CN106676074A - Method for inducing liver cell cells to be transformed into liver cancer stem cells - Google Patents
Method for inducing liver cell cells to be transformed into liver cancer stem cells Download PDFInfo
- Publication number
- CN106676074A CN106676074A CN201710159623.7A CN201710159623A CN106676074A CN 106676074 A CN106676074 A CN 106676074A CN 201710159623 A CN201710159623 A CN 201710159623A CN 106676074 A CN106676074 A CN 106676074A
- Authority
- CN
- China
- Prior art keywords
- cell
- liver
- cancer stem
- culture
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 62
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 62
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 34
- 230000001939 inductive effect Effects 0.000 title abstract description 4
- 210000005229 liver cell Anatomy 0.000 title abstract 2
- 210000004027 cell Anatomy 0.000 claims abstract description 86
- 229920002678 cellulose Polymers 0.000 claims abstract description 36
- 239000001913 cellulose Substances 0.000 claims abstract description 36
- 238000004113 cell culture Methods 0.000 claims abstract description 25
- 230000014509 gene expression Effects 0.000 claims abstract description 23
- 239000006285 cell suspension Substances 0.000 claims abstract description 22
- 102100032912 CD44 antigen Human genes 0.000 claims abstract description 21
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims abstract description 21
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 claims abstract description 17
- 102100040120 Prominin-1 Human genes 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 238000000684 flow cytometry Methods 0.000 claims abstract description 11
- 238000001262 western blot Methods 0.000 claims abstract description 11
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 claims description 18
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 claims description 18
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 claims description 15
- 206010028980 Neoplasm Diseases 0.000 claims description 15
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 claims description 15
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 14
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 claims description 14
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- 239000001301 oxygen Substances 0.000 claims description 14
- 230000006698 induction Effects 0.000 claims description 13
- 230000008859 change Effects 0.000 claims description 9
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 7
- 239000006143 cell culture medium Substances 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- 238000003757 reverse transcription PCR Methods 0.000 claims description 7
- 230000000118 anti-neoplastic effect Effects 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 210000004881 tumor cell Anatomy 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 239000012620 biological material Substances 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 239000001569 carbon dioxide Substances 0.000 claims 1
- 239000002771 cell marker Substances 0.000 abstract description 5
- 210000004748 cultured cell Anatomy 0.000 abstract description 5
- 238000003753 real-time PCR Methods 0.000 abstract description 4
- 206010059866 Drug resistance Diseases 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 2
- 238000003364 immunohistochemistry Methods 0.000 abstract 2
- 230000001413 cellular effect Effects 0.000 abstract 1
- 239000012466 permeate Substances 0.000 abstract 1
- 238000003752 polymerase chain reaction Methods 0.000 abstract 1
- 235000010980 cellulose Nutrition 0.000 description 26
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 20
- 239000000523 sample Substances 0.000 description 19
- 239000007788 liquid Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108010019160 Pancreatin Proteins 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 229940055695 pancreatin Drugs 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000003292 glue Substances 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000001079 digestive effect Effects 0.000 description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 206010019695 Hepatic neoplasm Diseases 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 229920000609 methyl cellulose Polymers 0.000 description 3
- 239000001923 methylcellulose Substances 0.000 description 3
- 235000010981 methylcellulose Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010016825 Flushing Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 238000010146 3D printing Methods 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 206010057573 Chronic hepatic failure Diseases 0.000 description 1
- 102000012804 EPCAM Human genes 0.000 description 1
- 101150084967 EPCAM gene Proteins 0.000 description 1
- 208000010334 End Stage Liver Disease Diseases 0.000 description 1
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101150057140 TACSTD1 gene Proteins 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229940063774 carbon dioxide 5 % Drugs 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000011444 chronic liver failure Diseases 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229920001821 foam rubber Polymers 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
Classifications
-
- 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
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/78—Cellulose
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for inducing liver cell cells to be transformed into liver cancer stem cells. The method comprises the following steps: adding human liver cancer cells with a single cell state into a culture medium, so as to prepare a cell suspension solution; dropwise adding the cell suspension solution into a porous cellulose bracket; after enabling the cell suspension solution to permeate into the porous cellulose bracket, putting the porous cellulose bracket into a cell culture box and culturing and adhering; then adding the culture medium into the cell culture box and culturing for a plurality of days to obtain three-dimensional cultured cells; detecting changes of expression conditions of liver cancer stem cell marker molecules EpCAM (Epithelial Cellular Adhesion Molecule), OCT4, CD44 and CD133 in the three-dimensional cultured cells through utilizing Western Blot, quantitative PCR (Polymerase Chain Reaction), flow cytometry and IHC (Immunohistochemistry) and (or) a microscope is used for observing; the transformed liver cancer stem cells are detected. The invention further discloses the liver cancer stem cells and application of the liver cancer stem cells. The liver cancer stem cells disclosed by the invention have good drug resistance.
Description
Technical field
Research field is learned the invention belongs to oncology and stem cell, and in particular to one kind induction HCC is dry thin to liver cancer
The method of dysuria with lower abdominal colic.
Background technology
Liver cancer is a kind of End-stage liver disease, and the state of an illness is dangerous, and case fatality rate is up to 80%, is the pernicious of serious harm human health
One of tumour.China is hepatopathy country occurred frequently, there is the people of all kinds of hepatopaths more than 100,000,000, annual newly 100 liver cancer of hair in the whole world at present
In, it is Chinese to have 55.There is research to think that the presence of tumor stem cell is the major reason that malignant tumour occurs.Liver cancer is dry thin
Born of the same parents can resist cancer treatment drugs, resist chemotherapy radiation, increase postoperative tumor recurrence and transfer probability.To sum up, how to open
Send suitable cell culture and screening technique and filter out liver-cancer stem cell to efficiently separate, so that it is used to onset of liver cancer mechanism,
The research of the aspect such as medicine and immunization therapy is subject matter to be solved by this invention.
The research of the past shows that EpCAM, OCT4, CD44 and CD133 are the most basic molecular markers of liver-cancer stem cell,
Attacked with HCC, the activity such as transfer is closely related.Therefore, the antineoplastic with liver-cancer stem cell as target spot how is developed
It is the important opportunity for treating liver cancer.However, the distribution proportion due to liver-cancer stem cell in liver cancer tissue is very low, thus how body
Outer culture liver-cancer stem cell, becomes liver-cancer stem cell technology development problem demanding prompt solution.
In recent years, Three-dimensional cell culture technology is increasingly ripe.Dimensional culture technology refers to by the carrier with three-dimensional structure
With cell co-incubation in vitro, cell is set to be migrated in the solid space of carrier bracket, grown, constituting has three-dimensional knot
The cell-vector compound of structure feature.At present, Three-dimensional cell culture technology is in oncobiology, cartilage and bone tissue, cyclic system
The fields such as system and heart, nervous system are obtained for extensive use, expand the breadth and depth of biomedical research.
Traditional two-dimentional cell culture system gradually lose original property due to being grown under cell in vitro condition of culture
Shape, does not often correspond with situation in organism, and zoopery is subject to inside and outside Various Complex factors, it is difficult to study
Single factors regulatory mechanism.Three-dimensional cell culture technology a kind of new skill then between two-dimentional cell culture and zoopery
Art, can maximum simulated in vivo environment, the intuitive and condition controllability of cell culture can be represented again.
The content of the invention
Goal of the invention：First purpose of the invention there is provided what a kind of induction HCC was converted to liver-cancer stem cell
Method.
It is a further object of the present invention to provide the liver cancer that induction HCC is obtained to the method that liver-cancer stem cell is converted
Stem cell.
It is a further object of the present invention to provide liver-cancer stem cell antineoplastic in-vitro screening or tumour cell body
Application in outer drug sensitive experiment.
Technical scheme：In order to solve the above problems, turn to liver-cancer stem cell the invention provides one kind induction HCC
The method of change, comprises the following steps：
1) human liver cancer cell of unicellular will be obtained, is added in culture medium, be made cell suspension；
2) cell suspension is added drop-wise on porous cellulose support, treats that cell suspension is penetrated into porous cellulose support
Portion, be placed in cell culture incubator cultivate attach after add culture medium, then be placed in a few days cultivated in cell culture incubator, obtain three-dimensional
Cultured cells；
3) using Western Blot, RT-PCR, flow cytometry and IHC, the liver tumour in dimensional culture cell is determined
The change of stem-cell marker molecule EpCAM, OCT4, CD44 and CD133 expression, and turned using the detection of microscope observational technique
Change gained liver-cancer stem cell.
Wherein, above-mentioned porous cellulose support is with cellulose as main material, or is aided with other biologies based on cellulose
Compatibility preferably various biomaterials.
Wherein, above-mentioned cellulosic material can be methylcellulose, hydroxypropyl cellulose, HPMC and cellulose
Ether-ether class material.
Wherein, above-mentioned porous cellulose support can be prepared with solution cast/particle leaching method, also can with fusion sediment method,
3D printing technique and stereolithography etc. prepare gained.
Wherein, above-mentioned porous cellulose support is the porous sponge-like vector of intercommunication, and aperture is 50~500 μm.
Wherein, the aperture of above-mentioned porous cellulose support is preferably 50-200 μm.The even aperture distribution, cell be easy into
In entering hole, and space is provided to be grown to cell microsphere.
Wherein, above-mentioned porous cellulose stiffness of support is 5~100kPa.
Wherein, above-mentioned porous cellulose stiffness of support is preferably 5~30kPa.
Wherein, above-mentioned HCC is HepG2, HUH7, PLC/PRF/5 or other type HCCs.
Wherein, corresponding culture medium used or the tumor stem cell without serum when above-mentioned culture medium is SMMC-7721 culture
Culture medium.
Wherein, above-mentioned condition of culture is 35-39 DEG C of temperature, oxygen content 3%-21%, carbon dioxide 5%.
Present disclosure also includes adopting the liver-cancer stem cell for converting obtain with the aforedescribed process.
Present disclosure also include by above-mentioned liver-cancer stem cell antineoplastic in-vitro screening or tumour cell
Application in In vitro chemo-drug sensitive test.
Beneficial effect：Relative to prior art, the present invention has advantages below：
1st, the present invention carries out dimensional culture using porous cellulose support to human liver cancer cell, directly obtains liver-cancer stem cell
Microballoon, thus obtained microsphere cell contains a large amount of EpCAM, OCT4, CD44 and CD133 positive cell, hence it is evident that the spy with liver-cancer stem cell
Property.
2nd, abductive approach of the invention is easy to operate, the pretreatment without carrying out complexity to cell, and cell will not be caused
Additional physical chemistry pressure, cell is in normal physiological condition.
3rd, method of inducing differentiation of the invention can farthest induce HCC to be converted into liver-cancer stem cell, be liver cancer
Diagnosis and treatment new thinking is provided.The inventive method yield is high, and timeliness is fast, and can not commodity in use stem cell culture product
Product, reduce Costco Wholesale.
4th, porous cellulose support is used in Induction Transformation method of the invention, three dimensional growth ring can be provided for cell
Border, the microenvironment in analogue body.
5th, the liver-cancer stem cell that the present invention is obtained has good resistant characterization to 5-FU and cis-platinum, can be used for antineoplastic
Thing antibiotics susceptibility test.
Brief description of the drawings
In HepG2, HUH7, PLC/PRF/5 cell of Fig. 1 Western Blot technology for detection two and three dimensions cultures
The protein expression level of EpCAM, OCT4, CD44 and CD133；
In HepG2, HUH7 cell of Fig. 2 Real time-PCR technology for detection two and three dimensions cultures EpCAM and
OCT4mRNA expressions；
EpCAM and CD44 expressions in HepG2 the and HUH7 cells of Fig. 3 IHC technology for detection two and three dimensions cultures；
The expression of CD44 and CD133 in the HepG2 cells of Fig. 4 Flow cytometry two and three dimensions cultures；
The expression water of CD44 and CD133 in the PLC/PRF/5 cells of Fig. 5 Flow cytometry two and three dimensions cultures
It is flat；
Fig. 6 microscopes observe HepG2 the and HUH7 cells of the two and three dimensions culture of embodiment 1 under the conditions of light field.
The resistance experiment of the HCC PLC/PRF/5 cells of Fig. 7 two and three dimensions cultures；
The resistance experiment of the hepatocellular carcinoma H22 cell of Fig. 8 two and three dimensions cultures.
Specific embodiment
The present invention is further described below.
Embodiment 1：A kind of method that induced liver tumour cell is converted to tumor stem cell
1) growth conditions are good, the HCC (HepG2, HUH7 and PLC/PRF/5) in exponential phase is used
0.25% pancreatin is digested, and 5min is digested in 37 DEG C, obtains the human liver cancer cell of unicellular, digestive juice of the removal containing pancreatin
Afterwards, add in the DMEM complete mediums containing 10% hyclone, be made cell suspension；
2) appropriate cell suspension is laid in Tissue Culture Dish, is placed in 37 DEG C, 5%CO2,20% oxygen content, cell
Incubator culture 7 days, compares as two-dimentional cultured cells.DMEM complete mediums are changed every other day；Wherein, the DMEM is trained completely
Foster base is the DMEM in high glucose culture medium for being added with 10% hyclone, 100U/ml penicillin and 100mg/ml streptomysins.
3) by 60 μ l cell suspensions, (cell density is 105Individual/ml) it is added dropwise on porous cellulose support, 37 DEG C are placed in,
5%CO2,20% oxygen adds DMEM complete mediums after attaching within 4 hours in cell culture incubator, is placed in 37 DEG C, 5%CO2,
20% oxygen, incubator culture 7 days, obtains dimensional culture cell.Example of spatial compartmentalizationis.The cellulose of the porous cellulose support
Predominantly methylcellulose, aperture is 50 μm, and rigidity is 15kPa；
4) cell that two and three dimensions culture is obtained is collected respectively, uses Western Blot, RT-PCR, flow cytometry
With the method such as IHC, determine under two dimension or three-dimensional cultivation condition, HCC marker molecule EpCAM, OCT4, CD44 and CD133 table
Up to the change of situation, and changed with micro- sem observation dimensional culture cell characterization.
The method that a kind of induced liver tumour cell of embodiment 2 is converted to tumor stem cell
1) growth conditions are good, the HCC (HepG2, HUH7 and PLC/PRF/5) in exponential phase is used
0.25% pancreatin is digested, and 5min is digested in 37 DEG C, obtains the human liver cancer cell of unicellular, digestive juice of the removal containing pancreatin
Afterwards, add in the DMEM complete mediums containing 10% hyclone, be made cell suspension；
2) appropriate cell suspension is laid in Tissue Culture Dish, is placed in 35 DEG C, 5%CO2,5% oxygen, cell culture incubator
Culture 7 days, compares as two-dimentional cell culture.The DMEM complete mediums are changed every other day；Wherein, the DMEM is cultivated completely
Base is the DMEM in high glucose culture medium for being added with 10% hyclone, 100U/ml penicillin and 100mg/ml streptomysins.
3) by 60 μ l cell suspensions, (cell density is 105Individual/ml) be added dropwise on porous cellulose support (37 DEG C are placed in,
5%CO2, DMEM complete mediums are added after attaching within 4 hours in 21% oxygen cell culture incubator, are placed in 35 DEG C, 5%CO2,5%
Cultivated 7 days in oxygen incubator, obtain dimensional culture cell.DMEM complete mediums are changed every other day.The porous cellulose support
Cellulose is mainly hydroxypropyl cellulose, and aperture is 100 μm, and rigidity is 30kPa；
4) cell that two and three dimensions culture is obtained is collected respectively, uses Western Blot, RT-PCR, flow cytometry
With the method such as IHC, determine under two dimension or three-dimensional cultivation condition, liver-cancer stem cell marker molecule EpCAM, OCT4, CD44 and CD133
The change of expression, and changed with micro- sem observation dimensional culture cell characterization.
The method that a kind of induced liver tumour cell of embodiment 3 is converted to tumor stem cell
1) growth conditions are good, the HCC (HepG2, HUH7 and PLC/PRF/5) in exponential phase is used
0.25% pancreatin is digested, and 5min is digested in 37 DEG C, obtains the human liver cancer cell of unicellular, digestive juice of the removal containing pancreatin
Afterwards, add in the DMEM complete mediums containing 10% hyclone, be made cell suspension；
2) appropriate cell suspension is laid in Tissue Culture Dish, is placed in 35 DEG C, 5%CO2,10% oxygen, cell culture
Case culture 7 days, compares as two-dimentional cell culture.Serum-free tumor stem cell culture medium is changed every other day；
3) by 60 μ l cell suspensions, (cell density is 105Individual/ml) be added dropwise on porous cellulose support (37 DEG C are placed in,
5%CO2, serum-free tumor stem cell culture medium is added after attaching within 4 hours in 21% oxygen cell culture incubator, is placed in 35 DEG C, 5%
CO2, cultivates 7 days in 10% oxygen incubator, obtains dimensional culture cell.Serum-free tumor stem cell culture medium is changed every other day.Should
The cellulose of porous cellulose support is mainly HPMC, and aperture is 500 μm, and rigidity is 100kPa；
4) cell that two and three dimensions culture is obtained is collected respectively, uses Western Blot, RT-PCR, flow cytometry
With the method such as IHC, determine under two dimension or three-dimensional cultivation condition, liver-cancer stem cell marker molecule EpCAM, OCT4, CD44 and CD133
The change of expression, and changed with micro- sem observation dimensional culture cell characterization.
Embodiment 4
1) growth conditions are good, the HCC (HepG2, HUH7 and PLC/PRF/5) in exponential phase is used
0.25% pancreatin is digested, and 5min is digested in 37 DEG C, obtains the human liver cancer cell of unicellular, digestive juice of the removal containing pancreatin
Afterwards, add in the DMEM complete mediums containing 10% hyclone, be made cell suspension；
2) appropriate cell suspension is laid in Tissue Culture Dish, is placed in 39 DEG C, 5%CO2,10% oxygen, cell culture
Case culture 7 days, compares as two-dimentional cell culture.Serum-free tumor stem cell culture medium is changed every other day；
3) by 60 μ l cell suspensions, (cell density is 105Individual/ml) be added dropwise on porous cellulose support (39 DEG C are placed in,
5%CO2, serum-free tumor stem cell culture medium is added after attaching within 4 hours in 21% oxygen cell culture incubator, is placed in 39 DEG C, 5%
CO2, cultivates 7 days in 10% oxygen incubator, obtains dimensional culture cell.Serum-free tumor stem cell culture medium is changed every other day.Should
The cellulose of porous cellulose support is mainly cellulose ether-esters, and aperture is 500 μm, and rigidity is 100kPa；
4) cell that two and three dimensions culture is obtained is collected respectively, uses Western Blot, RT-PCR, flow cytometry
With the method such as IHC, determine under two dimension or three-dimensional cultivation condition, liver-cancer stem cell marker molecule EpCAM, OCT4, CD44 and CD133
The change of expression.And changed with micro- sem observation dimensional culture cell characterization.
The Western Blot technology for detection of experimental example 1
The extraction of 1.1 total proteins
After two and three dimensions that embodiment 1 is obtained are cells trypsinised, washed 2 times in cold PBS, it is each to add
The 200 μ pre- Cold lysis buffers of l ice, ice bath 30min after mixing shakes 10s, fully cracking every 5min.Centrifugation 25min.Will be upper
Sorting is filled, and is stored in -20 degree standby.
The measure of 1.2 BCA method protein concentrations
1.2.1 according to the quantity of sample, the BCA reagent B of 1 volume are added by 50 volume BCA reagent As, configures appropriate BCA works
Make liquid, then fully mixed with pipette tips.
1.2.2 protein standard substance is completely dissolved, 10 μ l protein standard substances is taken and is diluted to 100 μ l, make its final concentration of
0.5mg/ml。
1.2.3 standard items are pressed into 0,1,2,4,8,12,16,20 μ l, is sequentially added in the standard sample wells of 96 orifice plates, use standard
Product dilution supplies the μ l of every hole 20.
1.2.4 plus in the sample well of 10 μ l testing samples to 96 orifice plates, with standard dilutions to 20 μ l.
1.2.5 each hole adds the BCA working solutions of 200 μ l, 37 degree of incubation 30min.
1.2.6 the OD value of determination sample and standard items in 520nm wavelength.Concentration standard according to protein standard substance is bent
Line, calculates the protein concentration of each group sample.
1.3 SDS-PAGE electrophoresis
1.3.1 the SDS sample-loading buffers for the protein lysate that 15 μ l are adjusted to same concentrations being added into 5 μ l are mixed, and are taken dry
Net Bio-Rad 1.5cm glass plates, by specification is installed on gum-making rack.
1.3.3 according to the form below prepares 10% separation gel：
1.3.4 carefully with pipette tips by separation gel implantation glass sheet separation, about accounting at 2/3rds glass plate height
Stop, adding several milliliters of distilled waters above afterwards, it is therefore an objective to prevent air to the inhibitory action of gel polymerisation.
1.3.5 after separation gel polymerization is completed, the distilled water that glue is covered above is outwelled, the liquid of exhaustion remaining of being tried one's best with filter paper
Body, should not carefully encounter separation gel.
1.3.6 according to the form below prepares 5% and concentrates glue, and injects the upper end of separation gel, and careful insertion is mutually fitted with sheet thickness
The sample comb answered, it is to avoid produce bubble.
1.3.2 after the polymerization of enriched layer glue, sample comb is carefully taken out, is subsequently adding the electrophoretic buffer of 1xTris-Gly,
Check whether there is leakage.
1.3.8 draw in appropriate amount of sample supernatant addition sample well, the albumen Marker of pre-dyed added in the hole by sample,
It is not added with the hole of Sample supernatants, adds 1xSDS sample-loading buffers to keep glue surface balance.
1.3.9 turn on the power, it is 80V that voltage is started setting up, after protein sample enters separation gel, voltage can bring up to
120V.With reference to the position of pre-dyed Marker, when purpose band enters gel optimal separation area (about the 2/3 of gel), stop
Electrophoresis.
1.4 transferring film
1.4.1 in advance by the 4 degree of precoolings of transferring film liquid.
1.4.2 pound and transfer box is opened on pallet, spread with having that transferring film buffer solution soaks near the inner face of cathode side
Hole dimension pad, puts three layers of filter paper for being soaked with transferring film buffer solution thereon, notes emptying bubble.
1.4.3 glass plate is carefully pried open, glue is placed in the pallet containing transferring film liquid, by the separation gel containing purpose band
Cut, be placed on filter paper after being steeped with transferring film immersion.
1.4.4 spread on gel through methyl alcohol and the wet pvdf membrane of transferring film immersion, there can not be bubble between glue and film.
1.4.5 put the Whatman filter paper of the dipped transferring film liquid of two-layer again on pvdf membrane, note emptying bubble.
1.4.6 second block of foam-rubber cushion is put, transfer folder is closed, is put into transfer groove, transferring film liquid is filled in groove.
1.4.7 turn on the power, 100V, 60min；
1.4.8 after transferring film terminates, take out pvdf membrane and perform mark, film is washed 10min*3 times with TBST.
1.5 closings, antigen-antibody reaction
1.5.1 closing：Pvdf membrane is put into plate, the confining liquid containing 5% skimmed milk power, shaking table concussion 1.5- is added
2h。
1.5.2 after closing terminates, film is washed 10min*3 times with TBST.
1.5.3 film is put into the plate containing primary antibody, 4 degree of shaking tables shake overnight incubation.
1.5.4 take out within second day, primary antibody is abandoned in suction, and TBST is washed 10min*3 times.
1.5.5 secondary antibody, room temperature shaker concussion reaction 1-2h are diluted with 5% skimmed milk power confining liquid.
1.5.6 after secondary antibody reaction terminates, secondary antibody is reclaimed.Then film 5-10min*3. is washed with TBST
1.6 colour developing
1.6.1 the two kinds of liquid of A, B in ECL chemical luminescence reagent kits are pressed 1：1 isometric mixing, is configured to working solution standby
With.
1.6.2 pvdf membrane is taken out from TBST, gets rid of unnecessary liquid, the film containing protein is face-up, put
In preservative film, appropriate working solution is added dropwise, is covered with preservative film, place in development folder, close development folder.
1.6.3 enter scotography, photographic film is placed in development folder, when adjusting exposure according to protein band intensity
Between, during film then is sequentially placed into developer solution, fixing solution, makes film development and be fixed.
The HepG2 of the two and three dimensions culture by the above-mentioned conventional Western Blot technologies of use in embodiment 1,
EpCAM, the protein expression level of OCT4, CD44 and CD133 are have detected in HUH7, PLC cell.As shown in Figure 1, dimensional culture bar
Under part, EpCAM in each cell, the protein expression level of OCT4, CD44 and CD133 has rising.It is embodied in dimensional culture
When, the Western Blot bands gray value of each molecule is higher than two-dimentional condition of culture.
The RT-PCR technology of experimental example 2 is detected
1st, RNA is extracted
The cell obtained under the 1.1 two and three dimensions condition of culture to the acquisition of embodiment 1 discards culture supernatant and adds 1ml pre-
Cold TRIzol, is blown and beaten to the cell liquid that cracking is without particle see-through look solution repeatedly with 1ml pipette tips；
1.2 are transferred in 1.5ml centrifuge tubes the cell liquid of above-mentioned abundant cracking, 5min are placed at room temperature so that core
Acid albumin compound is kept completely separate；
1.3 add chloroform (often adding 0.2ml chloroforms using 1ml TRIzol), cover tightly centrifuge tube lid, with lower vibration on hand
15s, solution is in milky white shape, without lamination, is stored at room temperature 3min；
1.4 12,000g, 4 DEG C centrifugation 15min；
1.5 take out centrifuge tube, and sample is divided into three layers：The lower floor of colourless supernatant water phase, middle white layer and pink
Organic phase；
1.6 careful colorless supernatant water of drawing mutually move to another centrifuge tube, and water phase volume is about Trizol reagents used
60%；
1.7 are added to isometric isopropanol to the supernatant water for obtaining, and gently mix, and are stored at room temperature 10min；12,000g,
4 DEG C of centrifugation 10min；
1.8 careful removal supernatants, slowly add the ethanol (water treated with DEPC is prepared) of 1ml 70%, gently along tube wall
It is light to mix；
1.9 12,000g, 4 DEG C centrifugation 10min；
1.10 it is careful exhaust supernatants, drying at room temperature precipitation 5min or so, it is appropriate depending on (can not dry in the air too dry, otherwise RNA will
Dissolving can be difficult), being precipitated without RNase water dissolving RNAs for 30~50 μ L is added, until completely dissolved in -70 DEG C of preservations.
2nd, the measure of RNA concentration and purity
During 2.1 take 5 μ L RNA samples to 595 μ 1 × TE of L Buffer, absorption value of the determination sample in 260nm and 280nm
It is determined that；
Concentration=the OD260 of 2.2 RNA × extension rate × 0.04 μ g/ μ L (cuvette optical path 1cm), OD260/280 exists
The purity of 1.8~2.1 RNA for being considered as extracting is very high.
3rd, the chains of cDNA first synthesis (10 μ L systems)：
3.1 reaction system
Flick ttom of pipe to mix solution, the of short duration centrifugations of 6 000rpm.
3.2 mixed liquors first 70 DEG C of dry baths 3 minutes before reverse transcriptase MMLV is added, ice-water bath is interior to managing immediately after taking-up
Outer temperature is consistent, then plus reverse transcriptase 0.5 μ l, 37 DEG C of water-baths 60 minutes.
3.3 take out after 95 DEG C of dry baths 3 minutes immediately, obtain reverse transcription end solution as cDNA solution, be stored in -80 DEG C and treat
With.
4 Real time-PCR are tested
4.1 in a certain order, and a sample gene does 2 multiple holes；
4.2 toward 0.1ml PCR pipes, sequentially add following component：
4.3 PCR amplification programs
The sample examination with computer for preparing, according to the different setting differential responses conditions of detection gene.The condition is routine
PCR experiment condition.Using Realtime-PCR technologies, in HepG2, HUH7 cell of the two and three dimensions culture of embodiment 1
Have detected EpCAM, and OCT4mRNA expressions.In the cell of dimensional culture, the expression quantity of EpCAM and OCT4 substantially rises
It is high.EPCAM mRNA expressions increase by 24 times in HepG2 cells, and the mRNA expressions of OCT4 increase by 74 times.In HUH7
In cell, the mRNA expressions of OCT4 increase by 24 times (Fig. 2).
The IHC technology for detection of experimental example 3
1. it is placed in 70 DEG C of baking ovens after cell climbing sheet fixation sample or dimensional culture section sample being carried out into paraffin section, is dried
Piece 2 hours, crosses graded ethanol dewaxing, is flushed three times with PBS, each 3min.
2. a certain amount of pH=6.0 citrate buffers are taken, is added in microwave box, heating using microwave is extremely seethed with excitement, by the water that dewaxes
Histotomy after change is placed in high-temperature resistance plastice slide holding frame, is put into the buffer solution for having seethed with excitement, middle-grade microwave treatment 10 minutes,
Take out microwave box flowing water it is naturally cool but, slide is taken out from buffer solution, with distilled water flushing twice first, afterwards with PBS rinse 2 ×
3min。
3. every section Jia 1 drip 3%H2O2, 10min is incubated at room temperature, to block the activity of endogenous peroxydase.
PBS rinses 3 × 3min.
4. every section is added dropwise 1 drop SABC unspecific staining blocking agent, and 10min is incubated at room temperature.
5. PBS liquid removed, and every section Jia 1 and drip corresponding first antibody that (extension rate is 1：500), 4 degree of overnight incubations.
6.PBS rinses 3 × 5min.Remove PBS liquid, every section Jia 1 drip polymer intensifier, 20min is incubated at room temperature.
PBS rinses 3 × 3min.
7. PBS liquid removed, and every section Jia 1 and drips enzyme mark anti-mouse/rabbit polymer, and 30min is incubated at room temperature.PBS flushings 3 ×
5min。
8. remove PBS liquid, every section Jia 1 drip Fresh DAB liquid (diaminobenzidine), basis of microscopic observation
10min。
9. haematoxylin is redyed, and 0.1%HCl differentiation, running water is rinsed, and oil blackeite, section is dried through gradient alcohol dehydration, diformazan
Benzene is transparent, neutral gum sealing, is observed after drying.
Using IHC technologies, be have detected in HepG2 the and HUH7 cells of the two and three dimensions culture of embodiment 1 EpCAM and
CD44 expressions.In the cell of dimensional culture, the expression quantity of EpCAM and CD44 is significantly raised (Fig. 3).It is thin that two dimension is cultivated
In born of the same parents, EpCAM and CD44 expression quantity is extremely low, it is virtually impossible to observed in stained slice, in the cell of dimensional culture, purpose dye
Color molecule positive rate is significantly raised.
The Flow cytometry of experimental example 4
1. HepG2 and PLC/PRF/5 cells are carried out two dimension and dimensional culture respectively, in culture bar same as Example 1
Same time is cultivated under part.
2. collected by trypsinisation cell, and clean remaining cell once with 1mL PBSs, all add 15ML pipes
In.For the cell of dimensional culture, porous methylcellulose rack mechanical is separated after pancreatin digestion so that cell mass be scattered into
In digestive juice, and support fragment is filtered out, obtain dimensional culture cell.
3. 1200rpm centrifugations 5min, removes supernatant, plus 5mL PBS re-suspended cells, and supernatant is abandoned in centrifugation again, weight
Again twice, last re-suspended cell is transferred in 1.5mL centrifuge tubes in 0.1mL PBS.
4. according to 1:500 add 1UL primary antibodies, are placed on vertical mixed liquor and are incubated at room temperature 1 hour.
5. 1200rpm, compact centrifuge centrifugation 5min, removes supernatant, plus 1mL PBS re-suspended cells, again from
The heart abandons supernatant, is repeated 3 times, and finally washed cell is resuspended in 0.1mL PBS.
6. by fluorescence secondary antibody according to 1:800 ratios are added in cell suspension, are incubated at room temperature 1 hour.
7. 1200rpm centrifugations 5min, removes supernatant, plus 1mL PBS re-suspended cells, and supernatant is abandoned in centrifugation again, weight
Multiple 3 times, last re-suspended cell is in 0.2mL PBS.
8. lucifuge is encased with masking foil, by cell addition streaming pipe, analyzed on flow cytometer.
Using flow cytometry, in HepG2 the and PLC/PRF/5 cells of two and three dimensions culture prepared by embodiment 1
Have detected the expression of CD44 and CD133.Collection cell concentration is 10,000 cells.Two dimension culture and the cell sample of dimensional culture
Originally the negative control of single dye secondary antibody is provided with, and single group for contaminating CD44 and single dye CD133, in unified experiment condition and inspection
On the premise of surveying parameter, after excluding false positive results, each group of visible dimensional culture is thin from the percent positive of flow cytometer detection
In born of the same parents, CD44 and CD133 expression quantity is significantly raised (Fig. 4~Fig. 5).
Experimental example 5
HepG2 the and HUH7 cells of the two and three dimensions culture of embodiment 1 are observed under the conditions of light field using microscope.By
Fig. 6 is visible, and the cell attachment of two dimension culture in culture dish bottom, unfold, and the cell of dimensional culture is then suspended in culture by form
Agglomerating growth in liquid, shows as multiple cell aggregations together, as microcell ball.
Experimental example 6
The liver-cancer stem cell of the PLC/PRF/5 cells of dimensional culture and HepG2 cells and two dimension are cultivated in 1.96 orifice plates
HCC PLC/PRF/5 and HepG2 cell are cultivated 72 hours after culture adds 5-FU or cis-platinum on the 4th day；Control
(control) be the dimensional culture for being added without corresponding antineoplastic liver-cancer stem cell and two dimension culture HCC；
2. above-mentioned two dimension and dimensional culture cell culture the 7th day, MTT solution (5mg/ml PBS are added per hole<PH=7.4>
With) 20 μ l；
2. continue to terminate culture after being incubated 4 hours, careful suction abandons culture supernatant in hole.Add 150 μ l DMSO per hole, shake
Swing 10 minutes, crystal is fully melted；
3. colorimetric：Selection 490nm wavelength, determines each hole absorbance value on enzyme linked immunological monitor, records result；
4., according to detection gained absorbance, cell viability (cell viability) under the conditions of different disposal is calculated.
The liver-cancer stem cell resistance experiment of the HCC and dimensional culture that carry out two-dimentional culture with this, can by Fig. 7 and Fig. 8
Know, the drug resistance of the liver-cancer stem cell of the dimensional culture is significantly larger than two-dimentional cultured cells.
Shown by result above experimental example 1~6, it is dry thin HCC can be converted into liver cancer using dimensional culture technology
Born of the same parents.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method that induction HCC is converted to liver-cancer stem cell, it is characterised in that comprise the following steps：
1）The human liver cancer cell of unicellular will be obtained, added in culture medium, be made cell suspension；
2）Cell suspension is added drop-wise on porous cellulose support, treats that cell suspension penetrates into porous cellulose internal stent, put
Cultivated in cell culture incubator and add culture medium after attaching, then be placed in a few days is cultivated in cell culture incubator, obtain dimensional culture
Cell；
3）The liver-cancer stem cell mark in dimensional culture cell is judged using Western Blot, RT-PCR, flow cytometry and IHC
The change of will molecule EpCAM, OCT4, CD44 and CD133 expression and as microscope observational technique detect conversion obtained by
Liver-cancer stem cell.
2. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
Porous cellulose support is stated with cellulose as main material, or to be aided with other biocompatibilities based on cellulose preferably various
Biomaterial.
3. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
It is the porous sponge-like vector of intercommunication to state porous cellulose support, and internal aperture is 50 ~ 500 μm.
4. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
Porous cellulose stiffness of support is stated for 5 ~ 100kPa.
5. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
HCC is stated for HepG2, HUH7, PLC/PRF/5 or other type HCCs.
6. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
Corresponding culture medium used or the tumor stem cell culture medium without serum when stating culture medium for SMMC-7721 culture.
7. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
Cultivation temperature is stated for 35-39 DEG C.
8. the method that a kind of induction HCC according to claim 1 is converted to liver-cancer stem cell, it is characterised in that institute
Condition of culture is stated for oxygen content 3%-21%, carbon dioxide content 5%.
9. the liver-cancer stem cell that the method conversion described in any one of claim 1 ~ 8 is obtained.
10. in-vitro screening or tumour cell In vitro chemo-drug sensitive test of the liver-cancer stem cell described in claim 9 in antineoplastic
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710159623.7A CN106676074B (en) | 2017-03-17 | 2017-03-17 | Method for in-vitro amplification of liver cancer stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710159623.7A CN106676074B (en) | 2017-03-17 | 2017-03-17 | Method for in-vitro amplification of liver cancer stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106676074A true CN106676074A (en) | 2017-05-17 |
| CN106676074B CN106676074B (en) | 2020-09-25 |
Family
ID=58828994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710159623.7A Expired - Fee Related CN106676074B (en) | 2017-03-17 | 2017-03-17 | Method for in-vitro amplification of liver cancer stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106676074B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977494A (en) * | 2017-06-03 | 2018-12-11 | 加乐生医股份有限公司 | A method of prediction curative effect of medication |
| CN110511867A (en) * | 2019-07-04 | 2019-11-29 | 福建医科大学 | A kind of fusion sediment type 3D printing chip for cell microsphere preparation |
| CN110862954A (en) * | 2018-08-28 | 2020-03-06 | 广州洁特生物过滤股份有限公司 | Cell culture method based on three-dimensional cell culture support |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571792A (en) * | 2012-07-25 | 2014-02-12 | 中国科学院大连化学物理研究所 | Method for in-vitro amplification of tumor stem cells |
| CN103756903A (en) * | 2014-01-10 | 2014-04-30 | 赵海涛 | Three-dimensional culture system for screening liver-caner chemotherapeutic drugs |
| CN106399228A (en) * | 2016-09-26 | 2017-02-15 | 南开大学 | Cell cross-linking agent material for three-dimensional hepatocyte sphere culture |
-
2017
- 2017-03-17 CN CN201710159623.7A patent/CN106676074B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571792A (en) * | 2012-07-25 | 2014-02-12 | 中国科学院大连化学物理研究所 | Method for in-vitro amplification of tumor stem cells |
| CN103756903A (en) * | 2014-01-10 | 2014-04-30 | 赵海涛 | Three-dimensional culture system for screening liver-caner chemotherapeutic drugs |
| CN106399228A (en) * | 2016-09-26 | 2017-02-15 | 南开大学 | Cell cross-linking agent material for three-dimensional hepatocyte sphere culture |
Non-Patent Citations (1)
| Title |
|---|
| MADHUSHREE BHATTACHARYA等: "Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture", 《JOURNAL OF CONTROLLED RELEASE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108977494A (en) * | 2017-06-03 | 2018-12-11 | 加乐生医股份有限公司 | A method of prediction curative effect of medication |
| CN110862954A (en) * | 2018-08-28 | 2020-03-06 | 广州洁特生物过滤股份有限公司 | Cell culture method based on three-dimensional cell culture support |
| CN110511867A (en) * | 2019-07-04 | 2019-11-29 | 福建医科大学 | A kind of fusion sediment type 3D printing chip for cell microsphere preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106676074B (en) | 2020-09-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106811411B (en) | A kind of method for building up of the human heart model based on micro-fluidic chip | |
| CN107012116A (en) | A kind of construction method of small intestine 3D organoids research BCRP mediate drug transshipment models and application | |
| JP5337169B2 (en) | Animal hepatocyte culture method | |
| Sun et al. | Maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation | |
| CN112553142B (en) | 3D organ of nasal mucosa epithelial cells and culture method and application thereof | |
| CN106676074A (en) | Method for inducing liver cell cells to be transformed into liver cancer stem cells | |
| CN102250840B (en) | Human pancreatic cancer cell line and its application | |
| CN106011072A (en) | Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction | |
| CN203411544U (en) | Aorta three-dimensional culture single cell separator | |
| CN108070561A (en) | A kind of isolation and culture method of the primary colon cancer cell of people | |
| CN115029243A (en) | Preparation method and preparation device of intestinal tract-blood vessel-immune co-culture organ chip | |
| CN105624037A (en) | Method for establishing in-vitro blood-brain barrier model based on micro-fluidic chip | |
| Li et al. | TiO2 nanopillar arrays coated with gelatin film for efficient capture and undamaged release of circulating tumor cells | |
| CN106367393A (en) | Mouse prostate cancer circulating tumor cell line and prostate cancer circulating tumor cell isolating and culturing method | |
| CN102559579A (en) | Novel multi-cell three-dimensional co-culture system for in-vitro detection of newly born blood vessel and kit thereof | |
| Takezawa et al. | A protein-permeable scaffold of a collagen vitrigel membrane useful for reconstructing crosstalk models between two different cell types | |
| CN108070560A (en) | A kind of isolation and culture method of the primary stomach cancer cell of people | |
| CN111596058A (en) | Kit and method for detecting FGFR gene mutation of peripheral blood circulation tumor cells of cholangiocarcinoma patient | |
| CN107541497A (en) | Pituitary adenoma cell strain and application thereof | |
| Zhou et al. | Degradable porous nanoflower substrate-embedded microfluidic device for capture, release and in situ manipulation of cancer cells | |
| TWI662130B (en) | Method for circulating tumor cells isolation | |
| CN116445284A (en) | Tumor cell-vascular endothelial cell in-vitro co-culture device and preparation method and application thereof | |
| Pozzobon et al. | Alveolar rhabdomyosarcoma decellularization | |
| CN105483087A (en) | Human bronchial epithelial cell strain HBE-TT | |
| CN109097332A (en) | A kind of inoculating cell fixing means based on chicken embryo tumor formation model |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200925 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |