CN117946977A - Human chronic granulocytic leukemia cell strain and application thereof - Google Patents
Human chronic granulocytic leukemia cell strain and application thereof Download PDFInfo
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Abstract
The invention discloses a human chronic granulocytic leukemia cell strain and application thereof, which is established by chronic granulocytic leukemia cells in chronic stage of the first strain internationally, is named as human chronic granulocytic leukemia cells YYXY-M6, and is preserved in China center for type culture Collection (university of Wuhan, china) for 24 months in 2023, with a preservation number of CCTCCNO: C2023219. The leukemia cell strain has an original cell morphology, is provided with three karyotypes of t (6:11) (q 25:q 23), del (11) (q 23) and normal karyotypes (46, XX), has a good in-vitro proliferation capacity, can be used for researching chronic granulocytic leukemia and transforming chronic phase into acute phase, can be used for researching chronic granulocytic leukemia occurrence and development mechanism research of the negative chronic granulocytic leukemia and individuation treatment of cell materials researched in vitro, can be used for researching chronic granulocytic leukemia and transforming chronic phase into acute phase in vitro and in vivo, and can be used for screening and evaluating medicines for treating chronic granulocytic leukemia and guiding clinical medicines.
Description
Technical Field
The invention relates to the fields of biology and oncology, and relates to a human chronic granulocytic leukemia cell strain, a construction method and application thereof.
Background
Chronic myelogenous leukemia (Chronic myeloid leukemia, CML) is a type of leukemia that is manifested by excessive proliferation of late and young granulocytes in the peripheral blood or bone marrow, inhibiting the normal hematopoietic system of the bone marrow, and is a chronic course of disease, clinically accompanied by hepatomegaly and splenomegaly. CML accounts for about 15% of adult leukemia, and the incidence rate in China is about 0.36/10 ten thousand. In the course of the disease, CML is divided into chronic, acceleration and acute phase, and if the chronic phase is untreated, the disease further progresses to the acceleration or acute phase, further turning into acute leukemia. Chronic myeloid leukemia has characteristic Philadelphia chromosome, and generates specific BCR-ABL fusion gene, and the application of the targeted BCR-ABL tyrosine kinase inhibitor in CML enables the overall survival rate of CML to reach more than 90% in 10 years, and CML is also a very few malignant tumors as chronic disease management.
CML generally has a good prognosis under the treatment targeted by the tyrosine kinase inhibitors, but once CML enters the acceleration or rapid change phase, the prognosis is very poor and the overall survival is less than 1 year. However, it is not clear how CML progresses from the chronic phase to the acceleration phase and the rapid phase. The CML patients who are regularly applied with the tyrosine kinase inhibitor have long natural course, and most patients can survive for a long time. It is therefore difficult to study the mechanism of CML progression from the chronic, acceleration, and rapid phase of a single patient. The construction of CML cell lines in different periods has important significance for the research of the mechanism of CML disease progression and the research and development of medicines. The K562 cell strain is a CML cell strain established by leukemia cells of patients in the acute stage of chronic granulocytic leukemia in 1976, is also an international second cell strain established by blood tumor, and is widely applied to research of pathogenesis, targeted drugs and animal models of CML at present. The K562 cell strain BCR-ABL fusion gene is positive, and provides a good cell tool for the discovery and preclinical research of the tyrosine kinase. Only 13 CML strains were successfully established in the follow-up period, but all the cell strains are established from the CML acute phase, and all the cell strains are positive for the BCR-ABL gene. Studies have shown that some patients with BCR-ABL fusion gene turn negative when CML enters the acceleration phase or the rapid change phase, and this part of patients have poorer prognosis and often lose sensitivity to the tyrosine kinase inhibitor. CML patients may exhibit acute myeloid leukemia, or acute lymphoblastic leukemia, or mixed cell leukemia after entering the acute phase, with poor overall prognosis. The key problems that current CML research and therapeutic fields need to solve are: 1. revealing the mechanisms of CML progression, particularly in the acute phase; 2. searching for a drug that prevents CML progression; 3. disclosed are mechanisms of CML tyrosine kinase inhibitor resistance. The cell strains in different periods are established, and the method has important significance for researching the pathogenesis, the progress mechanism, the drug resistance mechanism and the drug screening of CML. At present, CML cell strains are not established in China, and cell strains established in the CML chronic phase are not reported internationally.
Chromosomal karyotype abnormalities play an important role in the development and resistance of tumors. Among them, t (6, 11) translocations are considered as tumor-associated clones, where it has been shown that t (6:: 11) (q 27:: q 23) translocations play an important role in the occurrence of AML. At the same time, T (6:11) (q 27:q 23) translocation was also reported in T cell acute lymphoblastic leukemia and chronic eosinophilic leukemia, but no related report of T (6:11) (q 25:q 23) translocation has been found so far. The del (11) (q 23) chromosomal changes are found in myelodysplastic syndrome, turner syndrome, primary testicular sign, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute myeloid leukemia. The presence of del (11) (q 23) was found to be associated with poor patient prognosis in chronic lymphocytic leukemia. A patient with chronic neutrophilic leukemia has also been reported to have del (11) (q 23) unresponsive to hydroxyurea and cytarabine treatment. However, the specific mechanism of action of del (11) (q 23) is not yet known. At present, no blood tumor cell lines with t (6:11) (q 27:q 23) abnormality or del (11) (q 23) abnormality are internationally established, nor are blood tumor cell lines with t (6:11) (q 27:q 23) abnormality and del (11) (q 23) abnormality simultaneously established.
Because each patient has different genetic background and gene expression characteristics, tumors have different molecular mechanisms in different periods. The tumor cell strain retains the biological characteristics of the tumor, can be subjected to in vitro continuous subculture, can complete a plurality of experiments which cannot be carried out in vivo, and can overcome the defects that primary cells are limited in cell number and cannot be continuously proliferated in vitro for a long time. Provides an irreplaceable cell tool for the research of tumors. Therefore, CML cell lines in different periods are established, the progress of CML, particularly the mechanism of rapid change, is revealed by means of advanced molecular biology technology, and the cell model and the animal model are utilized to screen CML rapid change therapeutic drugs, so that the method has important significance for further improving the poor prognosis after CML progress.
Disclosure of Invention
The invention aims to provide a human chronic granulocytic leukemia cell strain established from human chronic granulocytic leukemia chronic stage cells, a construction method and application thereof.
The aim of the invention is realized by the following technical scheme: a human chronic granulocytic leukemia cell strain is named human chronic granulocytic leukemia cell YYXY-M6, and is preserved in China center for type culture Collection (CCTCC NO: C2023219) at 24 days 7 of 2023.
The invention also provides daughter cells of the human chronic granulocytic leukemia cell strain described above.
The human chronic granulocytic leukemia cell YYXY-M6 provided by the invention is established by chronic granulocytic leukemia chronic stage cells, and is expressed as primitive cell morphology, and the BCR-ABL gene is negative and is suspension cell.
The invention also provides the use of a human chronic granulocytic leukemia cell strain as described above, selected from any one or more of:
a. The mechanism research for the chronic phase to the acute phase of the chronic granulocytic leukemia;
b. The chromosome karyotype analysis after the acute phase of the chronic granulocytic leukemia is complex karyotype, and specifically comprises the following steps: t (6:11) (q 25:q 23), del (11) (q 23) and normal karyotype (46, XX) are subjected to mechanism research;
c. The method is used for the mechanism research of BCR-ABL negativity after the acute phase of chronic granulocytic leukemia;
d. Preparing a tumor cell model or preparing a tumor animal model; wherein the tumor cell model comprises daughter cells established in the cell line or cells established in the cell line by transfecting genes with fluorescent markers. The animal tumor model comprises an animal model of chronic granulocytic leukemia established by subcutaneous tumor-bearing or tail vein injection modeling.
E. screening and/or evaluating/preparing tumor therapeutic drugs in vitro; the method for screening tumor chemotherapeutic drugs can be as follows: and (3) adding different therapeutic drugs into the human chronic granulocytic leukemia cell YYXY-M6 culture medium, and observing the cytotoxicity of the drugs to obtain preliminary effective candidate drugs. Then, candidate drugs are applied to the cells, the half inhibition concentration (IC 50) of the screened effective drugs is calculated, the drugs with the lowest IC50 are selected to further act on an animal model, and compared with the survival time, the tumor size, the metastasis condition and the like of animals without application of drugs, the drugs for treating the chronic granulocytic leukemia are screened and obtained. Wherein, the animal model adopts an immunodeficiency mouse.
F. Developing tumor drug targets;
g. preparing a tumor diagnosis product;
h. In vitro screening of tumor biotherapeutic drugs/agents.
The invention also provides a construction method of the human chronic granulocytic leukemia chronic stage cell strain, which comprises the following steps: obtaining the blood separated by a blood cell separator of a fresh chronic stage chronic granulocytic leukemia patient. 5ml of the separated blood was added dropwise to a 15ml clean sterile centrifuge tube, to which 5ml of human peripheral blood lymphocyte separation solution was added in advance, and centrifuged at 2000rpm for 20 minutes. And (3) taking a leucocyte layer at the junction of lymphocyte separation liquid and plasma to a new 15ml sterile centrifuge tube after centrifugation, adding 5ml sterile 1xPBS pre-cooled at 4 ℃ to resuspend cells, and centrifuging for 2000 revolutions per 5 minutes. The supernatant was then discarded, and the cells were resuspended in 5mlIMDM complete medium (IMDM 90% + fetal bovine serum 10%), centrifuged, 1500 revolutions, and 5 minutes. The supernatant was discarded, 5mlIMDM complete medium was added and the cells resuspended. The automatic cell counter counts the cell density of the cell suspension, 1-2 x 10 8 cells are taken and added into a T25 cell culture flask, IMDM culture medium is added to 6 ml-8 ml for uniformly mixing the cells, and the mixed cells are placed into a 37 ℃ constant temperature and humidity incubator for culturing the cells. After 5-7 days, new IMDM complete medium is replaced by centrifugation, and then, according to the growth state of the cells, the new IMDM complete medium is replaced every 1 week until the cells start to proliferate before the cells do not start to proliferate.
The beneficial effects of the invention are as follows: the human chronic granulocytic leukemia cells are separated from peripheral blood of CML patients in a chronic period, and can realize stable proliferation in vitro and continuous passage under the condition of no stimulus factor in vitro, and the cells have stable shape in vitro and accord with clinical tumor biological characteristics. The human chronic granulocytic leukemia cell strain originates in CML chronic stage, the cell morphology after strain establishment shows original cell morphology, CD 13/CD 38/HLA-DR/TdT/CD 19/CD 20/CD 22 is expressed by flow typing, chromosome karyotype analysis is accompanied with t (6:11) (q 25:q 23), del (11) (q 23) and normal karyotype (46, XX) karyotype, and RT-PCR analysis is carried out to obtain the BCR-ABL fusion gene negative. The human chronic granulocytic leukemia cells can be used for the pathogenesis of CML, in particular the research on the mechanism of CML from chronic phase to acute phase. The cell can be used for analyzing the curative effect of the novel anti-leukemia drug and the combined scheme, screening and evaluating the leukemia drug, and can be used for guiding clinical medication.
Drawings
The invention is further illustrated by the following examples and figures;
FIG. 1 shows the results of primary and established cell lines of the human chronic myelogenous leukemia patient after staining with Rayleigh-Giemsa;
FIG. 2 is a graph showing the cell growth curves of the human chronic myelogenous leukemia cell line at different cell densities;
FIG. 3 shows the results of expression of a portion of the surface antigens of primary cells and established cell lines of the human chronic myelogenous leukemia patient; wherein A-D respectively correspond to CD13, CD38, HLA-DR, tdT surface antigens;
FIG. 4 shows the results of expression of another portion of the surface antigens of primary cells and established cell lines of the human chronic myelogenous leukemia patient; wherein A-D correspond to CD19, CD20, CD22, CD4 surface antigens, respectively;
FIG. 5 shows the results of a karyotype analysis of cells of the human chronic granulocytic leukemia cell line; wherein A-C corresponds to t (6:11) (q 25:q 23), 46, XX, del (11) (q 23) and normal karyotypes (46, XX), respectively;
FIG. 6 shows the expression level of BCR-ABL gene in primary cells and established cell lines of the human chronic myelogenous leukemia patient.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental procedure, in which specific conditions are not noted in the examples below, is generally followed by conventional conditions.
Example 1YYXY-M6 preparation of cell lines
Primary cell culture: peripheral blood isolated specimens of 5ml (female) of CML patients with clear chronic phase for diagnosis obtained from the hospitals of affiliated people at the university of Ningbo were immediately isolated for leukemia cells. In a biosafety cabinet, 5ml of blood sample is dripped into a 15ml sterile centrifuge tube, to which 5ml of lymphocyte separation liquid is added in advance, and centrifuged for 2000 revolutions for 20 minutes. After centrifugation, the buffy coat cells were removed to a new 15ml sterile centrifuge tube, 5ml sterile 1xPBS was added to resuspend the cells, and the cells were centrifuged for 2000 revolutions for 5 minutes. The supernatant was discarded, and cells were resuspended in 5mlIMDM complete medium (IMDM 90% + fetal bovine serum 10%), centrifuged, 1500 revolutions, and 5 minutes. The supernatant was discarded, 5mlIMDM complete medium was added and the cells resuspended. The cell counting plate counts the cells, 1x 10 8 cells are added into a 25cm culture flask, then IMDM culture medium is added to 6ml of mixed cells, and the mixed cells are placed into a 37 ℃ constant temperature and humidity incubator to culture the cells. After 1 week, cell debris was removed by low-speed centrifugation, and the culture was continued with replacement of the fresh IMDM complete medium. The medium was changed once a week later.
And (3) cell subculture: cells undergo apoptosis after 1 to 2 weeks of culture. The remaining non-apoptotic cells were in a non-proliferating, non-dead state, after which the medium was changed weekly. Cells began to proliferate and grew in suspension when cultured for 3 months. At this time, the cell culture medium is replaced every 48 to 72 hours and the passage is started, and the cells are passaged for more than 50 generations to present, so as to form immortalized cell strains.
In the invention, cells grow in a suspension state, single cells or masses grow, the cells are round or oval, the cell growth speed is stable, the cells are named as human chronic granulocytic leukemia cells YYXY-M6, and the cells are preserved in China center for type culture Collection (address: china, university of Wuhan, and the university of Wuhan) for 7 months and 24 days in 2023, and the preservation number is CCTCC NO: C2023219.
Example 2 biological Properties of human chronic granulocytic leukemia cell Strain and application
The invention adopts IMDM culture medium containing 10% fetal bovine serum to culture cell YYXY-M6, so that the cell can stably grow in vitro and stably pass. Cells were observed under a microscope to grow in suspension as single or in clusters, round or oval. Rui-Giemsa stained cells were acute leukemia primordial cells and nuclei were stained profoundly (FIG. 1). The cell strain which is established in the chronic stage of the chronic granulocytic leukemia patient is the first strain which is established internationally at present by expressing CD 13/CD 38/HLA-DR/TdT/CD 19/CD 20/CD 22/CD 4 (figures 3-4) through flow typing, carrying t (6:: 11) (q 25:: q 23), 46, XX, del (11) (q 23) and normal karyotype (figure 5), and carrying out RT-PCR analysis on the negative gene fusion of BCR-ABL. The cell strain can be used for researching pathogenesis from chronic stage to acute stage of chronic granulocytic leukemia, screening and/or evaluating/preparing tumor therapeutic drugs; developing tumor drug targets; preparing a tumor diagnosis product; screening for tumor biotherapeutic drugs/agents; development and detection of tumor-related bioengineering products. The method comprises the following steps:
Morphological observation
Cells of primary cells of a patient with chronic myelogenous leukemia and cultured YYXY-M6 cell line were taken 1×10 6 cells in 1.5mlEP tubes, centrifuged for 1500 revolutions for 5 min, and 10 μl of medium was added to resuspend the supernatant and then the cells were pushed. After the cell smear is dried, the cell smear is stained with gares-giemsa dye solution for 5 minutes, rinsed and dried. The cell morphology was observed under an inverted microscope, and as shown in FIG. 1A, primary cells of CML patients exhibited morphological characteristics of chronic stage of chronic granulocytic leukemia, cells were mainly mature granulocytes. As shown in FIG. 1B, YYXY-M6 cell lines showed large deep nuclear staining and mononuclear state, showing original cell morphology.
In vitro proliferation ability observation
Cells of the cultured YYXY-M6 cell line were plated in 96-well plates at a concentration of 1,2,4,8 x 10 5/ml, 100 μl of each well was plated at 0, 24, 48, 72, 96, 120 hours, 20 μl of cell proliferation reagent MTS was added respectively, and after 4 hours, the absorbance of the 96-well plates was measured at 492nm by using a microplate reader, and proliferation curves of the cells at different plating concentrations were plotted as shown in FIG. 2 using graphpad mapping software, and proliferation rates were related to cell plating densities.
Flow surface antigen inspection
The cultured cells were taken 1x 10 6, a total of 23 parts, packed in 23 clean sterile EP tubes, centrifuged at 1500 rpm for 5min, the supernatant discarded and the cells washed once with 1 xPBS. Centrifuging for 1500 turns, discarding supernatant after 5min, adding 100 μl of 1xPBS resuspended cells into each EP tube, adding no antibody into the 1 st tube, adding different surface antibodies into the 2 nd tube to the 23 rd tube respectively, adding 5 μl of each antibody in the sequence of ：CD11b/CD13/CD14/CD33/CD34/CD36/CD38/HLA-DR/CD2/CD3/CD4/CD7/CD8/CD10/CD19/CD20/CD22/CD117/TdT/CD64/CD66c/MPO,, incubating for 30min at room temperature, adding 1ml of 1xPBS, mixing uniformly, and staining after membrane rupture of intracellular antigens by using BD membrane rupture. Centrifuging for 1500 revolutions for 5min, and discarding supernatant. The expression level of cell surface antigen was measured by an up-flow analyzer after adding 300. Mu.l of 1xPBS resuspended cells per tube. As a result, the cell line expressed the CD13\CD38\HLA-DR\TdT\CD19\CD20\CD22\CD4 surface antigen as shown in FIGS. 3-4.
Chromosome karyotyping
The cultured cells are added with colchicine to make the final concentration to be 0.05g/ml, and are evenly shaken and then placed in a 37 ℃ incubator for 2-3 hours. The cells were harvested. 0.075mol/L KCl solution preheated to 37℃is slowly added along the tube wall, at least 8ml of which is placed in a 37℃incubator for 40min. Then, a cell suspension was prepared after adding a fixative. Cell chromosome banding was performed using the R banding method. The cell nucleus type is defined. As a result, as shown in FIGS. 5A-C, the cells of the cell line had t (6:: 11) (q 25:: q 23) (FIG. 5A), del (11) (q 23) (FIG. 5B) and normal karyotype (FIG. 5C).
BCR-ABL gene detection
Cell counting is carried out on leukemia cells and YYXY-M6 cell lines of a patient with chronic myelogenous leukemia, at least 1 x 10 7 cells are taken, centrifugation is carried out for 1500 turns/5 min, supernatant is discarded, cell clusters are left to be taken to a-80 ℃ ultralow temperature refrigerator, the subsequent BCR-ABL fusion gene is detected by a Dios Bei Ken medical detection third party platform, and the result is shown in figure 6, the primary cell BCR-ABL gene expression 83.94% of the patient with chronic myelogenous leukemia is shown in the specification, and the BCR-ABL gene expression is not detected by YYXY-M6 cell lines.
Cell STR identification
Cells of the cultured cell line and cells of the patient just separated are both sent to Shanghai ptera and organisms for genotyping at the STR site and the Amelogenin site of the cells. The results suggest that the cell lines are not matched with internationally existing cell lines, are unique, and are 100% completely matched with genotyping of the as-isolated cell STR site and the Amelogenin site of the patient, i.e., no cross contamination during proper cell-derived culture. Genotyping of the STR site and the Amelogenin site of the cell is shown in Table one.
Table one: genotyping results of STR site and Amelogenin site of cells
The above embodiments are intended to illustrate the present invention, not to limit it, and any modifications and changes made to the present invention within the spirit of the present invention and the scope of the appended claims fall within the scope of the present invention.
Claims (5)
1. A human chronic granulocytic leukemia cell strain, characterized in that the cell strain is named human chronic granulocytic leukemia cell YYXY-M6, which is preserved in China Center for Type Culture Collection (CCTCC) at the 24 th month of 2023, with the preservation number of CCTCC NO: C2023219.
2. The human chronic granulocytic leukemia cell strain according to claim 1, characterized in that said human chronic granulocytic leukemia cells YYXY-M6 are established from chronic granulocytic leukemia chronic stage cells, presenting primitive cell morphology, chromosome karyotype analysis being complex karyotypes, in particular: t (6:11) (q 25:q 23), del (11) (q 23) and normal karyotype (46, XX) are negative, and the cell strain is a suspension cell.
3. A progeny cell of the human chronic granulocytic leukemia cell strain of claim 1.
4. Use of a human chronic granulocytic leukemia cell strain according to claims 1-2 or a progeny cell according to claim 3, characterized in that it is selected from any one or more of the following:
a. The mechanism research for the chronic phase to the acute phase of the chronic granulocytic leukemia;
b. The chromosome karyotype analysis after the acute phase of the chronic granulocytic leukemia is complex karyotype, and specifically comprises the following steps: t (6:11) (q 25:q 23), del (11) (q 23) and normal karyotype (46, XX) are subjected to mechanism research;
c. The method is used for the mechanism research of BCR-ABL negativity after the acute phase of chronic granulocytic leukemia;
d. Preparing a tumor cell model or preparing a tumor animal model;
e. Screening and/or evaluating/preparing a tumor therapeutic agent;
f. Developing tumor drug targets;
g. preparing a tumor diagnosis product;
h. screening tumor biological therapeutic drugs/agents.
5. The use of claim 4, wherein the animal used in the tumor animal model is an immunodeficient mouse.
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