CN115232791A - In-vitro culture method and application of mouse liver cancer model cells - Google Patents
In-vitro culture method and application of mouse liver cancer model cells Download PDFInfo
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Abstract
The invention relates to an in vitro culture method of mouse liver cancer model cells, wherein the mouse is a C57BL/6 mouse, and the method comprises the following steps: (1) preparing Hepa1-6 cells for injecting mice; (2) Injecting the injected mouse with Hepa1-6 cells, monitoring the growth of the tumor volume of the mouse, and screening the mouse with the tumor volume reaching a preset volume to obtain a tumor tissue; (3) And treating the tumor tissue to obtain a mouse liver cancer model cell which is named as a Hepa1-6BL cell. The invention also provides a corresponding application.
Description
Technical Field
The invention relates to the technical field of liver cancer models, in particular to an in-vitro culture method of mouse liver cancer model cells and application thereof.
Background
Liver cancer is one of the most common cancers, and causes serious harm to human life and health, and the morbidity and mortality of the liver cancer are high for a long time. The exploration and diagnosis of the pathogenesis of liver cancer, the discovery of new targets and the research and development of drugs thereof are problems which need to be solved urgently.
Tumor tissue comprises a variety of cells, such as immune cells, fibroblasts, etc., and different cells secrete cytokines and growth factors, extracellular matrix, etc. The tumor immune microenvironment is infiltrated by a variety of immune cells, including CD4 + T、CD8 + T cells, natural killer cells, macrophages, monocytes, dendritic cells, regulatory T lymphocytes (tregs), etc. (Balkwill, capasso et al 2012). Different types of cells in tumor tissues, different ligands expressed by the cells, corresponding receptors and extracellular matrix interact to form a complex regulation system, and a tumor microenvironment is formed. Therefore, it is difficult for a simple in vitro tumor cell culture system to fully reveal a complex mutual regulatory network among different cells in a tumor microenvironment. Therefore, the establishment of a mouse liver cancer model becomes an important scheme for solving the problem and is also the basis for researching tumor development mechanisms and drug research and development.
With the gradual and deep understanding of the mutual process between tumor cells and the immune system of the organism in the process of tumor development, the tumor immune editing theory is gradually formed. Tumorigenesis progresses through mainly three stages of immune editing, immune clearance, immune balance and immune escape under the surveillance of the immune system (Gavin p. Dunn, allen t. Bruce et al 2002). The immunoediting process is complete when all the mutated cells are recognized and killed by immune cells and tumor cells are eliminated. However, some of the mutated cells may edit the autoimmunity or escape the surveillance of the immune system by affecting the tumor microenvironment, and the tumor cells and the immune cells are in a transient equilibrium state, and the tumor cells are in a state similar to dormancy under the stress of the immune system, and show clinically undetectable tumor, which is generally regarded as a tumor survival stage. With the continuous accumulation of mutations (such as DNA mutation and gene expression change), tumor cells can further generate a series of malignant phenotypes (such as no specific antigen, cell apoptosis resistance and the like) to avoid the monitoring of an immune system, and the tumor cells cannot be eliminated at the stage, so that the tumor cells resist to enter an escape stage, and form clinically detectable tumors. Tumor immunoeditors revealed the relationship of the immune system to tumorigenesis and progression. Therefore, the identification of the molecular mechanisms by which the immune system eliminates tumor cells and the immune escape of tumor cells is critical for the development of tumor immunotherapy (Gavin P Dunn, lloyd J Old et al 2004). Different types of cells in the tumor microenvironment play different roles in the occurrence, development and treatment of liver cancer, for example, tumor cells can express immunosuppressive factors, and macrophages with anti-tumor effects are converted into tumor-promoting macrophages. Because of the "self" and "non-self" distinguishing features of the body's immune system, studies of the interaction between tumor cells and immune cells in the tumor microenvironment can only be performed using isogenic tumor models. Therefore, the syngeneic mouse liver cancer model is the basis for researching the interaction between liver cancer cells and immune cells in a tumor microenvironment.
Common mouse models for the immunological study of liver cancer include a transplanted tumor model, a chemical carcinogen-induced liver cancer model and a genetic engineering liver cancer model. The test period of the liver cancer model induced by the chemical carcinogen is long, the model establishment needs enough technical experience, and the morbidity time is difficult to control. The genetically engineered liver cancer model needs a specific genetically engineered mouse, and compared with the liver cancer induced by a chemical carcinogen, the genetically engineered liver cancer model has the advantages of long experimental period and highest cost. The model of the subcutaneous liver cancer transplantation tumor has the advantages of simple and convenient operation, visual observation of the growth of the tumor, convenient detection of important data such as animal weight, tumor growth curve, tumor weight and the like. Therefore, the subcutaneous tumor model is the most commonly used tumor model in tumor research, tumor immunity research, drug research and the like at present.
Mouse hepatoma cells Hepa1-6 and the like are derived from C57L lead mice and can rapidly form subcutaneous nodules in immunodeficient mice and C57L lead mice. However, C57BL/6 mice are currently the most widely used mouse strain in oncology studies and tumor immunization studies. The gene background of the C57BL/6 mouse strain is different from that of the C57L lead-colored mouse, so that subcutaneous tumors built by the Hepa1-6 in wild type C57BL/6 mice with complete immune systems grow slowly and are even rejected by the immune systems of the C57BL/6 mice bodies.
The lack of C57BL/6 mouse isogenic liver cancer models severely limits liver cancer-related studies, particularly liver cancer immunomodulation and therapy-related studies.
Therefore, there is a need to establish a cell line (named Hepa1-6 BL) capable of rapidly establishing subcutaneous tumors and tail vein injection to form liver cancer in situ in the widely used Immune complete (Immune component) wild type C57BL/6 mice.
Disclosure of Invention
The invention provides an in vitro culture method of mouse liver cancer model cells and application thereof, aiming at solving the problems of the mouse liver cancer model building method.
In order to realize the aim, the invention provides an in vitro culture method of mouse liver cancer model cells, which is mainly characterized in that: the method comprises the following steps:
(1) Preparing a Hepa1-6 cell for injecting mice;
(2) Injecting the injection mouse with Hepa1-6 cells, monitoring the growth of the tumor volume of the mouse, screening the mouse reaching the preset tumor size, and obtaining a tumor tissue;
(3) And treating the tumor tissue to obtain a mouse liver cancer model cell which is named as a Hepa1-6BL cell.
Preferably, the step (1) is specifically:
(1-1) removing the Hepa1-6 cells frozen in liquid nitrogen; rapidly thawing and recovering at 37 ℃;
(1-2) opening a cell freezing tube, adding cell sediment into a 10ml centrifuge tube containing 5ml of culture solution, centrifuging at 4 ℃, and removing supernatant;
(1-3) 4ml of DMEM complete high sugar medium resuspended cell pellets, added uniformly to a 6cm cell culture dish, left at 37 ℃ and 5% CO 2 Culturing in an incubator;
(1-4) carrying out cell passage by pancreatin digestion when the cell density reaches 80% -90%;
(1-5) 1ml of complete medium was used to suspend the cells, and the cells were plated in a 6cm dish and cultured to be good, to obtain the Hepa1-6 cells for injection mouse.
Preferably, in the step (1-5), the cells are cultured to be good and visible under high power microscope: the cell has smooth edge, clear and visible outline, good light transmission inside the cell, strong refractivity, few particles and no vacuole.
Preferably, the step (2) is specifically:
(2-1) preparing a single cell suspension;
(2-2) injecting each mouse subcutaneously and raising;
(2-3) recording the cell injection mice as day 0, monitoring the growth of the tumor volume of the mice from day 7, and recording the tumor volume;
(2-4) screening mice reaching the preset tumor size to obtain tumor tissues.
Preferably, the step (3) is specifically:
(3-1) placing the tumor tissue into a 6-well plate containing digestive juice, and digesting;
(3-2) adding all tissues and digestive juice of the 6-well plate into a 50ml centrifuge tube containing a cell sieve for filtration and centrifugation;
(3-3) discarding the supernatant, adding DMEM to the complete high-glucose medium to resuspend the cell pellet, and plating it in a 6-well plate, standing at 37 ℃ and 5% CO 2 Culturing in an incubator to obtain the Hepa1-6BL cells.
The invention also provides application of the mouse liver cancer model cell obtained by the in vitro culture method in establishing a mouse liver cancer model.
The Hepa1-6BL cell and subcutaneous tumor model has the advantages that:
(1) The Hepa1-6BL cell has the following characteristics in vitro culture: the culture conditions and the culture method of the Hepa1-6BL cells are simple, the growth speed is high, and the proliferation rate is high. The expression of various immunoregulation related genes in the Hepa1-6BL cells is changed, the genes can be modified by a molecular biological method, the action and the mechanism of the genes in the development of liver cancer are researched at the molecular level and the cell level, and the interaction between the liver cancer cells and immune cells can be researched in vitro experiments. The results of the cellular level studies can be validated directly in the mouse model.
(2) The Hepa1-6BL liver cancer cell subcutaneous tumor model has the characteristics that:
the expression of the genes related to immunity in the Hepa1-6BL cells is changed, and the immune escape capability is enhanced. Compared with other liver cancer cell lines, the liver cancer cell line has the characteristics of high cell tumor formation rate, high subcutaneous tumor formation speed and uniform tumor generation in C57BL/6 mice with complete immune systems. In addition, the Hepa1-6BL hepatoma cell subcutaneous tumor model has the characteristics of easiness in establishment, short experimental period, low experimental cost and the like. The subcutaneous tumors of Hepa1-6BL have abundant immune cell infiltration, wherein macrophages are the main tumor infiltration immune cell subset, and the tumor infiltration of T cells and NK cells is less. The characteristics are similar to the characteristics of immune cell infiltration in tumor tissues of liver cancer patients, so the tumor microenvironment of the liver cancer patients can be reflected by the immune cell infiltration and the functions in the model. The model can be used for liver cancer immune escape, immune drug resistance and immunotherapy drug screening and research and development.
(3) The Hepa1-6BL cells form liver carcinoma in situ in a C57BL/6 mouse:
the Hepa1-6BL cells can be used for constructing a liver in situ cancer model in a wild type C57BL/6 mouse which is most widely used, and compared with a liver cancer model induced by a chemical carcinogen and a gene engineering liver cancer model, the Hepa1-6BL cells have the following 3 characteristics in forming liver in situ cancer in the C57BL/6 mouse:
1) The establishment of the Hepa1-6BL liver cancer in situ model does not need carcinogenic reagent induction and other reagent assistance, and has high safety in the whole experimental process.
2) The in-situ cancer can be established only by C57BL/6 background mice without constructing and breeding specific transgenic mice, and the model construction can be completed only by carrying out high-pressure tail vein injection once, so that the model establishment operation is simple, the whole experimental period is short, the research period is effectively shortened, and the research cost is reduced.
3) After C57BL/6 mice are injected, liver orthotopic tumors are formed within the time range of 3-4 weeks, the mice have stable morbidity time, small difference among individual mice and good repeatability, and the method is very suitable for the drug effect evaluation and mechanism research of the anti-liver cancer drugs.
Drawings
FIG. 1 is a flow chart of a mouse liver cancer model provided by the present invention.
FIGS. 2a and 2b are graphs of tumor growth after injection of cells into mice in example 1.
FIG. 3 is a flow chart of the single cell suspension culture in example 2.
FIG. 4 is a diagram of the short chain repeat (STR) pattern of Hepa1-6BL in example 2.
FIGS. 5a and 5b are graphs of tumor growth after injection of cells into mice in example 3.
FIGS. 6a to 6c are graphs showing the results of liver tissue Hepa1-6BL cell transfer in example 3.
Figure 7 is the volcano pattern obtained in example 4.
Fig. 8 is a graph of the GO functional enrichment analysis results obtained in example 4.
FIG. 9 is a graph of the functional enrichment analysis results of KEGG obtained in example 4.
FIGS. 10a and 10b are schematic diagrams of the differential genes of Hepa1-6 and Hepa1-6BL tumor cells.
FIG. 11 is a schematic diagram showing the analysis of the flow cytometry results in example 5.
FIG. 12 is a classification chart of different immune cell subsets in Hepa1-6BL tumor tissue.
Figure 13 is a thermogram of expression of top ten marker hypervariable genes in different cell subsets.
FIG. 14 is a schematic diagram of the ratios of different immune cell subsets in Hepa1-6BL tumor tissues.
Detailed Description
In order to more clearly describe the technical contents of the present invention, the following further description is made in conjunction with specific embodiments.
FIG. 1 shows a flow chart of a mouse liver cancer model provided by the present invention. The mouse liver cancer model cell mainly comprises the following steps: preparing a Hepa1-6 cell for an injection mouse; injecting the injection mouse with Hepa1-6 cells, monitoring the growth of the tumor volume of the mouse, and screening the mouse with the tumor volume reaching a preset volume to obtain a tumor tissue; and treating the tumor tissue to obtain a mouse liver cancer model cell which is named as a Hepa1-6BL cell. The mouse liver cancer model cell obtained by the in vitro culture method can be used for establishing a mouse liver cancer model.
Example 1
Establishing a model of subcutaneous tumor of a Hepa1-6 mouse
(1) Cell culture and preparation of injection mouse cells
The required materials are as follows: opening the ultra-clean bench in advance, and keeping the ultra-clean bench under ultraviolet radiation for 30 min; opening the water bath kettle in advance, and adjusting the temperature to 37 ℃ for later use; 0.25% EDTA tryptic digest, DMEM complete medium (1% penicillin, 1% streptomycin and 10% FBS), sterile PBS buffer was left at room temperature for use.
The cell culture comprises the following specific steps:
taking out cells from liquid nitrogen, rapidly placing in 37 deg.C water bath, and taking out cells from the water bath after 1-2 min;
opening a cell freezing tube in a super clean bench of a cell culture chamber, adding cell suspension into a 10ml centrifuge tube containing 5ml of culture solution, centrifuging for 4min at the rotating speed of 350g at the temperature of 4 ℃, and removing supernatant;
resuspending the cell pellet in 4ml of DMEM complete high sugar medium, adding uniformly to a 6cm cell culture dish, standing at 37 deg.C, 5% 2 Culturing in an incubator;
when the cell density reaches 80% -90%, carrying out cell passage in a super clean bench, and comprising the following steps: discarding cell culture supernatant, adding 3ml sterile PBS buffer solution to wash cells, discarding PBS, addingAdding 0.5ml of 0.25% EDTA pancreatin digest, completely covering the bottom of the cell culture dish, adding it to 37 ℃,5% 2 Digesting for 1min in the incubator, observing the cell morphology under an inverted microscope, adding 2ml of complete culture medium into a culture dish when partial cells fall or the cell morphology is changed, blowing and beating the cells at the bottom of the culture dish to enable the cells to fall completely, transferring the cells into a centrifuge tube, centrifuging for 4min at the rotation speed of 350g at the temperature of 4 ℃, and discarding the supernatant;
1ml of complete culture medium was resuspended in cells and 5X 10 was plated in a 6cm petri dish 5 Cell, 4ml culture system;
the cells are cultured to a good state, and can be seen under a high power microscope: the cell has smooth edge, clear and visible outline, good light transmission inside the cell, strong refractivity, few particles, no vacuole and the like.
The injection mouse cells were prepared as follows:
single cell suspension was prepared as above, 1ml of fetal calf serum-free 1640 medium was added to resuspend the cells, cell counting and trypan blue staining were performed, and the number of cells was adjusted to 1.0X10 8 Ml and ensures cell viability greater than 90%.
(2) Construction of mouse model
The required materials are as follows: c57BL/6J female Wild Type (WT) mice, week old about 8 weeks.
The specific steps for constructing the mouse model are as follows:
hepa1-6 cells (1.0x10) to be prepared in advance 8 Per mouse, 100ul of the solution was injected subcutaneously, and the mice were kept in an SPF barrier system, and were given normal food and drinking water supply, and every 2 days, and the mice were observed for good condition;
recording the cell injection mice as day 0, monitoring the tumor volume growth of the mice by using a vernier caliper every 2 days from day 7, and recording the size of the tumor by adopting a measurement mode of multiplying the length by the width;
most tumors were rejected around 15 days, only 1 mouse tumor grew faster, and 1 month after tumor cell injection, the tumor size reached 17mm x 10mm, with tumor growth curves as shown in fig. 2a and 2 b.
Example 2
Hepa1-6 tumor tissue treatment and Hepa1-6BL cell preparation
(1) Obtaining tumour tissue
1) The tumor volume is 170mm 2 Euthanizing the mice of (a);
2) Collecting tumor tissue in a clean bench, weighing 50mg of tumor tissue, placing the tumor tissue into a 6-hole plate containing 300ul of digestive juice (1 mg/ml of collagenase and 1 ug/ml of DNase), shearing the tumor tissue by using scissors, and supplementing the digestive juice to 1.5ml;
4) Placing the 6-hole plate on a 37 ℃ shaking table, rotating at 150RPM, and digesting for 45min;
5) Place the sterile cell sieve on a 50ml centrifuge tube in a clean bench, add 3ml PBS to wet the cell sieve cloth for use. Adding all tissues and digestive juice of a 6-hole plate into a 50ml centrifuge tube containing a cell sieve, grinding tumor tissues by using a piston handle of a 1ml syringe, adding PBS (phosphate buffer solution) to wash cells, and completely filtering the cells into the 50ml centrifuge tube;
6) Centrifuging at the rotating speed of 350g in a4 ℃ centrifuge for 4min;
7) Discarding supernatant, adding 4ml DMEM to complete high sugar medium to resuspend cell pellet, and spreading into 6-well plate, 4ml system, standing at 37 deg.C, 5% CO 2 Culturing in an incubator;
(2) Single cell suspension culture, see FIG. 3.
(3) Passage of Hepa1-6BL cells
1) Day 1: observing the cell morphology, and observing under a mirror: removing cell debris, suspension cells and adherent cells, wherein the color of the culture medium does not turn yellow, removing supernatant, washing the cells with sterile PBS for 3 times to remove suspension cells and cell debris, adding 2ml DMEM complete high-sugar medium, and culturing in an incubator at 37 deg.C and 5% CO2;
2) Day 2: observing the cell morphology, and observing under a mirror: and a small amount of suspension cells and most of adherent cells do not turn yellow in the culture medium, and the cell density reaches 90%. Cell passage: the supernatant was discarded and the cells were washed 3 times with sterile PBS to remove suspended cells; adding 0.5ml of 0.25% EDTA pancreatin digest to completely cover the bottom of the cell culture dish, and digesting for 30s; adding 2ml of complete culture medium into a culture dish, blowing and beating cells at the bottom of the culture dish to enable the cells to fall completely, transferring the cells into a centrifuge tube, centrifuging at the rotating speed of 350g for 4min at the temperature of 4 ℃, and discarding supernatant; adding 2ml of complete culture medium for resuspension, taking 250ul of cell suspension, and paving the cell suspension into a 6-hole plate, 2ml of culture system;
3) Day 3: observing the cell morphology, and observing under a mirror: a small amount of suspension cells and most of adherent cells, the color of the culture medium does not turn yellow, and the cell density reaches 50%;
(4) Hepa1-6BL short chain repeat (STR) pattern
A Hepa1-6BL short chain repeat (STR) pattern is shown in FIG. 4.
(5) Cryopreservation of Hepa1-6BL
1) The supernatant was discarded and 3ml of sterile PBS buffer was slowly added along the wall of the cell culture dish to wash the cells;
2) Discarding PBS, adding 0.5ml of 0.25% EDTA pancreatin digestive juice, completely covering the bottom of the cell culture dish, digesting for 30s, adding 2ml of complete culture medium into the culture dish, blowing and beating the cells at the bottom of the culture dish to completely drop the cells, transferring the cells into a centrifuge tube, centrifuging for 4min at the rotation speed of 350g at the temperature of 4 ℃;
3) Discarding supernatant, adding 1ml sterile serum-free cell freezing medium, mixing, transferring to cell freezing tube, and freezing at-80 deg.C with freezing box. After 24h, the frozen cells were transferred to liquid nitrogen for preservation.
(6) Resuscitation of Hepa1-6BL
1) Taking out cells from liquid nitrogen, rapidly placing in 37 deg.C water bath, and taking out cells from water bath after 1-2 min;
2) Opening a cell freezing tube in a super clean bench of a cell culture chamber, adding cell suspension into a 10ml centrifuge tube containing 5ml of culture solution, centrifuging for 4min at the rotating speed of 350g at the temperature of 4 ℃, opening a cover in the super clean bench, and discarding supernatant;
3) Adding 4ml of DMEM complete high sugar medium to resuspend the cell pellet, inoculating into 6cm cell culture dish, standing at 37 deg.C, 5% 2 Cultured in an incubator.
(7) Growth rate of Hepa1-6BL cells
When the state of the Hepa1-6BL cells is good, the growth speed is higher, the density reaches 90% on the 1 st day, and the ratio of 1:3, the cell density can reach 80-90% the next day.
Example 3
Establishing a Hepa1-6BL subcutaneous tumor model and a liver metastasis in-situ model
(1) Construction of mouse subcutaneous tumor model
1) C57BL/6J female WT mice were selected, aged about 8 weeks, and the animal feeding conditions were the same as those of Hepa1-6 model mice.
2) Preparing Hepa1-6BL cell suspension, adjusting the concentration of the cell suspension to 5.0x10 6 Perml, 100ul of subcutaneous injection per mouse, tumor volume was monitored as before.
3) The tumor growth curves are shown in fig. 5a and 5 b.
(2) Construction of mouse liver metastasis in-situ cancer model
1) Experimental mice C57BL/6J male WT mice were selected, and the week age was about 11 weeks, and the animal feeding conditions were the same as above.
2) Hepa1-6BL cells were prepared and the cell suspension concentration was adjusted to 5.0x10 5 Per mouse 2ml was injected intravenously over 8 seconds, euthanized on day 22, liver tissue harvested and tumor metastases assessed.
3) The results of Hepa1-6BL cell transfer in liver tissues are shown in FIGS. 6 a-6 c.
Example 4
Analysis of the difference between the transcriptome of Hepa1-6 and Hepa1-6BL cells and the difference between immunoregulatory genes
(1) Trizol extracts Hepa1-6 and Hepa1-6BL cell total RNA for transcriptome sequencing.
Significant difference genes of two different cell lines were compared by the DEseq2 software package of the R software.
As shown in FIG. 7, it is shown that there are 6031 genes with significant difference in transcription level in Hepa1-6 and Hepa1-6BL cells (the screening criteria of significant difference genes are adjusted p value <0.05, | log2FC | ≧ 1), wherein 2652 genes with up-regulated transcription level and 3479 genes with down-regulated transcription level are present in Hepa1-6BL compared with Hepa 1-6.
(2) And (4) performing functional enrichment analysis on the significant difference genes.
As shown in fig. 8, the Clusterprofiler software package of the R software was used to perform GO (Gene Ontology) function enrichment analysis, which indicates that the differential genes are mainly significantly enriched in Cell migration (Cell migration), cell chemotaxis (Cell chemotaxis), and other biological functions.
(3) The Cluster profiler software package of the R software is used for KEGG functional enrichment analysis, as shown in FIG. 9, it is shown that the differential genes are mainly and significantly enriched in related pathways such as Cytokine-Cytokine receptor interaction, cell adhesion molecules, IL17 signaling pathway (IL 17 signaling pathway), and the like. Alterations in these pathways indicate altered interactions with the immune system during development in Hepa1-6BL cell mice compared to Hepa1-6 cells.
(4) A list of immune Gene sets was obtained from the Gene Lists (immeport. Org) site and intersected with the difference genes of Hepa1-6 and Hepa1-6BL tumor cells, resulting in 347 significantly different immune genes (as shown in fig. 10 a).
An immunogene expression thermogram with significant differences was plotted using the R pheamap function. As shown in fig. 10b, it was demonstrated that there were more immune-related genes down-regulated in the Hepa1-6BL cell line; immunosuppressive genes S100a4, slc16a3, etc. are up-regulated in Hepa1-6 BL. The enhancement of the immune escape capacity of the Hepa1-6BL tumor cells is shown to be one of the main reasons for the Hepa1-6BL cells to rapidly form tumors. Comparison of the sequencing results of the transcriptome of Hepa1-6 and Hepa1-6BL is helpful for deeply revealing genes of Hepa1-6BL liver cancer cells, which are subjected to immune editing in mice, and genes related to tumorigenesis, tumor development and immune escape.
Example 5
Hepa1-6BL subcutaneous tumor immune cell infiltration assay
(1) Single cell suspension preparation
1) The tumor-bearing mice were euthanized, tumor tissue was removed, and weighed;
3) Putting 200mg of tumor tissue into a 12-hole plate with 300ul of tissue digestive juice, and shearing the tumor tissue by using scissors until the volume of the tissue digestive juice is 1.5ml;
4) Placing the 12-hole plate on a shaking table at 37 ℃, rotating at 150RPM, and digesting for 45min;
5) Placing the cell sieve on a 50ml centrifuge tube, and adding 3ml PBS to wet the cell sieve filter cloth for later use; adding all tissues and digestive juice of a 12-hole plate into a 50ml centrifuge tube containing a cell sieve, grinding tumor tissues by using a piston handle of a 1ml syringe, and adding PBS (phosphate buffer solution) to wash cells to enable the cells to completely enter the 50ml centrifuge tube;
6) Centrifuging at the rotating speed of 350g in a4 ℃ centrifuge for 4min;
7) Discard the application, add 1ml 2% FACS buffer (1 ml fetal bovine serum +50ml PBS) per 200mg tumor tissue to resuspend the cell pellet.
(2) Flow antibody staining and staining
1) Adding 150ul of sample into a 96-hole U bottom plate, precooling a centrifuge at 4 ℃, centrifuging for 4min at a rotating speed of 350g, and discarding the supernatant;
2) Diluting APCcy7-CD45.2 (500-fold), fc blockade (200-fold), 7AAD (200-fold) in a brown centrifuge tube using 2% FACS buffer as a dilution solution;
3) Adding 40ul of mixed antibody diluent into the step 1), blowing and beating the mixed cells by using a pipette gun for 2-3 times, dyeing for 20min in a dark place at 4 ℃, and adding 150ul of PBS for dilution and dyeing;
4) Centrifuging at 4 deg.C with a precooling centrifuge at 350g for 4min, and discarding the supernatant.
5) Adding the FACS buffer to the cell suspension at 200ul 2% and waiting for detection on a machine;
6) Detection was performed on a machine using an AriaIII flow cytometer.
7) Streaming data was analyzed using FlowJo _ V10 software, as shown in FIG. 11, CD45.2 + The proportion of cells was about 10%.
8) Sorting of CD45.2 in Hepa1-6BL tumor tissue Using Aria III according to 7) flow cytometry analysis + A living cell. Analysis of single cell transcriptome expression profiling of 9200 cells was achieved by 10X Genomics 3' transcriptome using short read length sequencing and microfluidic technologies. Gel Beads containing barcode information were combined with a mixture of cells and enzymes, and the heads were encapsulated by oil surfactant droplets In a microfluidic system to form GEMs (Gel Beads-In-Emulsions). The GEMs flow into the reservoir and are collected,the gel beads are dissolved to release the Barcode sequence, the cDNA fragments are reverse transcribed, and the sample is labeled.
9) The gel beads were broken and oil droplets were broken up and PCR amplification was performed using cDNA as a template. After the cDNA is broken into 200-300bp fragments by using cNDA enzyme, PCR amplification is carried out by adding a sequencing linker P3, a sequencing primer R1 and the like to obtain a DNA library.
10 High-throughput sequencing of the constructed libraries was performed using the paired-end sequencing mode of the Illumina sequencing platform. At the Read 1 end, 16bp of barcode information and 10bp of UMI information are contained for determining cells and quantifying expression quantity; at the Read2 end, a cDNA fragment is included for reference genomic alignment to determine the gene corresponding to the mRNA. Data quality statistics were performed on the raw data using 10 xgecomics official analysis software Cell Ranger and aligned to the reference genome.
11 For quality control of cells, gene expression levels and ratios, and mitochondrial expression, 9200 immune cells were clustered into 14 cell subsets using the software Sseurat software package, and the results are shown in fig. 12.
12 FIG. 13 is a thermogram of the expression of the top ten highly mutated marker genes in different cell subsets. The function of the Find markers of each cell subset is screened out by using the R software, the search software package, and the functions of the Find markers. Figure 13 is a thermogram of the expression of top ten hypervariable genes in each cell subpopulation. Establishing a marker gene library for each immune cell subset by consulting literature, and annotating and identifying the species of each cell subset according to tumor immunity theory, based on a hypermutation gene expression thermogram ranking top ten of each cell subset. The results of the identification of the cell subpopulations are annotated in fig. 13.
13 FIG. 14 shows the ratio of different immune cell subsets in Hepa1-6BL tumor tissues. After 9200 immune cells are classified, identified and annotated, the proportion of the cells of each cell subset in the whole immune cells of the Hepa1-6BL liver cancer tissue is calculated.
The research result shows that the proportion of macrophages with TAM characteristics in Hepa1-6BL liver cancer tissue immune cells is nearly 50 percent, the proportion of monocytes is 26 percent, and the proportion of basophils and CD8 cells is nearly 50 percent + T cells and CD4 + Of T cellsThe ratio is close to 5%, DC and NK cells account for nearly 3%, and neutrophils account for 1%. The types of cell subsets infiltrated by the liver cancer tissue of Hepa1-6BL and the proportion of the cell numbers thereof are completely and clearly shown for the first time through a single cell transcriptome sequencing result. The infiltration of immune cells in this model has a high similarity to the infiltration of immune cells in the tumor tissue of patients with liver cancer. The model can well reflect the tumor microenvironment of the liver cancer patients and is used for the research of liver cancer and immunology.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.
Claims (7)
1. An in vitro culture method of mouse liver cancer model cells, wherein the mouse is a C57BL/6 mouse, and the method comprises the following steps:
(1) Preparing a Hepa1-6 cell for an injection mouse;
(2) Injecting the injected mouse with Hepa1-6 cells, monitoring the growth of the tumor volume of the mouse, and screening the mouse reaching the preset tumor size to obtain a tumor tissue;
(3) And treating the tumor tissue to obtain a mouse liver cancer model cell which is named as a Hepa1-6BL cell.
2. The method for culturing mouse liver cancer model cells according to claim 1, wherein the step (1) comprises:
(1-1) taking out the Hepa1-6 cells frozen in liquid nitrogen; rapidly thawing and recovering at 37 ℃;
(1-2) opening a cell freezing tube, adding cell sediment into a 10ml centrifuge tube containing 5ml of culture solution, centrifuging at 4 ℃, and removing supernatant;
(1-3) 4ml of DMEM complete high sugar medium resuspended cell pellets, added uniformly to a 6cm cell culture dish, left at 37 ℃ and 5% CO 2 Culturing in an incubator;
(1-4) when the cell density reaches 80% -90%, carrying out cell passage by trypsinization;
(1-5) 1ml of complete medium was used to suspend the cells, and the cells were plated in a 6cm dish and cultured to be good, to obtain the Hepa1-6 cells for injection mouse.
3. The method for culturing mouse liver cancer model cells according to claim 2, wherein in the step (1-5), the cells are cultured to be good and visible under high power microscope: the cell has smooth edge, clear and visible outline, good light transmission inside the cell, strong refractivity, few particles and no vacuole.
4. The method for culturing mouse liver cancer model cells according to claim 1, wherein the step (2) comprises:
(2-1) preparing a single cell suspension;
(2-2) injecting each mouse subcutaneously and raising;
(2-3) recording the cell injection mice as day 0, monitoring the growth of the tumor volume of the mice from day 7, and recording the tumor volume;
(2-4) screening mice reaching the preset tumor size to obtain tumor tissues.
5. The method for culturing mouse liver cancer model cells according to claim 1, wherein the step (3) is specifically as follows:
(3-1) placing the tumor tissue into a 6-well plate containing digestive juice, and digesting;
(3-2) adding all tissues and digestive juice of the 6-well plate into a 50ml centrifuge tube containing a cell sieve for filtration and centrifugation;
(3-3) discarding the supernatant, adding DMEM to the complete high-glucose medium to resuspend the cell pellet, and plating it in a 6-well plate, standing at 37 ℃ and 5% CO 2 Culturing in an incubator to obtain the Hepa1-6BL cells.
6. Use of the mouse liver cancer model cell obtained by the in vitro culture method according to any one of claims 1 to 5 in establishing a mouse liver cancer model.
7. The use of claim 6, wherein said modeling mouse liver cancer comprises the establishment of subcutaneous tumors and the development of carcinoma in situ.
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| CN114009399A (en) * | 2021-10-28 | 2022-02-08 | 复旦大学附属华山医院 | Preparation and application of drug-resistant mouse and cell strain of liver cancer immune check point antibody |
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