CN102936600A - A549 nude mouse model of stably expressed luciferase and building and application thereof - Google Patents
A549 nude mouse model of stably expressed luciferase and building and application thereof Download PDFInfo
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Abstract
The invention relates to an A549 nude mouse model of stably expressed luciferase and building and application thereof. The nude mouse model leads firefly luciferase genes to human lung adenocarcinoma A549 cell strains through the gene recombination technology and lentivirus infection, tumor clone cell strains of the stably expressed luciferase are obtained through subcloning screening, and recombinational A549 cells are inoculated in a mouse to obtain the A549 nude mouse model. Compared with a traditional tumor model drug effect detection method, the A549 nude mouse model enables observation to be visual and convenient, can be used for somatoscopy without damaging animals, is reliable in result, and has good application prospect.
Description
Technical field
The invention belongs to nude mice model and foundation thereof and Application Areas, particularly a kind of A549 nude mice model of stably express luciferase and foundation thereof and application.
Background technology
Lung cancer is the No.1 killer who threatens China's people ' s health." China Health statistical yearbook in 2010 " according to Ministry of Health's issue, China's malignant tumour in 2009 ranks first in all disease death reasons, statistical information shows that in the malignant tumour of mortality ratio rank top ten, lung cancer ranks first, for the social development of China brings serious economical load.In recent years, treatment and the fundamental research of lung cancer have obtained large development, but at present the basis of lung-cancer medicament and preclinical study still be short of in a kind of reliable body that can assess more easily curative effect of medication, the in vitro study platform, this has seriously restricted basis and the preclinical study of lung cancer.The lung cancer animal model is the important means of lung cancer research, and the Lewis lung carcinoma model is that current clinical prodrug is tested the most frequently used model.The transplantation type lung cancer model is that human lung carcinoma cell is transplanted to the growth of animal interior generation, the tumour cell morphological feature of transplanting, karyomit(e) quantity, isozyme level etc. will remain unchanged, also roughly the same to clinical the sensitivity of anti cancer drugs, the common method that the transplantation type lung cancer model is set up subcutaneous (armpit, back, hind leg etc.) that to be skin be inoculated in nude mice or other immunodeficient mouses by lung carcinoma cell or tissue block is set up lung cancer model.This model modeling is simple, tumor formation rate is high, easily monitor the tumor growth situation, thus be commonly used to do the animal model of screening anticancer medicine, simultaneously this model also have that animal-origin is convenient, the implanted tumor cells growth rapidly, the doubling time is short, expend the advantages such as low.But the transplantation type lung cancer model of application needs to take the method for dissecting to be assessed to the assessment of curative effect of medication more at present, and complicated operation, be subject to the influence factor of manual operation large, is not easy to preclinical study.If can set up the visual lung cancer model of a kind of integral body, can directly carry out the growth transfer case of external Imaging: Monitoring assessment tumour, can be observed living animal, by the preclinical study of large convenience lung cancer, also can reduce the interference of human factor, improve the accuracy of assessment simultaneously.
The application of luciferase reporter gene (Luc) facilitates the structure of the whole visual animal model of lung cancer.Luciferase reporter gene mainly is divided into bacterial luciferase (Bacterial luciferase according to the source difference, BL), Photinus pyralis LUC (fireflyluciferase, FL) and the luciferase that is source such as starfish, luminous fish, luminous beetles, research at present the most extensively and that become commercial enzyme is BL and FL.The heterodimer that BL is comprised of 2 polypeptide subunits, relative molecular mass is about 79 * 10
3.At reductibility flavine (FMNH), the eight above long-chain fat aldehyde of carbon (RCHO) and oxygen molecule (O
2) while existing, launch blue green light (450 ~ 490nm).FL is comprised of single polypeptide chain, and relative molecular mass is (60 ~ 64) * 10
3, at Mg
2+, ATP, O
2Catalysis D-fluorescein (D-Luciferin) oxidative decarboxylation luminous (550 ~ 580nm) while existing.The fluorescein-luciferase system of Lampyridea is one of luminous model of setting up the earliest, this luminescent system adopts the luminous form of fluorescein substrate, do not need exciting light, there is higher sensitivity, in addition, light tissue that Photinus pyralis LUC sends absorbs few, is conducive to the deep tissue imaging, and light intensity and labeled cell quantity are linear dependence.Build the carrier for expression of eukaryon of luciferase and be transfected in cancer cells by gene engineering method, make it express Photinus pyralis LUC, and then the cancer cells that utilizes expressing luciferase is seeded to animal and can builds whole visual tumor model, can, by the method for external imaging, animal body be carried out somatoscopy and not damage animal.
Slow virus infection has stronger advantage than other rotaring transfecting modes, have that pattern of infection is wide, efficiency is high, can infect Unseparated Cell, and can make the goal gene stable integration to host genome thereby can long-term expression, the characteristics such as immune response is little, be a kind of efficient transgenosis instrument.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of A549 nude mice model of stably express luciferase and sets up and application, with traditional tumor model effect of drugs detection method, compare, observe intuitive and convenient more, and can carry out somatoscopy and not damage animal, result is more reliable, has a good application prospect.
The A549 nude mice model of a kind of stably express luciferase of the present invention, described tumor model is by adopting gene recombination technology and slow virus infection that firefly luciferase gene is introduced in human lung adenocarcinoma A549 cell strain, screen the tumour clonal cell line that obtains the stably express luciferase by subclone, then adopt the A549 cell of recombinating to be inoculated in the mouse structure and form.
The establishment method of the A549 nude mice model of a kind of stably express luciferase of the present invention comprises:
(1) take the plasmid that carries firefly luciferase gene that builds early stage goes out the cDNA of luciferase as template amplification, insert in slow virus carrier for expression of eukaryon PRRL-CMV plasmid construction recombination plasmid Luc-PRRL-CMV expression plasmid by the method for gene clone:
Design firefly luciferase gene Auele Specific Primer
(this primer is: upstream primer: CCTTCTAGAATGGAAGATGCCAAAAACATTAAG; Downstream primer: CCTGTCGACTTACACGGCGATCTTGCCGCCCTTC);
The clone of luciferase gene and the structure of lentiviral vectors: use this Auele Specific Primer pcr amplification firefly luciferase gene and be cloned in the PCR-Blunt cloning vector and transform competent escherichia coli cell, after plasmid extraction, using gene recombination method insert in slow virus carrier for expression of eukaryon PRRL-CMV empty plasmid by luciferase goal gene fragment and transform competent escherichia coli cell;
Carry out the exactness of the expression vector of double digestion and order-checking confirmation structure after plasmid extraction;
(2) slow virus infection method imports the luciferase foreign gene step of lung cancer cell line:
Intestinal bacteria amplification fluorescent element enzyme lentiviral vectors and slow virus packaging plasmid, and purifying is measured concentration; Will
The packing of luciferase lentiviral vectors: the luciferase Lentiviral, with after the slow virus packaging plasmid mixes, with transfection reagent cotransfection 293T cell, the supernatant that contains Photinus pyralis LUC slow virus particle with generation, is determined the titre of slow virus particle;
Virus infection: take human lung adenocarcinoma A549 as parent cell, infected with above-mentioned viral supernatant;
Amplification is built and is: carry out the screening of cell subclone, detect the luciferase expression situation in monoclonal cell, filter out the recombinant cell strain of stably express luciferase reporter gene and carry out enlarged culturing;
(3) tumour subcutaneous injection method:
The recombinant human lung adenocarcinoma cell obtained is resuspended in to (200 μ l) in sodium chloride solution after digestion, in nude mice by subcutaneous inoculation (2 * 10
6) this recombinant human lung adenocarcinoma cell, monitor propagation situation under cell skin, obtain the A549 cell strain of stably express luciferase.
Gene recombination method in described step (1) is that enzyme is cut and is connected; Wherein, enzyme is cut to be specially and is adopted Xba I and Sal I double digestion.
Luciferase Lentiviral in described step (2) and the mass ratio of slow virus packaging plasmid are 1:3,1:6 or 1:9.
Described slow virus packaging plasmid comprises that third generation slow virus copies needed three gene: gag, pol and rev.
Transfection reagent in described step (2) is the MV40 transfection reagent.
Determine the titre of slow virus particle in described step (2) by the ELISA method; Carry out the screening of cell subclone by the cell limiting dilution assay; Detect the luciferase expression situation in monoclonal cell by fluor tester or viable cell imager.
Sodium chloride solution mass percent concentration in described step (3) is 0.9%.
Monitor propagation situation under cell skin by vernier caliper measurement and living imaging instrument in described step (3).
Nude mice model of the present invention is applied to observe the result for the treatment of of growing state and the antitumor drug of tumour.
Selected animal, except nude mice, can be also the SCID mouse.
Beneficial effect
The present invention compares with traditional tumor model effect of drugs detection method, observes intuitive and convenient more, and can carry out somatoscopy and not damage animal, and result is more reliable, has a good application prospect.
The accompanying drawing explanation
Figure 1A is Photinus pyralis LUC Luciferase lentiviral vectors schematic diagram;
Figure 1B is plasmid enzyme restriction evaluation figure;
The packing that Fig. 2 is the Luciferase lentiviral vectors; Wherein, A be goal gene plasmid and slow virus packaging plasmid by 1:3 transfection 293T cell, B be goal gene plasmid and slow virus packaging plasmid by 1:6 transfection 293T cell, C is that goal gene plasmid and slow virus packaging plasmid are by 1:9 transfection 293T cell;
Fig. 3 infects A549 cell 72h for the slow virus supernatant after packing;
The A549 cell monoclonal screening that Fig. 4 is stably express Luciferase; Wherein, A is that the Luciferase active level is analyzed (RLU), and B is the viable cell imaging;
The A549 clone biological property analysis (scratch test) that Fig. 5 is stably express Luciferase;
Become the knurl experiment in the A549 cell paste that Fig. 6 is stably express Luciferase; Wherein, A is the living animal imaging, and B is living animal imaging quantitative analysis, and C is tumor growth curve;
Become the knurl experiment in the A549 cell paste that Fig. 7 is stably express Luciferase; Wherein, A is rear the 12nd day living imaging for the treatment of, and B is the living imaging quantitative analysis, and C is different number of days tumor growth curve after treating.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
The clone of step 1. luciferase gene and the structure of lentiviral vectors:
After design Photinus pyralis LUC (Firefly-Luciferase) gene-specific primer, take and carry the Luciferase gene plasmid as template (green the skies company), pcr amplification goes out Luciferase cDNA.The Luciferase gene of amplification is successfully inserted to the PCR-Blunt carrier, after the transformed competence colibacillus Bacillus coli cells, plasmid is little takes out, after Xba I and Sal I double digestion, the rubber tapping purifying reclaims the Luciferase gene fragment, this fragment is connected with slow virus carrier for expression of eukaryon PRRL-CMV empty plasmid (Backbone), after the transformed competence colibacillus Bacillus coli cells, plasmid is little takes out, double digestion is identified and sequencing result all proves the success of Luciferase-PRRL-CMV expression vector establishment, through order-checking, confirms sequence entirely true (about 1700bp)
（ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAA）(See Figure 1), and large-scale plasmid extraction, concentration measured after the standby position -80 ℃.
The packing of step 2. luciferase lentiviral vectors and virus titer are measured:
Intestinal bacteria amplification fluorescent element enzyme lentiviral vectors and slow virus packaging plasmid, and purifying is measured concentration.
The luciferase lentiviral vectors prepares with MV40 transfection reagent transfection HEK293T cell.
Luciferase-PRRL-CMV plasmid and slow virus packaging plasmid are pressed respectively to 1:3,1:6,1:9 transfection 293T cell, and result demonstration Luciferase-PRRL-CMV plasmid and slow virus packaging plasmid are pressed the best (see figure 2) of 1:6 transfection 293T cell effect.Collect the 293T cells and supernatant in transfection 48 with after 72 hours ,-80 ℃ of centrifugal removal cell debris postposition are standby.With the GFP-PRRL-CMV plasmid vector as positive control.HIV p24 ELISA method is determined the titre of slow virus particle.
People A549 lung adenocarcinoma cell is gone down to posterity in two anti-DMEM in high glucoses containing 10wt% calf serum and 1wt% cultivation.Fetching is counted the tumour cell in vegetative period, and the 0.25wt% trypsin is containing EDTA) digestion, press 2 * 10 after accurate metering
5Individual cells/well is planted in six orifice plates, when the cytogamy degree reaches 70 ~ 80%, polybrene is to substratum in dilution, making its final concentration is 8 μ g/ml, then infect the A549 lung adenocarcinoma cell with luciferase slow virus supernatant with best titre, do positive control with GFP slow virus supernatant simultaneously, after infection, 48h, 72h and 96h detect GFP expression in cell with inverted fluorescence microscope respectively, and normally going down to posterity, it is rear by luciferase expression situation in fluor tester and viable cell imager detection cell to cultivate.With the positive contrast of GFP, the slow virus supernatant infects after the A549 cell 48h and can be observed most cells and be the GFP positive expression, 72h increases, 96h reaches the optimum regime (see figure 3), by this condition, carries out the screening that cell that the Luciferase-PRRL-CMV plasmid transfection obtains can be used for the stable cell strain of high expression level luciferase.
Screening and the foundation of the lung adenocarcinoma cell system of step 4. stably express luciferase:
With limiting dilution assay elder generation A549 lung adenocarcinoma cell after the luciferase slow virus infection of 10cm culture dish plantation different concns, after being cultured to single cell clone and forming, under mirror, 48 monoclonal cell to 96 orifice plates of picking continue clone's cultivation (each monoclonal cell is established multiple hole), detect respectively the expression of luciferase in 48 monoclonal cells with fluor tester and viable cell imager, choose luciferase expression strong, the cell clone (as No. 3 single cell clones in Fig. 4) that Growth of Cells is all right, and adopt scratch test to confirm that cell (luc-A549), than biological characteristics before transfection (propagation and transfer ability etc.), (see figure 5) does not occur obviously to change, A549 cell to the stably express luciferase carries out enlarged culturing, making for tumor model.
The foundation of the lung adenocarcinoma cell tumor model of step 5. stably express luciferase:
The A549 lung adenocarcinoma cell (Luc-A549) of stably express luciferase is incubated at containing in 10wt% calf serum and the two anti-DMEM in high glucoses of 1wt% to 37 ℃, 5%volCO
2The amplification of going down to posterity under culture condition.Fetching is counted the tumour cell in vegetative period, and 0.25% trypsin is containing EDTA) digestion, medical 0.9wt% sodium chloride solution is resuspended, and centrifuge washing is adjusted concentration of cell suspension standby after accurate metering.Choose the naked J mouse hypodermic inoculation of female Balb/c in 6 ~ 8 week age Luc-A549 cell (1 * 10
6Individual/200 μ l).Under the SPF condition, normal the raising also becomes the knurl situation in the routine observation body.At postvaccinal the 5th, 10,15,20,25 days major axis by the vernier caliper measurement tumour (a) and minor axis (b), press formula: V=0.5 * ab
2, calculate gross tumor volume (mm
3), detected the luminous intensity (photon/sec/cm of in-vivo tumour with small animal living body imager (German BERTHOLD, NightOWL II LB983) at postvaccinal the 7th, 14,21 days
2/ steridian), with 0.4% vetanarcol (40mg/kg) intraperitoneal injection of anesthesia nude mice, press again 15mg/kg body weight abdominal injection D-fluorescein sylvite (D-Luciferin Potassium Salt) working fluid, after 10-15 minute, nude mice is lain on the back and carry out IMAQ and analysis on small animal living body imager Stage microscope, can observe structure expression Luciferase the A549 cell the C57BL/6J mouse interior tumor luminous intensity with the growth number of days strengthen gradually, and tumor growth curve shows tumour luminous intensity and gross tumor volume curve positive correlation (see figure 6).
The Luc-A549 C57BL/6J mouse tumor model gross tumor volume that step 6. is to be built reaches 100mm
3Left and right is used for pharmacological agent by the animal random packet.
GP TH is as follows: (1) physiological saline control group; (2) Gemcitabine group (injection in every three days once for abdominal injection, 60mg/kg nude mice).Each organized continued treatment 3 weeks, measured respectively gross tumor volumes at the 3rd, 6,9,12,15 days that treat, and detected the luminous intensity of tumour with the living imaging instrument at the 12nd day, with this, determined growth and the medication effect of tumour.The living imaging quantitative analysis results shows, between control group and treatment group, the luminous intensity of tumour is different, and, with different number of days tumor growth curve positive correlation after treatment, illustrate that A549 cell fluorescence element enzyme tumor model can be used for reflecting the result for the treatment of (see figure 7) of antitumor drug.
Claims (10)
1. the A549 nude mice model of a stably express luciferase, it is characterized in that: described nude mice model is by adopting gene recombination technology and slow virus infection that firefly luciferase gene is introduced in human lung adenocarcinoma A549 cell strain, screen the tumour clonal cell line that obtains the stably express luciferase by subclone, then adopt the A549 cell of recombinating to be inoculated in the mouse structure and form.
2. the establishment method of the A549 nude mice model of a stably express luciferase comprises:
(1) design firefly luciferase gene Auele Specific Primer; Use this Auele Specific Primer pcr amplification firefly luciferase gene and be cloned in the PCR-Blunt cloning vector and transform competent escherichia coli cell, after plasmid extraction, use gene recombination method that luciferase goal gene fragment is inserted in slow virus carrier for expression of eukaryon PRRL-CMV empty plasmid and transformed competent escherichia coli cell, carry out double digestion after plasmid extraction and the exactness of the expression vector that the confirmation of checking order builds;
(2) intestinal bacteria amplification fluorescent element enzyme lentiviral vectors and slow virus packaging plasmid, and purifying is measured concentration; The luciferase Lentiviral, with after the slow virus packaging plasmid mixes, with transfection reagent cotransfection 293T cell, the supernatant that contains Photinus pyralis LUC slow virus particle with generation, is determined to the titre of slow virus particle; Take human lung adenocarcinoma A549 as parent cell, infected with above-mentioned viral supernatant; Carry out the screening of cell subclone, detect the luciferase expression situation in monoclonal cell, filter out the recombinant cell strain of stably express luciferase reporter gene and carry out enlarged culturing;
(3) the recombinant human lung adenocarcinoma cell obtained is resuspended in sodium chloride solution after digestion, at nude mice by subcutaneous, inoculates this recombinant human lung adenocarcinoma cell, monitor propagation situation under cell skin, obtain the A549 cell strain of stably express luciferase.
3. the establishment method of the A549 cell strain of a kind of stably express luciferase according to claim 2, it is characterized in that: the Auele Specific Primer in described step (1) is:
Upstream primer: CCTTCTAGAATGGAAGATGCCAAAAACATTAAG;
Downstream primer: CCTGTCGACTTACACGGCGATCTTGCCGCCCTTC;
Gene recombination method is that enzyme is cut and is connected; Wherein, enzyme is cut to be specially and is adopted Xba I and Sal I double digestion.
4. the establishment method of the A549 nude mice model of a kind of stably express luciferase according to claim 2, it is characterized in that: the luciferase Lentiviral in described step (2) and the mass ratio of slow virus packaging plasmid are 1:3,1:6 or 1:9.
5. according to the establishment method of the A549 nude mice model of the described a kind of stably express luciferase of claim 2 or 4, it is characterized in that: described slow virus packaging plasmid comprises that third generation slow virus copies needed three gene: gag, pol and rev.
6. the establishment method of the A549 nude mice model of a kind of stably express luciferase according to claim 2, it is characterized in that: the transfection reagent in described step (2) is the MV40 transfection reagent.
7. the establishment method of the A549 nude mice model of a kind of stably express luciferase according to claim 2, is characterized in that: the titre of determining the slow virus particle in described step (2) by the ELISA method; Carry out the screening of cell subclone by the cell limiting dilution assay; Detect the luciferase expression situation in monoclonal cell by fluor tester or viable cell imager.
8. the establishment method of the A549 nude mice model of a kind of stably express luciferase according to claim 2, it is characterized in that: the sodium chloride solution mass percent concentration in described step (3) is 0.9%.
9. the establishment method of the A549 nude mice model of a kind of stably express luciferase according to claim 2, is characterized in that: in described step (3), by vernier caliper measurement and living imaging instrument, monitor propagation situation under cell skin.
10. the application of the A549 nude mice model of a stably express luciferase as claimed in claim 1 is characterized in that: described tumor model is applied to observe the result for the treatment of of growing state and the antitumor drug of tumour.
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| CN103468643A (en) * | 2013-09-13 | 2013-12-25 | 十堰市人民医院 | Human colonic cancer SW480 cell line capable of stably expressing luciferase and construction method of human colonic cancer SW480 cell line |
| CN105039410A (en) * | 2015-08-26 | 2015-11-11 | 苏州大学附属第一医院 | Method for establishing inflammation-based pancreatic cancer animal model |
| CN105462932A (en) * | 2015-12-29 | 2016-04-06 | 同济大学苏州研究院 | Tumor metastasis resistance medicine screening cell model constructed by adopting lentivirus technology and application method thereof |
| CN107723312A (en) * | 2017-09-07 | 2018-02-23 | 中国人民解放军第二军医大学 | The foundation and application of mice lung cancer original position lotus knurl irradiation model |
| CN108707626A (en) * | 2018-04-10 | 2018-10-26 | 中国科学院微生物研究所 | A kind of preparation method of the monoclonal cell system of detectable pyrogen |
| CN112369369A (en) * | 2020-11-09 | 2021-02-19 | 嘉兴学院 | Construction method and application of human melanoma cell A375/DR5 stable-transgenic nude mouse transplantation tumor model |
| CN113621578A (en) * | 2021-08-10 | 2021-11-09 | 上海南方模式生物科技股份有限公司 | Cytotoxic T cell tracking system and animal model construction method and application |
| CN117402828A (en) * | 2023-04-13 | 2024-01-16 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Human lung cancer brain metastasis cell line and application thereof |
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| CN103468643A (en) * | 2013-09-13 | 2013-12-25 | 十堰市人民医院 | Human colonic cancer SW480 cell line capable of stably expressing luciferase and construction method of human colonic cancer SW480 cell line |
| CN105039410A (en) * | 2015-08-26 | 2015-11-11 | 苏州大学附属第一医院 | Method for establishing inflammation-based pancreatic cancer animal model |
| CN105039410B (en) * | 2015-08-26 | 2018-05-01 | 苏州大学附属第一医院 | A kind of method for building up of the pancreas carcinoma animal model with inflammatory basis |
| CN105462932A (en) * | 2015-12-29 | 2016-04-06 | 同济大学苏州研究院 | Tumor metastasis resistance medicine screening cell model constructed by adopting lentivirus technology and application method thereof |
| CN107723312A (en) * | 2017-09-07 | 2018-02-23 | 中国人民解放军第二军医大学 | The foundation and application of mice lung cancer original position lotus knurl irradiation model |
| CN108707626A (en) * | 2018-04-10 | 2018-10-26 | 中国科学院微生物研究所 | A kind of preparation method of the monoclonal cell system of detectable pyrogen |
| CN108707626B (en) * | 2018-04-10 | 2021-10-01 | 中国科学院微生物研究所 | Preparation method of monoclonal cell line capable of detecting pyrogen |
| CN112369369A (en) * | 2020-11-09 | 2021-02-19 | 嘉兴学院 | Construction method and application of human melanoma cell A375/DR5 stable-transgenic nude mouse transplantation tumor model |
| CN113621578A (en) * | 2021-08-10 | 2021-11-09 | 上海南方模式生物科技股份有限公司 | Cytotoxic T cell tracking system and animal model construction method and application |
| CN113621578B (en) * | 2021-08-10 | 2023-11-28 | 上海南方模式生物科技股份有限公司 | Cytotoxic T cell tracing system, and construction method and application of animal model |
| CN117402828A (en) * | 2023-04-13 | 2024-01-16 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Human lung cancer brain metastasis cell line and application thereof |
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