CN105331584A - Human hepatoma cell line having drug tolerance or sensitivity to sorafenib and application of human hepatoma cell line - Google Patents
Human hepatoma cell line having drug tolerance or sensitivity to sorafenib and application of human hepatoma cell line Download PDFInfo
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- CN105331584A CN105331584A CN201410384329.2A CN201410384329A CN105331584A CN 105331584 A CN105331584 A CN 105331584A CN 201410384329 A CN201410384329 A CN 201410384329A CN 105331584 A CN105331584 A CN 105331584A
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Abstract
The invention discloses a human hepatoma cell line having drug tolerance or sensitivity to sorafenib and application of the human hepatoma cell line. The human hepatoma cell line having drug tolerance to the sorafenib is named as LIXC-004-SR and is preserved with preservation number of CCTCC NO: C2013181; and the cell line having sensitivity to the sorafenib is named as LIXC-004-NA and is preserved with preservation number of CCTCC NO: C2013180. The human hepatoma cell line is stable in character and is capable of achieving multiple passages stably, wherein the LIXC-004-SR cell line has an effect of drug tolerance on the in vivo therapy of the sorafenib, which serves as a first-tier drug for liver cancer; the cell line is the first liver cancer cell model which is drug-tolerant to sorafenib acquired in vivo therapy and is also a rare liver cancer model of acquired tumor angiogenesis increase; and the cell line not only provides novel experimental materials closer to the biological characteristics of the liver cancer in clinical field for the study of the liver cancer but also offers an effective model for researching the resistance mechanism and the resistance reversal of the sorafenib.
Description
Technical field
The invention belongs to clone field, particularly Xarelto is had to Bel7402 and the application thereof of resistance or susceptibility.
Background technology
Primary hepatocarcinoma have morbidity example high, prognosis is poor and case fatality rate high, is the one of common malignant tumour.According to up-to-date statistic data, the liver cancer whole world is new sends out number of cases about 74.8 ten thousand, death 69.6 ten thousand, and wherein half occurs in China.Its morbidity example and mortality ratio occupy the 5th respectively, the 2nd in male tumor patient; The 7th and the 6th (JemalA, BrayF, CenterMM, FerlayJ, WardE, FormanD.Globalcancerstatistics, 2011.CACancerJClin2011 is occupy respectively in female cancer patients; 61 (2): 69-90. Chen Jianguos, Chen Wanqing, Zhang Siwei, Zheng Rongshou, Zhu Jian, Zhang Yonghui. Chinese 2003-2007 onset of liver cancer rate and mortality analysis. Chinese epidemiology magazine 2012; 33 (6): 547-53.).
Primary hepatocarcinoma onset is hidden, and lack classical symptom in early days, early diagnosis is more difficult, disease is fast, and rapidly, when making a definite diagnosis, Most patients has reached Locally Advanced or distant metastasis occurs progress, treatment difficulty, prognosis is very poor, seriously threatens the healthy of the people and life security.Excision and liver transplantation are the methods for the treatment of of liver cancer first-selection, but it has been late period constantly that most of patient (60-70%) makes a definite diagnosis, lose operative treatment chance, other treatment scheme can only be adopted, comprise local ablation, hepatic artery interventional therapy (chemotherapy and embolism), radiotherapy, systemic treatment (targeted drug Xarelto, combined treatment based on cell toxicity medicament Zorubicin and oxaliplatin), Chinese medicine and immunotherapy etc., wherein chemotherapy plays an important role.But traditional chemotherapy regimen (as Zorubicin, platinum medicine etc.) is difficult to the lifetime effectively extending advanced liver cancer patient.
Xarelto (sorafenib, trade(brand)name: Nexavar, Nexavar) is a kind of Mutiple Targets inhibitor, and it can suppress the kinase whose activity of Raf, blocks the kinase whose phosphorylation of downstream ERK, thus the increment of inhibition tumor cell; It goes back target vascular therapy endothelial growth factor receptor (Vascularendothelialgrowthfactorreceptor simultaneously, and platelet derived growth factor receptor (plateletderivedgrowthfactorreceptor VEGFR), thus the angiogenesis of Tumor suppression PDGFR).Xarelto can suppress the in-vitro multiplication of hepatoma cell line, and can suppress the tumor growth in vivo of these cells.In two large-scale III phase clinical experiments (SHARP and Oriental), the treatment of Xarelto all significantly improves Overall survival and the disease developing time of patient.Therefore, U.S. FDAs in 2007 and European EAMA in succession ratify it and are used for the treatment of inoperable hepatocellular carcinoma in late period, and within 2008, Chinese SFDA ratifies it for liver cancer treatment, are the clinical standard chemotherapeutic agents for end-stage hepatocellular liver cancer patient at present.
But the result for the treatment of of Xarelto is ofer short duration, the Overall survival of patient's several months can only be extended; Most patients is accepting the administration initial stage, performance good result; But after long-term treatment, tumour there will be resistance, and regrows, progression of disease, Endodontic failure.Therefore, set up the liver cancer model to Xarelto resistance, and explore relevant resistance mechanism, there is earth shaking scientific research value and clinical meaning.At present, the starting stage is in the resistance mechanism research of Xarelto, and concentrates on external medicine-resistant cell line model, the animal model of resistance in body be there is no and clearly report.There is research team's Xarelto at cell levels long term in hepatoma cell line, inducing cell resistance occurs, and compare with parental cell, find external resistance mechanism, find that the activation of the signal paths such as Akt and STAT-3 appears in mdr cell, and the overactivity of EGFR/HER-3 acceptor, or occur that mesenchymal-epithelial transforms (epithelial-mesenchymaltransition, EMT) etc., but the outer inducible resistance cell of comparing bulk is not in vivo to the reactivity of Xarelto.And in vivo in model, have research team to be inoculated in immunodeficient mouse body by Bel7402 Hep3B, carry out Xarelto long term administration, in inductor, resistance produces; But they find that this resistance is instability but can produces reverse.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly deficient for the current clinical front liver cancer model to Xarelto resistance, be difficult to further investigation liver cancer cell and this technical barrier is developed to the resistance mechanism of Xarelto and reversing drug, provide Bel7402 and the application thereof Xarelto to resistance or susceptibility.
The present invention solves the problems of the technologies described above one of adopted technical scheme: a kind of Bel7402 Xarelto to resistance, and it is deposited in China typical culture collection center, and deposit number is CCTCCNO:C2013181.
Bel7402 of the present invention preferably also comprises the daughter cell system of Bel7402 as above.
The present invention solves the problems of the technologies described above two of adopted technical scheme: human liver cancer cell as above ties up to the purposes prepared in the reagent making immune deficiency Mammals generation liver cancer.
The reagent producing liver cancer in immune deficiency Mammals of the present invention is the reagent of this area routine, the preparation method of this reagent is preferably for be suspended in all kinds of SOLVENTS by Bel7402 of the present invention and to get final product, described solvent is this area Conventional solvents, be preferably HBSS damping fluid, the formula of described HBSS damping fluid is: containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs.
Reagent of the present invention is preferably for the preparation of liver cancer animal model.The preparation method of wherein said liver cancer animal model is this area ordinary method, preferably comprises the following steps: the described reagent making immune deficiency Mammals produce liver cancer is inoculated in immune deficiency deficient animals, raises, obtain liver cancer animal model.Wherein said immune deficiency Mammals is the immune deficiency Mammals of this area routine, it is preferably immunodeficient mouse, described immunodeficient mouse is preferably nude mouse or Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse), be preferably Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse), wherein said nude mouse is preferably the nude mouse of NU/NU strain.
The mode of wherein said inoculation is the vaccination ways that this area routine uses.Described vaccination ways preferably comprises subcutaneous puncture inoculation, inoculates in in-situ inoculating or scrotum film, is preferably subcutaneous puncture inoculation.The cell concentration of described inoculation is preferably 1.0 ~ 2 × 10
7individual cell, is preferably 5.0 × 10
6individual cell.The treatment of income earner's liver cancer animal model to Xarelto has resistance, is Xarelto treatment resistance people liver cancer animal model.
The application method of people's liver cancer animal model of the present invention is preferably, income earner's liver cancer animal model is utilized to detect the suppression liver cancer activity of testing compound, testing compound is applied to people's liver cancer animal model, observes and measure body weight and the tumor growth situation of animal; The testing compound causing people's liver cancer animal model liver cancer symptom to be improved after using or to cure has suppression liver cancer activity.
The method that wherein said testing compound is used is the application process of this area routine, preferably comprises: one or more modes in tail vein injection, abdominal injection, gavage, oral and tumor by local medication are applied to the tumor animal of liver cancer.Reference examples in described experiment is preferably: use simultaneously not containing testing compound solvent application in liver cancer tumor animal in contrast.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of testing compound suppresses the detection method of liver cancer activity, this detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the testing compound suppressing liver cancer activity, wherein said cell model is Bel7402 as above.
The detection method of testing compound suppression liver cancer activity of the present invention is the detection method of this area routine, and described detection method preferably comprises the following steps:
(1) Bel7402 of the present invention or its daughter cell are inoculated in 96 porocytes to cultivate in plate hole, cultivate 24 hours;
(2) testing compound is diluted to different concns and is applied to cell, compound effects passes through the vigor measuring cell after 72 hours, calculate the testing compound of different concns to the Proliferation Ability ability of cell, calculate the half-inhibition concentration of testing compound, in order to judge the suppression liver cancer activity of testing compound.
Wherein the inoculum size of step (1) described Bel7402 or its daughter cell is preferably 5000/ hole.Wherein the detection method of the half-inhibition concentration of step (2) described testing compound preferably comprises ATP biloluminescence method or mtt assay, and detection method of the present invention is preferably ATP biloluminescence method.Described ATP biloluminescence method detects a kind of homogeneous detection method of viable count object in culture by carrying out quantitative assay to the ATP in cell, wherein said ATP is the metabolic important indicator of viable cell, and described ATP biloluminescence method can utilize commercially available detection kit to carry out.
The preparation method of cell model of the present invention is this area customary preparation methods, and the preparation method of wherein said cell model preferably comprises the following steps:
(1) obtain fresh clinical people's operation of liver cancer Operated Specimens, cut into fritter, subcutaneous puncture seeded with mammalian;
(2) percutaneous puncture-inoculation is after 70 ~ 90 days, is put to death by tumor animal, and take out tumor tissues, subcutaneous vaccination is to new immunodeficient mouse;
(3) after tumour is formed, be divided into two groups at random, one group is that (the concrete composition of solvent is 12.5% polyoxyethylenated castor oil (Cremphor) to Vehicle controls group, 12.5% ethanol and 75% sterilized water, dosage is 10ml/ kg animal weight, every day is administered once, gavage); One group is Xarelto administration group (administering mode is 40 mg/kg the weight of animals, and every day is administered once, gavage);
(4) from the mice with tumor of Vehicle controls group and the treatment resistance mice with tumor of Xarelto administration group, be separated tumor tissues, carry out original cuiture and the Secondary Culture of cancer cells.
Income earner's hepatoma cell line, to the treatment resistance of Xarelto, utilizes this clone can prepare Xarelto interior therapeutic drug-resistant cell model.
Fresh clinical people's operation of liver cancer Operated Specimens wherein described in step (1), preferably treatment process is: the fritter cutting into 20 ~ 50mg.Wherein said Mammals is the Mammals of this area routine, is preferably immunodeficient mouse or nude mouse, and described immunodeficient mouse is preferably Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse).The processing mode of wherein said fresh clinical people's liver cancer Operated Specimens is preferably: with fresh HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing.
Primary culture method wherein described in step (4) is the primary culture method of conventional mammalian cell.Described primary culture method preferably comprises the following steps: tumor tissues is cut into fritter, inserts in culturing bottle, in 37 DEG C of incubator 5%CO
2cultivate under condition; Next day, culturing bottle is slowly overturn and keeps flat, DMEM/F12 nutrient solution is added (containing 10ng/ml recombinant human epidermal growth factor in bottle, 10 μ g/ml recombinant human insulins, 6.7ng/ml Sodium Selenite, 5.5 μ g/ml Transferrins,iron complexess, 2 μ g/ml thanomins, 0.1% bovine serum albumin, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), quiescent culture.
Secondary Culture method wherein described in step (4) is the Secondary Culture method of conventional mammalian cell.Described passage cultural method preferably comprises the following steps: inhale and abandon old nutrient solution, in bottle, add 0.05% fresh trypsin solution, after cell detachment, add fresh DMEM/F12 nutrient solution, careful piping and druming, makes it to depart from bottle wall and forms cell suspension; On bottle wall, become circle but the cell do not come off on a small quantity, hang culturing bottle surface gently with sterile Cell Scraper, collect whole cell, centrifugal, be inoculated in new culturing bottle respectively; After passage to the 5th generation, nutrient solution is replaced by RPMI1640 nutrient solution (containing 10% foetal calf serum, 10 μ g/ml recombinant human insulins, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B).
The present invention solves the problems of the technologies described above four of adopted technical scheme: a kind of Bel7402 Xarelto to susceptibility, and it is deposited in China typical culture collection center, and deposit number is CCTCCNO:C2013180.
Bel7402 of the present invention preferably also comprises the daughter cell system of Bel7402 as above.
The present invention solves the problems of the technologies described above five of adopted technical scheme: human liver cancer cell as above ties up to the purposes prepared in the reagent making immune deficiency Mammals generation liver cancer.
The wherein said reagent producing liver cancer in immune deficiency Mammals that makes is the reagent of this area routine, and the preparation method of reagent thereof recorded of technical scheme two is identical as described above for the preparation method of this reagent.The purposes of wherein said reagent is preferably for utilizing this reagent to prepare liver cancer animal model.
Wherein said reagent is preferably for the preparation of liver cancer animal model.The preparation method of wherein said liver cancer animal model is the preparation method of this area ordinary method, described liver cancer animal model, and the liver cancer animal model preparation method that liver cancer cell vaccination ways and technical scheme two are above recorded is identical with liver cancer cell vaccination ways.The treatment of income earner's liver cancer animal model to Xarelto has susceptibility, is to have Xarelto therapeutic sensitivity people liver cancer animal model.The application method of the liver cancer animal model that the application method of wherein said people's liver cancer animal model and technical scheme two are above recorded is identical.
The present invention solves the problems of the technologies described above six of adopted technical scheme: a kind of testing compound suppresses the detection method of liver cancer activity, this detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the testing compound suppressing liver cancer activity, wherein said cell model is Bel7402 as above.
Testing compound of the present invention suppresses the detection method of liver cancer activity to be the detection method of this area routine, and described detection method is identical with three detection methods recorded of technical scheme above.
The preparation method of cell model of the present invention is this area customary preparation methods, and the preparation method of wherein said cell model is identical with the preparation method of above three cell models recorded of technical scheme.
One treatment of strain Bel7402 to Xarelto of gained of the present invention has susceptibility, utilizes this clone to prepare and has sensibility tumor model to Xarelto interior therapeutic.
Wherein said testing compound suppresses the detection method of liver cancer activity to be the detection method of this area routine, and the detection method described in three of described detection method and technical scheme is above identical.The hepatoma model utilizing described Bel7402 to prepare gained has susceptibility to Xarelto, and gained hepatoma model is hepatoma model Xarelto to susceptibility.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: human hepatocellular carcinoma cell line proterties provided by the invention is stablized, and Absorbable organic halogens repeatedly goes down to posterity, for liver cancer research provides the new experiment material closer to clinical tumor biological characteristics.Two kinds of human hepatocellular carcinoma cell line provided by the invention have Tumor formation, successfully can prepare liver cancer animal model.LIXC-004-NA and LIXC-004-SR two strain cell has identical short-movie section tumor-necrosis factor glycoproteins collection of illustrative plates (STR), and they derive from same liver cancer patient; Wherein LIXC-004-NA tumour is responsive to the interior therapeutic of Xarelto, and LIXC-004-SR tumour is to the interior therapeutic resistance of Xarelto.LIXC-004-SR tumour is the first liver cancer model Xarelto being obtained to resistance in gonosome, also be the rare liver cancer model that acquired induced tumor vasculogenesis improves, because Xarelto can be bred and vasculogenesis by inhibition tumor cell simultaneously, therefore the liver cancer model to resistance in Xarelto body only having foundation effectively stable and the liver cancer model to sensitivity in Xarelto body, Xarelto could be simulated as far as possible in the clinical mode of action, and study generation and the Effect path of resistance on this basis, there is great clinical value and significance of scientific research.Therefore two strain human liver cancer cells provided by the invention are drug-resistance mechanism exploration and the reversing drug exploitation of Xarelto, provide effective research model.
biomaterial preservation information
Two kinds of human hepatocellular carcinoma cell line of the present invention, are deposited in China typical culture collection center on November 20th, 2013 and (are called for short: CCTCC), preservation address: Wuhan University of Wuhan City of Hubei China province postcode 430072.Culture name is called: human liver cancer cell LIXC-004-NA, and its deposit number is CCTCCNO:C2013180.Culture name is called: human liver cancer cell LIXC-004-SR, and its deposit number is CCTCCNO:C2013181.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, characteristic sum beneficial effect of the present invention is described.
Fig. 1 behaves the long-term vivo medicine-feeding experimental result picture of source liver cancer animal model.
Fig. 2 is the morphological observations figure (100 ×) of LIXC-004-NA cell and LIXC-004-SR cell.The photo that Fig. 2 (A) is LIXC-004-NA cell; The photo that Fig. 2 (B) is LIXC-004-SR cell.
Fig. 3 is the chromosome analysis result figure of LIXC-004-NA cell and LIXC-004-SR cell.Fig. 3 (A) for oily mirror 1000 × under the photo of LIXC-004-NA cell chromosome; Fig. 3 (B) for oily mirror 1000 × under the photo of LIXC-004-SR cell chromosome; The chromosome profiling that Fig. 3 (C) is LIXC-004-NA cell and LIXC-004-SR cell.
Fig. 4 is LIXC-004-NA cell and LIXC-004-SR cell doubling time graphic representation.Fig. 4 (A) is LIXC-004-NA cell doubling time graphic representation; Fig. 4 (B) is LIXC-004-SR cell doubling time graphic representation.
Fig. 5 is LIXC-004-NA cell and LIXC-004-SR cell cycle analysis result figure.Fig. 5 (A) is LIXC-004-NA cell analysis figure; Fig. 5 (B) is LIXC-004-SR cell analysis figure.
Fig. 6 is LIXC-004-NA cell and LIXC-004-SR Immunohistochemistry result figure.Fig. 6 (A) is LIXC-004-NA control antibodies (200 ×); Fig. 6 (B) is LIXC-004-NA cytokeratin (200 ×); Fig. 6 (C) is LIXC-004-NA vimentin (200 ×); Fig. 6 (D) is LIXC-004-SR control antibodies (200 ×); Fig. 6 (E) is LIXC-004-SR cytokeratin (200 ×); Fig. 6 (F) is LIXC-004-SR vimentin (200 ×).
Fig. 7 be LIXC-004-NA cell and LIXC-004-SR Transplanted cells knurl body in Xarelto evaluating drug effect experimental result picture.Fig. 7 (A) for LIXC-004-NA cell in nude mouse (NU/NU strain) body through solvent or Xarelto treatment tumour evaluating drug effect result figure; Fig. 7 (B) is control group and treatment group LIXC-004-NA tumor weight detected result figure; Fig. 7 (C) for LIXC-004-SR cell in nude mouse (NU/NU strain) body through solvent or Xarelto treatment tumor growth curve figure; Fig. 7 (D) is control group and treatment group LIXC-004-SR tumor weight detected result figure.
Fig. 8 is the histopathology result figure of LIXC-004-NA cell and LIXC-004-SR cell nude mice model tumour.Fig. 8 (A) is LIXC-004-NA Vehicle controls group photo (100 ×); Fig. 8 (B) is LIXC-004-NA Xarelto administration group photo (100 ×); Fig. 8 (C) is LIXC-004-SR Vehicle controls group photo (100 ×); Fig. 8 (D) is LIXC-004-SR Xarelto administration group (100 ×) photo.
Fig. 9 is the CD34 vasculogenesis dyeing of LIXC-004-NA cell and LIXC-004-SR cell nude mice model tumour.Fig. 9 (A) is LIXC-004-NA Vehicle controls group photo (100 ×); Fig. 9 (B) is LIXC-004-NA Xarelto administration group photo (100 ×); Fig. 9 (C) is LIXC-004-SR Vehicle controls group photo (100 ×); Fig. 9 (D) is LIXC-004-SR Xarelto administration group photo (100 ×); The micro-vessel area comparative result figure that Fig. 9 (E) is LIXC-004-NA and LIXC-004-SR tumour.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Xarelto is bought from LCLaboratory company of the U.S..
Polyoxyethylenated castor oil (Cremphor) is bought from Sigma Co., USA, and cell culture reagent is all bought from American I nvitrogen company.
The foundation of embodiment 1LIXC-004-NA and LIXC-004-SR clone
SCID mouse: 5 SCID mouse, male, body weight 16.0 ± 1.0 grams, in 5 weeks ages of mouse, raises in SPF environment.SCID mouse is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..
NU/NU mouse: 50 NU/NU mouse, male, body weight 20.0 ± 1.0 grams, in mouse 7-8 in age week, raises and SPF environment.NU/NU mouse is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..
Fresh clinical liver cancer Operated Specimens (female, 39 years old, primary hepatocarcinoma, nationality: the Chinese, native place: Nantong City is obtained from Nantong tumour hospital.Preoperatively do not make chemotherapy, pathological diagnosis result is: low differentiated hepatocellular liver cancer), immerse immediately (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) in the aseptic HBSS damping fluid of precooling.In Biohazard Safety Equipment, with this fresh aseptic HBSS wash buffer sample, be cut into the fritter of 20 ~ 50mg, percutaneous puncture-inoculation SCID mouse armpit dorsal sc.Inoculate after 30 days, find have tumor nodules save inoculation position is subcutaneous by touching, lesser tubercle starts growth in inoculation after 40 days obvious, and to when inoculating latter 60 days, gross tumor volume is more than 300mm
3.
Long-term vivo medicine-feeding experiment: SCID mouse is at subcutaneous puncture inoculated tumour block after 70 ~ 90 days, lotus knurl SCID mouse excess carbon dioxide gas anesthesia is put to death, aseptic dissection, take out tumor tissues, with HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing tumor tissue 3 times, remove reticular tissue and necrotic tissue; With aseptic operation blade, tumor tissues is sheared into about 15-20mm
3fritter, percutaneous puncture-inoculation NU/NU mouse (nude mouse) armpit dorsal sc.Inoculate after 15 days, inoculation position subcutaneous tumor nodules joint start growth, when tumor growth is to 100-250mm
3time, select animal and carry out random packet administration.One group is Vehicle controls group, and concrete composition is 12.5% polyoxyethylenated castor oil (Cremphor), 12.5% ethanol and 75% sterilized water, and dosage is 10ml/ kg animal weight, and every day is administered once, gavage; One group is Xarelto administration group, and dosage is 40mg/kg the weight of animals, and every day is administered once, gavage.Investigate the weight of animals and gross tumor volume two to three times weekly, when gross tumor volume reaches 1500-2000mm
3time, to euthanizing animals, terminate experiment.Gross tumor volume is according to following formulae discovery: gross tumor volume (mm
3)=major diameter (mm) × wide footpath (mm)
2× 0.5.The results are shown in Figure 1, people source tumor cells of hepatocellular carcinoma inoculation nude mouse in after, Vehicle controls group tumor growth is very fast; Xarelto, at the treatment initial stage, has good reactivity, inhibits the growth of tumour; But after long term administration (80-100 days), Partial tumors occurs Drug-resistant, restarts growth.
Original cuiture: lotus knurl nude mouse (NU/NU strain) excess carbon dioxide gas anesthesia is put to death, aseptic dissection, take out tumor tissues, carry out original cuiture, method is as follows: use HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing tumor tissue 3 times, removes reticular tissue and necrotic tissue; With aseptic operation blade, tumor tissues is sheared into about 1mm3 fritter; The tissue block inoculating needle sheared is sent into culturing bottle, and evenly puts, interval 0.5cm, builds bottle cap; Overturn culturing bottle gently, at the bottom of making bottle upwards, in 37 DEG C of incubator 5%CO
2cultivate under condition; Next day, culturing bottle is slowly overturn and keeps flat, 10mlDMEM/F12 nutrient solution is added (containing 10ng/ml recombinant human epidermal growth factor in bottle, 10 μ g/ml recombinant human insulins, 6.7ng/ml Sodium Selenite, 5.5 μ g/ml Transferrins,iron complexess, 2 μ g/ml thanomins, 0.1% bovine serum albumin, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), quiescent culture; Original cuiture changes liquid once in every 3 days, removes floating tissue block and residual hemocyte.
Secondary Culture: the cell grown in by tissue block is covered with after bottom culturing bottle, carry out passage, concrete steps are as follows: inhale and abandon old nutrient solution, 0.05% trypsin solution that 1ml is fresh is added in bottle, rinse adherent layer gently, suction is abandoned, add 0.05% trypsin solution that 1ml is fresh again, hatch in 37 DEG C of incubators, observe tenuigenin retraction, intercellular substance increase, after cell detachment, add the DMEM/F12 nutrient solution that 3ml is fresh, careful piping and druming, makes it to depart from bottle wall and forms cell suspension; On bottle wall, become circle but the cell do not come off on a small quantity, hang culturing bottle surface gently, collect whole cell with sterile Cell Scraper, centrifugal, counting, is inoculated in new culturing bottle respectively.After passage to the 5th generation, nutrient solution is progressively replaced by RPMI1640 nutrient solution (containing 10% foetal calf serum, 10 μ g/ml recombinant human insulins, 100U/ml penicillin G, 100 μ g/ml Vetstreps), Growth of Cells is good, form is comparatively homogeneous, more than subculture in vitro separately to 50 generation.
In the present invention, derive from the original cuiture of long term administration group drug-resistant tumor in Vehicle controls group and Xarelto body and cultured cell line respectively all in Epithelial, cell attachment growth under inverted microscope, be uniformly dispersed, differ in size, shape is class spindle body sample, and cellular form is comparatively homogeneous, contactless suppression, primary growth speed is comparatively slow; Along with the increase of passage number, the speed of growth is progressively accelerated.The clone called after LIXC-004-NA obtained will be cultivated from Vehicle controls group tumour, cultivate the clone called after LIXC-004-SR obtained from Xarelto vivo medicine-feeding group drug-resistant tumor; Gained two kinds of cells lie in and are deposited in China typical culture collection center (CCTCC), preservation address on November 22nd, 2013: Hubei China province wuchang, wuhan Luo Jia Shan Wuhan University postcode 430072.Culture title behaviour source liver cancer cell LIXC-004-NA, its deposit number is CCTCCNO:C2013180; Culture title behaviour source liver cancer cell LIXC-004-SR, its deposit number is CCTCCNO:C2013181.
The biological characteristics of embodiment 2LIXC-004-NA and LIXC-004-SR cell and application
The present invention adopts the RPMI1640 nutrient solution containing foetal calf serum and Regular Insulin to cultivate embodiment 1 gained LIXC-004-NA cell and LIXC-004-SR cell, can external long term growth go down to posterity with stable.When cell reaches 20 generations more than, cell quality is stablized gradually, and relevant biology, genetics and tissue-derived qualification show until the 50th generation all had identical stable proterties.Through experimental observation and checking, the LIXC-004-NA cell of growth in vitro is similar with LIXC-004-SR cellular form, in Epithelial, under inverted microscope, cell attachment growth, is uniformly dispersed, differs in size, shape is class spindle body sample, loses contact growth-inhibiting, in malignancy.Genetics research confirms that two strain cells are heteroploid, and Numerical and structural chromsomal aberrations is serious, meets the genetics characteristics of malignant tumour.Two strain cells have identical short-movie section tumor-necrosis factor glycoproteins collection of illustrative plates (STR), prove that two strain cell deriveds are in same patient tumors.Two strain cells can form tumour in nude mouse (NU/NU strain) body, have tumorigenicity.Wherein, responsive to the treatment of Xarelto in LIXC-004-NA transplantation tumor body, tumor growth is stagnated; And the treatment resistance to Xarelto in LIXC-004-SR transplantation tumor body.This is the liver cancer model to Xarelto acquired resistance in the first body, is also the rare liver cancer model that acquired tumor-blood-vessel growth improves.Two strain human liver cancer cells are that the drug-resistance mechanism of Xarelto is explored and reversing drug exploitation, provide effective research model.Specific experiment method is as described below:
A. long-term vivo medicine-feeding experiment
Specific experiment step asks for an interview embodiment 1.Two strain clones are established, called after LIXC-004-NA clone and LIXC-004-SR clone respectively by long-term vivo medicine-feeding experiment.
B. morphological observation
Under the culturing bottle cultivating LIXC-004-NA cell and LIXC-004-SR cell is placed in inverted microscope, take pictures under bright field.The results are shown in Figure 2, wherein Fig. 2 (A) photo that is LIXC-004-NA cell; The photo that Fig. 2 (B) is LIXC-004-SR cell.As can be seen from photo shown in Fig. 2, under inverted microscope, two strain cellular fories are similar, and cell attachment grows, and be uniformly dispersed, differ in size, shape is Polygons, and in class spindle body sample, contactless suppression, in malignancy.
C. chromosomal qualification
In the LIXC-004-NA cell and LIXC-004-SR cell of logarithmic phase, add colchicine, make its final concentration be 0.4 μ g/ml, then in 37 DEG C of incubators, continue cultivation 4 hours.Gather the cell of metaphase, be fixed with stationary liquid, then cell suspension dripped on the microscope slide of precooling, with the dyeing of Giemas staining fluid, in counted under microscope chromosome number.The results are shown in Figure 3, wherein Fig. 3 (A) for oily mirror 1000 × under the photo of LIXC-004-NA cell chromosome; Fig. 3 (B) for oily mirror 1000 × under the photo of LIXC-004-SR cell chromosome; The chromosome profiling that Fig. 3 (C) is LIXC-004-NA cell and LIXC-004-SR cell.As can be seen from Fig. 3 chromosome morphology in central authorities and submetacentric chromosome, distinguishable go out this be human archeocyte karyomit(e).At random the division phases caryogram of cell is counted, and draw according to Frequence Analysis (Originlab7.5) computing: the modal number of discovery LIXC-004-NA cell is 55 ± 4, and the modal number of LIXC-004-SR cell is 85 ± 4.The karyomit(e) of LIXC-004 cell is relatively concentrated in mode region (46.83%), and the chromosome number of LIXC-004-SR substep in cell colony comparatively disperses, the chromosome number of individual cells, from 28 to 116, only has the cell chromosome number of 27.78% to concentrate on mode region.All there is obvious polyploidization feature in the karyomit(e) of two cells, Numerical and structural chromsomal aberrations is serious, meets the genetics characteristics of malignant tumour.
D. short-movie section tumor-necrosis factor glycoproteins (STR) qualification
STR is also called microsatellite DNA, refer on karyomit(e), by several base pair as core unit (2-6 base pair), the class DNA sequence dna (multiplicity is more than 10 ~ 60 time, and gene fragment is below 400 base pairs) that tandem sequence repeats is formed; The number of times that each core unit repeats there will be individual difference, thus the allelotrope that formation sheet segment length is different.Therefore, the multiplicity of one group of STR sequence is almost unique in Different Individual, is individual gene identities feature, is also that cytobiology is to cell identity and the main method identified of originating.
Collect LIXC-004-NA cell and the LIXC-004-SR cell of fresh culture, with AxyPrep genomic dna small volume of reagent box (purchased from the AxyPrep of Axygen company
tMmultisourceGenomicDNAMiniprepKit, article No. is AP-MN-MS-GDNA) extract cell genomic dna, fluorescently-labeled primer is held to carry out pcr amplification (primer sequence is in table 1 and table 2) with 5 ', products therefrom is checked order, calculates the repeat number of each short-movie section tumor-necrosis factor glycoproteins.The Amelogenin that wherein table 1 is recorded is sex chromosome specific position, its sequence is as shown in SEQ ID NO:1 and SEQIDNO:2, what table 2 was recorded is primer sequence and the sequencing result in autosomal STR site, and the sequence of described primer is as shown in SEQ ID NO:3 ~ SEQIDNO:18.In American type culture collection (ATCC) database, carry out STR sequence retrieval, do not find identical STR detected result; And two strain cells have intimate identical STR collection of illustrative plates, show that LIXC-004-NA cell and LIXC-004-SR cell are the tumours deriving from same patient.
The primer sequence in the heterosomal STR site of table 1 and sequencing result
The primer sequence in the autosomal STR site of table 2 and sequencing result
E. cytokinetics
The cell of taking the logarithm vegetative period, after trysinization, resuspended with fresh culture.Inoculate the cell density of 2000 cells/well in 37 DEG C of CO
2cultivate in incubator, take out one block of plate respectively at cultivation 12,24,36,48,72 and 96 hours six time points, add CTG.Measuring condition surveys noclilucence value, analyzes and calculates its doubling time.The observed value of 4 days is gathered drafting cell growth curve, and as shown in Figure 4, wherein Fig. 4 (A) is LIXC-004-NA cell doubling time graphic representation to result; Fig. 4 (B) is LIXC-004-SR cell doubling time graphic representation.Calculated by cytokinetics, the population doubling time of LIXC-004-NA cell and LIXC-004-SR cell is respectively 32.37 hours and 24.67 hours.
F. the cell cycle is detected
Utilize the method that Propidium iodide marks, by flow cytometer, the relative content of DNA in cell is measured, can the per-cent of analysis of cells cycle each phase.Clean re-suspended cell with PBS after peptic cell, fix with ice ethanol ,-20 DEG C of preservations of spending the night.Centrifugal and with PBS cleaning and re-suspended cell, add RNA enzyme re-suspended cell, 37 DEG C of conditions digest 30 minutes.Removing RNA enzyme, add Propidium iodide, 4 DEG C of lucifuges dye 30 minutes.Proceed to flow cytometer detection pipe, the analysis of cells cycle.Result shows, and in LIXC-004-NA cell, the G1 phase is 65.87%, the S phase be 22.81%, the G2/M phase is 11.32%; And in LIXC-004-SR cell the S phase on the high side, the G1 phase was 48.87%, S phase be 40.69%, G2/M phase is 10.44%, and as shown in Figure 5, wherein Fig. 5 (A) is LIXC-004-NA cell analysis figure to result; Fig. 5 (B) is LIXC-004-SR cell analysis figure.
G. immunohistochemical methods protein expression
LIXC-004-NA cell and the inoculation of LIXC-004-SR cell are cultivated on the cover slip, after cytochrome oxidase isozymes, fixes with 4% formaldehyde, carry out immunohistochemical staining (DAB development process).Result shows, and cytokeratin and vimentin dyeing are the positive, and as shown in Figure 6, wherein Fig. 6 (A) is LIXC-004-NA control antibodies (200 ×) to result; Fig. 6 (B) is LIXC-004-NA cytokeratin (200 ×); Fig. 6 (C) is LIXC-004-NA vimentin (200 ×); Fig. 6 (D) is LIXC-004-SR control antibodies (200 ×); Fig. 6 (E) is LIXC-004-SR cytokeratin (200 ×); Fig. 6 (F) is LIXC-004-SR vimentin (200 ×).This result shows this feature clone all being met to low differentiated hepatocellular liver cancer.
Pharmacodynamic evaluation in the animal body of h. Xarelto
By tumor cell inoculation in nude mouse (NU/NU strain) body, after tumour grows, carry out random packet administration, detection of drugs, to the restraining effect of tumor growth in vivo, is the important indicator evaluating antitumor drug drug effect.Respectively enlarged culturing two strain hepatoma cell line LIXC-004-NA and LIXC-004-SR, digests collecting cell with trypsin solution, centrifugally abandons supernatant; With fresh serum-free RPMI1640 nutrient solution re-suspended cell rinsing, centrifugally abandon supernatant; With serum-free RPMI1640 nutrient solution re-suspended cell fresh on a small quantity, count and adjust cell density 5 × 10
7/ ml, and matrigel (buying from U.S. company BD) is according to 1:1 Homogeneous phase mixing, is inoculated into subcutaneous (concrete product are NU/NU mouse) (5 × 10 of nude mouse (NU/NU strain) of immune deficiency
6/ 0.2ml/mouse).Treat that tumor growth is to 100-250mm
3, choose animal and carry out random packet administration.One group is Vehicle controls group, concrete composition is 12.5% polyoxyethylenated castor oil (Cremphor) (buying from Sigma Co., USA), 12.5% ethanol and 75% sterilized water, and dosage is 10ml/kg the weight of animals, every day is administered once, gavage; One group is Xarelto administration group, and dosage is 40mg/kg the weight of animals, and every day is administered once, gavage; Often group is 8-10 tumor-bearing mice.Investigate the weight of animals and gross tumor volume two to three times weekly, when gross tumor volume reaches 1500-2000mm
3time, to euthanizing animals, terminate experiment, peel off tumor tissues, weigh fixing.
According to formula gross tumor volume (mm
3)=major diameter (mm) × wide footpath (mm) 2 × 0.5, calculates gross tumor volume.Medication effect is evaluated: T/C%=[(T-T with formula T/C%
0)/(C-C
0)] × 100%; If administration group tumor regression, T/C%=[(T-T
0)/C
0] × 100%.Wherein, T is administration group gross tumor volume, T
0for D
0it administration group gross tumor volume; C is control group gross tumor volume, C
0for D
0it control group gross tumor volume.
Gross tumor volume detected result such as Fig. 7 shows, wherein Fig. 7 (A) for LIXC-004-NA cell in nude mouse (NU/NU strain) body through solvent or Xarelto treatment tumour evaluating drug effect result figure; Fig. 7 (B) is control group and treatment group LIXC-004-NA tumor weight detected result figure; Fig. 7 (C) for LIXC-004-SR cell in nude mouse (NU/NU strain) body through solvent or Xarelto treatment tumor growth curve figure; Fig. 7 (D) is control group and treatment group LIXC-004-SR tumor weight detected result figure.It is subcutaneous that result shows that LIXC-004-NA and LIXC-004-SR cell is inoculated into nude mouse (NU/NU strain), can form tumour.It is subcutaneous that LIXC-004-NA cell is inoculated into nude mouse (NU/NU strain), and inoculate and carry out grouping administration after about 20 days, administration is after about 50 days, and control group gross tumor volume reaches about 1500mm
3, terminate experiment; Carry out interior therapeutic with Xarelto, effectively can suppress the tumor growth of LIXC-004-NA tumour, inhibiting rate (T/C ratio) is about 75%.LIXC-004-SR cell inoculation nude mouse (NU/NU strain) is subcutaneous, and growth is very fast, and inoculate and carry out grouping administration after 7 days, administration is after about 20 days, and control group gross tumor volume reaches about 1500mm
3, terminate experiment.
The interior Xarelto of body is treated, and can not suppress the tumor growth of LIXC-004-NA tumour completely, and T/C% is 109.47%.According to anti-tumor in vivo drug evaluation principle, when control group tumor growth is more than 1000mm
3time, be considered as tumor growth good, reliable experiment result; And experimentally animal welfare protection philosophy, nude mouse (NU/NU strain) carries out the experiment of lotus knurl, and gross tumor volume should not exceed 20% of animal own wt, namely 20g × 20%=4g (about 4000mm
3), close to this standard, euthanasia animal should be put to death in time.This experiment in vivo is at least repeated three times, and all closely, in the Xarelto body that therefore this clone can be described, resistance is stable and irreversible to each result.
I. the pathology qualification of tumour
By the tumour that LIXC-004-NA and LIXC-004-SR in above-mentioned h step produces, comprise control group and Xarelto drug treatment group, carry out that formaldehyde is fixed, specimens paraffin embedding slices and H & E dye, as shown in Figure 8, wherein Fig. 8 (A) is LIXC-004-NA Vehicle controls group photo (100 ×) to result; Fig. 8 (B) is LIXC-004-NA Xarelto administration group photo (100 ×); Fig. 8 (C) is LIXC-004-SR Vehicle controls group photo (100 ×); Fig. 8 (D) is LIXC-004-SR Xarelto administration group (100 ×) photo.Visible LIXC-004-NA cell and LIXC-004-SR cell can form tumour, similar in nude mouse (NU/NU strain) body, and Xarelto treatment does not also obviously change the weave construction of tumour.Microscopic observation, tumour cell arrangement is fine and close, and nucleus engrain, visible split coil method cell under the visual field, through pathological diagnosis, be all differentiated hepatocellular liver cancer, result is as shown in table 3.
Table 3 pathological diagnosis result
J. the immunohistochemical staining display vasculogenesis of tumour
By the tumour embedding wax block that the LIXC-004-NA clone prepared in above-mentioned i step and LIXC-004-SR clone produce, comprise control group and Xarelto drug treatment group, carry out the immunohistochemical staining of CD34.CD34 antigen is a kind of transmembrane protein of high glycosylation, and its molecular weight is 90-120kD, and it is optionally expressed in hemopoietic stem cell, progenitor cell and Surface of Vascular Endothelial Cells, is the mark of conventional instruction blood vessels in tumors density.After CD34 immunohistochemical staining, with the section of Aperio system scan, ImageScope software is utilized to carry out data analysis; Multiple visuals field of random selecting section, according to formula: micro-vessel area (MicrovesselArea, MVA)=CD34 positive cell/(CD34 positive cell+CD34 negative cells), calculate micro-vessel area, evaluate the vasculogenesis in tumour, carry out data analysis with T-test.
As shown in Figure 9, wherein Fig. 9 (A) is LIXC-004-NA Vehicle controls group photo (100 ×) to CD34 coloration result; Fig. 9 (B) is LIXC-004-NA Suo Feini administration group photo (100 ×); Fig. 9 (C) is LIXC-004-SR Vehicle controls group photo (100 ×); Fig. 9 (D) is LIXC-004-SR Xarelto administration group photo (100 ×); The micro-vessel area comparative result figure that Fig. 9 (E) is LIXC-004-NA and LIXC-004-SR tumour.The above results shows, a large amount of vasculogenesis is had in gained tumour, irregular distribution is disorderly, tube chamber is irregular, occur narrow, expansion or distortion, tube wall is thin, structure is imperfect, even only have one deck endotheliocyte, lack basilar membrane and be centered around around blood vessel, and the smooth muscle cell of the control blood pressure of normal operation.In LIXC-004-NA control group tumour, micro-vessel area is CD34 cell positive rate is 2.586% ± 1.122%, and in Xarelto treatment group, only having 0.853% ± 0.300%, micro-vessel area significantly declines (P<0.001).In the control group tumour of LIXC-004-SR clone, micro-vessel area is increased to 4.879% ± 0.665%, and the control group tumour of LIXC-004-NA clone of comparing is significantly increased (P<0.001); And the generation (4.291% ± 1.129%, P=0.243) of the treatment of the Xarelto blood vessels in tumors that LIXC-004-SR clone can not be suppressed to produce.
The above results shows, in the transplantation tumor that LIXC-004-SR clone generates, its vessel density is significantly higher than the transplantation tumor that LIXC-004-NA clone generates; The vivo medicine-feeding of Xarelto significantly suppress the vasculogenesis in LIXC-004-NA transplantation tumor; But could not suppress the vasculogenesis of LIXC-004-SR transplantation tumor, the above results display Xarelto is relevant with to tumor vascular restraining effect to the restraining effect of tumour.Therefore LIXC-004-NA clone provided by the invention has significant Xarelto therapeutic sensitivity, and LIXC-004-SR clone has significant Xarelto treatment resistance.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. Xarelto is had to a Bel7402 for resistance, it is characterized in that, it is deposited in China typical culture collection center, and deposit number is CCTCCNO:C2013181.
2. the daughter cell system of Bel7402 as claimed in claim 1.
3. human liver cancer cell as claimed in claim 1 or 2 ties up to the purposes prepared in the reagent making immune deficiency Mammals generation liver cancer.
4. apply as claimed in claim 3, it is characterized in that, described immune deficiency Mammals is immunodeficient mouse.
5. the detection method of a testing compound suppression liver cancer activity, it is characterized in that, this detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the testing compound suppressing liver cancer activity, wherein said cell model is Bel7402 as claimed in claim 1 or 2.
6. Xarelto is had to a Bel7402 for susceptibility, it is characterized in that, it is deposited in China typical culture collection center, and deposit number is CCTCCNO:C2013180.
7. the daughter cell system of Bel7402 as claimed in claim 6.
8. human liver cancer cell as claimed in claims 6 or 7 ties up to preparation and makes immune deficiency Mammals produce purposes in the reagent of liver cancer.
9. apply as claimed in claim 8, it is characterized in that, described immune deficiency Mammals is immunodeficient mouse.
10. the detection method of a testing compound suppression liver cancer activity, it is characterized in that, this detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the testing compound suppressing liver cancer activity, wherein said cell model is Bel7402 as claimed in claims 6 or 7.
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