Mouse prostate cancer primary cell as well as construction and culture method and application thereof

Abstract

The invention relates to the technical field of primary cell lines, and discloses a mouse prostate cancer primary cell and a construction and culture method and application thereof, the mouse prostate cancer primary cell is named as mPCPU, and the cell adheres to the wall and is fusiform; the cells aggregate to form a cavity similar to a prostate cavity; the multiplication speed is high, and the multiplication time is about 24 hours. By separating and culturing C57 / BL6 mouse prostate cancer tissues, the mouse prostate cancer primary cell line is successfully established, and the cell line has stable passage capacity and can be used for research on generation and development molecular mechanisms of prostate cancer, especially research on gene functions in immune totipotent mice.

Description

Technical Field

[0001] The present invention relates to the technical field of primary cell lines, in particular to primary cells of mouse prostate cancer and construction, culture methods and applications thereof. Background Art

[0002] Prostate cancer is the most common malignant tumor in the urinary system. Its incidence rate ranks second among male tumors worldwide and is a major disease that endangers men's life and health. The tumor microenvironment plays an indispensable role in the occurrence and development of tumors. However, due to ethical restrictions, the study of the tumor microenvironment of prostate cancer is mainly carried out in mouse models. Due to the differences between humans and mice, when analyzing molecular mechanisms, especially when analyzing the interaction between tumor cells and tumor microenvironment, there is a lack of stable mouse primary prostate cancer cell models for research. The present invention separates mouse prostate cancer tissue tumor cells for in vitro culture to obtain a stable mouse prostate cancer primary cell line. Summary of the invention

[0003] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a primary mouse prostate cancer cell and a construction, culture method and application thereof, which has the advantage of providing a stable passage primary mouse prostate cancer cell line.

[0004] (II) Technical solution In order to achieve the above-mentioned purpose of providing a stable passage mouse prostate cancer primary cell line, the present invention provides the following technical solution: mouse prostate cancer primary cells, named mPCPU, grow adherently and have a spindle-shaped morphology; the cells aggregate to form a cavity similar to the prostate cavity; the proliferation rate is fast, with a doubling time of about 24 hours.

[0005] The cells are isolated and cultured from prostate tumor tissue of spontaneous prostate cancer gene-engineered mice with C57 / BL6 mouse genetic background.

[0006] This cell line was used for the construction of gene knockdown cell lines, cell proliferation experiments, cell invasion experiments, and co-culture experiments with immune cells.

[0007] (III) Beneficial effects Compared with the prior art, the present invention provides a mouse prostate cancer primary cell and its construction, culture method and application, which have the following beneficial effects: The present invention successfully established a mouse prostate cancer primary cell line (mPCPU) by isolating and culturing C57 / BL6 mouse prostate cancer tissue. The cell line has stable passage ability and can be used to study the molecular mechanism of prostate cancer occurrence and development, especially the study of gene function in immune omnipotent mice. It can also be used for cell biology, biochemistry, and molecular biology research on prostate cancer, and has important value in the fields of gene function verification, drug screening and sensitivity research, and cell behavior analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 , pictures of primary mouse prostate cancer cells; Figure 2 , Mouse prostate cancer primary cell proliferation experiment; Figure 3 , construct mouse prostate cancer primary cell knockdown Rela cell line and control cell line; Figure 4 , invasion experiment of mouse prostate cancer primary cell knockdown Rela cell line and control cell line; Figure 5 , mouse prostate cancer primary cell knockdown Rela cell line and control cell line conditioned culture medium and mouse-derived MDSCs co-culture experiment. DETAILED DESCRIPTION

[0009] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0010] The present invention separates and cultures a mouse prostate cancer primary cell line from prostate tumor tissue of a spontaneous prostate cancer gene-engineered mouse with a C57 / BL6 mouse genetic background. The primary cell line has the characteristics of immortalization and rapid proliferation. A variety of cell experiments and cell phenotype analysis can be performed on the cell line, and the interaction between prostate cancer cells and immune cells can also be studied. This is further described below in conjunction with specific examples.

[0011] Embodiment 1: Mouse prostate cancer primary cell culture, passaging, cryopreservation, recovery and morphological analysis ( Figure 1 ).

[0012] 1) mPCPU cell culture and passaging.

[0013] Cultured in RPMI-1640 containing 10% FBS, cultured in a cell culture incubator at 37°C containing 5% CO2. When the cells were 80-90% confluent, the culture medium was discarded, washed once with PBS, digested with 0.25% EDTA-trypsin (about 1 minute at 37°C), and digested with RPMI-1640 containing 10% FBS. The detached cells were transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 3 minutes at room temperature, the supernatant was discarded, the cells were resuspended, and subcultured at 1:4.

[0014] 2) Cryopreservation of mPCPU cells.

[0015] The cell morphology and growth status are good. When the confluence reaches about 80%, discard the culture medium in the culture dish, wash once with pre-warmed PBS, add 0.25% EDTA-trypsin to digest, wait for the cells to fall off, terminate the digestion with RPMI-1640 containing 10% FBS, and transfer the cell suspension to a 15ml centrifuge tube. Centrifuge at 1000rpm at room temperature for 3 minutes, discard the supernatant, resuspend the cells with freezing solution (10% DMSO and 90% FBS), put them in a freezing box and transfer them to a -80℃ refrigerator for freezing. After 24 hours, transfer them to liquid nitrogen for long-term storage.

[0016] 3) mPCPU cell recovery.

[0017] Take out the cryovial containing cells from liquid nitrogen or -80℃ refrigerator, quickly put it into 37℃ warm water, gently shake the cryovial until the liquid is completely melted, transfer the cell suspension into 6ml pre-warmed RPMI-1640 culture medium, centrifuge at 1000rpm for 3 minutes at room temperature, discard the supernatant, resuspend with 10% FBS RPMI-1640 cell culture medium, transfer to a cell culture dish, and culture in a cell culture incubator at 37℃ containing 5% CO2.

[0018] 4) mPCPU cell morphology.

[0019] The cells are adherent, spindle-shaped, have strong migration ability, and spontaneously aggregate to form a cavity (see Figure 1 ).

[0020] Embodiment 2: Proliferation phenotype analysis of primary mouse prostate cancer cells ( Figure 2 ).

[0021] 1) Test method steps mPCPU cells were plated into 96-well plates at a density of 3000 cells / well and cultured in a cell culture incubator at 37°C. After the cells adhered to the wall, 10μL 5mg / ml MTT was added to each well and incubated at 37°C for 2 hours. The upper culture medium was discarded, and the time of the first addition of MTT was set to zero. MTT was then added every 24 hours for three days. On the last day, after discarding the supernatant, 100μL dimethyl sulfoxide (DMSO) was added to each well to dissolve purple crystalline formazan, incubated at 37°C for 10 minutes, and its light absorption value was measured at a wavelength of 490nm using an enzyme-linked immunosorbent assay. Since the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble purple crystalline formazan and deposit it in the cells, while dead cells do not have this function, the concentration of purple crystalline formazan can indirectly reflect the number of living cells.

[0022] 2) Test results mPCPU cells have a rapid proliferation rate, reproducing one generation in about 24 hours (see Figure 2 ).

[0023] Embodiment three: Construction of Rela knockdown cells and control cells in mouse prostate cancer primary cells ( Figure 3 ).

[0024] 1) Construction of shRela plasmid.

[0025] Synthesize shRela sequence (forward sequence 5'-ccggGGAGTACCCTGAAGCTATAACctcgagGTTATAGCTTCAGGGTACTCCtttttg-3' and reverse sequence 5'-aattcaaaaaGGAGTACCCTGAAGCTATAACctcgagGTTATAGCTTCAGGGTACTCC-3'); The shRela sequence was inserted into the pLKO.1 vector, then transformed into E. coli for amplification, and the pLKO.1-shRela plasmid was purified and used for subsequent lentivirus packaging after correct sequencing.

[0026] 2) Encapsulation of lentivirus and knockdown of Rela cell construction.

[0027] When the 293FT cells in the 10cm cell culture dish are confluent to 80%, pLKO.1-shRela / pLKO.1-shC, VSV-G and gal-pol are transfected into 293FT cells. After 6-8 hours of transfection, fresh DMEM medium containing 10% FBS is replaced, and lentivirus is collected after 72 hours and infects mPCPU cells. After 48 hours of infection, puromycin is added to select positive cells, and after all negative control cells die, about 5-7 days, fresh RPMI-1640 medium containing 10% FBS is replaced and culture is continued.

[0028] 3) Western blotting to verify the knockdown efficiency of Rela ( Figure 3 ).

[0029] Use RIPA lysis buffer to lyse mPCPU shC and shRela cells. The cell lysate was transferred into an EP tube and lysed on ice for 30 minutes, then centrifuged at 12,000 rpm and 4°C for 30 minutes. The precipitate was discarded, the supernatant was taken, and BCA protein quantification was performed. 5x Loading Buffer was added, mixed, and placed in a 95°C metal bath for 5 minutes. The samples were stored at -20°C.

[0030] 20 μg of total protein was subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the membrane was transferred to a 0.45 μm PVDF membrane. After blocking with 5% milk for 1 hour, the membrane was incubated with primary antibodies of Rela and β-Actin at 4°C overnight. After washing with PBST, the membrane was incubated with secondary antibodies of the corresponding species at room temperature for 2 hours. After washing with PBST, the membrane was developed.

[0031] 4) Real-time fluorescence quantitative PCR verification of Rela knockdown efficiency ( Figure 3 ).

[0032] When the confluence of mPCPU shC and shRela cells reached 90%, the culture medium was discarded, and the cells were washed twice with PBS. Then 1 ml of Trizol was added to the culture dish to lyse the cells. After all the cells fell off, they were transferred to a 1.5 ml EP tube and placed at room temperature for 5 minutes until the cells were completely lysed.

[0033] Add 1 / 5 volume of chloroform, mix thoroughly, let stand at room temperature to separate the layers, then centrifuge at 4°C and 12000rpm for 20 minutes. After the centrifugation, take the upper colorless transparent liquid, add an equal volume of isopropanol, mix thoroughly, centrifuge at 4°C and 12000rpm for 15 minutes. After the centrifugation, discard the supernatant, keep the white precipitate at the bottom, wash twice with 75% ethanol, discard the supernatant, and when the precipitate at the bottom becomes translucent, add DEPC water to dissolve it, and measure the RNA concentration with a spectrophotometer. It can only be stored at -80°C for a long time.

[0034] 1 μg of total RNA was reverse transcribed into cDNA, and then fluorescent quantitative PCR was performed using the Novozyme SYBR qPCR Master Mix kit. The primer sequences are as follows: β-Actin: 5'-CTTCGTTGCCGGTCCACAC-3' and 5′-GCCTCGTCACCCACATAGG-3′; Rela: 5'-GAGTCTCCATGCAGCTACGG and TTTCGGGTAGGCACAGCAAT-3'.

[0035] Embodiment 4: Analysis of invasion phenotype of mouse prostate cancer primary cells shRela cells and shC cells ( Figure 4 ).

[0036] 1) Test methods and procedures.

[0037] Matrigel without growth factors was diluted with pre-cooled PBS at a ratio of 1:8. 100 μL of the dilution was added to an 8 μm transwell chamber and placed in a 37 ℃ cell culture incubator until the matrix gel solidified.

[0038] Take cells with good morphology and growth status when the confluence reaches 80%, discard the culture medium, wash once with PBS, and then add 0.25% trypsin for digestion. After the cells fall off, add 10% FBS RPMI-1640 culture medium to stop the process, centrifuge and discard the supernatant, resuspend the cells in PBS and centrifuge again and discard the supernatant, resuspend the cells in FBS-free RPMI-1640 culture medium and count the cells, take 1×105 cells, resuspend them in 100μL culture medium, add them to the transwell chamber coated with matrix gel, and add 500μL of 10% FBS RPMI-1640 culture medium under the chamber.

[0039] After 16-20 hours, the chamber was taken out and fixed with 4% paraformaldehyde for 10 minutes, then the paraformaldehyde was discarded, and the cells were washed with PBS for 3 times, 3 minutes each time, and then stained with 0.1% crystal violet for 10 minutes. The cells above the chamber that did not penetrate the polycarbonate membrane were wiped off with a cotton swab, and the excess crystal violet was washed off with running water. The cells that penetrated the polycarbonate membrane were the infiltrated cells, and the cells were photographed under a microscope and the number of cells was counted.

[0040] 2) Test results mPCPU cells have strong invasive characteristics, and knocking down Rela weakens the invasive ability of cells ( Figure 4 ).

[0041] Embodiment five: Mouse prostate cancer primary cells shRela cells and shC cells secrete cytokines to recruit myeloid-derived immunosuppressive cells (MDSCs) ( Figure 5 ).

[0042] 1) MDSCs cell isolation.

[0043] MDSCs were isolated from prostate tumor tissues of mice with prostate cancer using the MDSCs magnetic bead isolation kit. In short, the prostate tumor tissue was cut into pieces of about 2 mm3 and digested into single cells. The supernatant was discarded after centrifugation, and the cells were resuspended in buffer. Then Ly6G Biotion antibody was added, and the cells were incubated at 4°C in the dark for 10 minutes. Then the buffer was added and the supernatant was discarded after centrifugation. Then anti-Biotion beads were added and mixed, and the cells were incubated at 4°C in the dark for 15 minutes. The cells were washed with buffer and centrifuged and the supernatant was discarded. Then the cells were resuspended in buffer and passed through the LS column. The filtrate was collected for later use. The cells in the washed LS column are Ly6G positive cells.

[0044] The filtrate was centrifuged and the supernatant was discarded. After the cells were resuspended, Gr1 Biotion was added and incubated at 4°C in the dark for 10 minutes. After that, the cells were rinsed with buffer, centrifuged and the supernatant was discarded. After resuspending, anti-Biotion beads were added and mixed, and incubated at 4°C in the dark for 15 minutes. Then, the cells were rinsed with buffer, centrifuged and the supernatant was discarded. After resuspending, the cells were passed through the MS column. The cells retained in the MS column were Ly6G-negative and Ly6C-positive cells.

[0045] The two groups of cells were combined to form MDSCs cells.

[0046] 2) Collection of conditioned medium.

[0047] The same number of shRela cells and shC cells were cultured in 10% FBS RPMI-1640 medium for 24 hours, the culture medium was collected, filtered with a 0.22 μm filter membrane, and stored at -80°C.

[0048] 3) MDSCs recruitment assay.

[0049] 1×10 6 MDSCs were inoculated into the transwell chamber, 500 μL of conditioned culture medium was added to the bottom of the chamber, and the chamber was placed in a 37°C cell culture incubator for culture. After 6 hours, the culture medium at the bottom was collected for flow cytometry counting.

[0050] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Mouse prostate cancer primary cells, named mPCPU, characterized by: The cells grow adherently and have a spindle-shaped morphology; The cells aggregate to form a cavity similar to the prostate cavity; The proliferation rate is fast, with a doubling time of approximately 24 hours.

2. A method for constructing the mouse prostate cancer primary cells as claimed in claim 1, characterized in that: The cells are isolated and cultured from prostate tumor tissue of spontaneous prostate cancer gene-engineered mice with C57 / BL6 mouse genetic background.

3. A use of the mouse prostate cancer primary cells as claimed in claim 1, characterized in that: Used for in vitro cell biology, biochemistry and molecular biology research of prostate cancer; Used for studying the interaction between tumor cells and tumor microenvironment.

4. A use of the mouse prostate cancer primary cells as claimed in claim 1 in drug screening and sensitivity studies, comprising: Used for drug cytotoxicity testing; Used for sensitivity analysis and mechanism of action research of prostate cancer therapeutic drugs.

5. An application of the mouse prostate cancer primary cell as claimed in claim 1 in gene function research, comprising: Used for the construction of gene knockdown or overexpression cell lines; Used for verification and analysis of target gene function.

6. A use of the mouse prostate cancer primary cells as claimed in claim 1 in cell behavior analysis, comprising: Used for the study of cell proliferation ability; Used for the study of cell migration and invasion ability.

7. Use of the mouse prostate cancer primary cells as claimed in claim 1 in the study of their interaction with tumor microenvironment cells.

8. A method for culturing the mouse prostate cancer primary cells according to claim 1, characterized in that: The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

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