CN110438081B - Malignant clone cell strain of mantle cell lymphoma and establishing method and application thereof - Google Patents
Malignant clone cell strain of mantle cell lymphoma and establishing method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of tumor cell line establishment, and particularly relates to a tumor malignant clone cell line with an important abnormal developmental differentiation stage of mantle cell lymphoma, and an establishment method and application thereof. The invention provides a human mantle cell lymphoma malignant clone cell strain containing early malignant clones, named as human mantle cell lymphoma malignant clone cell strain JeKo-1-spherioid, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2018264. The invention uses mature JeKo-1-partial cell strain as the inducing object to obtain the malignant clone cell strain containing the main malignant differentiation stage of mantle cell lymphoma by adopting a continuous culture natural purification method, and the obtained cell strain is highly matched with the primary cells of a patient.
Description
Technical Field
The invention belongs to the technical field of tumor cell line establishment, and particularly relates to a malignant tumor clone cell line with an important abnormal developmental differentiation stage of mantle cell lymphoma and an establishment method thereof.
Background
The tumor is composed of cell populations with malignant clone proliferation at different stages, wherein the most important to the generation and development of the tumor is the development of the malignant clone, particularly the clone at the early stage, and the cell populations have important significance for further comprehensive and objective research on the tumor. The focus of tumor research focuses on the malignant clonal evolution law among cells in the stage of differentiation and dysplasia, not only further deepens the understanding of tumor occurrence, but also has profound influence on the diagnosis and treatment, prognosis judgment and drug development of tumors.
At present, the study of the evolution of malignant clones is still poorly defined, while the study of mantle cell lymphoma is just in its infancy. Classical research protocols: 1. carrying out differentiation and screening of dry markers through cell strains and clinical specimens; 2. studies were performed by side population cell analysis in combination with tumor markers. Although recent studies report that the starting cells of Hodgkin Lymphoma (HL) are a group of cells expressing CD20 (mature B cell surface marker), CD27 (memory B cell antigen) and stem cell marker (ALDH), the rules of clone evolution in non-hodgkin lymphoma (NHL) are rarely confirmed by studies. Analyzing the origin of malignant clone of lymphoma from cells at different stages of hematopoietic system provides further understanding for the research of pathogenesis of lymphoma.
The primary cell of B-cell lymphoma is currently under the following hypotheses: (1) derived from common lymphoid progenitor Cells (CLP). (2) originated from GC. Sehmitz et al detected two tumor cells with the same SHM in 1 lymphoma patient, one was DLBCL and the other was HL; however, DLBCL cells also had p53 gene mutation, suggesting that a part of tumor cells secondarily generated p53 inactivation after GC SHM, resulting in a new malignant clone. There are only two reports on mantle cell lymphoma, both demonstrating that the marker for early stage progenitor cells of mantle cell lymphoma is a CD19 negative population of cells.
As can be seen from the above description, the major bottleneck in the current limited tumor research is the very low proportion of cells in the early stage of the cell model studied by the inventor and the lack of cell models containing the stages of tumor differentiation and dysplasia consistent with the patient. Previous research has made many beneficial attempts: 1. collecting the suspension spheres in the serum-containing culture medium and culturing the suspension spheres in the serum-free culture medium; 2. directly transferring the tumor cells from a serum culture medium to a serum-free culture medium for culture; 3. performing flow type continuous multi-generation sorting on the tumor marker; 4. flow cytometric sorting by multi-passage side group. The biggest problem of the above research is to establish a pure early stage single cell component cell line as far as possible, neglecting the fact that the differentiation of the dry early malignant clone tumor cells is continuously generated, the malignant clones at each stage of the tumor are separately and separately treated, the cell model research has certain sidedness, and the objective research should use the cell model consistent with the malignant clones in vivo as the research object. Moreover, the above studies also show the following drawbacks: the culture difficulty is high, and the cell activity is poor, so that a stable cell line containing malignant clones in various differentiation stages of tumors, particularly early stages, which can be used for experiments is difficult to obtain.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects, the invention adopts a continuous culture and natural purification method to obtain a malignant clone cell strain containing the main malignant differentiation stage of mantle cell lymphoma by taking a mature JeKo-1-parent cell strain as an induction object, and the obtained cell strain is highly matched with primary cells of a patient.
The technical scheme of the invention is as follows:
the first technical problem to be solved by the invention is to provide a human mantle cell lymphoma malignant clone cell strain containing early malignant clones, named JeKo-1-spherioid, which is preserved in China Center for Type Culture Collection (CCTCC) in 12 months and 21 days in 2018, wherein the preservation number is CCTCC NO: c2018264, the preservation address is: wuhan, wuhan university, zip code: 430072.
the human mantle cell lymphoma malignant clone cell strain JeKo-1-spherioid is established by taking JeKo-1-parent (purchased from cell banks of Chinese academy of sciences in 2015) as mother cells and adopting a continuous culture and natural purification method.
The human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid has the following biological characteristics:
1) The human mantle cell lymphoma malignant clone cell strain JeKo-1-spherioid has strong in vitro cloning capacity, and p is less than 0.0001;
2) The human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid is highly inosculated with clinical specimens, and the biological characteristics of in vivo and in vitro tumors are highly inosculated with the clinical specimens;
3) CD19 negative cells in the human mantle cell lymphoma cell strain JeKo-1-partial account for 0.34% +/-0.07%; the proportion of CD19 negative cells in the human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid is 88.97% + -3.50%.
The human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid of the invention has the following biological characteristics: strong in vitro cloning ability (p < 0.0001), high expression of oncogene and xerosis gene: bcl-9, bcl-2, bcl-11b, sox2, CD133, c-myc, CCND1, and bmi-1 (P < 0.0001), CD19/IgM subclone type highly matched clinical specimens, and in vivo tumor biology characteristics highly matched clinical specimens.
The second technical problem to be solved by the present invention is to provide the method for establishing the human mantle cell lymphoma malignant clonal cell strain, wherein the method uses the human mantle cell lymphoma cell strain JeKo-1-parent as an induction object, and adopts a continuous culture natural purification method to establish the human mantle cell lymphoma malignant clonal cell strain JeKo-1-spherioid.
Further, the continuous culture natural purification method comprises the following steps:
the method comprises the following steps: after the human mantle cell lymphoma cell strain JeKo-1-parent is recovered, culturing the cell strain until logarithmic growth phase for passage, and continuously passing the cell strain until 10-20 generations, so that a large number of irregular multicellular spheroids are obtained, the phenomenon of single suspension cell death is accompanied, and the activity of the whole cells is poor;
step two: and (3) continuously culturing all the cells of the irregular multicellular spheroids formed in the step one in the original culture system, wherein the ratio of 1: 2-5 times of density passage, continuously culturing for 5-10 times, and converting cells from irregular spheres into regular multicellular spheres;
step three: and continuously culturing the regular multicellular spheroids formed in the step two for 5-10 generations to obtain the human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid.
Furthermore, the obtained human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid can be cultured in vitro for a long time after being frozen and recovered, and the activity is good.
Preferably, the culture solution in the first step, the second step and the third step is RPMI-1640 culture medium, wherein the culture medium contains 10-20% of fetal bovine serum and 1-2% of penicillin and streptomycin.
Preferably, the cell culture temperature in the first step, the second step and the third step is 37 ℃, and CO is contained in the culture atmosphere 2 The volume concentration of (3) is 5%.
The third technical problem to be solved by the invention is to provide the application of the malignant clone cell strain of the mantle cell lymphoma: the human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid is used for basic and clinical researches on malignant clone evolution, pathogenesis, drug resistance mechanism, biological targeted drugs and construction of mantle cell lymphoma animal models.
The invention has the beneficial effects that:
compared with the prior art, the human mantle cell lymphoma malignant clone cell strain JeKo-1-spherioid provided by the invention contains a large amount of CD19 negative early-stage malignant clone cells, the proportion of the CD19 negative early-stage cells is increased to be more than 80% from < 1% of the original state, the CD19 negative early-stage malignant clones of two groups of cells which are not discovered before, namely CD 19-/IgM-and CD19-/IgM +, which are earlier stages, exist in the group of cells due to the amplification of the CD19 negative, the cell model is highly matched with a clinical source sample in biological characteristics, and the cell model is highly inosculated and displays great advantages in clone balling, B cell differentiation and development and dry gene expression, in vivo tumorigenesis, in vivo hemorrhage infection and extramedullary hematopoiesis biological characteristics. Thereby providing a more reliable malignant clone research model for further uncovering the pathogenesis, clinical staging, prognosis judgment and even the research of biological target treatment of the disease.
Drawings
FIG. 1 shows the different growth patterns observed under microscope (Olympus IX 53) for two human mantle cell lymphoma cell lines; FIG. 1a (JeKo-1-parent) is a graph showing the growth pattern of the JeKo-1-parent used in the examples, and FIG. 1b (JeKo-1-seed) is a graph showing the growth pattern of the JeKo-1-parent obtained in the examples observed under a microscope (Olympus IX 53); as can be seen from fig. 1: more than 99% of the JeKo-1-partial cell strains mainly grow on single suspended cells, and less than 1% of the JeKo-1-partial cell strains grow on multi-cell spheres; the JeKo-1-sphenoid cell strain obtained by the invention takes more than 90 percent of multi-cell spheres with larger volume and regular morphology as a main growth mode.
FIG. 2 shows the difference in expression of CD 19-negative early-stage differentiation antigens in two mantle cell lymphoma cell lines; FIG. 2a is JeKo-1-parent and FIG. 2b is JeKo-1-parent obtained in the example; as can be seen from fig. 2: CD 19-negative cells of the JeKo-1-partial cells account for only 0.34% + -0.07%; the proportion of CD19 negative cells in the newly established JeKo-1-sphenoid cells reaches up to 88.97% + -3.50% (figure 2-4), which is 261 times higher than that of CD 19-cells in JeKo-1-partial cells.
FIG. 3 is a JeKo-1-sphenoid cell STR test obtained in the example of the present invention, which indicates that the JeKo-1-sphenoid cell is derived from the JeKo-1-parent cell strain, is not mixed with other human cells, excludes unstable biological characteristics caused by cell contamination from other cell sources, and is a reliable cell model.
FIG. 4 shows the expression of the malignant clones CD19 and IgM of the JeKo-1-sphenoid and the mantle cell lymphoma cells of the clinical specimens obtained in the present invention; i.e. the expression map of the JeKo-1-spherioid gene and the JeKo-1-parent gene; as can be seen from fig. 3: three sub-populations of cells with different immunophenotypes were finally found by CD19/IgM combinatorial antibodies: CD19-/IgM-, CD19-/IgM +, CD19+/IgM +, and it is consistent with 4 clinical specimens (Pt 1, pt2, pt3, pt 4).
FIG. 5 is a graph showing the difference between the expression of the JeKo-1-heterologous gene in the examples of the present invention and the expression of the JeKo-1-parent gene in the examples; as can be seen from fig. 4: the qrt-pcr experiment shows that the transcription factor Oct2 of mature B cells is low expressed by the Jeko-1-sphenoid cells, and the result is consistent with the result that CD19 negative immature B cells are enriched by Jeko-1-sphenoid cell strains; high expression oncogenes and sternness genes: bcl-9, bcl-2, bcl-11b, sox2, CD133, c-myc, CCND1 and bmi-1, with low expression of bcl-11a and nanog, and JeKo-1-sphenoid is therefore a more research-advantageous malignant clonal cell model.
FIG. 6 is a graph showing the difference between the cloning balling capacities of JeKo-1-sphenoid and JeKo-1-parent established in the examples of the present invention; as can be seen from fig. 5: the Jeko-1-sphenoid cell strain forms more cloning balls (p is less than 0.0001) than the Jeko-1-partial cell strain, so that the Jeko-1-sphenoid cell strain has stronger in-vitro cloning balling capacity.
Detailed Description
The first technical problem to be solved by the invention is to provide a malignant clone cell strain of mantle cell lymphoma containing early malignant clones, named JeKo-1-sphenoid, which is preserved in China Center for Type Culture Collection (CCTCC) in 12 and 21 months in 2018, wherein the preservation number is CCTCC NO: C2018264, and the preservation address is as follows: wuhan, wuhan university, postcode: 430072.
the second technical problem to be solved by the invention is to provide the method for establishing the human mantle cell lymphoma malignant clone cell strain, wherein the method takes a human mantle cell lymphoma cell strain JeKo-1 as an induction object and adopts a continuous culture and natural purification method to establish the human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid.
Further, the continuous culture natural purification method comprises the following steps:
the method comprises the following steps: after the human mantle cell lymphoma cell strain JeKo-1-parent is recovered, the cell strain is cultured to a logarithmic growth phase for passage, and under the microscope (20X), the observation is carried out on about 1 percent of multicellular spheroids which are visible under the background of mainly single suspension cells, when the cell strain is continuously passed to 10-20 generations, a large number of irregular multicellular spheroids appear on the cell, the phenomenon of death of the single suspension cells is accompanied, and the activity of the whole cell is poor;
step two: and (4) continuously culturing all the cells forming irregular spheres in the first step in the original culture system in a ratio of 1: 10-1: 2, carrying out density passage, continuously culturing for 5-10 generations, and converting cells from irregular spheres into regular multicellular spheres;
step three: and (3) continuously culturing the regular multicellular spheres formed in the step two for 5-10 generations, freezing and restoring the regular multicellular spheres for long-term in-vitro culture, and ensuring that the activity is good and can meet the experimental requirements.
The invention is further illustrated by the following examples. The examples of the present invention are only for illustrating the present invention and not for limiting the present invention, and the simple modification of the method of the present invention based on the idea of the present invention is within the scope of the claimed invention.
Examples
1. Establishment of malignant clone cell strain JeKo-1-sphenoid of human mantle cell lymphoma
A malignant clone cell strain of human mantle cell lymphoma containing early-stage malignant clone is named JeKo-1-sphenoid, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2018264.
JeKo-1-parenta in the examples of the present invention was purchased from the cell bank of Chinese academy of sciences.
The establishment method of the malignant clone cell strain JeKo-1-spherioid of the human mantle cell lymphoma comprises the following steps: the method is characterized in that a malignant clone cell strain JeKo-1-spherioid of the human mantle cell lymphoma is established by adopting a continuous culture natural purification method on the basis of a JeKo-1-parenta cell strain, wherein the continuous culture natural purification method comprises the following steps:
1) Verifying whether the genetic engineering method is reasonable or not for human mantle cell lymphoma cell strain JeKo-1-parent, culturing in RPMI-1640 medium containing 20% fetal calf serum, 1% penicillin and streptomycin at 5% CO 2 Culturing at 37 deg.C;
2) Culturing to logarithmic growth phase according to the ratio of 1:10, under the microscope observation, the small multicellular spheroids with the volume of less than 1 percent can be seen on the background that single suspension cells are mainly observed, when the cells are continuously passaged to 10-20 generations, a large number of multicellular spheroids with irregular volumes appear, the phenomenon of single suspension cell death is accompanied, and the whole cell activity is poor;
3) All cells forming irregular spheres were continued to be cultured in the original culture system at a ratio of 1:2, carrying out the proportional passage, continuously culturing for 5-10 generations, and converting cells from irregular spheres into multi-cell spheres with obviously increased regular volumes;
4) The multi-cell spheres are continuously cultured for 5-10 generations to obtain JeKo-1-sphenoid, and the JeKo-1-sphenoid is frozen for resuscitation and long-term in vitro culture, and the activity is good, so that the experimental requirements can be met; the characteristics of the JeKo-1-sphenoid cell strain obtained by continuously culturing the multicellular spheroids obtained in the step 3) for more than 100 generations are not changed, which shows that the cell strain established by the invention has stable characteristics.
2. Biological characteristic detection of malignant clone cell strain JeKo-1-sphenoid of human mantle cell lymphoma
1. Morphological Observation of JeKo-1-sphenoid cell line
The cell morphology was observed under an inverted microscope using the human mantle cell lymphoma cell strain JeKo-1-parenta in the logarithmic growth phase and the malignant clonal cell strain JeKo-1-sphenoid obtained in example, and the results are shown in FIG. 1: compared with the JeKo-1-partial cell strain, the growth of single suspended cells is more than 99 percent, and a few multicellular spheroids less than 1 percent exist; the JeKo-1-sphenoid cell strain takes more than 90% of multicellular spheres with larger volume and regular morphology as a main growth mode.
2. Expression of CD19 in JeKo-1-sphenoid
CD 19-negative cells of the JeKo-1-partial cells account for only 0.34% + -0.07%; the proportion of CD19 negative cells in the newly established JeKo-1-sphenoid cells reaches 88.97% + -3.50% (figure 2), and is increased by 261 times compared with the CD19 negative cells in the JeKo-1-parental cells.
3. STR, EB virus, chlamydia and mycoplasma detection of JeKo-1-sphenoid cells
The JeKo-1-sphenoid cells are proved to be derived from the JeKo-1-parent cells through STR detection of Chinese type culture collection, are not mixed with other human cells, have no pollution and are reliable and pure cell models (figure 3).
4. CD19, igM immunophenotypic test for JeKo-1-sphenoid and clinical specimens
Preparing target cells with excellent logarithmic growth phase state into cell suspension; four clinical samples of clinical samples Pt1, pt2, pt3 (all bone marrow mononuclear cells) and Pt4 (peripheral blood mononuclear cells) of clinically confirmed IV-stage mantle cell lymphoma patients were subjected to Ficoll density gradient centrifugation to extract mononuclear cells; adding 5-20ul of flow antibody to be detected according to the BD flow antibody operating instruction, reacting for 15 minutes at 4 ℃ in a dark place, and detecting by a BD LSR flow cytometer.
Three immunophenotypically distinct cell subsets were found by CD19/IgM combinatorial antibodies: CD19-/IgM-, CD19-/IgM +, CD19+/IgM + and it was consistent with 4 clinical specimens (Pt 1, pt2, pt3, pt 4) (FIG. 4).
4. Detection of JeKo-1-sphenoid Gene expression
And taking target cells in the logarithmic growth phase, extracting RNA and reversing the RNA into cDNA according to the operation instruction of a TaKaRa kit, and adding primers for detection on a computer. Confirmed by qrt-pcr experiments: the transcription factor Oct2 of the mature B cells is low expressed by the Jeko-1-sphenoid cells, and the result is consistent with that of CD19 negative immature B cells enriched by the Jeko-1-sphenoid cell strain and CD19 positive mature B cells low expressed; high expression oncogenes and sternness genes: bcl-9, bcl-2, bcl-11b, sox2, CD133, c-myc, CCND1 and bmi-1, low-expressing bcl-11a and nanog (FIG. 5), and thus JeKo-1-sphenoid is a more research-advantageous malignant clonal cell model.
5. JeKo-1-parent and JeKo-1-sphenoid methylcellulose cloning balling experiments:
(1) JeKo-1-parent group and JeKo-1-spheres group, wherein three compound dishes are arranged in each group;
(2) Cell: 1000 per 35mm dish;
(3) A culture system: methocult H4435:1.1ml/35mm dish, 0.1ml cell suspension, 1.2ml total;
(4) The culture conditions are as follows: the culture conditions of the same cell lines were 5% by weight, CO2, 37 ℃;
(5) Counting: the number of clonal spheres was counted under the mirror after 2 weeks (note: only if the number of clonal sphere cells was greater than 40 was counted) and recorded experimentally.
The results confirmed that: the Jeko-1-sphenoid cell strain forms more cloning balls (p is less than 0.0001) than the Jeko-1-partial cell strain, so that the Jeko-1-sphenoid cell strain has stronger in-vitro cloning balling capacity.
6. NOD/SCID in vivo tumor formation experiment
(1) Grouping: jeKo-1-parent group and JeKo-1-sphenoid group, two groups;
(2) NOD/SCID mice: mice of 8-12 weeks, weight 20-30 g/mouse, 4 mice per group; all NOD/SCID mice for experiments are subjected to preliminary experiment results without special treatment such as rays and the like according to the random grouping experiment principle; all feeding articles and experimental articles contacting NOD/SCID mice are subjected to autoclaving treatment; all animal experiment-related procedures were performed strictly following the SPF laboratory-related experimental regulations.
(3) Number of cells injected: selecting JeKo-1-parent and JeKo-1-spheres cells with good growth state in logarithmic growth phase respectively, washing for 2 times by PBS, adjusting the cell density to 1 × 106/cell to meet the experiment requirement, subpackaging in a sterile EP tube and placing in an ice box;
(4) And (3) injection: under the condition of SPF experimental animals, the animal surgical instruments are sterilized under high pressure and the sterility is ensured, all animal experiments are carried out in a sterile workbench, and the aseptic operation principle is paid attention to in the whole process. Using a sterile medical cotton swab to dip 75 percent alcohol, carefully and quickly disinfecting abdominal skin and hair of a mouse, sucking 100ul of prepared cell suspension in an EP tube, injecting target cells into an abdominal cavity, and accurately and softly acting;
(5) The termination time of the experiment: the mice are discomfortable such as emaciation and bleeding and the like, and the experiment is stopped in time;
(6) The sacrifice mode is as follows: painless sacrifice by anesthesia with pentobarbital sodium 3 times higher than the conventional dose;
(7) Collecting organs: keeping sternum, thymus, brain, heart and lung, liver, kidney, spleen, adrenal gland, stomach, pancreas and intestinal tract in a super clean bench, subpackaging in sample tubes filled with 4% paraformaldehyde, respectively staining with H & E, and performing immunohistochemistry on spleen;
(8) Results evaluation system: number of neoplastic organs, number of organs that develop infection and hemorrhage.
Table 1 shows the difference in the in vivo tumorigenic capacity between JeKo-1-sphenoid and JeKo-1-parent; as can be seen from Table 1: the results of the verification of the biological characteristics of in vivo tumors suggest that the JeKo-1-sphenoid cell strain has stronger in vivo tumorigenesis, higher infection and bleeding generation capacity, and is consistent with the biological characteristics of easy secondary infection bleeding of clinical patients.
TABLE 1
While the invention has been described in conjunction with the embodiments described above, it will be apparent to those skilled in the art that various modifications may be made to the embodiments described above without departing from the spirit and scope of the claims.
Claims (3)
1. A human mantle cell lymphoma malignant clone cell strain containing early malignant clone is named as human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2018264.
2. The application of a malignant clone cell strain JeKo-1-sphenoid of human mantle cell lymphoma in establishing a mantle cell lymphoma animal model; the human mantle cell lymphoma malignant clone cell strain JeKo-1-sphenoid is the human mantle cell lymphoma malignant clone cell strain according to claim 1.
3. The human mantle cell lymphoma malignant clone cell strain JeKo-1-spherioid is used for the basic and clinical research of malignant clone evolution, pathogenesis, drug resistance mechanism and biological targeted drugs; the human mantle cell lymphoma malignant clone cell line JeKo-1-spherioid is the human mantle cell lymphoma malignant clone cell line of claim 1.
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