CN117625549A - Rat hydrodynamic liver cancer cell line and application thereof - Google Patents
Rat hydrodynamic liver cancer cell line and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a rat hydrodynamic liver cancer cell line and application thereof. The invention provides a rat hydrodynamic liver cancer cell line, which has the preservation number of GDMCCNO of 63760. The invention applies the rat hydrodynamic liver cancer cell line in the field of biological medicine. The rat liver cancer cell line RMEC-9M cell line is a Wistar rat liver cancer cell line with high proliferation and high metastasis tendency, can form tumors under the skin and liver in situ in nude mice or Wistar rats, and has rapid tumor growth and no restrictive growth. The cell line can be used for extensive liver cancer related research.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a rat hydrodynamic liver cancer cell line and application thereof.
Background
Primary liver cancer is a common malignant tumor of the digestive system, and hepatocellular carcinoma (hepatocellular carcinoma, hereinafter referred to as "liver cancer") is a main pathological type of primary liver cancer, accounting for 75% -85%. The liver cancer treatment strategy is to carry out multidisciplinary comprehensive treatment according to different clinical stages, but the overall treatment response rate of the liver cancer is low, the recurrence rate is high, and the treatment resistance is easy to generate no matter the treatment is carried out by single local treatment or local combined system treatment.
In order to clarify the characteristics of liver cancer cells, search for key pathogenesis and potential biomarkers, develop new therapeutic targets, and currently, there is a great need to perfect an animal experiment platform for liver cancer. Liver cancer models and cell models for mice are relatively perfect at present, but rats have incomparable operational advantages compared with mice in the fields of local interventional therapy, ablation and surgical treatment of liver cancer. The Chinese patent No. 202310598588.4 discloses a strategy for constructing a rat primary liver cancer model by utilizing tail vein hydrodynamic technology, which is used for realizing the generation of the rat primary liver cancer driven by a key driving gene for the first time and providing a proper experimental material for researching the generation and treatment of the liver cancer. However, due to the fact that the related tail vein hydrodynamic liver cancer modeling technology is high in difficulty, the model construction period is long, the cost is high, quantitative production is difficult to achieve, due to the fact that heterogeneity among animal individuals is large, randomness exists in the tumor occurrence position of the hydrodynamic liver cancer model, and homogenization of the tumor ending is difficult to achieve. Therefore, constructing the corresponding rat liver cancer cell line for in vitro research and site-directed subcutaneous or in situ tumorigenesis is a choice with good cost performance. In the prior art, the existing primary culture and establishment method of liver cancer cells of human or mouse origin generally relates to a complex cell dispersion method, which comprises a plurality of steps such as enzymolysis digestion, filtration centrifugation and the like, and a complex tissue primary treatment process and a culture medium of special additives are needed, and the method has the advantages of high culture success rate, high initial cell purity, relatively good activity, complex operation, quite high requirements on a platform and no promotion. Aiming at the aspects of rat liver cancer cell lines and the establishment method thereof, the prior rat liver cancer cell lines are recently reported to be difficult to obtain in China, and most cell lines have the defect of self-limited growth in vivo, so that the general rule of liver cancer development cannot be simulated.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a rat hydrodynamic liver cancer cell line and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a rat hydrodynamic liver cancer cell line, which has the accession number GDMCC NO. 63760. The cell line is named as a rat liver cancer cell line RMEC-9M and is preserved in the Guangdong province microorganism strain collection center (GDMCC) at the 8 th month of 2023, and the preservation address is No. 100 Mitsui university in the View district of Guangzhou, guangdong province.
The established rat liver cancer cell line RMEC-9M is a Wistar rat liver cancer cell line with high proliferation and high metastasis tendency, can form tumors under the skin and liver in situ in nude mice or Wistar rats, and has rapid tumor growth and no restrictive growth. The cell line can be used for extensive liver cancer related research.
As a preferred embodiment of the rat hydrodynamic liver cancer cell line of the present invention, the cell line overexpresses MYC, EMX1, CTNNB1 and luciferase.
In a second aspect, the invention provides a construction method of a rat hydrodynamic liver cancer model, comprising the following steps:
injecting the plasmid composition into tail vein of rat, and transforming into liver cells of rat to obtain hydrodynamic liver cancer model of rat; the plasmid composition is pT3-EF1A-MYC-P2A-Emx1-IRES-Luciferase, pT3-N90-beta-catenin and pCMV (CAT) T7-SB100.
As a preferred embodiment of the construction method of the present invention, the pT3-EFIA-MYC-P2A-Emx1-IRES-Luciferase, pT-N90-beta-catenin, pCMV (CAT) T7-SB100 has a concentration of (5-20): 1-4. Mu.g/ml; preferably, the concentration is 10:10: 2. Mu.g/ml.
In a third aspect, the invention provides a culture method of a rat hydrodynamic liver cancer cell line, which adopts an improved mechanical adherence method to culture primary liver cancer cells and adopts a gradient digestion method to purify and construct the liver cancer cells.
As a preferred embodiment of the culture method according to the present invention, the method comprises the steps of:
cutting active liver cancer tissues in the rat hydrodynamic liver cancer model, cutting into fragments, suspending with pure fetal bovine serum, transferring to a culture flask, and standing; turning over the culture bottle up and down, and standing; the liquid is sucked and removed, the culture flask is turned over, and the complete culture medium is added for incubation and culture.
The steps are operated at low temperature, under aseptic conditions.
In a fourth aspect, the present invention applies the rat hydrodynamic liver cancer cell line in the field of biological medicine.
In a fifth aspect, the present invention applies the rat hydrodynamic liver cancer cell line in constructing a liver cancer cell model or an animal model.
In a sixth aspect, the invention applies the rat hydrodynamic liver cancer cell line in preparing, screening or evaluating liver cancer therapeutic drugs. The hepatoma cell line RMEC-9M was inoculated subcutaneously or in situ in nude mice or Wistar rats and tumor progression was followed by in vivo imaging.
In a seventh aspect, the invention uses the rat hydrodynamic liver cancer cell line as an experimental material in liver cancer basic or clinical research.
Compared with the prior art, the invention has the beneficial effects that:
1. the rat liver cancer cell line RMEC-9M cell line has the advantages of good tumor cell morphology, high purity and quick proliferation, retains the characteristics of hydrodynamic driving genes, overcomes the problem of limited growth of in-situ or subcutaneous transplantation tumor of most of the rat liver cancer cell lines in the past, is a Wistar rat liver cancer cell line with high proliferation and high metastasis tendency, can form tumors under the skin and liver in-situ tumors of nude mice or Wistar rats, and has the advantages of quick tumor growth and no occurrence of the situation of limited growth. The method can realize the modeling of the models including subcutaneous tumor formation, liver in-situ tumor formation and the like, and the tumors have the characteristics of high proliferation and high metastasis, can meet the construction requirements of various Wistar rat liver cancer animal models, and opens up a new way for the construction of cells and animal models for liver cancer intervention and treatment. The cell line can be used for extensive liver cancer related research.
2. Compared with traditional chemical drugs and poison induced primary liver cancer of rats, the source of the liver cancer of the rats adopted by the invention is hydrodynamic liver cancer driven by specific genes, has the advantages of single and controllable gene background and similar biological characteristics of the cell line and the primary liver cancer tissue, and can construct a rat liver cancer library and a cell library platform according to different driving gene combinations.
3. Compared with the existing primary culture method of liver cancer cells, the technical scheme of the invention has simple operation, no special experimental conditions, short molding time, low cost and pure quality of the cultured cell line, and can reproduce the genome and biological characteristics of the original molding liver cancer cells.
Drawings
FIG. 1 shows cell separation, culture and purification;
in FIG. 1, (a) primary cell culture (scale bar 500 μm); (b) pre-purification cell populations (scale bar 500 μm); (c) Culturing more than 20 generations of RMEC-9M cell line (scale bar 100 μm) after separation and purification; (d) Culturing more than 20 generations of RMEC-9E cell line (scale bar 100 μm) after separation and purification; (e) Cytoskeletal immunofluorescence of the RMEC-9M cell line after purification (scale bar 20 μm); (f) After purification, the cytoskeletal immunofluorescence of the RMEC-9E cell line (scale bar 20 μm).
FIG. 2 is a representation of the characteristics of the RMEC-9 liver cancer cell line;
in FIG. 2, (a) a report experiment of RMEC-9 cell luciferase gene before purification; (b) Reporting experiments of luciferase genes of the purified RMEC-9M cell line and the RMEC-9E cell line; (c) Normal liver tissue and liver tumor tissue of original RMEC-9 rat and protein expression characteristics of purified RMEC-9M, RMEC-9E cell line.
FIG. 3 shows the tumor formation of RMEC-9M liver cancer cell line in Wistar rats;
in fig. 3, (a) Wistar rats were imaged in vivo 2 weeks after subcutaneous tumor growth; (b) general conditions after 2 weeks of subcutaneous tumor in Wistar rats; (c) After 2 weeks of subcutaneous tumor formation and in situ liver tumor formation in Wistar rats, the tumors were of general origin.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: construction of rat hydrodynamic liver cancer model
Referring to the method in the Chinese patent No. CN 202310598588.4, a rat hydrodynamic liver cancer model is constructed by adopting pT3-EF1A-MYC-P2A-Emx1-IRES-Luciferase (modified by adedge # 129775), pT3-N90-beta-catenin (adedge # 31785) and pCMV (CAT) T7-SB100 (adedge # 34879) plasmid combinations. Wherein, the ratio of pT3-EF1A-MYC-P2A-Emx1-IRES-Luciferase, pT3-N90-beta-catenin to pCMV (CAT) T7-SB100 is 10:10:2 mug/ml concentration configuration, a Wistar rat primary liver cancer model driven by MYC, EMX1 and CTNNB1 genes was constructed.
After conventional hydrodynamic modeling of rats, continuous in vivo imaging was performed to monitor tumor development and progression. Through tumor occurrence and progress evaluation, a model No. 9 rat is preferred, and the liver tumor signal is determined to be in a continuous and rapid growth trend through in vivo imaging monitoring, and the rat is determined to have multiple nodular lesions with ascites due to intrahepatic blood enrichment by Doppler ultrasonic examination. Through general dissection and pathological confirmation, the rat is multiple liver cancer with intrahepatic metastasis, extensive peritoneal metastasis and cancerous ascites.
The Rat was selected as a representative for primary isolated culture of hydrodynamic liver cancer cells of the Rat and cell line construction, named RMEC-9 (Rat-MYC-Emx 1-CTNNB1- # 9).
Example 2: primary cell culture of rat liver cancer
The improved tissue block mechanical adherence method is adopted to culture the primary cells of liver cancer. The method comprises the following specific steps:
MYC, EMX1 and CTNNB1 three genes drive Wistar rat hydrodynamic liver cancer to model for 9 weeks laterPreferably model No. 9 rat, after the rat is sacrificed by carbon dioxide anesthesia, the body surface is sufficiently sterilized and the liver is dissected by aseptic manipulation. The liver tumor sampling needs to be carried out by taking care of avoiding obvious blood vessels and envelope areas, avoiding bleeding and necrotic tissues, cutting fish-meat liver cancer tissues with the size of soybean, transferring to a sterile dish on ice, carefully removing adhered fibrous tissues and blood clots, and washing with normal saline. Tumor tissue was cut to about 1mm with a sterile blade 3 Pieces of the size, the tissue pieces were suspended with 1ml of pure fetal bovine serum, transferred to a conventional T25 flask, and allowed to stand in the incubator for 5-10 minutes. And then the culture flask is turned up and down rapidly, the tumor tissue blocks are still adsorbed at the bottom of the flask due to tension, most of culture medium flows to the top of the flask, and the incubator stands for 10-20 minutes. Carefully sucking the culture medium, turning the flask upside down again, adhering the tissue block to the bottom of the flask, adding appropriate amount of DMEM medium (hereinafter referred to as complete medium) containing 10% fetal calf serum and 1% penicillin-streptomycin, and placing in a flask containing 5% CO 2 Subsequent cultures were performed in an incubator at 37 ℃. The material drawing process is operated on ice, and the whole process is aseptic.
After overnight incubation of the tissue mass, the primary cells were observed with a phase contrast microscope. The visible tissue block is attached to the bottom of the culture bottle, a small amount of cells climb out from the periphery, at the moment, tumor cells are in an active moving state, and the cells are visible to divide, but are not vigorous, no special intervention is needed, and the tumor cells continue to return to the incubator for culture. The cell migration and crawling-out time depends on the biological characteristics of the sampled tumor cells, and is usually observed for 3-14 days by continuous culture, so that the cells are migrated and crawled out around a plurality of tissue blocks in a culture flask, the tissue blocks gradually collapse and become smaller, the cell areas of the adherent cells are larger and larger, a circular cell island (figure 1 a) taking the tissue blocks as the center is formed, similar to a large cell colony, the fact that the cell island is internally mixed with tumor-related fibroblasts, vascular endothelial cells and tumor cells to jointly grow is possibly observed, the growth of primary cells is temporarily not interfered, and the continuous observation is carried out. If the culture medium turns yellow, the culture medium should be sucked, the tissue blocks and cells with weak adhesion are rinsed gently by PBS, and fresh complete culture medium is added again for continuous culture. If a large amount of tissues and cell clusters float or the culture medium becomes turbid meat chyme red, the pollution of cells is indicated, and timely harmless treatment is required.
Example 3: purification and establishment of rat liver cancer cell
After the primary cells of the liver cancer of the rat are successfully cultured, the support of the primary tissue matrix is lost, and the in vitro culture adherence and proliferation capacity of the primary cells are weaker, so that after the primary cell culture is successfully established, the primary cells are subjected to conventional digestion, centrifugation and resuspension by trypsin, and then are subjected to bottle-changing culture according to a 1:1 passage. Since no special culture medium is used for culture or drug screening, fibroblasts or other cells are usually mixed in the cultured primary liver cancer cells (FIG. 1 b). If the proliferation activity of the taken tumor cells is not strong, no human intervention is applied, and after continuous passage, the fibroblasts are easy to appear to become dominant populations to displace the growth of the tumor cells.
By subculturing the first three generations of cells, the reaction characteristics of different cells to digestive enzymes in the enzyme digestion process can be basically clarified: usually, the fibroblast is digested and wall removed firstly, while the liver cancer cell with interstitial feature is removed, while the typical epithelial-like feature liver cancer cell is digested and wall removed only for a long time (usually more than 3-5 minutes), the difference between the primary culture and the primary culture is obvious, and the difference can be used for separating the fibroblast from the liver cancer tumor cell subgroup with different characteristics by adopting a method of multiple difference digestion. The differential digestion passage is repeated for a plurality of times, and the separation and purification of liver cancer cells can be completed within 10 generations of primary culture. After purification, the liver cancer cells can be continuously and stably passaged for more than 20 generations, and the liver cancer cells are considered to be successfully established.
By using the enrichment method of differential digestion, the Wistar rat RMEC-9 cell line provided by the invention is finally separated and purified to form a RMEC-9M cell sub-line (subgroup of the mesenchymal morphological characteristics) and a RMEC-9E cell sub-line (subgroup of the epithelial morphological characteristics) which can be stably passaged. Cell growth morphology of the cell lines of the two subpopulations was greatly different (FIGS. 1 c-f). Wherein, the epithelial type RMEC-9E cell line grows in a compact hepatocyte epithelial-like characteristic, and the mesenchymal type RMEC-9M cell line grows in a more obvious mesenchymal type cell characteristic than RMEC-9E. At present, two cell lines of RMEC-9M and RMEC-9E cultured in a laboratory are continuously passaged for more than half a year, and can still keep vigorous division proliferation capacity after in vitro passaging for more than 50 generations, and mycoplasma pollution is detected negatively conventionally.
If the liver cancer cells and the fibroblasts are still not completely separated finally through repeated differential digestion operations, the situation usually occurs that the proliferation activity of the collected primary liver cancer cells is not vigorous but the fibroblast is excessively vigorous, and the final purification of the cells depends on a single cell cloning method. The adaptive medium may be harvested in advance to increase the success rate of monoclonal cell culture prior to determining that monoclonal cell culture is to be performed. Specifically, the homologous cells to be cultured are brought to a semi-confluent state, the culture solution is changed, the culture is further cultured for 24 hours, all the culture solution is sucked out, the centrifugation is carried out for 10 minutes at 4000 revolutions per minute, and the adaptive culture medium is filtered through a 0.22 mu m filter for standby. Taking a bottle of primary liver cancer cells in logarithmic growth phase, conventionally digesting, centrifuging and counting, and re-suspending the cells by using an adaptive culture medium. The plates were graded by limiting dilution using 96 well plates and placed in an incubator for culture. The following day under an inverted microscope, since the proliferation capacity of primary cultured hepatoma cells was not vigorous, not only single cell wells were considered potential targets, but all cell-dispersed wells should be considered potential monoclonal targets. Culturing for several days, observing under an inverted microscope, marking to form a single clone hole by only single cells, culturing until the cell clone reaches more than 50 cells, and performing conventional liquid exchange. After the cell clone number reaches a certain degree, the cell clone number can be gradually transferred from a 96-hole plate to a 48-hole plate, a 24-hole plate, a 12-hole plate and a 6-hole plate, and finally the cell clone number enters a conventional cell passage until stable passage is carried out for more than 20 generations, and the establishment of a purified rat liver cancer cell line is successful.
The RMEC-9M and RMEC-9E cells of the invention have been continuously passaged for half a year, and can still maintain vigorous division proliferation ability after in vitro passaging for 50 generations.
Example 4: application of rat liver cancer cell
In the early stage of primary culture, the obtained RMEC-9 cell line is subjected to a cell luciferase gene reporting experiment (figure 2 a), and the result proves that the primary cultured cells retain the characteristics of the expression of the original hydrodynamic liver cancer luciferase gene. Further, cell lines enriched for different characteristics were continuously screened using differential digestion enrichment, and finally an RMEC-9M cell line with significant interstitial characteristics (FIG. 1E) and an RMEC-9E cell line with significant epithelial characteristics (FIG. 1 f) were isolated. And selecting RMEC-9M and RMEC-9E cell lines which are stably passaged for more than 20 generations, and comparing with normal liver and primary liver cancer tissues of rats through a cytoluciferase gene report experiment (figure 2 b) and a Western-blot (figure 2 c) experiment, the RMEC-9M cell with interstitial type characteristics more remarkably reserves characteristic expression genes of the primary dynamic liver cancer tissues, including over-expression MYC, EMX1, CTNNB1 genes and luciferase genes. Whereas the epithelial-like characteristic RMEC-9E cells retain only a partial characterization of the primitive hydrodynamic liver cancer tissue.
The established RMEC-9M cell line is a Wistar rat liver cancer cell line with high proliferation and high metastasis tendency. Taking RMEC-9M cells in logarithmic growth phase, conventionally digesting, counting, taking 2-10×10 6 Individual cells were resuspended in 1-3:1 volume with PBS matrigel and Wistar rats were subjected to subcutaneous or hepatic in situ neoplasia. 2 weeks after cell inoculation, in vivo imaging suggests that Wistar rat subcutaneous tumors grew rapidly. After 2 weeks of in situ inoculation of the liver with cells, a large tumor was noted in the liver area, the tumor grew rapidly, and no restricted growth was observed (FIGS. 3 a-c).
The invention selects the model No. 9 Rat as the representative for primary isolated culture and cell construction of the Rat hydrodynamic liver cancer cells, and marks the model as a Wistar Rat hydrodynamic (MYC-Emx 1-CTNNB 1) liver cancer cell line interstitial type, and RMEC-9M (Rat-MYC-Emx-CTNNB 1- # 9-Mesenchyal_subtype). The cell line is named as a rat liver cancer cell line RMEC-9M, and is preserved in the microorganism strain collection (GDMCC) of Guangdong province at the 8 th month of 2023, the preservation number is GDMCC NO. 63760, and the preservation address is Mitsui No. 100 university in the View district of Guangzhou province. The Wistar rat tail vein hydrodynamic liver cancer cell line RMEC-9M is a Wistar rat liver cancer cell line with high proliferation and high transfer tendency, is an important supplement for the existing rat liver cancer cell line and liver cancer animal model, and can be used for constructing models of immunodeficient mice such as nude mice, subcutaneous and in-situ liver cancer of Wistar rats and the like. The model has the characteristics of single cell line gene background, simple construction operation and low cost, and can be used for constructing more liver cancer model experiment platforms aiming at the whole mutation spectrum of the multi-cell line of rats or mice on the basis of the model in future.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A rat hydrodynamic liver cancer cell line, characterized in that the cell line has a deposit number of GDMCCNO 63760.
2. The rat hydrodynamic liver cancer cell line of claim 1, wherein the cell line overexpresses MYC, EMX1, CTNNB1, and luciferase.
3. The construction method of the rat hydrodynamic liver cancer model is characterized by comprising the following steps of:
injecting the plasmid composition into tail vein of rat, and transforming into liver cells of rat to obtain hydrodynamic liver cancer model of rat;
the plasmid composition is pT3-EF1A-MYC-P2A-Emx1-IRES-Luciferase, pT3-N90-beta-catenin and pCMV (CAT) T7-SB100.
4. The method according to claim 3, wherein the pT3-EFIA-MYC-P2A-Emx1-IRES-Luciferase, pT-N90-beta-catenin and pCMV (CAT) T7-SB100 are used in a concentration of (5-20): 1-4. Mu.g/ml.
5. A method for culturing the hydrodynamic liver cancer cell line of rat features that the primary liver cancer cell is cultured by improved mechanical adhesion method and the gradient digestion method is used to purify and construct liver cancer cell line.
6. The culture method according to claim 5, comprising the steps of:
cutting active liver cancer tissue in the rat hydrodynamic liver cancer model according to claim 3, cutting into fragments, suspending with pure fetal bovine serum, transferring to a culture flask, and standing; turning over the culture bottle up and down, and standing; sucking and removing liquid, turning over a culture bottle, adding a complete culture medium, and incubating and culturing;
the steps are operated at low temperature, under aseptic conditions.
7. Use of the rat hydrodynamic liver cancer cell line of claim 1 or 2 in the field of biological medicine.
8. Use of the rat hydrodynamic liver cancer cell line of claim 1 or 2 as a liver cancer cell model or animal model.
9. Use of the rat hydrodynamic liver cancer cell line of claim 1 or 2 in the preparation, screening or evaluation of a liver cancer therapeutic drug.
10. Use of the rat hydrodynamic liver cancer cell line of claim 1 or 2 as an experimental material in liver cancer basal or clinical research.
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