001 TABLE 1 Summary results of SNP and InDel mutation sites in ZYXY-M2 cells Mutation Mutation Gene name site Domain type JAK2 — CALR — MPL — LNK — TET2 — ASXL1 C20: 31022959 T < C Exon-non-synonymous M IDH1 C2: 209120640 C < T Upstream M IDH2 — EZH2 — DNMT3A — CBL — RAS — KRAS — HRAS — NRAS — IKZF1 C7: 50430033 A < G Exon-non-synonymous M TP53 C17: 7579472 G < C Exon-non-synonymous WT/M SF3B1 — SRSF2 — U2AF1 — FLT3 C13: 28611358 C < T Exon-non-synonymous WT/M C13: 28624294 G < A Exon-non-synonymous WT/M C13: 28674628 T < C Exon-non-synonymous WT/M TET1 C10: 70332580 A < G Exon-non-synonymous WT/M C10: 70332672 T < G Exon-non-synonymous WT/M C10: 70332862 C < T Exon-non-synonymous WT/M C10: 70445539 A < G Exon-non-synonymous WT/M C10: 70405855 A < G Exon-non-synonymous WT/M Note: M means that only mutant type is detected in the cell; WT/M means that both wild type and mutant type of the cell are detected.

[0041] Cell STR Identification

[0042] The cultured cells were sent to Shanghai Blowing for genotyping of the STR locus and Amelogenin site. The results suggested that the cell strain was unique because it did not match the existing cell strains in the world. Furthermore, the genotyping matching degree with STR locus and Amelogenin locus of the cell just isolated from the patient was up to 97%, and they were cells from the same source, that is, the culture process of the cell source was correct and there was no cross-contamination. The genotyping of STR and Amelogenin loci in cells is shown in Table 2.

TABLE-US-00002 TABLE 2 Genotyping results of STR locus and Amelogenin locus of cells STR information of test cells Test cell name: ZYXY-M2 Loci Allele1 Allele2 Allele3 D5S818 10 14 D13S317 8 9 D7S820 10 12 D16S539 11 13 VWA 16 18 TH01 7 9.3 AMEL X Y TPOX 8 11 CSF1PO 12 13 D12S391 17 19 FGA 21 22 D2S1338 23 25 D21S11 29 29 D18S51 14 16 D8S1179 14 15 D3S1358 16 17 D6S1043 11 14 PENTAE 14 15 D19S433 14 14 PENTAD 10 11 D1S1656 13 17

[0043] The above examples are used to explain, rather than 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.