CN109321528A - Chinese B7-H5 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up - Google Patents
Chinese B7-H5 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up Download PDFInfo
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- CN109321528A CN109321528A CN201811089103.4A CN201811089103A CN109321528A CN 109321528 A CN109321528 A CN 109321528A CN 201811089103 A CN201811089103 A CN 201811089103A CN 109321528 A CN109321528 A CN 109321528A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
Abstract
The invention discloses a kind of Chinese B7-H5 to express positive pancreatic cancerous cell line sipanc-1076 method for building up, the following steps are included: 1. collections of specimens, the configuration of 2.Organoid organoid complete medium, 3. tissue digestion, 4. cancer of pancreas Organoid organoid is established and culture, the passage of 5. cells and culture.Beneficial effect of the present invention: the Chinese human pancreatic cancer cell sipanc-1076 biological property that the present invention constructs is ductal adenocarcinoma of pancreas, the unique features that this other commercialization human pancreatic cancer cell with B7-H5 positive expression do not have, and its Tumor formation is good, can be applied to the research such as pancreatic tumour immunologic escape and Chinese's human pancreatic cancer-associated genes pedigree analysis.
Description
Technical field
The present invention relates to a kind of cancer of pancreas primary tumor cells to build the method for being, mainly a kind of Chinese B7-H5 expression
Positive pancreatic cancerous cell line sipanc-1076 method for building up.
Background technique
Ductal adenocarcinoma of pancreas (Pancreatic ductal adenocarcinoma, PDAC) is that the most common pancreas is swollen
Tumor, early stage shift, and symptom is unobvious, and majority has been advanced stage when discovery, and it is past to make a definite diagnosis rear five year survival rate for poor prognosis
Toward less than 5%, referred to as " king of cancer "[1-3], positioned at the 6th of China's male cancer patient's death rate[4].Past basis and
Used pancreatic carcinoma is in clinical research mostly from external ATCC cell bank, such as Panc-1, Bxpc-3,
Miapaca-2, ASPC-1 etc., cell line derive from white people's Pancreas cancer patients, for China's cancer of pancreas research, Ke Nengcun
In level differences such as certain population structure, genes.Due to the weary blood supply of cancer of pancreas itself[5]And more tumour associated fibroblast cells
The characteristic of (cancer associated fibroblasts, CAF) enrichment, traditional Tumor cell build system, method in pancreas
Application in gland cancer is extremely limited.
Summary of the invention
It is an object of the invention to overcome the shortcomings of the prior art, and it is positive to provide a kind of Chinese B7-H5 expression
Pancreatic carcinoma sipanc-1076 method for building up discloses new Chinese humanization ductal adenocarcinoma of pancreas (human
Pancreatic ductal adenocarcinoma) cell line and its build system, method.
The object of the present invention is achieved by the following technical solutions.This Chinese B7-H5 expresses positive pancreatic cancer
Cell line sipanc-1076 method for building up, this method comprises the following steps:
(1), collection of specimens and tissue block is made；
(2), tissue digestion: above-mentioned small tissue blocks are resuspended with 5mlPBS, and the IV Collagen Type VI of 700ul aqua sterilisa configuration is added
Enzyme, the sterile CaCl of working concentration 1437u/ml, 25ul2Solution, working concentration 1M, 25-30mgBSA powder, 37 DEG C of shaking table,
200 revs/min, after being incubated for 15 minutes, above-mentioned tissue suspension is taken out from shaking table, 70um grades of strainer filtering removings do not digest tissue
Block, after adding 10mlPBS to wash the cell suspension of filtration, 1500 revs/min 4 DEG C are centrifuged 5 minutes, are discarded supernatant, and addition 5ml splits red
Liquid cracks 5 minutes on ice, and again plus 10mlPBS is washed, and 1500 revs/min 4 DEG C are centrifuged 5 minutes, stand-by after discarding supernatant；
(3), cancer of pancreas Organoid organoid is established and cultivated: the previous day thaws low growth factor matrigel in 4 DEG C.
500ul liquid matrix glue is taken, cancer of pancreas primary cell precipitating is resuspended, after matrigel solidification, is placed in slide chamber or culture plate
In, add 5ml complete medium；Circular ring shape organoid structure is grown at 4-6 days, in matrigel；At 10-12 days, matrix is taken out
Glue, 10mlPBS repeated flushing, pancreatin digestive group matter glue, 70um grades of strainer filtering removings do not digest tissue block, by the cell of filtration
After suspension adds 10mlPBS to wash, 1500 revs/min 4 DEG C are centrifuged 5 minutes, are discarded supernatant；
(4), above-mentioned cell precipitation: being added new sterile cell maintenance medium by cell passage and culture, and culture medium uses
The configuration of IMDM culture medium, 10%FBS containing final concentration (v/v%) and the dual anti-each 100mg/ml of penicillin/streptomycin, place incubator
Culture, condition of culture are 37 DEG C, 5%CO2(v/v%), routine observation cells survival situation, cell are covered with to 70-80% through pancreas
25cm is transferred to after enzymic digestion2Culture bottle subculture.
The collection of specimens: asepsis collects fresh pancreatic tumour sample, immerses 20ml ordinary culture medium immediately, general
Logical culture medium is configured using RPMI 1640,10%FBS containing final concentration (v/v%) and the dual anti-each 100mg/ of penicillin/streptomycin
Ml, safe ventilation cabinet is interior to use 20mlPBS repeated flushing tissue, cleans and removes the visible clot of naked eyes and necrotic tissue, cut-off
The big little tumour of diameter 1*1*1-1.5*1.5*1.5cm, is shredded as 1mm3Size tissue block.
The configuration of Organoid organoid complete medium: AdDMEM/F12 culture medium, 10mMHEPES, 2mMGlutamax,
The dual anti-each 100mg/ml of penicillin/streptomycin, 1 multiplies B27,1mg/ml primary cell antibiotic primocin, 1mM N- acetyl half
Cystine, 50% (v/v) Wnt3a conditioned medium, 10% (v/v) RSPO1 conditioned medium, 10% (v/v) Noggin condition
Culture medium, 50ng/ml epidermal growth factor EGF, 10nM gastrin, 100ng/ml Fibroblast growth factor 10,10mM Buddhist nun gram acyl
Amine, 0.5uMA83-01.
The invention has the benefit that the Chinese human pancreatic cancer cell sipanc-1076 biological characteristics that the present invention constructs
Sign is ductal adenocarcinoma of pancreas, this other commercialization human pancreatic cancer cell with B7-H5 positive expression do not have only
Feature, and its Tumor formation is good, can be applied to pancreatic tumour immunologic escape and Chinese's human pancreatic cancer-associated genes pedigree point
The research such as analysis.
Detailed description of the invention
Fig. 1 is that cancer of pancreas Organoid organoid establishes schematic diagram.
Fig. 2 is form under the common light microscopic of sipanc-1076 cell line (under different object lens multiples).
Fig. 3 is the surface Flow cytometry sipanc-1076 B7-H5 expression (primary antibody mouse anti human
B7-H5,clone5B6-2；Secondary antibody APC goat anti mouse IgG1, k；isotypemouseIgG1,k).
Specific embodiment
Below in conjunction with attached drawing, the present invention will be described in detail:
Of the invention builds system, method:
1. Specimen origin: sample is derived from 2nd Affiliated Hospital Zhejiang University School of Medicine liver and gallbladder pancreas 67 years old male patient of surgery,
Pathological diagnosis: ductal adenocarcinoma of pancreas, TNM stage: T3N0M0。
The configuration of 2.Organoid organoid complete medium: AdDMEM/F12 culture medium, 10mMHEPES,
2mMGlutamax, the dual anti-each 100mg/ml of penicillin/streptomycin, 1 multiplies B27, and (b27 is the mixture being much formulated, the concentration of producer
It is 100 times of working concentration, use when is diluted to 1 times of use), 1mg/ml primary cell antibiotic (primocin), 1mM
N-acetylcystein, 50% (v/v) Wnt3a conditioned medium, 10% (v/v) RSPO1 conditioned medium, 10% (v/v)
Noggin conditioned medium, 50ng/ml epidermal growth factor (EGF), 10nM gastrin, 100ng/ml fibroblast growth factor
10,10mM niacinamides, 0.5uMA83-01[6]。
3. collection of specimens: asepsis collects fresh pancreatic tumour sample, immerses 20ml ordinary culture medium (RPMI immediately
1640 configurations, 10%FBS containing final concentration (v/v%) and the dual anti-each 100mg/ml of penicillin/streptomycin), make in safe ventilation cabinet
With 20mlPBS repeated flushing tissue, the visible clot of naked eyes and necrotic tissue are cleaned and removed, diameter 1*1*1-1.5*1.5* is taken
The big little tumour of 1.5cm, is shredded as 1mm3Size tissue block.
4. tissue digestion: above-mentioned small tissue blocks being resuspended with 5mlPBS, the IV Collagenase Type of 700ul aqua sterilisa configuration is added
(working concentration 1437u/ml), the sterile CaCl of 25ul2Solution (working concentration 1M), 25-30mgBSA powder, 37 DEG C of shaking table,
200 revs/min, after being incubated for 15 minutes, above-mentioned tissue suspension is taken out from shaking table, 70um grades of strainer filtering removings do not digest tissue
Block, after adding 10mlPBS to wash the cell suspension of filtration, 1500 revs/min 4 DEG C are centrifuged 5 minutes, are discarded supernatant, and addition 5ml splits red
Liquid cracks 5 minutes on ice, and again plus 10mlPBS is washed, and 1500 revs/min 4 DEG C are centrifuged 5 minutes, stand-by after discarding supernatant.
5. cancer of pancreas Organoid organoid is established and culture: the previous day is by low growth factor matrigel (GFR
Matrigel it) thaws in 4 DEG C.500ul liquid matrix glue is taken, cancer of pancreas primary cell precipitating is resuspended, after matrigel solidification, is set
In slide chamber or culture plate, 5ml complete medium is added.About 5 days or so (at 4-6 days), annulus can be grown in matrigel
Shape organoid structure.At about 10-12 days, matrigel, 10mlPBS repeated flushing, pancreatin digestive group matter glue, 70um grades of strainers are taken out
It is filtered to remove and does not digest tissue block, after adding 10mlPBS to wash the cell suspension of filtration, 1500 revs/min 4 DEG C are centrifuged 5 minutes, are abandoned
Remove supernatant.
6. cell passage and culture: by above-mentioned cell precipitation be added new sterile cell maintenance medium (IMDM culture medium configure,
10%FBS containing final concentration (v/v%) and the dual anti-each 100mg/ml of penicillin/streptomycin), and placement incubator culture (37 DEG C, 5%
CO2(v/v%)), routine observation cells survival situation, cell, which is covered with to 70-80%, to be transferred to 25cm after pancreatin digests2Training
Bottle subculture is supported, passage in general 4-5 days or so is primary.The sipanc-1076 passage number alreadyd exceed for 50 generations at present.
In addition, for tumour immunity research, aforementioned commercial human pancreatic cancer cell its surface B7-H5 expression is in
Existing feminine gender, and strong positive expression is presented in this plant of its B7-H5 of sipanc-1076.Therefore, the Chinese human pancreas cancer that the present invention establishes is thin
Born of the same parents system is for studying state's human pancreas cancer pathogenesis, the biological properties such as tumor immune escape, invasion transfer, and the anti-pancreas of exploitation
Gland cancer treatment new drug etc. has particularly significant meaning.
It is understood that it will be understood by those skilled in the art that being subject to technical solution of the present invention and inventive concept
It all should fall within the scope of protection of the appended claims of the present invention with replacement or change.
Claims (3)
1. a kind of Chinese B7-H5 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up, it is characterised in that: the party
Method includes the following steps:
(1), collection of specimens and tissue block is made；
(2), tissue digestion: above-mentioned small tissue blocks are resuspended with 5mlPBS, and the IV Collagenase Type of 700ul aqua sterilisa configuration is added,
The sterile CaCl of working concentration 1437u/ml, 25ul2Solution, working concentration 1M, 25-30mgBSA powder, 37 DEG C of shaking table, 200
Rev/min, after being incubated for 15 minutes, above-mentioned tissue suspension is taken out from shaking table, 70um grades of strainer filtering removings do not digest tissue block, will
After the cell suspension of filtration adds 10mlPBS to wash, 1500 revs/min 4 DEG C are centrifuged 5 minutes, discard supernatant, and 5ml is added and splits red liquid ice
Upper cracking 5 minutes, again plus 10mlPBS is washed, and 1500 revs/min 4 DEG C are centrifuged 5 minutes, stand-by after discarding supernatant；
(3), cancer of pancreas Organoid organoid is established and cultivated: the previous day thaws low growth factor matrigel in 4 DEG C.It takes
Cancer of pancreas primary cell precipitating is resuspended in 500ul liquid matrix glue, after matrigel solidification, is placed in slide chamber or culture plate,
Add 5ml complete medium；Circular ring shape organoid structure is grown at 4-6 days, in matrigel；At 10-12 days, matrigel is taken out,
10mlPBS repeated flushing, pancreatin digestive group matter glue, 70um grades of strainer filtering removings do not digest tissue block, the cell of filtration are hanged
After liquid adds 10mlPBS to wash, 1500 revs/min 4 DEG C are centrifuged 5 minutes, are discarded supernatant；
(4), above-mentioned cell precipitation: being added new sterile cell maintenance medium by cell passage and culture, and culture medium is trained using IMDM
Feeding basigamy is set, 10%FBS containing final concentration (v/v%) and the dual anti-each 100mg/ml of penicillin/streptomycin, places incubator culture,
Condition of culture is 37 DEG C, 5%CO2(v/v%), routine observation cells survival situation, cell is covered with to 70-80% to be digested through pancreatin
After be transferred to 25cm2Culture bottle subculture.
2. Chinese B7-H5 according to claim 1 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up,
It is characterized by: the collection of specimens: asepsis collects fresh pancreatic tumour sample, immerses 20ml Nostoc commune Vanch immediately
Base, ordinary culture medium are configured using RPMI 1640, and 10%FBS containing final concentration (v/v%) and penicillin/streptomycin are dual anti-each
100mg/ml, safe ventilation cabinet is interior to use 20mlPBS repeated flushing tissue, cleans and remove the visible clot of naked eyes and downright bad group
It knits, takes the big little tumour of diameter 1*1*1-1.5*1.5*1.5cm, shredded as 1mm3Size tissue block.
3. Chinese B7-H5 according to claim 1 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up,
It is characterized by: Organoid organoid complete medium configures: AdDMEM/F12 culture medium, 10mMHEPES,
2mMGlutamax, the dual anti-each 100mg/ml of penicillin/streptomycin, 1 multiplies B27,1mg/ml primary cell antibiotic primocin,
1mM N-acetylcystein, 50% (v/v) Wnt3a conditioned medium, 10% (v/v) RSPO1 conditioned medium, 10% (v/
V) Noggin conditioned medium, 50ng/ml epidermal growth factor EGF, 10nM gastrin, 100ng/ml fibroblast growth factor
10,10mM niacinamides, 0.5uMA83-01.
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Cited By (4)
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| CN110317790A (en) * | 2019-07-22 | 2019-10-11 | 中山大学孙逸仙纪念医院 | A method of separation and in vitro culture Tissues of Human Adenocarcinoma of Pancreas organoid |
| CN110402892A (en) * | 2019-04-30 | 2019-11-05 | 梁廷波 | Selectivity knocks out the method for building up of the spontaneous cancer of pancreas mouse model of 1 molecule of pancreatic epithelial cells programmed death ligand |
| CN112961819A (en) * | 2020-12-01 | 2021-06-15 | 保信亚太生物科技（深圳）有限公司 | Method for constructing bocavirus small intestine epithelial organoid infection model |
| CN114426949A (en) * | 2022-02-25 | 2022-05-03 | 重庆嘉士腾生物科技有限公司 | Culture medium for establishing pancreas or pancreatic cancer organoid, method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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Application publication date: 20190212 |