CN105925530A - High-metastasis pancreatic cancer cell strain and construction method and application thereof - Google Patents
High-metastasis pancreatic cancer cell strain and construction method and application thereof Download PDFInfo
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- 206010061902 Pancreatic neoplasm Diseases 0.000 title claims abstract description 77
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 title claims abstract description 77
- 208000008443 pancreatic carcinoma Diseases 0.000 title claims abstract description 55
- 201000002528 pancreatic cancer Diseases 0.000 title claims abstract description 28
- 238000010276 construction Methods 0.000 title claims abstract description 8
- 206010027476 Metastases Diseases 0.000 title abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000012216 screening Methods 0.000 claims abstract description 11
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- 238000012546 transfer Methods 0.000 claims description 18
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- 238000013508 migration Methods 0.000 claims description 5
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Abstract
The invention discloses a high-metastasis pancreatic cancer cell strain and a construction method and application thereof. The pancreatic cancer cell strain is named as human pancreatic cancer cell line BxPC-3M8, and a preservation number is CCTCC NO: C2016119. By repetition of a Transwell penetrating experiment of a pancreatic cancer cell strain BxPC-3 for eight times, the cancer cell line BxPC-3M8 is obtained by screening. The cell line has high metastasis and invasiveness, can be used for preparation of mammal pancreatic cancer models, researches on pancreatic cancer cell metastasis mechanisms, extraction of markers of pancreatic cancer metastasis and recurrence, screening and assessing of pancreatic cancer treating medicines, development of pancreatic cancer recurrence detection kits and the like, has high scientific research and production application values and is expected to generate high scientific research benefits, economic benefits and social benefits.
Description
Technical field
The present invention relates to biology and oncology, particularly relate to a kind of high transfer pancreas cancer cell strain and construction method thereof and application.
Background technology
Cancer is first big " killer " of serious threat our people's life and health, it has also become public health problem urgently to be resolved hurrily！Current research points out China's cancer new cases and death to increase year by year, wherein within 2015, there are about 429.2 Wan Xinfa invasive cancer cases, newly sends out about 12000 examples average every day；Having 281.4 ten thousand cancer mortality cases, average every day about 7500, people died from cancer.Cancer of pancreas is one of common malignant tumor of digestive tract.Cancer of pancreas sickness rate in the world in ascendant trend year by year, rejuvenation, the course of disease is short, progress is fast, poor prognosis, mortality rate height are cancer of pancreas clinic and biological property.The annual morbidity of China's cancer of pancreas is 5.1/10 ten thousand, relatively significantly raises before 20 years.Owing to lacking early diagnosis and effective Therapeutic Method, the median survival interval of advanced pancreatic cancer only has 3～4 months, and 5 years survival rates of Pancreas cancer patients, less than 5%, are known as again " king of cancer ".
Research it turned out the underlying cause of death that neoplasm metastasis is Pancreas cancer patients.Cancer of pancreas invasion and attack, the research of metastasis always are hot issue, and set up the important method that good cell experiment model is research cancer of pancreas Invasion and Metastasis mechanism.But lacking preferable in vitro study cell model at present, restriction researcher furthers investigate cancer of pancreas transfer further, need foundation badly and identify the highest transfer pancreatic cancer cell model.
Tumor cell has heterogeneity.The tumor cell subgroup incomplete same by biological characteristics and Invasion and Metastasis power forms.And the invasive ability of tumor depends on the cell of minority height transfer ability in heterogeneous maternal colony, by repeatedly screening, some high transfer subsystem cell can settle out.Meanwhile, inadaptable and unstable subbreed is removed, it is thus achieved that be the more stable subbreed with stronger invasive ability.This provides preferable cell model for research tumor invasion and metabasis mechanism.Major part researcher was all to filter out tumor cell subbreed by the method for screening in animal body in the past.But this method is the longest, operation easier is big, and poor repeatability, success rate is relatively low.
But lacking preferable in vitro study cell model at present, restriction researcher furthers investigate cancer of pancreas transfer further.Set up and identify the highest transfer pancreatic cancer cell model, contributing to screening the crucial target spot of cancer of pancreas relapse and metastasis, providing New Policy for successfully developing the clinical treatment means of anti-recurrence transfer, have important scientific meaning and clinical value！
Summary of the invention
It is an object of the invention to provide one and there is high transitivity and invasive pancreas cancer cell strain.
A kind of pancreas cancer cell strain, named human pancreatic cancer cell BxPC-3M8, it is preserved in the China typical culture collection center being positioned at Wuhan, China Wuhan University, preservation date is on June 3rd, 2016, and preserving number is: CCTCC NO:C2016119.
Invention further provides the daughter cell of described pancreas cancer cell strain.Described daughter cell remains the characteristic of parental generation pancreatic carcinoma BxPC-3M8 substantially or all, is also to have high transitivity and invasive.
Present invention also offers the construction method of a kind of described pancreas cancer cell strain, described construction method is Transwell migration test method, and initiator cell strain is pancreatic cancer cell BxPC-3, and test number of repetition is 8 times.
Transwell cell is put in culture plate, little indoor deserve to be called room, lower room is claimed in culture plate, upper indoor splendid attire culture supernatants, lower indoor splendid attire lower floor culture fluid, levels culture fluid is separated by with polycarbonate membrane, by cell kind in upper indoor, owing to polycarbonate membrane has permeability, the composition in lower floor's culture fluid can have influence on the cell of indoor, such that it is able to the impact of the composition cell growth studied in lower floor's culture fluid, motion etc..By parental generation pancreatic cancer cell is repeated several times Transwell migration test, it is possible to screening domestication obtains the pancreatic cancer cell of high transfer.
BxPC-3 cell is derived from a Japanese women cancer of pancreas biopsy specimen, Tan MH et al. the human pancreatic cancer cell set up in nineteen eighty-two, is cell model conventional in the research of current cancer of pancreas.STR (STR) is the simple repeated sequence being widely distributed in eukaryotic gene group, generally uses PCR amplification, and amplified production detects by electrophoretic analysis and according to size separation allele.Being analyzed by STR detection and find, BxPC-3M8 and the complete homology of parental generation BxPC-3, there is not the cross-contamination of other cells, cellular morphology and growth rate does not has significant difference.But the migration of BxPC-3M8 and invasive ability are significantly stronger than BxPC-3 cell.
Present invention also offers the application in preparing mammalian pancreas cancer model of the described pancreas cancer cell strain.Described mammal is nude mouse or nude rat.By the described pancreatic cancer cell BxPC-3M8 of certain cell quantity being inoculated in nude mouse or the position such as subcutaneous, the abdominal cavity of nude rat or tail vein, it is thus achieved that the animal model of high transitivity cancer of pancreas.
Present invention also offers the application in extracting the mark of cancer of pancreas transfer and recurrence of the described pancreas cancer cell strain.By comparing research with low transitivity pancreatic cancer cell, it appeared that the molecular marker of high transfer cancer of pancreas, the application such as follow-up drug development can be carried out for this molecular marker.
Present invention also offers the application in screening and assessment treatment pancreatic cancer drug of the described pancreas cancer cell strain.First, by adding different pharmaceutical in described pancreatic cancer cell BxPC-3M8 culture medium, observation of cell state changes, it is thus achieved that preliminary effective drug candidate.Then, drug candidate being administered to above-mentioned high transitivity cancer of pancreas animal model, observe and the survival period of non-dispenser treated animal, tumor size, transfer case etc., screening obtains the medicine of potential treatment cancer of pancreas.
Present invention also offers the application in exploitation cancer of pancreas recurrence detection kit of the described pancreas cancer cell strain.Proteomic techniques can be used, com-parison and analysis parental generation BxPC-3 cell and the differential expression of high transfer pancreatic carcinoma BxPC-3M8 secretory protein, filter out the secretory protein relevant to cancer of pancreas hepatic metastases, developing the detection kit of detection height transfer cancer of pancreas specific secretory protein expression, this test kit can be used for detecting cancer of pancreas transfer and recurrence.
The present invention wears film experiment by pancreas cancer cell strain BxPC-3 carries out for 8 times Transwell repeatedly, screening has obtained BxPC-3M8 cell line, this cell line has high transitivity and aggressive, can be used and prepare mammalian pancreas cancer model, research pancreatic cancer cell metastasis, extract cancer of pancreas transfer and the mark of recurrence, screening and assessment treatment pancreatic cancer drug, the purposes such as exploitation cancer of pancreas recurrence detection kit, there is the value of higher research and production application, it is contemplated that good scientific research, economic and social benefit can be produced.
Accompanying drawing explanation
Fig. 1 is BxPC-3 and BxPC-3M8 cellular morphology detection figure in embodiment 2；
Fig. 2 is the growth curve testing result figure of BxPC-3 and BxPC-3M8 cell in embodiment 4；
Fig. 3 is the scratch experiment results contrast figure of BxPC-3 and BxPC-3M8 cell in embodiment 5；
Fig. 4 is the migration Comparison of experiment results figure of BxPC-3 and BxPC-3M8 cell in embodiment 6；
Fig. 5 is the Matrigel results contrast figure of BxPC-3 and BxPC-3M8 cell in embodiment 7.
Detailed description of the invention
Experiment material:
Cell strain: pancreas cancer cell strain BxPC-3, pancreatic cancer cell BxPC-3M8.
Reagent consumptive material: RPMI-1640 (Hangzhou Ji Nuo biological medicine technology company limited), 0.25% pancreatin (Hangzhou Ji Nuo biological medicine technology company limited), hyclone (Gibco company), Giemsa staining kit (bio tech ltd is built up in Nanjing), 60mm and 100mm culture dish (Corning company), 6 holes and 24 hole Transwell cells (aperture 8 μm, corning company), matrigel (Matrigel).
Basic culture solution: containing the RPMI-1640 of 10% hyclone.
Serum-free medium: RPMI-1640.
Instrument: just putting microscope, inverted microscope, cell culture incubator, superclean bench, haemocytometer.
Embodiment 1 BxPC-3M8 Establishment of Cell Line
Pancreas cancer cell strain BxPC-3 RPMI-1640 (serum-free medium) is prepared 1 × 106/ mL single cell suspension, is then that the upper room of 6 orifice plate Transwell cells of 8 μm adds single cell suspension described in 1mL in aperture, and lower room adds 2mL basic culture solution, after cultivating 48h, collects the cell through Transwell cell barrier film, amplification cultivation.After repetition aforesaid operations 8 times, it is thus achieved that BxPC-3M8 cell.
Embodiment 2 morphocytology detects
100,000 BxPC-3 and BxPC-3M8 cells are inoculated in 60mm culture dish, culture fluid based on culture medium, cultivate 72 hours, to 60%～70% adherent rate, removing culture fluid, PBS washs 2 times, Giemsa dyes (fully according to test kit operating instruction), is just putting basis of microscopic observation, is taking pictures.
As it is shown in figure 1, compare with parental cell BxPC-3, the difference such as the cell size of BxPC-3M8, cell outline is little, but cell clone relative loose, and intercellular substance is bigger.
Embodiment 3 BxPC-3M8 cell STR detects analysis
Entrusting China typical culture collection center that BxPC-3M8 cell carries out STR detection to analyze, detection method is as follows: extract the DNA of cell to be checked, uses GoldeneyeTM20A STR composite amplification reagent kit expands, 20 known STR bit points and gender-specific genes Amelogenin are carried out detection by ABI 3100 type genetic analyzer continuously analyze, to determine the Species origin of cell to be detected, judging whether its cell exists cross-contamination, testing result is as shown in table 1.
Table 1
| Labelling (Marker) | Allele 1 (Allele1) | Allele 2 (Allele2) |
| D19S433 | 13 | 16.2 |
| D5S818 | 11 | 11 |
| D21S11 | 29 | 29 |
| D18S51 | 12 | 12 |
| D6S1043 | 12 | 12 |
| AMEL | X | X |
| D3S1358 | 14 | 16 |
| D13S317 | 11 | 11 |
| D7S820 | 10 | 13 |
| D16S539 | 9 | 11 |
| CSF1PO | 13 | 13 |
| Penta D | 14 | 14 |
| D2S441 | 12 | 14 |
| vWA | 14 | 18 |
| D8S1179 | 13 | 13 |
| TPOX | 8 | 8 |
| Penta E | 12 | 14 |
| TH01 | 9 | 9 |
| D12S391 | 19.3 | 20 |
| D2S1338 | 17 | 19 |
| FGA | 20 | 21 |
The conclusion of examining report is:
1, all there is not triallelic phenomenon at each locus in BxPC-3M8 cell line, does not find human cell's cross-contamination in cell.
2, in the STR data base of BxPC-3M8 cell line STR data and U.S. ATCC, German and Japanese JCRB, data compare, and all find the cell matched with its cell typing 100% in JCRB, DSMZ cell bank, and Cell Name is BxPC-3.
Embodiment 4 cell growth curve detects
Respectively 50,000 BxPC-3 and BxPC-3M8 cells are inoculated in 100mm culture dish, culture fluid based on culture medium, cultivate 72～96 hours, to 70%～90% adherent rate, change fresh medium, 0.25% trypsinization, collect cell, cell counting, the single cell suspension of preparation 10000/mL concentration, inoculation 5mL single cell suspension in every hole is to 30 60mm culture dishs respectively, and every total cell number is 50000.Change culture fluid according to situation, it is ensured that sufficient nutritional labeling, respectively at 24,48,72,96,120,144,168,196 hours, digest 3 ware cell countings, average, draw growth curve.
Result is as in figure 2 it is shown, BxPC-3M8 with BxPC-3 growth curve substantially overlaps, BxPC-3M8 Yu BxPC-3 proliferation rates in vitro does not has significance to distinguish.
Embodiment 5 scratch experiment
500,000 BxPC-3 and BxPC-3M8 cells are inoculated in 6 orifice plates, culture fluid based on culture medium, when length is to 100% degrees of fusion, changes serum-free medium, with 200 aseptic μ L rifle head cuts, wash 2 times, remove suspension cell, change serum-free medium and cultivate, choose scratch width suitable, the zone marker that border is regular, takes pictures every 24 hours basis of microscopic observation, compares the difference of two kinds of cellular level locomotivities.
Result is as it is shown on figure 3, serum-free culture is after 24 hours, and the cut gap width of BxPC-3M8 cell is significantly less than BxPC-3 cell, illustrates that the motor capacity of the horizontal direction of BxPC-3M8 cell is significantly stronger than BxPC-3 cell.
Embodiment 6 Cell migration assay
Digestion BxPC-3 and BxPC-3M8 cell, produces 5 × 10 with serum-free medium5/ mL single cell suspension, adds 200 μ L single cell suspensions in the upper room of 24 orifice plate Transwell (aperture is 8 μm), and lower room adds 600 μ L basic culture solutions.Within 24 hours, removing ventricular cell on Transwell, Gimsa dyes, and extracts Transwell barrier film, is just putting basis of microscopic observation, and take pictures after mounting.
As shown in Figure 4, under similarity condition, BxPC-3M8 cell is significantly more than BxPC-3 cell through the cell of Transwell barrier film to result, illustrates that the transfer ability of BxPC-3M8 cell is significantly stronger than BxPC-3 cell.
Embodiment 7 cell invasion is tested
Mixing by 1: 5 with matrigel (matrigel) and serum-free medium, each Transwell cell (24 orifice plates, aperture is 8 μm) adds 30 μ L mixed liquors, uniform fold barrier film, and 37 DEG C of hatchings are coated 4 hours, standby.Digestion BxPC-3 and BxPC-3M8 cell, produces 5 × 10 with RPMI-16405/ mL single cell suspension, on the Transwell being coated with matrigel (matrigel), room adds 200 μ L single cell suspensions, and lower room adds 600 μ L basic culture solutions.After 48 hours, removing ventricular cell on Transwell, Gimsa dyes, and extracts Transwell barrier film, is just putting basis of microscopic observation, and take pictures after mounting.
Result is as it is shown in figure 5, under similarity condition, BxPC-3M8 cell is significantly more than BxPC-3 cell through the number of Transwell barrier film (matrigel (matrigel) is coated).Illustrate that the invasive ability of BxPC-3M8 cell is significantly stronger than BxPC-3 cell.
Claims (8)
1. pancreas cancer cell strain, it is characterised in that named human pancreatic cancer cell BxPC-3M8, preserving number is: CCTCC NO:C2016119.
2. the daughter cell of pancreas cancer cell strain as claimed in claim 1.
3. the construction method of pancreas cancer cell strain as claimed in claim 1, it is characterised in that described construction method is Transwell migration test method, and initiator cell strain is pancreatic cancer cell BxPC-3, test number of repetition is 8 times.
4. pancreas cancer cell strain application in setting up mammalian pancreas cancer model as claimed in claim 1.
Apply the most as claimed in claim 4, it is characterised in that described mammal is nude mouse or nude rat.
6. the pancreas cancer cell strain as claimed in claim 1 application in extracting the mark of cancer of pancreas transfer and recurrence.
7. the pancreas cancer cell strain as claimed in claim 1 application in screening or assessment treatment pancreatic cancer drug.
8. the pancreas cancer cell strain as claimed in claim 1 application in exploitation cancer of pancreas recurrence detection kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107794279A (en) * | 2017-08-21 | 2018-03-13 | 中国科学院昆明动物研究所 | A kind of method for establishing pancreatic cancer models in wild type adult mouse with slow virus |
| CN108866001A (en) * | 2018-08-02 | 2018-11-23 | 郭世伟 | Human pancreas cancer neural invasion cell line |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250840A (en) * | 2010-05-18 | 2011-11-23 | 上海睿智化学研究有限公司 | Human pancreatic cancer cell line and its application |
| CN103131667A (en) * | 2011-11-26 | 2013-06-05 | 复旦大学附属肿瘤医院 | Pancreatic cancer cell line with lymphatic channel high migration activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102250840A (en) * | 2010-05-18 | 2011-11-23 | 上海睿智化学研究有限公司 | Human pancreatic cancer cell line and its application |
| CN103131667A (en) * | 2011-11-26 | 2013-06-05 | 复旦大学附属肿瘤医院 | Pancreatic cancer cell line with lymphatic channel high migration activity |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107794279A (en) * | 2017-08-21 | 2018-03-13 | 中国科学院昆明动物研究所 | A kind of method for establishing pancreatic cancer models in wild type adult mouse with slow virus |
| CN107794279B (en) * | 2017-08-21 | 2019-12-17 | 中国科学院昆明动物研究所 | method for establishing pancreatic cancer model in wild-type adult mouse by using lentivirus |
| CN108866001A (en) * | 2018-08-02 | 2018-11-23 | 郭世伟 | Human pancreas cancer neural invasion cell line |
| CN108866001B (en) * | 2018-08-02 | 2023-05-02 | 郭世伟 | Human pancreatic cancer neuro-infiltration cell line |
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