CN114457017B - Mouse fibroblast tumor cell strain HXLyAF-KBM and application thereof - Google Patents
Mouse fibroblast tumor cell strain HXLyAF-KBM and application thereof Download PDFInfo
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Abstract
The invention discloses a mouse fibroblast tumor cell strain HXLyAF-KBM and application thereof, wherein the cell strain is derived from a bone marrow microenvironment in a human lymphoma cell nude mouse transplantation tumor model, and the cell name is mouse lymphoma related fibroblast tumor cell HXLyAF-KBM, and the preservation number is CCTCC NO: c2021106, the preservation date is 2021, 12 and 09, and the preservation unit is China center for type culture Collection. The cell strain is a bone marrow-derived lymphoma related mouse fibroblast tumor cell strain which is established for the first time at home and abroad, and can be applied to the construction of a mouse fibroblast tumor animal model, a malignant bone marrow microenvironment model, a 3D tumor culture system and organoid system model and a bone marrow microenvironment and lymphoma progression and drug resistance relation model.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a mouse fibroblast tumor cell strain HXLyAF-KBM and application thereof.
Background
The tumor microenvironment interacts with tumor cells, affecting the occurrence, development and prognosis of tumors. Cancer-related fibroblasts (CAFs) are the main cellular components constituting the microenvironment of solid tumors, have high heterogeneity, plasticity and multidirectional differentiation potential, and play a complex and important role in the occurrence, development, metastasis, chemotherapy resistance and malignancy prognosis of solid tumors by remodelling the tumor microenvironment. CAFs are a cell with high heterogeneity, and it is currently believed that the precursor cells of CAFs are mainly of 7 sources: bone marrow mesenchymal stem cells, resident fibroblasts in tissues, astrocytes, epithelial cell mesenchymal transition, endothelial cell mesenchymal transition, fibroblasts and other cells such as pericytes, smooth muscle cells, adipocytes and tumor stem cells, etc., and therefore, various CAFs precursor cells exist in the bone marrow microenvironment.
Since CAFs of different sources play different roles in the progression of tumors, the construction of CAFs cells of different sources contributes to the deep exploration of tumor pathology. However, until nearly 10 years, the role of CAFs in hematological tumors has not been of interest. To date, the invention patent with publication number of CN110699324A discloses a leukemia-related mouse fibroblast tumor cell strain derived from nude mice subcutaneous transplantation tumor, no report of tumor-related fibroblast tumor, no report of related cell strain, and no report of malignant transformation of lymphoma bone marrow infiltration induced bone marrow microenvironment CAFs. Although the invention patent publication No. CN112980768A provides a method for establishing a novel liver fibroblast strain, liver tumor-related fibroblasts derived from patients with colon cancer liver metastasis only provide a basic identification of primary cells passaged to the third generation.
Disclosure of Invention
The invention aims to provide a mouse fibroblast tumor cell strain HXLyAF-KBM, which is a mouse fibroblast tumor cell strain related to bone marrow-derived lymphomas, and the cell strain is a mouse fibroblast tumor cell strain related to bone marrow-derived lymphomas, which is established for the first time at home and abroad. Therefore, the invention also provides application of the cell strain in constructing a mouse fibroblast tumor animal model, a malignant bone marrow microenvironment model, a 3D tumor culture system and organoid system model, and a bone marrow microenvironment and lymphoma progression and drug resistance relation model.
The invention is realized by the following technical scheme: the mouse fibroblast tumor cell strain HXLyAF-KBM is derived from the bone marrow microenvironment in a human lymphoma cell nude mouse transplantation tumor model, and the cell name is the mouse lymphoma related fibroblast tumor cell HXLyAF-KBM, and the preservation number is CCTCC NO: c2021106, the preservation date is 2021, 12 and 09, and the preservation unit is China center for type culture Collection. The storage unit address is in the eight-channel 299-number university school (first appendage face of university of Wuhan) in Wuchang district of Wuhan, hubei province, and the university of Wuhan collection center.
The bone marrow-derived mouse fibroblast tumor cell strain is derived from a human anaplastic large cell lymphoma cell Karpas299 nude mouse transplantation tumor model, is taken from mouse bone marrow, and is a bone marrow-derived lymphoma related mouse fibroblast tumor cell strain. The primary cells of the cell strain are derived from a model mouse of human anaplastic large cell lymphoma transplantation tumor; taking bone marrow cells, inoculating and culturing in vitro, and removing suspension cells to obtain adherent growth cells; after 20 passages of cells, the morphology is mainly polygonal star, so that the cells are in a fusiform shape, large in nucleus, clear in nucleolus, rich in cytoplasm, provided with slender protrusions, non-contact inhibition, nondirectional in growth, capable of cross overlapping growth and clustered when the cells are dense; stable unlimited passage in vitro, and high tumorigenicity; designated as mouse fibroblast tumor cell HXLyAF-KBM.
The invention also provides application of the mouse fibroblast tumor cell strain HXLyAF-KBM in constructing a mouse fibroblast tumor animal model, in constructing a malignant bone marrow microenvironment model, in constructing a 3D tumor culture system and an organoid system model, and in constructing a bone marrow microenvironment and lymphoma progression and drug resistance relation model. Provides a new thought and a new target point for the mechanism and treatment of lymphoma bone marrow infiltration, and explores a new method for treating the relapse refractory lymphoma by targeting CAFs.
The mouse fibroblast tumor cell strain HXLyAF-KBM has the following biological characteristics:
is a cell of mouse origin by transcriptome sequencing and STR analysis.
The result of the western blotting detection shows that: the cell highly expresses alpha-SMA, vimentin, HSP47, FAP, beta-catenin, PDGFR-alpha and PDGFR-beta which are fibroblast-derived tumors.
HXLyAF-KBM cells are grown in an adherence manner in an RPMI 1640 complete culture medium, the shape of a polyhedron is mainly that the shape of a spindle is visible, the nucleus is large, the cytoplasm is rich, the slender protuberance is arranged, the in vitro culture growth is good, and the culture is carried out according to the volume of 3 multiplied by 10 5 /55cm 2 The method comprises the steps of density inoculation, passage once in 3-4 days, in vitro continuous culture for more than 9 months, passage for more than 70 generations, population multiplication for more than 280 generations, uniform cell morphology, stable proliferation kinetics and stable genetic characteristics in the continuous passage process, and good state; the characteristics are unchanged after the freezing and the recovery by liquid nitrogen or ultra-low temperature, and the immortalized cell strain is obtained.
Drawing a growth curve of HXLyAF-KBM cells by using a trypan blue staining cytometry method, calculating the cell multiplication time to be 17-20 hours, ensuring stable proliferation rate, and observing a division phase, wherein the cells still adhere after division; the cell lines were confirmed to have proliferation kinetic stability.
The HXLyAF-KBM cells are super 4 times somatic cells and have cell cycle stability by adopting a PI staining method and chromosome karyotype analysis.
Compared with the prior art, the invention has the following advantages:
(1) The invention relates to a cell strain HXLyAF-KBM of a mouse fibroblast tumor related to bone marrow-derived lymphoma, which is established for the first time at present, has high tumorigenicity, and can be used for constructing a mouse fibroblast tumor animal model, a malignant bone marrow microenvironment model, a 3D tumor culture system and an organoid system model.
(2) The invention can apply the mouse fibroblast tumor cell strain HXLyAF-KBM in the study of lymphoma microenvironment, construct a relation model of the progress and drug resistance of the myeloma microenvironment and lymphoma, acquire the related data of the fibroblastic tumor metaplasia caused by lymphoma cells, acquire the related data of the relation of the lymphoma related fibroblast tumor and lymphoma microenvironment, acquire the pathophysiological mechanism of lymphoma marrow infiltration, and acquire the related data of lymphoma chemotherapy drug resistance and the like in the lymphoma treatment process.
Drawings
FIG. 1 is a view of HXLyAF-KBM cells under an inverted phase contrast microscope (. Times.200).
FIG. 2 is a diagram of the ultrastructural view of HXLyAF-KBM cells under transmission electron microscopy (x 6000).
FIG. 3 is a graph of HXLyAF-KBM cell growth.
FIG. 4 is a diagram of HXLyAF-KBM cell cycle.
FIG. 5 shows the Western blotting detection of the expression patterns of HXLyAF-KBM cells alpha-SMA, vimentin, FAP, HSP47, beta-catenin, PDGFR-alpha, PDGFR-beta.
FIG. 6 is a graph of an experiment of inguinal lymphadenectasis in HXLyAF-KBM cell nude mice.
FIG. 7 is a diagram of a subcutaneous tumor formation experiment in HXLyAF-KBM cell nude mice.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Example 1: isolation and construction of mouse fibroblast tumor cell strain HXLyAF-KBM
(1) Human anaplastic large cell lymphoma cells Karpas299 in logarithmic growth phase were prepared at a ratio of 8×10 6 Mu.l/100 mu.l/mouse, inoculated under nude of female Balb/c (nu/nu) mice (8 weeks old, weight 25.41 g), after 67 days of inoculation, mice developed a progressive weight loss, and were sacrificed to dissect at a weight of 20.02g, bilateral femur and tibia bone marrow were harvested to make cell suspensions, and the cells were resuspended in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Thermo Co.), and placed at 37℃and saturated humidity with 5% CO 2 Culturing in an incubator. The adherent cells grow after 4 days, the supernatant and the suspension cells are removed, the liquid is changed every 2-3 days, the suspension cells in the culture dish are basically removed after 16 days, the adherent cells grow in clusters, the adherent cells are digested and passaged by 0.25% pancreatin-EDTA (Hyclone company), after that, the passaged once every 7-10 days, the cell morphology is uniform before 70 days after inoculation, the cell proliferation is accelerated, the cell proliferation is obviously rapid after 190 days, and the cells are mainly in a long fusiform shape or an irregular polygon.
(2) Initial density of HXLyAF-KBM cells passaged 3X 10 5 Per 55cm 2 And when the cells proliferate to 80-90% and fuse, the cells are passaged 1:6-10.
(3) Changing liquid 48 hours before freezing HXLyAF-KBM cells, making the cells in logarithmic phase, counting the cells, transferring the cells into a centrifuge tube under aseptic condition, centrifuging for 5 minutes at 1200 rpm, discarding supernatant, adding frozen liquid, and adjusting the cell concentration to 3×10 6 Mixing the above materials at a ratio of one ml, transferring into sterile freezing tube, and standing at 4deg.C for 30min; -20 ℃,60min; overnight at-80 ℃, the next day, transfer into liquid nitrogen.
(4) During recovery, the frozen storage tube is taken out from liquid nitrogen, quickly placed in warm water at 37 ℃, after frozen storage is melted, the cell suspension is transferred into a centrifuge tube added with 5ml of RPMI 1640 culture medium, gently mixed evenly, centrifuged for 5 minutes at 1200 rpm, the supernatant is discarded, the RPMI 1640 complete culture medium is added into cell sediment, mixed evenly, transferred into a culture dish, and placed at 37 ℃ and saturated humidity and 5% CO 2 Incubator cultureAnd (5) nourishing.
(5) The HXLyAF-KBM cells are repeatedly subjected to the freezing and recovering steps (3) and (4), so that the growth is stable, the biological characteristics of the cells are not affected, the cells are seen to be mainly in a polygonal star shape under a microscope, the fusiform is seen, the adherent growth is realized, and the cell multiplication time is maintained at 17-20 hours.
In the above steps, the formulation of the RPMI 1640 complete medium adopts 90% RPMI 1640 medium and 10% fetal bovine serum. The frozen stock solution was prepared using 45% RPMI 1640 medium, 50% fetal bovine serum and 5% DMSO (Sigma).
Example 2: growth and biological characterization of mouse fibroblast tumor cell line HXLyAF-KBM
(1) Cell morphology observation:
taking HXLyAF-KBM cells in logarithmic growth phase, observing the shape of the living cells under an inverted microscope, as shown in figure 1, growing the cells in an adherent manner, wherein the shape is mainly a polygonal star shape, and the cells can be seen to be fusiform, have large nuclei, clear nucleolus, are not inhibited by contact, can grow in a cross overlapping manner and are clustered when the cells are dense; 3% glutaraldehyde is pre-fixed, 1% osmium tetroxide is re-fixed, and the ultra-microstructure of the cells is observed under a JEM-1400FLASH perspective electron microscope stained with uranium acetate and lead citrate, as shown in figure 2, the nuclei are irregular, have a notch, clear nuclear membrane, obvious nucleolus, nuclear pit, increased cytoplasmic rough endoplasmic reticulum, abundant free ribosomes, visible endoplasmic reticulum, mitochondria and other organelles.
(2) Growth curve and doubling time determination:
centrifuging to collect cells in logarithmic growth phase, and adjusting cell concentration to 2×10 3 1.9 cm/1 2 Inoculating into 24-well plate, placing at 37deg.C and 5% CO 2 、21% O 2 Cells were counted daily with trypan blue staining for 8 days in an incubator. And drawing a cell growth curve by taking time as an abscissa and the number of cells as an ordinate, and calculating the cell multiplication time to be 17-20 hours as shown in figure 3, wherein the cell strain is repeatedly passaged, the population multiplication times are more than 280 generations, and the proliferation kinetic stability is still maintained.
(3) PI staining monitors cell cycle:
harvesting1×10 6 Washing the individual cells with PBS (pre-cooled phosphate buffer solution) at 4 ℃ for 2 times, fixing overnight with 75% ethanol at 4 ℃ for 2 times with PBS at 4 ℃ and dyeing PI (polyimide) at room temperature for 10min in a dark manner, and detecting G of the cells by a flow cytometer 1 /G 0 S, G 2 The ratio of phase/M is shown in FIG. 4, and HXLyAF-KBM cells are near tetraploid cells, and can maintain the cell cycle stability after repeated passage.
(4) STR detection confirmed HXLyAF-KBM cells as mouse-derived cells.
TABLE 1 STR site typing of HXLyAF-KBM cells
(5) The western blotting detection result shows that: the cells highly express alpha-SMA, vimentin, HSP47, FAP, beta-catenin, PDGFR-alpha, PDGFR-beta, which are fibroblast-derived tumors, as shown in figure 5.
The above results show that lymphoma cells can lead to fibroblast tumor metaplasia.
Example 3: construction of mouse fibroblast tumor animal model
In particular to a method for constructing a mouse fibroblast tumor animal model by using a mouse fibroblast tumor cell strain HXLyAF-KBM, which adopts a nude mouse tumor experiment, and the specific experimental process is as follows:
HXLyAF-KBM cells in logarithmic growth phase were grown at 3X 10 6 Mu.l/200 mu.l/mouse, inoculated subcutaneously in nu/nu (4 weeks old, body weight 15-17 g) nude region of female Balb/c mice; after 4-12 days of inoculation, single or double-sided inguinal lymph node growth is seen, as shown in FIG. 6; the nude mice can be subcutaneously touched with rice-sized tumor after 17 days of inoculation, and the tumor volume is 80mm after 40 days 3 As shown in fig. 7.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (5)
1. The mouse fibroblast tumor cell strain HXLyAF-KBM related to the bone marrow-derived lymphoma is characterized in that: the cell name is mouse lymphoma related fibroblast tumor cell HXLyAF-KBM, and the preservation number is CCTCC NO: c2021106, the date of deposit is 2021, 12, 09, china center for type culture Collection;
the cell strain is obtained by taking a model mouse bilateral femur and tibia internal bone marrow from a human lymphoma cell nude mouse transplantation tumor model constructed by human anaplastic large cell lymphoma cells Karpas 299.
2. The use of the mouse fibroblast tumor cell strain HXLyAF-KBM related to the bone marrow-derived lymphoma in constructing a mouse fibroblast tumor animal model.
3. The application of the mouse fibroblast tumor cell strain HXLyAF-KBM related to the bone marrow-derived lymphoma in constructing a malignant bone marrow microenvironment model.
4. The use of the mouse fibroblast tumor cell strain HXLyAF-KBM related to the bone marrow-derived lymphoma in constructing a 3D tumor culture system and an organoid system model.
5. The use of the mouse fibroblast tumor cell strain hxleaf-KBM related to the bone marrow-derived lymphoma of claim 1 in constructing a model of bone marrow microenvironment and lymphoma progression and drug resistance relationship.
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