CN117736993A - Human pancreatic cancer cell line PDAC-X3 and application thereof - Google Patents
Human pancreatic cancer cell line PDAC-X3 and application thereof Download PDFInfo
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Abstract
A human pancreatic cancer cell line PDAC-X3 and application thereof belong to the field of microbial animal cell lines. The provided pancreatic cancer cell line is a human pancreatic cancer cell line PDAC-X3, and the preservation number CCTCC No: C2023381. the human pancreatic cancer cell line PDAC-X3 can be used as a cell model for the research of the occurrence, development or metastasis mechanism of pancreatic cancer. Can also be used for establishing pancreatic cancer animal models. Can be applied to preparing pancreatic cancer screening prevention and treatment medicines. Can be used as a cell model for researching occurrence and development mechanism, cell morphology and function abnormality, drug research and development and the like of pancreatic cancer. The newly established cell line can keep the characteristics similar to those of the primary tumor to the greatest extent, and the result obtained by using the cell line for research is closest to the actual condition of a human body, thereby providing a basis for researching the pathogenesis and treatment method of pancreatic cancer and the like.
Description
Technical Field
The invention belongs to the field of microbial animal cell lines, and particularly relates to a human pancreatic cancer cell line PDAC-X3 and application thereof.
Background
Pancreatic cancer is a malignant solid tumor with extremely poor prognosis, and the incidence rate of the pancreatic cancer is in an annual rising situation at home and abroad. The number of deaths and cases caused by pancreatic cancer has doubled from 1990 to 2017, and the incidence of pancreatic cancer may continue to rise as the population ages. Pancreatic cancer is expected to be the second leading cause of cancer-related death worldwide in 2030. The clinical characteristics of hidden onset, high malignancy, high invasiveness and the like of pancreatic cancer lead to the advanced stage of most patients at the time of treatment, and the patients can not be treated by surgery along with the metastasis of adjacent or distant organs. Chemotherapy, represented by gemcitabine, is still currently the primary treatment regimen for pancreatic cancer patients, but drug resistance issues are prominent, which makes drug treatment limited and patient prognosis still poor. Around the pathogenesis of pancreatic cancer, many fundamental studies have been conducted with open-ended time, but clinical transformation studies of key targets in the relevant pathogenesis, including therapeutic strategy studies, remain relatively slow to develop. Pancreatic cancer requires more transformation studies to drive actual clinical diagnosis and prolong the survival of pancreatic cancer patients.
Human tumor cell lines, particularly those with complete data and follow-up, are important tools in tumor biology research. Since the 80 s of the 20 th century, several pancreatic cancer cell lines were established and identified, represented by three scientists in the united states, the japanese and the korean countries, but few chinese-derived pancreatic cancer cell lines were reported.
Cell lines from pancreatic primary tumors have tremendous roles in exploring biology and bile duct tumorigenesis, detecting drug sensitivity, developing molecular therapeutic targets, and studying drug resistance mechanisms. A complete pool of tumor cell lines should reflect the diversity of tumor phenotypes and be able to provide cell lines of different tumor heterogeneity; in view of the different etiologies of pancreatic tumors and their etiologic related genetic variations, it is important to use appropriate preclinical models reflecting these characteristics.
In 1986, the first pancreatic cancer cell line was established domestically. Provides an important cell experimental model for basic and clinical research of national pancreatic cancer. However, as the number of in vitro passages increases, some of the unique biological characteristics of the cell line gradually change or disappear, and even characteristics that the originating cell does not have. In addition, many cell lines are detected with cross-contamination of cells, the most common sources of contaminating cells being HeLa, T-24 and M14, and erroneous results from studies using these cells. The national experiment cell resource sharing service platform finds that a plurality of tumor cell lines established by domestic scholars are cross-contaminated when carrying out identity authentication in the processes of collecting, arranging, quality control and preserving cells. A total of 46 samples of 37 lines of cells were collected from different areas and different laboratories, and each of the samples was subjected to species identification and STR spectrum analysis. The cell cross contamination phenomenon is found to be serious, and the error rate reaches 62.6%. And many thousands of papers based on these contaminated cells are in our country each year. Cell lines that are misidentified, contaminated, genetically-bleaching, and clonally evolved are important reasons for the inability of many studies to replicate and affect everyone engaged in the cell study.
The newly established cell line can maintain the characteristics similar to those of the primary tumor to the greatest extent, and the result obtained by using the cell line for research is closest to the actual condition of a human body. Because of different genetic backgrounds, it is necessary to build a disease model specific to Chinese in order to better study the disease of Chinese. For the above reasons, the continuous establishment of new cell lines and the elimination of old cell lines have become an important link in the study of pancreatic cancer pathogenesis and treatment methods.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a human pancreatic cancer cell line PDAC-X3 and application thereof.
The pancreatic cancer cell line provided by the invention is a human pancreatic cancer cell line PDAC-X3, and the preservation number is as follows: cctccc No: C2023381.
the human pancreatic cancer cell line PDAC-X3 can be used as a cell model for the research of the occurrence, development or metastasis mechanism of pancreatic cancer. The human pancreatic cancer cell line PDAC-X3 can also be used to model animals of pancreatic cancer.
The invention adopts a surgical excision specimen of 73 years old male pancreatic cancer, adopts a mixed digestion of type II collagenase and neutral proteinase to perform primary culture, and establishes a pancreatic cancer cell line named PDAC-X3 through a cell culture technology. Has been preserved in 2023, 11 and 27 days, the preservation unit: china Center for Type Culture Collection (CCTCC), collection address: mail code 430072, deposit number of university of Wuhan in Wuhan, china: cctccc No: C2023381.
the cell strain has the following biological characteristics:
1. the cell grows by adherence, is inhibited in a non-contact way, and can generate superposition growth phenomenon.
2. Cell doubling time was approximately 50h.
3. Cell STR results showed: PDAC-X3 cells were consistent with STR results from tumor tissue of the same patient, cells were derived from the same tumor sample, and were not contaminated with other cells during culture.
4. Chromosome analysis suggests: 75% of the cell lines are sub-2 ploid karyotypes, 25% of the cell lines are sub-tetraploid karyotypes, and the cell lines have more chromosome deletion, ectopic and derivative phenomena.
5. BALB/C nude mice were inoculated subcutaneously with this cell at a rate of 33% of the tumor formation, and the histological properties of the transplanted tumors were similar to those of the primary tumors.
6. Drug sensitivity results: PDAC-X3 is resistant to oxaliplatin, ic50=18.86 μmol/L; sensitivity to fluorouracil, ic50= 7.109 μmol/L; sensitive to gemcitabine, ic50=0.029 μmol/L; sensitivity to paclitaxel, ic50=0.0046 μg/mL.
The human pancreatic cancer cell line PDAC-X3 can be applied to preparation of pancreatic cancer screening and preventing medicines.
The human pancreatic cancer cell line PDAC-X3 can be used as a cell model for researching occurrence and development mechanisms, cell morphology and function abnormality, drug research and the like of pancreatic cancer. The cell line is different from the existing pancreatic cancer cell line, has the improvement that the newly established cell line can keep the characteristics similar to those of the primary tumor to the greatest extent, and the research result carried out by using the cell line is closest to the actual condition of human body, thereby providing a basis for researching the pathogenesis of pancreatic cancer, the treatment method and the like.
Drawings
FIG. 1 shows the pathological results of PDAC-X3 cell-derived tumor tissue. The scale in the figure is 50. Mu.m.
FIG. 2 is a view of the morphology of PDAC-X3 cells under a microscope (. Times.100). The scale in the figure is 100. Mu.m.
FIG. 3 shows the growth curve of PDAC-X3 cells. The abscissa is time and the ordinate is cell number.
FIG. 4 shows the result of chromosome analysis of PDAC-X3 cells.
FIG. 5 shows the results of a subcutaneous oncological assay for PDAC-X3 cell immunodeficient mice.
FIG. 6 shows pathological section results (. Times.200) of transplanted tumors. The scale in the figure is 50. Mu.m.
FIG. 7 shows the results of PDAC-X3 drug sensitivity experiments. Wherein a is oxaliplatin, b is 5-FU, c is gemcitabine, and d is paclitaxel.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the following examples. The experimental methods described in the examples below, unless otherwise specified, are all conventional.
Establishment and identification of human pancreatic cancer cell line PDAC-X3.
1. Establishment of human pancreatic cancer cell line PDAC-X3
Tumor tissues of pancreatic cancer patients are clinically collected and subjected to primary culture after being digested by mixed enzymes, so that a pancreatic cancer cell line capable of continuous passage is successfully established, and the cell characteristics are kept stable until more than 60 generations are reached. As can be seen from fig. 1, the pathological outcome of this patient is medium-low differentiated adenocarcinoma.
2. Biological characterization of human pancreatic cancer cell line PDAC-X3
1. Cell morphology: after the cell growth is stable and transferred, living cell observation is carried out on the cultured cells; the monolayer cells grown on the coverslip were fixed with 95% ethanol by volume fraction, stained with H & E, and observed with a light microscope. The results show that: cells were found to be arranged as epithelial cells under a phase contrast microscope, and the cells grew on the wall with overlapping growth (FIG. 2).
2. Short fragment repeat (STR) identification
The short tandem repeat sequence is also called microsatellite DNA, and refers to a DNA sequence formed by tandem repeat (the number of times of repeat is 10-60 times, and the gene fragment is below 400 base pairs) on a chromosome, wherein a plurality of base pairs are taken as core units (2-6 base pairs); individual differences in the number of repetitions per core unit can occur, resulting in alleles of differing fragment lengths. Thus, the number of repetitions of a set of STR sequences is almost unique among individuals, and is a genetic identity characteristic of individuals, and is also the primary method of cell biology to identify cell identity and origin.
Freshly cultured PDAC-X3 cells and tumor tissue samples of the same patient were collected, genomic DNA was extracted, STR detection service was provided by Souzhou authentication Biotechnology Inc., PCR amplification was performed using 5' -end fluorescent-labeled primers, and the resulting products were sequenced, analyzing the number of sequence repeats including the 20 STR sites of Amelogenin, THO1, TPOX, D13S317, vWA, D16S539, D5S818, CSF1PO, and D7S820, etc. The above sequences were compared with databases of cell banks such as ATCC, DSMZ, etc., and the same genetic map was not returned. The STR results were consistent for PDAC-X3 cells and tumor tissue of the same patient, indicating that the cells were derived from the same tumor sample (table 1).
TABLE 1
3. Cell growth curve assay: taking 18 th generation cells in logarithmic growth phase to obtain 1×10 5 Per ml of single cell suspension, then plated in 96-well plates at 100 ul/well, 3 multiplex wells were set. After 24 hours, the OD value at 490nm wavelength was measured by starting the dosing, and the solution was changed every day for 9 days. After the end of the experiment, a growth curve was drawn and the doubling time was calculated according to the formula td=t×lg2/lg (N1/N0). The results show that: the growth of the cells is slow on the 1 st day after the adherence of the cells, the cells start to grow exponentially on the 2 nd day, and the cells can grow in a superposition way under the condition of sufficient nutrition when the culture medium is replaced every day, and the phenomenon of non-contact inhibition is avoided; the average doubling time of the cells was about 50h (FIG. 3).
4. Chromosome analysis: the 35 th generation cells in logarithmic growth phase were taken for karyotyping. Cells were subjected to colchicine for 2h, hypotonic with 0.075mol/L KCl, methanol-glacial acetic acid fixation, ice-wet sheet dripping, aged at room temperature, treated with pancreatin, and stained with Giemsa for banding analysis. The results show that: the cell line is 75% of subtetraploid karyotype and 25% of subtetraploid karyotype, and has more chromosome deletion, ectopic and derivative phenomena. The present cell line is seen to be characteristic of malignant cells (FIG. 4).
5. In vivo nodulation experiments in immunodeficient mice: 3 female BALB/C mice of 4 weeks old were subcutaneously injected with 1X 10 parts of the right forelimb 6 The nude mice were sacrificed by cervical dislocation after 4 weeks after the cells were cultured, transplanted tumors were removed, formalin fixed, paraffin embedded sections, H&E, dyeing, observing under a mirror, and photographing to record experimental data. The results show that: 1 week after subcutaneous inoculation of the cells, the transplanted tumors were seen to grow out with a rate of 33% (fig. 5).
6. Pathological section of tumor: tumor tissue was routinely fixed in formalin, paraffin embedded, sectioned and H & E stained. The results show that: the transplanted tumor grew vigorously, the nucleus increased, deeply stained, and the atypical appearance was evident, and its histological morphology was similar to that of the clinical specimens (fig. 6).
7. Drug sensitivity experiment: logarithmic growth of PDAC-X3 cells (P36) was taken and trypsinized to prepare a single cell suspension. 10000 cells per well per 100ul were seeded into 96-well plates and 6 wells per group were replicated. After the cells are attached, the anti-tumor drugs with different concentrations, oxaliplatin, 5-FU, gemcitabine and paclitaxel are added into the experimental group, and the corresponding drug dissolution solution is added into the control group. After 72h of drug action, the complete medium was replaced with 100uL serum-free medium containing 10% (v/v) CCK 8. After 2h, the OD at 450nm was measured. As can be seen from fig. 7, PDAC-X3 is resistant to oxaliplatin with ic50=18.86 μmol/L; sensitivity to fluorouracil, ic50= 7.109 μmol/L; sensitive to gemcitabine, ic50=0.029 μmol/L; sensitivity to paclitaxel, ic50=0.0046 μg/mL. Oxaliplatin is shown in FIG. 7 as panel a, 5-FU is shown in FIG. 7 as panel b, gemcitabine is shown in FIG. 7 as panel c, and paclitaxel is shown in FIG. 7 as panel d.
The above-described embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (4)
1. The human pancreatic cancer cell line PDAC-X3 is characterized in that the preservation number is CCTCC No: C2023381.
2. use of a human pancreatic cancer cell line PDAC-X3 according to claim 1 for the construction of a cellular model of pancreatic carcinogenesis and progression.
3. Use of a human pancreatic cancer cell line PDAC-X3 according to claim 1 for the construction of an animal model of pancreatic cancer.
4. The use of a human pancreatic cancer cell line PDAC-X3 as defined in claim 1 in the preparation of a pancreatic cancer screening and controlling medicament.
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