CN112094890A - Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo - Google Patents
Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo Download PDFInfo
- Publication number
- CN112094890A CN112094890A CN201910524004.2A CN201910524004A CN112094890A CN 112094890 A CN112094890 A CN 112094890A CN 201910524004 A CN201910524004 A CN 201910524004A CN 112094890 A CN112094890 A CN 112094890A
- Authority
- CN
- China
- Prior art keywords
- tumor cells
- injection
- embryos
- embryo
- zebra fish
- 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.)
- Pending
Links
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 75
- 241000252212 Danio rerio Species 0.000 title claims abstract description 68
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000035755 proliferation Effects 0.000 title claims abstract description 26
- 210000002257 embryonic structure Anatomy 0.000 claims abstract description 74
- 210000004027 cell Anatomy 0.000 claims abstract description 73
- 238000002347 injection Methods 0.000 claims abstract description 46
- 239000007924 injection Substances 0.000 claims abstract description 46
- 238000003753 real-time PCR Methods 0.000 claims abstract description 23
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 230000004720 fertilization Effects 0.000 claims abstract description 8
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 8
- 238000002054 transplantation Methods 0.000 claims abstract description 7
- 238000011158 quantitative evaluation Methods 0.000 claims abstract description 6
- 238000010367 cloning Methods 0.000 claims abstract description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 22
- 238000011161 development Methods 0.000 claims description 7
- 230000018109 developmental process Effects 0.000 claims description 7
- 230000002062 proliferating effect Effects 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 230000006378 damage Effects 0.000 claims description 6
- 238000000520 microinjection Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 206010058314 Dysplasia Diseases 0.000 claims description 2
- 230000013020 embryo development Effects 0.000 claims description 2
- 230000036244 malformation Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 2
- 101150112014 Gapdh gene Proteins 0.000 claims 1
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000003814 drug Substances 0.000 description 15
- 229940079593 drug Drugs 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 201000011510 cancer Diseases 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 8
- 238000011282 treatment Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 206010064571 Gene mutation Diseases 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229940072040 tricaine Drugs 0.000 description 3
- FQZJYWMRQDKBQN-UHFFFAOYSA-N tricaine methanesulfonate Chemical compound CS([O-])(=O)=O.CCOC(=O)C1=CC=CC([NH3+])=C1 FQZJYWMRQDKBQN-UHFFFAOYSA-N 0.000 description 3
- 210000003771 C cell Anatomy 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 241000212749 Zesius chrysomallus Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 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 1
- 241000997865 Histrio histrio Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004836 empirical method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000000270 postfertilization Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to a method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryos, which is characterized in that the tumor cells are subjected to fluorescence labeling before injection, the zebra fish embryos carrying the fluorescent tumor cells are selected for further culture 1 day after injection, and the human tumor cell genes are amplified 3 to 7 days after injection by a quantitative PCR method to evaluate the cloning and proliferation conditions of the human tumor cells in the zebra fish embryos. The injection period of the tumor cells which are subjected to the pre-fluorescence labeling is 48 hours after fertilization, and the injection part is the perivitelline space region. The injection volume of each embryo is 10nL, and the number of injected tumor cells is 300-500. In quantitative evaluation, several embryos are selected as a group, and the human gene content is detected by quantitative PCR. Day 3 to 7 after injection is an effective time window to assess whether human tumor cells proliferate within the embryo. The invention is beneficial to the practical application of the zebra fish PDX (patient-derived cell transplantation) model.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryos, which is characterized in that the tumor cells are subjected to fluorescence labeling before injection, the zebra fish embryos carrying the fluorescent tumor cells are selected for further culture 1 day after injection, and the human tumor cell genes are amplified by a quantitative PCR method at a window period of 3 to 7 days (equivalent to 5 to 9 days after fertilization) after injection to evaluate the cloning and proliferation conditions of the human tumor cells in the zebra fish embryos.
Technical Field
It is generally accepted that tumorigenesis is due to the cumulative effects of multiple gene mutations. Because of the diversity of gene mutations in tumor patients, existing tumor-targeted drugs can only exert curative effects on cancer patients carrying certain types of specific gene mutations. Therefore, aiming at different tumor patients, targeted clinical drugs are found in the existing tumor drug library in time, and a personalized treatment scheme for the cancer patients is established, thereby being beneficial to realizing accurate tumor treatment.
After a patient is diagnosed with a malignant tumor, cancer cells isolated from the patient are transplanted into an embryo of an early-stage development of a model animal such as a mouse or zebrafish, and it is possible that the cancer cells continue to proliferate maliciously in the early-stage embryo of the model animal. This model established by xenografting tumor cells from patients into animal embryos is called the pdx (patient derived xenograde) model. If tumor drugs are treated with the PDX model established in the method, which targeted drugs can effectively kill or inhibit cancer cells of the patient can be distinguished, so that a personalized treatment scheme for the cancer patient can be established.
How to evaluate whether the targeted tumor drug can effectively inhibit the tumor cell proliferation is the key point for establishing the personalized treatment scheme of the cancer patient by utilizing the PDX model. The most intuitive evaluation method is that the tumor cells are marked by fluorescence, and if the fluorescence brightness of the PDX model embryo is enhanced in the development process after the tumor cell transplantation, the malignant proliferation of the tumor cells can be judged; in this case, if the target drug for treating tumor is added, the fluorescence intensity is not increased or even disappears, and then the target drug can be judged to have the effect of inhibiting or killing tumor cells, so that the method can be applied to personalized treatment.
Fluorescence labeling is achieved by two methods. One is to stably recombine a fluorescent reporter gene in tumor cells by using a virus. When the reporter gene is stably recombined into the tumor cell, the fluorescent reporter gene is also increased by a certain factor due to the replication of DNA with the malignant proliferation of the tumor cell. However, this method is suitable for technical studies, but obviously not suitable for clinical application. Because the appropriate treatment regimen must be selected in time once a patient has been diagnosed with a malignant tumor, the window of time for targeting effectiveness of a given test drug in the clinic is limited, and such limited window of time is clearly insufficient to support stable transduction of a virus carrying an expressible fluorescent reporter gene in tumor cells isolated from the patient.
Another common strategy for labeling tumor cells is to directly stain the tumor cells with a fluorescent dye. However, this method also has the obvious disadvantage that the fluorescent-labeled dye does not increase with the proliferation of the cells, and furthermore the fluorescent dye remains on the non-proliferating cell debris. Therefore, with this labeling method, direct observation of tumor cell proliferation cannot be achieved.
In addition, the damage of the allogeneic cell transplantation to the animal embryo cells is large, and in practice, a considerable number of embryos die during the subsequent development of the embryos due to mechanical damage at the time of cell transplantation, and thus cannot be used as an object of evaluation by themselves.
In fact, the most accurate indicator of cell proliferation is the replication of the genomic DNA of the patient's cells. Therefore, whether the tumor cells colonize and proliferate in the model animal embryo such as zebrafish can be evaluated by detecting the change of the amount of the genomic DNA of the patient in the PDX model.
In order to solve the technical problem of how to evaluate whether the tumor cells are colonized and proliferated after being transplanted into the embryos of model animals such as zebra fish and the like, the invention provides a method for quantitatively evaluating the proliferation of the tumor cells after being transplanted into the embryos of the zebra fish. The method is characterized in that tumor cells are subjected to fluorescent labeling by using fluorescent dye, the labeled tumor cells are counted and injected into zebra fish embryos by microinjection according to a certain cell number, after the injected embryos develop, the zebra fish embryos which develop relatively normally and carry the fluorescent labels are selected under a fluorescent microscope as PDX model embryos, partial embryos are collected successively in different development stages, genome DNA templates are prepared rapidly, quantitative PCR amplification is carried out on tumor cell genes, and finally whether the allograft tumor cells effectively proliferate in the zebra fish embryos is evaluated according to the quantitative PCR result.
As a screening strategy of personalized drugs, another half of PDX embryos are taken for targeted tumor drug treatment, and finally, whether the candidate tumor drug can inhibit or kill the growth and/or proliferation of tumor cells in the animal embryo in a targeted manner is evaluated according to a quantitative PCR result by adopting an evaluation method similar to that for untreated drug embryos.
Reference to the literature
Hidalgo M，et al.(2014)Patient-derived xenograft models：An emerging platform for translational cancer research.Cancer Discov 4：998-1013.
Rita Fiora，et al.(2017)Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.Proc Natl Acad Sci U S A.114(39)：E8234-E8243.
Zhao C，et al.(2011)A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors.PLoS One 6：e21768.
Amatruda JF，et al.(2002)Zebrafish as a cancer model system.Cancer Cell 1：229-231.
Welker AM，et al.(2016)Correction：Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specifc characteristics and tumor cell heterogeneity.Dis Model Mech.9(9)：1063-5.
Disclosure of Invention
Tumor cells from a patient are transplanted into an animal embryo to manufacture a PDX model, and then tumor targeted drugs are added into the PDX model to observe whether the targeted drugs can inhibit malignant proliferation of the tumor cells, so that the targeted drugs capable of inhibiting or killing the tumor cells of the patient in a targeted manner are screened, and accurate medical treatment of the cancer patient is realized. This idea has been reported for a long time and was first tried on mice. However, since the immune system of mice has been effective very early, the PDX model of mice needs to be created based on knockout of the immune genes of mice. Zebrafish immunity to heterologous cells has not been established early in embryonic development, and much research has focused on establishing PDX models using zebrafish embryos. Although many reports prove that various tumor cells can be successfully colonized and proliferated in the zebra fish embryo, due to the large technical difficulty and poor repeatability, a technical method for quickly and accurately quantitatively evaluating whether the tumor cells can be colonized and proliferated in the zebra fish embryo has not been successful. The invention discloses a method for quantitatively evaluating the proliferation of tumor cells transplanted into zebra fish embryos, which is characterized in that the tumor cells are subjected to fluorescent labeling before injection, the zebra fish embryos carrying the fluorescent tumor cells are selected for further culture 1 day after injection, and the cloning and proliferation conditions of human tumor cells in the zebra fish embryos are evaluated by amplifying human tumor cell genes through a quantitative PCR method 3 to 7 days (equivalent to 5 to 9 days of embryos after fertilization) after injection.
The most basic premise for successful establishment of the PDX model is that the tumor cells transplanted by the cells can successfully colonize in the embryo of the zebra fish. To achieve this goal, we fluorescently label tumor cells. After the marked fluorescent cells are injected into the zebra fish embryos in a microinjection mode, after the injected embryos develop for 24 hours, the injected embryos are screened under a fluorescence destruction mirror, and only the injected embryos which are relatively normal in development and have fluorescence are selected to be continuously cultured for further analysis. The screening is the key for the subsequent quantitative evaluation of the proliferation of the tumor cells in the PDX model. Without this screening, embryos that may cause unsuccessful injection, embryos with injected cells that have died in the host embryo, or embryos with severe dysplasia (malformation) due to injection damage, etc. are counted in the base number for evaluating whether tumor cells are normally proliferating, thereby affecting the accuracy of the subsequent evaluation of whether tumor cells can proliferate in zebrafish embryos. Therefore, in order to quantitatively evaluate whether the tumor cells can be colonized and proliferated in the zebra fish embryos or not, the invention discloses that the tumor cells are firstly subjected to fluorescent labeling, then injected and screened after injection so as to select the zebra fish embryos which are successfully injected as subsequent further experiments.
Injected tumor cells may have died for a number of reasons, one of the main reasons being the number of cells injected, too much or too little will severely affect the colonization of cells within the embryo. In the present invention, the number of injected cells is 300 to 500, which is a preferable number. Second, the injection volume is too large, which can cause mechanical damage to the embryo, resulting in the death of the embryo. In the present invention, we disclose that the preferred injection volume is 10nl, corresponding to a cell density of 3-5X 104/μl。
After 24 hours (24hpi) of tumor cell development after injection into zebrafish embryos, only the surviving tumor cells can be used as the base number for further quantitative evaluation on whether the zebrafish embryos proliferate or not. This time point of 24hpi was chosen because human tumor cells can divide almost once at the embryo-injected feeding temperature (34 ℃), and non-viable cells are expected to die and lyse after 24 hours. Therefore, randomly preserving a portion of 24hpi embryos and confirming the number of human tumor cells in the 24hpi embryos by quantitative PCR as the basis for future proliferation of tumor cells is one of the technical features disclosed in the present invention. On the basis, other embryos screened and retained in the same batch are allowed to continue to develop, partial embryos are randomly selected at different development time points of 48hpi (or 2dpi after injection), 3dpi, 4dpi, 5dpi, 6dpi, 7dpi, 8dpi, 9dpi, 10dpi and the like, and then the number of human tumor cells in the embryos is confirmed by quantitative PCR.
To achieve quantitative evaluation of human tumor cells in the PDX model, we selected a plurality of embryos that fluoresce after injection, one group, preferably 2 groups, for 3 groups, and added ultramicro genomic DNA template preparation reagents (one-step method reagents, tokyo yao shunity biotechnology limited) into each group, rapidly prepared genomic DNA templates according to the specification requirements, and then performed quantitative PCR to detect the content of human genes. The invention also discloses a feature of randomly selecting a few embryos (containing only trace amount of human cell genome DNA) to directly carry out quantitative PCR amplification.
By quantitative PCR evaluation, the present invention also discloses that zebrafish embryos 3 to 7 days after injection (corresponding to zebrafish fries 5 to 9 days after fertilization) are an effective time window to evaluate whether human tumors are proliferating in the PDX model.
Detailed Description
Example 1: quantitative evaluation of colonization and proliferation conditions of human colon cancer cell HCT116 and human non-small cell lung cancer cell A549 after transplantation to zebra fish embryo
Materials and methods (Experimental methods in the examples, unless otherwise specified, are all conventional methods)
Cell line: human colon cancer cell HCT116, human non-small cell lung cancer cell A549
HCT116 cells were formulated with complete medium: a bag of DMEM powder, high glucose (Gibco), 3.7g NaHCO was weighed in addition3(Sigma), add ultrapure water (18.2 megohm) to a constant volume of 1L, filter and then autoclave for 30min at 121 ℃ and filter in a clean bench. To 500mL of the filtered liquid, 50mL of fetal bovine serum (PAN) and 5mL of penicillin-streptomycin (10,000U/mL) were added, mixed, dispensed into 50mL centrifuge tubes, sealed with a sealing membrane, and stored in a refrigerator at 4 ℃ for further use.
Culture medium for a549 cells: ham's F-12K (Kaighn's) Medium (Gibco).
1×PBS(NaCl 8.0g，KH2PO4 0.2g，Na2HPO42.9g, KCl 0.2g, and ultrapure water to constant volume of 1L, adjusting pH to 7.4),
0.25% pancreatin (Sigma)
Cfse (invitrogen): the stock concentration was 10mM (prepared by dissolving dry powder in 90. mu.L of anhydrous DMSO).
Preparing an injection embryo genome DNA template: ultramicro genomic DNA template preparation kit (Nanjing Yao Shunyuan Biotechnology Co., Ltd.).
Tricaine (Sigma, 400mg of Tricaine powder was weighed out and added to about 97mL of ultrapure water, adjusted to pH 7.0 with 1M Tris (pH 9.0)
A microinjection instrument: product of Freon japonica company
Egg Water: (0.24g NaCl, 400. mu.L methylene blue dissolved in 1L deionized water),
the water-proof incubator is constant in temperature of 34 ℃.
Experimental and empirical method
1. Cell recovery frozen cells HCT116 and A549 were removed from a-80 ℃ freezer one tube each and quickly placed in a 37 ℃ water bath and shaken vigorously. The thawed cells were placed in a 15mL sterile centrifuge tube, centrifuged at 1,000rcf for 3min, the supernatant aspirated, 1mL fresh complete medium added, resuspended thoroughly, then transferred to a 10cm diameter sterile cell culture dish containing 9mL, cells were shaken according to the "Mi" method, and the dish was placed in a 37 ℃ cell culture incubator (5% CO2) for culture. After 12 hours the waste was aspirated and the culture was continued with 10mL fresh complete medium.
2. After the cells are passaged again for 24h, the cells are microscopically observed to grow over about 80% of the area of the bottom of the culture dish. The old culture was first aspirated off with a vacuum suction pump, the cells were washed twice with 1mL of 1 XPBS, then aspirated off with a vacuum suction pump, 1mL of 0.25% pancreatin (Sigma) was added, the bottom was covered, the mixture was digested in an incubator at 37 ℃ for 1min, and after digestion, 2mL of complete medium was added to stop digestion. The above liquid was transferred to a 15mL centrifuge tube, centrifuged at 1,000rcf for 3min, and the supernatant was discarded. Add 2mL of complete medium to the centrifuge tube and blow 5-8 times. 2 new dishes were taken, 9mL of complete medium was added to each dish, and 1mL of cell suspension was added to each dish. Mixing, and culturing the two dishes of cells in a 37 ℃ cell culture box (5% CO 2).
3. CFSE staining of cells were observed to have grown over about 90% of the area of the bottom of the dish. First, old culture medium was aspirated by a vacuum suction pump, cells were washed twice with 1mL of 1 XPBS, then aspirated by a vacuum suction pump, 300. mu.L of 0.25% pancreatin (Sigma) was added, the bottom was covered, the mixture was digested in an incubator at 37 ℃ for 1min, and after digestion, 600. mu.L of complete medium was added to terminate digestion. The above liquid was transferred to a 15mL sterile centrifuge tube, centrifuged at 1,000rcf for 3min, and the supernatant discarded. The cells were then washed three times with 1mL of 1 XPBS and then aspirated by a vacuum suction pump. 2mL of 1 XPBS was added to the tube to resuspend the cells and the cell density was controlled at 5-10X 106and/mL. CFSE stock (10mM) was first diluted to 100. mu.M in 1. mu.L of 99. mu.L DMSO and then mixed as 1: 100 to the suspension 20. mu.L of diluted CFSE was added. Incubate in dark (wrapped in aluminum foil) for 10min at room temperature. Next, 4-fold precooled complete medium was added to the mixture, and a total of 8mL was used to stop the reaction and incubated on ice for 5 min. After completion, the cells were centrifuged at 1,000rcf for 3min and the supernatant was discarded. The cells were washed three more times with 2mL of complete medium.
4. Cell counting labeled HCT116 cells were resuspended in 2mL of 1 × PBS, 10 μ L of the suspension was taken and diluted 10-fold with 1 × PBS 90 μ L, after mixing, 10 μ L of the diluted suspension was aspirated into two counting zones of a hemocytometer and counted under a microscope: counting zone 1: (66+53+73+ 53)/4X 104＝6.125×106Per mL; counting zone 2: (50+40+52+ 51)/4X 104＝4.825×106and/mL. Taking the average value, the average cell density of the cell suspension is 5.47X 106/mL。
Another plate of A549 in a 10cm dish was resuspended in 1mL of 1 XPBS, 10. mu.L of the resuspended solution was diluted 3-fold by adding 1 XPBS 20. mu.L of the resuspended solution, and then cell counting was performed: counting zone 1: (79+67+72+ 49)/4X 3X 104＝2.003×106Per mL; counting zone 2: (39+52+58+ 57)/4X 3X 104＝1.545×106and/mL. The average cell density was about 1.774X 106/mL。
5. Cell injection transgenic zebrafish line Tg (gata 1: dsRed) zygotes were collected as usual, and after the embryos had grown to 48hpf (hours post fertilization), they were anesthetized with tricaine and placed on gel plates made of 2.5% agarose. Adjusting the cells to be injected to 3-5 × 10 with 1 × PBS4Density of/. mu.L. In addition, microinjection capillary glass needles were prepared and microinjected under a dissecting scope into the perivitelline space (PVS) region of 48hpf zebrafish embryos. The injection volume is about 10 mu L, and the number of injected cells is about 300-500 per zebra fish embryo.
6. Preparation of genomic DNA template for quantitative analysis of human tumor cell colonization and proliferation in zebra fish embryo the injected embryo was cultured in an incubator at 34 ℃. After the injected embryos are developed for 24 hours or 1 day (d) (1dpi), an Olympus type fluorescence microscope is used for observing the condition that the injected embryos carry green fluorescence, the embryos which obviously carry the green fluorescence are selected (figure 1, the colonization condition observation after the fluorescence-labeled HCT116 cells are injected into the zebra fish embryos, the fluorescence existence condition at different time after the green fluorescence-labeled cells are injected into the embryos can be seen after the injection for 5 days, but the fluorescence is obviously weakened, the red fluorescence indicates red blood cells), then 3 groups of embryos (from 1dpi) are randomly collected at each time according to the specified time, two embryos in each group are placed together in a 0.2ml PCR tube, the water solution is absorbed as much as possible, and then an ultramicro genome DNA template preparation reagent (one-step reagent, Yashunyu bioscience, Inc. of Nanjing) is added into the PCR tube for 3.24 muL. After rapid centrifugation, the PCR tube was placed in a PCR apparatus, and the following procedure was performed at 65 ℃ for 40min, 95 ℃ for 5min, and 16 ℃ for infinity to prepare a genomic DNA template. The prepared template is stored at-20 ℃ for later use. Note: the zygote formed by fertilization of zebrafish is about 0.7mm in diameter, about 0.35mm in radius, and the volume of single embryoV＝4/3×3.14×(0.035 cm)3＝0.1795mL＝0.18μL。
7. Quantitative PCR detection of the relative number of human cells colonized in zebrafish embryos A quantitative PCR reaction system (10. mu.L: containing 5. mu.L of 2 XSSYBR Green reagent (ABI), 0.3. mu.L of forward primer GAPDH2F (primer sequence: GGTCTCCTCTGACTTCAACAGC; 10. mu.M), 0.3. mu.L of reverse primer GAPDH2R (primer sequence: AGAGTTGTCAGGGCCCTTTTTCT; 10. mu.M), 2. mu.L of genomic DNA template (embryonic genomic DNA template samples from different days after injection), and 2.4. mu.L of ultrapure water were prepared as follows.
After the sample is added, the machine is arranged, and the instrument is an ABI StepOne Plus fluorescent quantitative PCR instrument. The PCR reaction program is: 2min at 50 ℃, 2min at 95 ℃, 40 × (15 s at 95 ℃, 1min at 60 ℃).
8. Results 1 quantitative PCR assay results of colonization and proliferation conditions in zebra fish embryos after transplantation of human tumor cell HCT116 into zebra fish embryos:
| sample number | 1dpi | 2dpi | 3dpi | 4dpi | 8dpi | 10dpi |
| Ct value of group 1 | 26.83948 | 27.38704 | 28.00272 | 29.12993 | 34.98957 | 35.49986 |
| Ct value of group 2 | 27.19987 | 34.42479 | 33.50132 | 30.95564 | 34.21007 | undetermined |
| Ct value of group 3 | 28.14873 | 33.16358 | 28.64276 | 34.92621 | 35.55998 | undetermined |
| Ct mean. + -. standard deviation | 27.40±0.68 | 31.66±3.75 | 30.05±3.01 | 31.67±2.96 | 34.92±0.68 | --- |
From the quantitative PCR detection results of HCT116 cells injected into zebra fish embryos at different times, the Ct value obtained by quantitative PCR detection after injection gradually increases, which indicates that the HCT116 cells cannot effectively survive and colonize after being transplanted into zebra fish bodies. Therefore, the HCT116 tumor cell line is not suitable for constructing a PDX model of zebrafish embryos.
Result 2 quantitative PCR detection result of colonization and proliferation condition in zebra fish embryo after human tumor A549 is transplanted into zebra fish embryo:
| sample number | 3dpi | 7dpi | 9dpi |
| Ct value of group 1 | 33.89175 | 29.44403 | 33.89489 |
| Ct value of group 2 | 31.91060 | 29.49757 | 32.89433 |
| Ct value of group 3 | 34.16014 | 29.63000 | 31.51370 |
| Ct mean. + -. standard deviation | 33.31±1.23 | 29.52±0.10 | 32.77±1.20 |
According to the quantitative PCR detection results of A549 injected into the zebra fish embryo in different times, the non-small cell lung cancer cell A549 can effectively survive 3 to 7 days after being transplanted into the zebra fish body, and the Ct of 7dpi is reduced by 3.39 compared with 3 dpi. Approximately 1-fold proliferation in zebrafish embryos corresponding to every 24h injection of human a549 tumor cells. However, after 7 days, the tumor cells are not proliferated any more, and the Ct value is obviously increased, which indicates that the cells in the zebra fish embryo are reduced. Thus, a549 cells are suitable for use in constructing a PDX model of zebrafish embryos that effectively colonize the contents of the zebrafish embryo with a window period of proliferation of 3 to 7 days post-injection (i.e., 5 to 9 dpf).
9. The immunohistochemistry experiment confirms that the human cells proliferate in the zebra fish embryo to confirm the conclusion that the A549 cells can colonize and proliferate in the zebra fish given by quantitative PCR, and the injected cells are further observed whether to have the proliferation capacity by a KI-67 antibody (specifically recognizing dividing human cells) immunofluorescence method, namely, the zebra fish embryo is detected whether to have the existence of KI-67 positive cells by the immunofluorescence method 3 days after the injection.
Three days after injection of a549 cells, 8 zebra fish fries were harvested into 1.5mL centrifuge tubes, washed three times with 1 × PBS, and 1 × PBS was aspirated. 1mL of 4% PFA was added and fixed at 4 ℃ overnight. The next day 4% PFA was aspirated and 1mL of 30% sucrose was added and allowed to settle overnight at 4 ℃. Then frozen at-20 ℃ for more than 2h with OCT (SAKURA Tissue-Tek) at room temperature. Then, the slices were sliced with a Leica cryomicrotome to a thickness of 8 μm, and the cut slices were left to dry at room temperature for more than 30 min.
Three discs in the middle were selected and washed three times with 1 × PBS. Blocking solutions of 10% goat serum and 2% BSA/1 XPBST were prepared, added to the tissue-containing area and incubated for 1h at room temperature.
Primary anti-KI-67 was added to the blocking solution at 1: 200 and incubated overnight at 4 ℃.
The following day was washed three times with 1 × PBS. Adding secondary antibody Alex568 cy3 (1: 500), and incubating for 1h at room temperature in the dark; then washed three times with 1 × PBS. Next, DAPI (1: 5000) was added to the blocking solution, and incubated for 5min in the dark at room temperature. Wash three times with 1 XPBS. Spin-drying the glass slide, dripping 50% of glycerol and sealing the nail polish.
After drying, a ZEISS LSM880 high-precision laser confocal microscopic imaging system is used for photographing and observing. The results indicated that KI-67 positive cells were present in the yolk sac region of the zebrafish embryo three days after a549 injection (fig. 2, a549 cells proliferating in zebrafish embryo, cells at red spots indicated by arrows are proliferating a549), indicating the ability of non-small cell lung cancer cells to colonize and proliferate in zebrafish bodies.
FIG. 1 is a graph showing the results of colonization of zebrafish embryos injected with green fluorescently labeled HCT116 cells. The green shows the existence of fluorescence of the fluorescence labeled cells at different time (dpi: days after injection) after the injection of the embryos, and the fluorescence can still be seen after the injection for 5 days, but is obviously weakened; red fluorescence indicates red blood cells.
FIG. 2 is a graph showing evidence of proliferation of injected A549 cells in zebrafish embryos. The arrows indicate that the cells are proliferating a549 at red spots.
Claims (6)
1. A method for quantitatively evaluating the proliferation of tumor cells transplanted into the embryo of zebra fish is characterized by carrying out fluorescent labeling on the tumor cells before injection, selecting the zebra fish embryo carrying the fluorescent tumor cells 1 day after injection for further culture, and evaluating the cloning and proliferation conditions of the human tumor cells in the zebra fish embryo by amplifying human tumor cell genes by a quantitative PCR method in a window period of 3 to 7 days after injection (which is equivalent to the embryo 5 to 9 days after fertilization).
2. The feature of claim 1, wherein the fluorescent substance for labeling tumor cells is any fluorescent substance capable of labeling living cells. And fluorescently labeled tumor cellsThe cell density is 3-5 × 104mu.L was used for microinjection of zebrafish embryos.
3. The method of claim 1, wherein the zebrafish embryo used for injection has a development period of 48hpf (48 hours after fertilization) and the injection site is the perivitelline space (PVS) region. The injection volume per embryo is 10nL, and the number of the injected tumor cells is 300-500 per embryo.
4. The feature of claim 1, wherein the embryo selection criteria 24h (or 1 day, 1dpi) after injection is to select only those injected embryos under the fluoroscope that have relatively normal embryo development and fluorescence for further analysis. Unsuccessfully injected embryos (no fluorescence), embryos with injected cells that have died in the host embryo (no fluorescence), or embryos with severe dysplasia (malformation) due to injection damage are not selected.
5. The feature of claim 1, wherein the embryos selected by fluorescence for subsequent analysis are arbitrarily selected within a specified time after injection, optionally several embryos in one group, preferably 2 embryos in one group, preferably 3 embryos in one group, and ultra-micro genomic DNA template preparation reagents are added to each group to rapidly prepare genomic DNA templates, and then quantitative PCR is performed to detect the content of human genes, in order to achieve quantitative evaluation of human tumor cells in PDX (patient-derived cell transplantation) model. Preferably, the GAPDH gene content is detected.
6. The feature of claim 1, zebrafish embryos 3 to 7 days after injection (corresponding to zebrafish fries 5 to 9 days after fertilization) are an effective time window to evaluate whether human tumor cells are proliferating in the PDX model.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910524004.2A CN112094890A (en) | 2019-06-17 | 2019-06-17 | Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910524004.2A CN112094890A (en) | 2019-06-17 | 2019-06-17 | Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112094890A true CN112094890A (en) | 2020-12-18 |
Family
ID=73748595
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910524004.2A Pending CN112094890A (en) | 2019-06-17 | 2019-06-17 | Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112094890A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716650A (en) * | 2021-01-25 | 2021-04-30 | 南开大学 | Robotized zebra fish main vein microinjection method |
| CN113767879A (en) * | 2021-09-08 | 2021-12-10 | 苏州木芮生物科技有限公司 | Cancer cell xenograft zebra fish model, and construction method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105121657A (en) * | 2012-12-02 | 2015-12-02 | 生物医学核心有限公司 | Accelerated prediction of cancer progression and response to treatment |
| CN108338991A (en) * | 2017-01-22 | 2018-07-31 | 南京艾莫瑞生物科技有限公司 | A kind of gastric cancer heterograft zebra fish model, its construction method and the application in patient source |
| US20180288982A1 (en) * | 2012-08-13 | 2018-10-11 | InnoGenomics Technologies, LLC | Method for measuring tumor burden in patient derived xenograft (pdx) mice |
| CN108841869A (en) * | 2018-05-31 | 2018-11-20 | 上海交通大学医学院附属瑞金医院 | construction method and application of zebra fish NK/TC L tumor model |
| CN108967358A (en) * | 2018-05-31 | 2018-12-11 | 上海交通大学医学院附属瑞金医院 | Construction method and application of zebra fish F L tumor model |
| CN109479814A (en) * | 2018-11-14 | 2019-03-19 | 苏州致诺优生物医学有限公司 | The screening model of anti-lung cancer tumour medicine |
| CN109744199A (en) * | 2017-11-08 | 2019-05-14 | 南京艾莫瑞生物科技有限公司 | A kind of tumour cell heterograft zebra fish model, its construction method and application |
-
2019
- 2019-06-17 CN CN201910524004.2A patent/CN112094890A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180288982A1 (en) * | 2012-08-13 | 2018-10-11 | InnoGenomics Technologies, LLC | Method for measuring tumor burden in patient derived xenograft (pdx) mice |
| CN105121657A (en) * | 2012-12-02 | 2015-12-02 | 生物医学核心有限公司 | Accelerated prediction of cancer progression and response to treatment |
| CN108338991A (en) * | 2017-01-22 | 2018-07-31 | 南京艾莫瑞生物科技有限公司 | A kind of gastric cancer heterograft zebra fish model, its construction method and the application in patient source |
| CN109744199A (en) * | 2017-11-08 | 2019-05-14 | 南京艾莫瑞生物科技有限公司 | A kind of tumour cell heterograft zebra fish model, its construction method and application |
| CN108841869A (en) * | 2018-05-31 | 2018-11-20 | 上海交通大学医学院附属瑞金医院 | construction method and application of zebra fish NK/TC L tumor model |
| CN108967358A (en) * | 2018-05-31 | 2018-12-11 | 上海交通大学医学院附属瑞金医院 | Construction method and application of zebra fish F L tumor model |
| CN109479814A (en) * | 2018-11-14 | 2019-03-19 | 苏州致诺优生物医学有限公司 | The screening model of anti-lung cancer tumour medicine |
Non-Patent Citations (2)
| Title |
|---|
| CHENGJIAN ZHAO等: "A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors", 《PLOSONE》, vol. 6, no. 7, pages 21768 * |
| 陈超;李海涛;: "骨髓间充质干细胞移植对胃癌模型大鼠肿瘤细胞的影响", 中国组织工程研究, no. 41, pages 6158 - 6163 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716650A (en) * | 2021-01-25 | 2021-04-30 | 南开大学 | Robotized zebra fish main vein microinjection method |
| CN112716650B (en) * | 2021-01-25 | 2023-03-03 | 南开大学 | Robotized zebra fish main vein microinjection method |
| CN113767879A (en) * | 2021-09-08 | 2021-12-10 | 苏州木芮生物科技有限公司 | Cancer cell xenograft zebra fish model, and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11866767B2 (en) | Simultaneous spatio-temporal measurement of gene expression and cellular activity | |
| AU2015280326B2 (en) | On-slide staining by primer extension | |
| US8921643B2 (en) | Method for acquiring genetically identical gamete from lethal fish haploid-derived germ cell via germ line chimera | |
| CN1671742A (en) | Use of HMGB1 in the treatment of tissue damage and/or to promote tissue repair | |
| CN112094890A (en) | Method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryo | |
| JP4522587B2 (en) | Methods for introducing heterologous cells into fish | |
| Amberg et al. | Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers | |
| CN1879892B (en) | Method for establishing human extranodal rhinoid NK/T cell lymphoma animal model | |
| JP2021099248A (en) | Non-alcoholic steatohepatitis marker and use thereof | |
| WO2017114007A1 (en) | Pml gene and rara gene detection probe, preparation method therefor, and test kit | |
| KR20180024262A (en) | Prediction method for using tissue origin of gliblastoma detecting genitic mutation and animal model for gliblastoma | |
| US20240101965A1 (en) | Ex vivo tumor angiogenesis model | |
| CN102943308A (en) | Echinococcus granulosus developmental-stage secretory protein expression gene chip | |
| US20220040334A1 (en) | High-throughput screening methods of senescence-antagonizing substances and systems thereof | |
| You et al. | Functional single cell selection and annotated profiling of dynamically changing cancer cells | |
| CN113767879A (en) | Cancer cell xenograft zebra fish model, and construction method and application thereof | |
| KR20140049993A (en) | Dsrna/dna hybrid genome replication intermediate of metakaryotic stem cells | |
| CN106755099B (en) | Method for screening medicine for promoting pancreatic islet beta cell function in vivo | |
| Yi et al. | Cytocapsular tube network-tumor system is an integrated physical target for the highly effective and efficient pharmacotherapy of solid cancers | |
| Ribau | Patient-derived xenotransplantation (PDX) of human tumor tissues | |
| JP2003061666A (en) | Technique for multistage differential cloning and cell growth control gene | |
| TWI703215B (en) | A method of in vitro diagnostic for prediction of drug efficacy | |
| CN106138045A (en) | Tumor microenvironment is handled to eliminate method and the preparation of drug resistance of tumor by targeting mTOR | |
| EP1826560B1 (en) | Methods for introducing heterologous cells into fish | |
| KR20150022451A (en) | Probes for discriminating Aphididae living in Compositae, DNA chip and kit for discriminating Aphididae living in Compositae, and method for discriminating Aphididae living in Compositae using the same |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201218 |
|
| WD01 | Invention patent application deemed withdrawn after publication |