Human immortalized pancreatic cancer fibroblast as well as preparation method and application thereof

Abstract

The invention relates to the field of biology, and provides a human immortalized pancreatic cancer fibroblast as well as a preparation method and application thereof.The human immortalized pancreatic cancer fibroblast comprises three cell strains which are all primary fibroblast cells extracted from tumor tissues of pancreatic ductal adenocarcinoma patients, the human pancreatic cancer fibroblast cell strain CAFPC-2209131, the human pancreatic cancer fibroblast cell strain CAFPC-2209132 and the human pancreatic cancer fibroblast cell strain CAFPC-2303221 are classified and named as the human pancreatic cancer fibroblast cell strain CAFPC-2209131, the human pancreatic cancer fibroblast cell strain CAFPC-2209132 and the human pancreatic cancer fibroblast cell strain CAFPC-2303221 respectively, the human pancreatic cancer fibroblast cell strain CAFPC-2209132 and the human pancreatic cancer fibroblast cell strain CAFPC-2303221 are all Cell STR identification and immunofluorescence detection prove that the cell strain is a fibroblast cell strain. After the tumor-associated fibroblast and pancreatic cancer cells are co-cultured, the proliferation ability of the tumor cells is obviously enhanced, and the sensitivity of the tumor cells to a chemotherapeutic drug gemcitabine is reduced. A new thought is provided for diagnosis and treatment of pancreatic cancer, and based on the pancreatic cancer in-vitro co-culture model established in vitro, the application has important significance for researching the effect and related mechanisms of fibroblasts in the pancreatic cancer tumor microenvironment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a pancreatic cancer fibroblast strain, in particular to a human immortalized pancreatic cancer fibroblast strain, a screening culture method thereof and application thereof. BACKGROUND

[0002] Pancreatic cancer is one of the common malignant tumors in the digestive tract. Due to its high malignancy, the five-year survival rate is less than 10%, and it is known as the "King of Cancer" in the field of tumors, and is one of the malignant tumors with the worst prognosis.

[0003] The stromal component of pancreatic cancer accounts for nearly 90% of tumor tissue, mainly composed of cancer-associated fibroblasts (CAFs), immune cells and endothelial cells and some molecules secreted by them, so the stromal component largely determines the occurrence and development of pancreatic cancer tumor tissue. Among them, cancer-associated fibroblasts, as the most abundant stromal cells, promote the malignant transformation of epithelial cells through cell-to-cell contact and the secretion of various cytokines, proteases and the like, and have an important regulatory effect on the interface components, participate in promoting cancer angiogenesis, tumor proliferation and metastasis, and construct an inhibitory immune microenvironment, and play an important role in the progression of pancreatic cancer that cannot be ignored.

[0004] Pancreatic cancer fibroblasts play an important role in the basic research of pancreatic cancer. By constructing an in vitro co-culture system with pancreatic cancer cells, the growth environment of the interaction between pancreatic cancer and stroma can be more fully restored, and the mechanism of tumor occurrence and development can be explored from the perspective of tumor microenvironment, thereby providing new ideas for early diagnosis, drug screening and treatment in clinical practice.

[0005] Therefore, screening and obtaining stable immortalized pancreatic cancer fibroblast strains will have important significance and value for the research and clinical decision-making of human pancreatic cancer. SUMMARY

[0006] The present application is directed to the above problems, the first purpose is to provide a human immortalized pancreatic cancer fibroblast, a total of three cell strains, the three cell strains are preserved in the China Center for Type Culture Collection, and the preservation numbers are CCTCC NO:C2023117, CCTCC NO:C2023118 and CCTCC NO:C2023119 respectively; the second purpose is to provide a preparation method of the human immortalized pancreatic cancer fibroblast; and the third purpose is to provide application of the human immortalized pancreatic cancer fibroblast in constructing an animal model and drug screening.

[0007] In order to achieve the above purposes, the technical solutions adopted by the present application are as follows:

[0008] In the first aspect of the present application, three human immortalized pancreatic cancer fibroblast cell strains are provided, which are all primary fibroblast cells extracted from tumor tissues of pancreatic ductal adenocarcinoma patients and are classified and named as human pancreatic cancer fibroblast cell strain CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221, respectively, and have been preserved in the China Center for Type Culture Collection, with the corresponding preservation numbers of CCTCC NO: C2023117, CCTCC NO: C2023118 and CCTCC NO: C2023119, respectively.

[0009] In the second aspect of the present application, a preparation method of human immortalized pancreatic cancer fibroblast cells is provided, in which an immortalized pancreatic cancer related fibroblast cell strain is established by using in vitro cell culture technology, and the specific culture steps are as follows:

[0010] A, primary culture

[0011] The pancreatic cancer tumor tissue is cut into small pieces of 3mm x 3mm and quickly placed in the tissue preservation solution and on ice. 3 After the tumor cells are washed with PBS, the tumor tissue is cut into small pieces of 1mm 3 , and the tumor tissue digestion solution is used to digest at 37℃ for 15 minutes. After the digestion is completed and the tissue pieces are settled, the cell suspension is transferred to a centrifuge tube. The remaining tissue is repeatedly digested twice with the tumor tissue digestion solution and combined in the centrifuge tube. After multiple digestions, the cell suspension after tissue digestion is collected, filtered through a 70μm filter, and the cell precipitate is washed with PBS twice until the cell debris is removed. The cells are resuspended with RPMI-1640 complete medium and counted, and cultured in a 37℃, 5% carbon dioxide incubator for 4-5 days. After the cell density in the culture dish reaches 80%-90%, trypsin digestion is used for 1 minute (the adhesion ability of fibroblasts is weaker than that of tumor cells, and tumor cells and fibroblasts can be separated according to different digestion times), and then complete medium is added to terminate the digestion. The cell suspension is transferred to a sterile centrifuge tube, centrifuged at 800r / min, and the supernatant is discarded. RPMI-1640 complete medium is added to resuspend the cells, and the cells are uniformly blown and transferred to a culture dish for culture.

[0012] B, subculture

[0013] Fibroblast cells were cultured in Petri dishes using RPMI-1640 complete medium, and when the cell density reached 80%-90%, the cells were subcultured; under sterile conditions in a clean bench, the culture medium in the Petri dish was aspirated, trypsin was used for digestion for 1 min, then complete medium was added to terminate digestion, the cells were blown several times, and the cell suspension was transferred into a sterile centrifuge tube and centrifuged at 800 r / min, then the supernatant was discarded, RPMI-1640 complete medium was added to resuspend the cells, the cells were blown evenly, and then transferred into a Petri dish for culture, with a subculture ratio of 1:2-1:3.

[0014] C. Fibroblast immortalization

[0015] Primary fibroblasts were infected with pGMLV-SV40T plasmid packaged lentivirus, and after the cells were stably subcultured, 2 μg / ml puromycin was used to screen positive cells; the unimmortalized cells were subcultured for no more than 20 generations, while the immortalized cells could be stably subcultured and maintained proliferative ability, and could grow stably and continuously without any cell growth factors and stromal cells, and then immortalized pancreatic cancer fibroblasts were screened.

[0016] D. Freezing

[0017] Fresh RPMI-1640 complete medium was replaced 24 hours before freezing, so that the cells were in the exponential growth phase; under sterile conditions, the culture medium in the Petri dish was aspirated, trypsin was used for digestion, then complete medium was added to terminate digestion, the cells were blown several times, the cell suspension was transferred into a sterile centrifuge tube, centrifuged, then the supernatant was discarded, RPMI-1640 medium was added, the cell concentration was adjusted to 1×10 6 ~2×10 6 / ml, the cell suspension was aliquoted into EP tubes, each tube contained 1 ml of cell suspension, about 1×10 6 ~2×10 6 cells, the supernatant was discarded after centrifugation, 1 ml of freezing solution was added to the cell pellet, the cells were blown evenly, transferred into a sterile freezing tube, 4°C for 30 minutes, -20°C for 60 minutes, and -80°C overnight, and the next day it was transferred into liquid nitrogen.

[0018] E. Thawing

[0019] The frozen tube was taken out of the liquid nitrogen and quickly placed in 37°C warm water, and when the frozen material in the frozen tube melted into a liquid, the cell suspension was aspirated into a centrifuge tube and centrifuged, the supernatant was discarded, RPMI-1640 complete medium was added to the cell pellet, the cells were blown evenly to a single cell suspension, transferred into a Petri dish, and cultured in a cell incubator at 37°C, 5% CO2, and 95% humidity.

[0020] In the above steps, the components of the tumor tissue digestion solution are as follows: DMEM / F-12, 5 mg / ml Collagenase XI, 10.5 μM Y27632, 10 μg / mL DNAse I;

[0021] The composition of the RPMI-1640 complete culture medium is as follows: RPMI-1640 culture medium, 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin;

[0022] The RPMI-1640 culture medium is 11875093 purchased from Sigma Company, wherein L-glutamine is added;

[0023] The cryopreservation solution is prepared before use and contains 90% fetal bovine serum and 10% DMSO.

[0024] The CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cell strains of the present application can grow in vitro for a long time and can be subcultured indefinitely, the cell growth state is good, the cells grow adherently, do not depend on any cell growth factor and stromal cells for continuous growth, can tolerate freezing and recovery, and there is no report on such cell strains in the current domestic and foreign literatures.

[0025] The three cell strains of the present application are confirmed to be fibroblast cell strains through cell STR identification and immunofluorescence detection. The STR genotype detection result indicates that the DNA typing of the three cell strains is not found in the comparison of cell line STR data in the ATCC, DSMZ, JCRB and RIKEN databases, confirming that the fibroblast cell lines of the present application are a new cell line that has not been reported. The doubling time of the three cell strains is 72 hours, and the proliferation speed is moderate.

[0026] In a third aspect, the present application provides the application of the human immortalized pancreatic cancer fibroblast cells. The cell strains are used as experimental objects to analyze the characteristics of the cells such as morphology, genetics, immunophenotype and malignant potential of tumor cells, thereby providing an effective and stable cell model for further studying the pathogenesis and treatment scheme of pancreatic cancer.

[0027] Specifically, the present application provides the application of the human immortalized pancreatic cancer fibroblast cells in constructing a pancreatic cancer animal model or drug screening for pancreatic cancer. After the tumor-related fibroblasts are co-cultured with pancreatic cancer cells, the proliferation ability of the tumor cells is obviously enhanced, and the sensitivity of the tumor cells to the chemotherapy drug gemcitabine is reduced.

[0028] In a fourth aspect, the present application provides a method for constructing a pancreatic cancer animal model. The suspension of any one of the human immortalized pancreatic cancer fibroblast cell strains described above is subcutaneously inoculated into a nude mouse, and a human pancreatic cancer animal model is obtained after the nude mouse is cultured for a certain period of time.

[0029] In a fifth aspect, the present application provides a method for screening drugs for pancreatic cancer, in a first mode, a test compound is applied to human immortalized pancreatic cancer fibroblasts in vitro culture, and the test compound that causes the number of human pancreatic cancer fibroblasts to decrease or die is a candidate compound for treating pancreatic cancer; in a second mode, a test compound is administered to the animal model constructed above, and the test compound that reduces tumor volume or improves mouse survival rate is a candidate compound for treating pancreatic cancer.

[0030] The beneficial guarantees and effects of the present application are as follows:

[0031] The CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cell strains of the present application can grow long-term and be passaged indefinitely in vitro, have good cell growth state, grow adherently, do not depend on any cell growth factors and stromal cells for sustained growth, can tolerate freezing and recovery, and no such cell strains have been reported in the literature at home and abroad.

[0032] Through cell STR identification and immunofluorescence detection, it is confirmed that the cell strain is a fibroblast strain, the doubling time of the cell strain is 72 hours, and the proliferation speed is moderate. After co-culturing the tumor-related fibroblasts with pancreatic cancer cells, the proliferation ability of the tumor cells is significantly enhanced, and the sensitivity of the tumor cells to the chemotherapeutic drug gemcitabine is reduced.

[0033] The present application provides a new idea for the diagnosis and treatment of pancreatic cancer, and based on the in vitro co-culture model of pancreatic cancer established in vitro, it has important significance for studying the role of fibroblasts in the pancreatic cancer tumor microenvironment and the related mechanism.

[0034] Preservation information of the biological material sample of the present application:

[0035] (1) Classification and naming: human pancreatic cancer fibroblast strain CAF_PC-2209131

[0036] Preservation unit: China Center for Type Culture Collection

[0037] Address: Wuhan University, Wuhan, China, postcode 430072

[0038] Preservation date: June 14, 2023

[0039] Preservation number: CCTCC NO: C2023117;

[0040] (2) Classification and naming: human pancreatic cancer fibroblast strain CAF_PC-2209132

[0041] Preservation unit: China Center for Type Culture Collection

[0042] Address: Wuhan University, Wuhan, China, 430072

[0043] Date of deposit: June 14, 2023

[0044] Accession No.: CCTCC NO: C2023118

[0045] (3) Taxonomic Name: Human pancreatic cancer fibroblast cell strain CAF_PC-2303221

[0046] Depository: China Center for Type Culture Collection

[0047] Address: Wuhan University, Wuhan, China, 430072

[0048] Date of deposit: June 14, 2023

[0049] Accession No.: CCTCC NO: C2023119 BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Fig. 1 is a 20-fold microscopic morphological chart of the cell strain, (A) CAF_PC-2209131 cell strain, (B) CAF_PC-2209132 cell strain, (C) CAF_PC-2303221 cell strain;

[0051] Figure 2 Fig. 2 is a column chart of gene expression amount detected in genetic characterization of CAF_PC-2209131 cell strain: A is the expression difference of KRAS, CDKN2A, TP53, SMAD4 in CAF_PC-2209131 compared with PANC-1 cells, B is the expression difference of EPCAM in epithelial cells compared with CAF_PC-2209131, C is the expression difference of ACTA2 in HFF-1 cells compared with CAF_PC-2209131;

[0052] Figure 3 Fig. 3 is a column chart of gene expression amount detected in genetic characterization of CAF_PC-2209132 cell strain: A is the expression difference of KRAS, CDKN2A, TP53, SMAD4 in CAF_PC-2209132 compared with PANC-1 cells, B is the expression difference of EPCAM in epithelial cells compared with CAF_PC-2209132, C is the expression difference of ACTA2 in HFF-1 cells compared with CAF_PC-2209132;

[0053] Figure 4Figure 1 is a column chart of genetic characterization of CAF_PC-2303221 cell strain: A is the expression difference of KRAS, CDKN2A, TP53 and SMAD4 in CAF_PC-2303221 compared with PANC-1 cells, B is the expression difference of EPCAM in CAF_PC-2303221 compared with epithelial cells, and C is the expression difference of ACTA2 in CAF_PC-2303221 compared with HFF-1 cells;

[0054] Figure 5 Figure 3 is the α-SMA immunofluorescence staining chart of three cell strains: (A) is the α-SMA immunofluorescence staining chart of CAF_PC-2209131, from left to right are α-SMA (red), nucleus (blue) and the combined results; (B) is the α-SMA immunofluorescence staining chart of CAF_PC-2209132, from left to right are α-SMA (red), nucleus (blue) and the combined results; (C) is the α-SMA immunofluorescence staining chart of CAF_PC-2303221, from left to right are α-SMA (green), nucleus (blue) and the combined results;

[0055] Figure 6 Figure 4 is the CFSE detection of tumor cells after PANC-1 cells were co-cultured with three cell strains: (A) co-cultured with CAF_PC-2209131 cell strain, (B) co-cultured with CAF_PC-2209132 cell strain, and (C) co-cultured with CAF_PC-2303221 cell strain;

[0056] Figure 7 Figure 5 is the IC50 detection of tumor cells after PANC-1 cells were co-cultured with three cell strains: (A) co-cultured with CAF_PC-2209131 cell strain, (B) co-cultured with CAF_PC-2209132 cell strain, and (C) co-cultured with CAF_PC-2303221 cell strain. DETAILED DESCRIPTION

[0057] The embodiments of the present application will be described in detail below with the examples of the present application. The following examples are implemented on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0058] Example 1: Obtaining of CAF_PC-2209131 cell strain

[0059] 1. Primary culture

[0060] The tumor tissue of 0.5 cm 3The tumor tissue was cut into small pieces of 3 mm x 3 mm, quickly placed into a tissue preservation solution, and placed on ice; after the tumor cells were washed with 5 ml of PBS, the tumor tissue was cut into small pieces of 1 mm 3 After the digestion was completed, the cell suspension was transferred to a 15 mL centrifuge tube after the tissue pieces were allowed to settle, and the remaining tissue was repeatedly digested twice by adding 3 mL of the tumor tissue digestion solution.

[0061] The cell suspension after tissue digestion was collected and filtered using a 70 μm filter screen. The cell precipitate was washed twice with 5 mL of PBS to remove cell debris. The cells were resuspended in RPMI-1640 complete medium and counted. The cells were cultured in a 37°C, 5% carbon dioxide incubator for 4-5 days. After the cell density in the culture dish reached 80%-90%, the cells were trypsinized for 1 minute (fibroblasts have weaker adhesion ability than tumor cells, and tumor cells and fibroblasts can be separated according to different digestion times). After the digestion was terminated by adding complete medium, the cells were blown several times, the cell suspension was transferred to a sterile centrifuge tube, centrifuged at 800 r / min, the supernatant was discarded, RPMI-1640 complete medium was added, the cells were resuspended, and the cells were transferred to a culture dish after being blown evenly.

[0062] 2. Subculture

[0063] The fibroblasts were cultured in RPMI-1640 complete medium in a culture dish. After the cell density reached 80%-90%, the cells were subcultured. In a sterile environment on a clean bench, the culture medium in the culture dish was removed, the cells were trypsinized for 1 minute, the digestion was terminated by adding complete medium, the cells were blown several times, the cell suspension was transferred to a sterile centrifuge tube, centrifuged at 800 r / min, the supernatant was discarded, RPMI-1640 complete medium was added, the cells were resuspended, and the cells were transferred to a culture dish after being blown evenly. The subculture ratio was 1:2-1:3.

[0064] 3. CAF_PC-2209131 cell immortalization

[0065] The primary CAF_PC-2209131 cells were infected with a pGMLV-SV40T plasmid packaged lentivirus. After the cells were stably subcultured, 2 μg / ml puromycin was used to screen positive cells. The unimmortalized cells were subcultured for no more than 20 generations, while the immortalized cells could be stably subcultured and maintained their proliferation ability, and could grow stably without any cell growth factors and stromal cells.

[0066] 4. Cryopreservation and recovery

[0067] Cryopreservation: Replace the medium with fresh RPMI-1640 complete medium 24 hours before cryopreservation to ensure the cells are in the exponential growth phase. Under aseptic conditions in a laminar flow hood, aspirate the medium from the culture dish, digest with trypsin for 1 minute, add complete medium to stop digestion, pipette the cells several times, transfer the cell suspension to a sterile centrifuge tube, centrifuge at 800 rpm, discard the supernatant, add RPMI-1640 complete medium, and adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 6 -2×10 6 / ml, dispense 1ml of cell suspension into EP tubes, approximately 1×10⁶ cells / ml. 6 -2×10 6 Count the cells, centrifuge again at 800 rpm, discard the supernatant, add 1 ml of cryopreservation solution to the cell pellet, mix well, transfer to a sterile cryovial, incubate at 4°C for 30 minutes, -20°C for 60 minutes, and -80°C overnight, then transfer to liquid nitrogen the next day.

[0068] Resuscitation: Remove the cryovial from liquid nitrogen and quickly place it in 37°C warm water. After the frozen contents in the cryovial have thawed into a liquid, transfer the cell suspension to a centrifuge tube and centrifuge at 800 rpm for 5 minutes. Discard the supernatant, add RPMI-1640 complete culture medium to the cell pellet, and mix well until a single-cell suspension is formed. Transfer the suspension to a culture dish and incubate in a cell culture incubator at 37°C, 5% CO2, and 95% humidity.

[0069] The culture medium formulations involved in this primary culture process are as follows:

[0070] RPMI-1640 complete medium (RPMI-1640 complete medium containing 10% fetal bovine serum): 1640 medium, 10% fetal bovine serum, penicillin 100 units / ml, streptomycin 100ug / ml;

[0071] RPMI-1640 medium: purchased from Sigma, 11875093 (with added L-glutamine);

[0072] Cryopreservation solution (prepared fresh before use): The cell cryopreservation solution contains 90% fetal bovine serum and 10% DMSO.

[0073] Example 2: Identification of genetic characteristics of cell lines

[0074] 1. Cell morphology observation

[0075] After cell digestion and counting, take 10 4 Cells were seeded in 24-well plates, and after 24 hours of cell adhesion, cell morphology was observed under a microscope. Figure 1(A)-(C) show the morphology of CAF_PC-2209131, CAF_PC-2209132, CAF_PC-2303221 cell lines under 20 times magnification. The three fibroblast cell lines have spindle-shaped or irregular triangular cell bodies with ovoid nuclei and cytoplasmic processes, and grow in a radial pattern.

[0076] 2. Cell STR identification

[0077] Logarithmic growth phase fibroblasts were collected and counted; 10 6 cells were taken, washed twice with PBS, and the cell precipitate was collected after centrifugation at 1000 r / min for 3 minutes. The cell precipitate was sent to Wuxi Xingrui and Application Biotechnology Co., Ltd. for STR genotype detection. DNA was extracted using Axygen's genomic extraction kit, amplified using a 21-STR amplification protocol, and the STR sites and gender gene Amelogenin were detected on an ABI 3730XL genetic analyzer. The specific results are shown in Table 1 below.

[0078] The results suggest that the DNA typing of the three cell lines did not match any cell lines in the STR data of the ATCC, DSMZ, JCRB, and RIKEN databases, confirming that the fibroblast cell lines invented in this study are a new cell line that has not been reported.

[0079] Table 1. Genotyping results of STR sites and Amelogenin sites of cells

[0080]

[0081] 3. Cell genetic characterization detection

[0082] When isolating CAFs, contaminating cancer cells (such as epithelial cells and mesenchymal cells) were detected and excluded. Using tumor tissue-specific gene mutations (such as KRAS, CDKN2A, TP53, and SMAD4 mutant genes in pancreatic cancer), epithelial cell markers such as EPCAM, their expression possibilities were also excluded, and human HFF-1 fibroblast cell lines were used as positive controls to detect the expression of α-SMA, and compared by qPCR. The results were consistent with expectations, and the isolated CAF_PC-2209131, CAF_PC-2209132, CAF_PC-2303221 fibroblast cell lines were extracted, and compared with pancreatic cancer cell line PANC1, KRAS, CDKN2A, TP53, and SMAD4 were lowly expressed, and compared with epithelial cells, EPCAM was also lowly expressed, and compared with human HFF-1 fibroblast cell lines, α-SMA (ACTA2) was highly expressed Figures 2-4 , which confirmed that the cells were fibroblasts.

[0083] 4. CAF_PC-2209131 cell immunofluorescence detection

[0084] 5 x 10 4 Fibroblasts were plated on cell slides and incubated at 37°C, 5% CO2, 95% humidity in a cell incubator for 12 hours. After fixation with 4% paraformaldehyde and membrane rupture with 0.1% Tween 20, the cells were incubated with α-SMA antibody for 12 hours, and secondary antibody staining was performed using Alexa Fluor 488 antibody. Finally, DAPI dye was used for nuclear staining.

[0085] The results showed that the CAF_PC-2209131, CAF_PC-2209132, and CAF_PC-2303221 fibroblast cell lines were positive for α-SMA expression. Figure 5 ).

[0086] The above STR identification results and immunofluorescence staining results confirm that the above three cell lines are fibroblasts.

[0087] Example 3: Co-culture experiment of human immortalized fibroblasts and PANC-1 cells

[0088] 1. CAF_PC-2209131, CAF_PC-2209132, and CAF_PC-2303221 cell proliferation detection after co-culture with PANC-1 cells

[0089] Logarithmic growth CAF_PC-2209131, CAF_PC-2209132, and CAF_PC-2303221 cells were collected and co-cultured with human pancreatic cancer cell line PANC-1 cells. PANC-1 cells were labeled with CFSE dye, and the FITC fluorescence intensity of the labeled PANC-1 cells was detected by flow cytometry. CAF_PC-2209131, CAF_PC-2209132, and CAF_PC-2303221 were not treated.

[0090] CAF_PC-2209131, CAF_PC-2209132, and CAF_PC-2303221 cells were mixed with PANC-1 cells at a ratio of 1:5, and the cell concentration was adjusted to 1 x 10 6 / ml. After thorough mixing by blowing, 2 x 10 5 The co-cultured cells were seeded in a 6-well plate, and the control group was only seeded with PANC-1 cells. After 72 hours of culture, the change in FITC fluorescence intensity was detected by flow cytometry.

[0091] The results show that the FITC fluorescence intensity of PANC-1 cells co-cultured with CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cells respectively is obviously lower than that of the control group Figure 6 , which indicates that tumor-associated fibroblasts can promote the proliferation of pancreatic cancer tumor cells.

[0092] 2. Gemcitabine drug sensitivity experiment of CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cells co-cultured with PANC-1 cells

[0093] Logarithmic growth of CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cells and PANC-1 cells was collected, and CAF_PC-2209131, CAF_PC-2209132 and CAF_PC-2303221 cells were mixed with PANC-1 cells at a ratio of 1:5, and the cell concentration was adjusted to 1×10 6 / ml. After mixing uniformly by blowing, 1×10 4 The co-cultured cells were inoculated in a 96-well plate, and the control group was only inoculated with PANC-1 cells. After 24 h of culture, the chemotherapeutic drug gemcitabine was added for drug sensitivity experiment; the gemcitabine concentration was set as 0, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50 and 100 uM, respectively. After adding the drug, the cells were incubated in an incubator for 72 h, then 10 ul of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance at 450 nm was determined by an enzyme-labeled instrument, and a curve of drug concentration and cell activity ratio was obtained. The change of gemcitabine drug sensitivity of pancreatic cancer cell line PANC-1 in co-culture was compared; the results show that the sensitivity of tumor cells to the chemotherapeutic drug gemcitabine is reduced Figure 7 .

[0094] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A human immortalized pancreatic carcinoma fibroblast, characterized in that, The cell strains of the cells are preserved in China Typical Culture Collection Center, and the preservation numbers are CCTCC NO: C2023117, CCTCC NO: C2023118 and CCTCC NO: C2023119 respectively.

2. The method of claim 1 for preparing human immortalized pancreatic cancer fibroblasts, characterized in that, The method comprises the following steps: A. Primary culture The pancreatic cancer tumor tissue is cut into small pieces and then quickly placed into a tissue preservation solution and placed on ice. After the tumor cells are washed with PBS, the tumor tissue is further cut into smaller pieces. The tissue digestion solution is used for multiple times of digestion, and then the cell suspension after tissue digestion is collected. The 70 μm filter screen is used for filtration, and the cell precipitate is washed with PBS until the cell debris is removed. The cell suspension is resuspended by adding RPMI-1640 complete culture medium and counted. The cells are cultured in a 37°C, 5% carbon dioxide incubator for 4-5 days. When the cell density in the culture dish reaches 80%-90%, trypsin digestion is performed, and then complete culture medium is added to terminate the digestion. The cell suspension is transferred into a sterile centrifuge tube, centrifuged, and the supernatant is discarded. RPMI-1640 complete culture medium is added to resuspend the cells. After uniform blowing and beating, the cells are transferred into a culture dish for culture. B. Subculture The fibroblasts are cultured in an RPMI-1640 complete culture medium in a culture dish. When the cell density reaches 80%-90%, subculture is performed. Under sterile conditions, the culture medium in the culture dish is aspirated, trypsin digestion is performed, complete culture medium is added to terminate the digestion, the cell suspension is transferred into a sterile centrifuge tube, centrifuged, and the supernatant is discarded. RPMI-1640 complete culture medium is added to resuspend the cells. After uniform blowing and beating, the cells are transferred into a culture dish for culture. The subculture ratio is 1:2-1:

3. C. Fibroblast immortalization The primary fibroblasts are infected with the plasmid packaged lentivirus. After the cells are stably subcultured, positive cells are screened by using puromycin. The unimmortalized cells are subcultured for no more than 20 generations. The immortalized cells can be stably subcultured and maintain the proliferation ability, and can continuously and stably grow without relying on any cell growth factor and stromal cells. The immortalized pancreatic cancer fibroblasts are screened.

3. The method for preparing human immortalized pancreatic cancer fibroblasts according to claim 2, characterized in that, The method further comprises the steps of freezing and recovering: The freezing step is as follows: Fresh RPMI-1640 complete medium was replaced 24 hours before freezing, so that the cells were in exponential growth phase; the culture medium in the culture dish was aspirated under sterile conditions, and trypsin was used to digest the cells, and complete medium was added to terminate the digestion, and the cells were blown several times, and the cell suspension was transferred into a sterile centrifuge tube, the supernatant was discarded after centrifugation, and RPMI-1640 medium was added to adjust the cell concentration to 1×10 6 2×10 6 / ml, and the cell suspension was aliquoted into EP tubes, 1 ml of cell suspension per tube, about 1×10 6 2×10 6 cells, the supernatant was discarded after centrifugation, 1 ml of freezing solution was added to the cell precipitate, and the cells were blown evenly, transferred into a sterile freezing tube, 4°C for 30 minutes, -20°C for 60 minutes, -80°C overnight, and transferred into liquid nitrogen the next day; Recovery: The freezing tube is taken out from the liquid nitrogen and quickly placed in 37°C warm water. After the freezing material in the freezing tube is melted into a liquid, the cell suspension is sucked into a centrifuge tube and centrifuged. The supernatant is discarded, and the cell precipitate is added with RPMI-1640 complete culture medium. After uniform blowing and beating, the single cell suspension is transferred into a culture dish and cultured in a 37°C, 5% CO2, 95% humidity cell culture box.

4. The method of claim 2 for the preparation of human immortalized pancreatic cancer fibroblasts, The method is characterized in that: Wherein, in step A, pancreatic cancer tumor tissue 0.5 cm 3 , cut into 3 mm x 3 mm small pieces and quickly put into tissue preservation solution and placed on ice; after PBS washing tumor cells, the tumor tissue is continuously cut into 1 mm 3 small pieces, When the tumor tissue is digested, the components of the tumor tissue digestion solution are as follows: DMEM / F-12, 5 mg / ml Collagenase XI, 10.5 μM Y27632 and 10 μg / mL DNAse I. The digestion condition is as follows: first 37°C digestion for 15 minutes. After the digestion is completed and the tissue pieces are settled, the cell suspension is transferred into a centrifuge tube. The remaining tissue is repeatedly digested twice by adding the tumor tissue digestion solution, and then combined into the centrifuge tube. In step C, the primary fibroblasts were infected with the lentivirus packaged with the pGMLV-SV40T plasmid, and after the cells were stably passaged, 2 μg / ml puromycin was used to screen the positive cells.

5. The method of claim 2 or 3, wherein the human immortalized pancreatic cancer fibroblasts are prepared by the following steps: wherein The composition of the RPMI-1640 complete medium is as follows: RPMI-1640 medium, 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin; The trypsin digestion time is 1 minute. The centrifugal conditions of all centrifugal steps are 800 r / min.

6. The method of claim 3, wherein the human immortalized pancreatic cancer fibroblasts are prepared by the following steps: wherein The RPMI-1640 medium is 11875093 purchased from Sigma Company, to which L-glutamine is added; The cryopreservation solution is prepared before use, containing 90% fetal bovine serum and 10% DMSO.

7. The use of the human immortalized pancreatic cancer fibroblasts of claim 1 in constructing an animal model of pancreatic cancer or in screening drugs for pancreatic cancer.

8. A method of constructing a pancreatic cancer animal model, characterized by: The human immortalized pancreatic cancer fibroblasts of claim 1 are subcutaneously inoculated into nude mice to obtain an animal model of human pancreatic cancer after the nude mice are cultured for a certain period of time.

9. A method for screening a drug for pancreatic cancer, characterized by, A test compound is applied to the human immortalized pancreatic cancer fibroblasts in vitro or to the animal model constructed in claim 8; The test compound that causes the number of human pancreatic cancer fibroblasts to decrease or die, or the test compound that reduces the tumor volume or improves the survival rate of the mice is a candidate compound for treating pancreatic cancer.

Images