CN104414771A - Establishment and detection method of multidrug resistance lung cancer nude mouse transplantation tumor model - Google Patents

Establishment and detection method of multidrug resistance lung cancer nude mouse transplantation tumor model Download PDF

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CN104414771A
CN104414771A CN201310369232.XA CN201310369232A CN104414771A CN 104414771 A CN104414771 A CN 104414771A CN 201310369232 A CN201310369232 A CN 201310369232A CN 104414771 A CN104414771 A CN 104414771A
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季旭明
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Abstract

The invention relates to an establishment and detection method of a multidrug resistance lung cancer animal model. The establishment method comprises the following steps: a, performing cell culture and inoculation passage among mice; b, performing the inoculation passage among the mice and establishing the model. The establishment method comprises the following steps: a, observing the growth situation of a lung cancer multidrug resistance nude mouse transplanted tumor; b, observing the morphology of the lung cancer multidrug resistance nude mouse transplanted tumor; c, detecting the drug resistance of the lung cancer multidrug resistance nude mouse transplanted tumor; d, performing semi-quantitative treatment on multidrug resistance gene mdr1 and multidrug resistance associated protein gene mrp messenger ribonucleic acid mRNA of the lung cancer multidrug resistance nude mouse transplanted tumor; e, expressing the multidrug resistance protein P-gp and the multidrug resistance associated protein gene MRP of the lung cancer multidrug resistance nude mouse transplanted tumor. The method has the advantages of stable drug resistance of the lung cancer nude mouse tumor transplantation model, convenience in observation of superficial tumor and capability of embodying complete biological characteristics, namely organizational structure and functional characteristic of human lung carcinoma, and the ideal is a more ideal multidrug resistance tumor model.

Description

A kind of foundation of multidrug resistance Pulmonary carcinoma nude mice Transplanted tumor model and detection method
Technical field
The present invention relates to animal pharmacological experiment technical field, particularly a kind of foundation of multidrug resistance lung cancer animal models methodand detection method.
Background technology
The multidrug resistance (Multidrug Resistance, MDR) of tumor is the main cause causing lung cancer chemotherapy failure, is also lung cancer therapy urgent need to solve the problem.According to relevant statistics, in tumor annual death rate, what belong to inwardness multidrug resistance accounts for 61%, and what belong to Acquired lung infection accounts for 33%, and namely the tumor patient cause of the death of more than 90% is relevant with drug resistance.Pulmonary carcinoma MDR Forming Mechanism is very complicated, and to generation, it has carried out a lot of exploration both at home and abroad, and therefore, the foundation of the multidrug resistance model of pulmonary carcinoma is very important.Nude mouse (Nude is called for short nude mice) has become indispensable experimental animal model in Medical Biology research field at present.Particularly in oncology, immunology, medicine and the experiment such as the safety evaluatio of biological product and the screening of effective medicine, it has special value.The congenital T lymphocyte immunity defect of nude mice and the feature of inbred line, considerably reduce individual variation, becomes and build the comparatively ideal carrier of tumor model.Nude Mouse Model can the Natural growth process of direct modeling people in-vivo tumour, and can observe the interaction of tumor and host.Long Term Passages is convenient simultaneously, and success rate is high, is a kind of important method studying tumor.The Nude Mouse Model be successfully established is the advantageous platform carrying out drug screening.
Summary of the invention
The object of the invention is to set up the stable nude mice adenocarcinoma of lung Transplanted tumor model of drug resistance, for carry out resistance mechanism research and reversing drug screening humanized animal is provided model, and can the diagnosis of expansive approach in tumor class major disease further, the conversion of Prevention Technique.The present invention realizes like this; A kind of method for building up of multidrug resistance lung cancer animal models:
A, first to carry out between cell culture and Mus inoculation and go down to posterity, human A459 lung cancer cell line and drug-resistant cell strain A549/DDP, routine passage is cultivated, and the cell of trophophase of taking the logarithm is for subsequent use;
Between b, Mus, inoculation is gone down to posterity, Modling model, female inbred lines nude mouse, 4 ~ 5 week age, body weight 15g ~ 17g, raises, free intake water and food under the qualified environment of no-special pathogen SPF level animal, each experiment is all carried out in aseptic superclean bench, under aseptic condition, take the logarithm trophophase tumor cell A549 and A549/DDP, prepares cell suspension, nude mice right oxter inoculation 0.2ml/ only, sets up first generation human A549 cell lines and persister A549/DDP Nude Mouse Model; Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, and A549, A549/DDP tumor block taken respectively, be placed in glass grinding device, add RPMI-160 culture medium, sampling proves not pollute, and prepares tumor cell suspension, carry out cell counting, nude mice right oxter inoculation, sets up second filial generation nude mice model; Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, and A549, A549/DDP tumor block taken respectively, be placed in glass grinding device, add RPMI-160 culture medium, sampling proves not pollute, and prepares tumor cell suspension, carry out cell counting, nude mice right oxter inoculation, sets up third generation nude mice model; Be passaged to for 3 generations, per generation 6 between Mus; Moulding property detection is carried out by the 4th generation nude mice A549 transplanted tumor group and A549/DDP transplanted tumor group.
The detection method of the lung cancer in nude mice multidrug resistance model set up according to the method for building up of above-mentioned multidrug resistance lung cancer animal models:
A, Mdr In Lung Cancer transplanted tumor in nude mice upgrowth situation are observed, 1st ~ 3 generation nude mice all in transplanting after 15 ± 6 days, namely within about 9-21 days, tumor is gone out, 5mm can be grown to about 3 weeks diameters, 4th generation A549/DDP Xenografts in nude mice success rate is 100%, average latency is 7 ± 1.35 days, i.e. 5.65-8.35 days; Average every day, the speed of growth was 102.23 ± 31.66mm3, and two groups of tumor volumes extend in time and increase gradually;
B, Mdr In Lung Cancer transplanted tumor in nude mice morphological observation, perusal: observation the 4th generation transplanted tumor is shown in that tumor body is in local nodal-like growth, smooth surface, not easily movable, regard sb. as an outsider when collecting transplanted tumor and see in yellow-white, angiogenic growth is had to distribute, with surrounding tissue sharpness of border, without adhesion, section pinkiness, central authorities are even hemorrhagic necrosis or liquefaction, light microscopy checking: transplanted tumor PD adenocarcinoma feature in presenting under mirror, A549/DDP transplanted tumor is then many in solid lumps, ovalize or spindle shape, the large engrain of core, in moderate heteromorphism, misaligned, kernel is obvious.Oncocyte is in invading profit growth, and downright bad less, interstitial has more blood capillary to be formed;
C, Mdr In Lung Cancer transplanted tumor in nude mice Resistance detection, get nude mice drug resistance transplanted tumor, and screen cloth method isolated cell carries out IC to primary cell and transplanted tumor cell 50detect, the IC of primary A549 cell and A549/DDP cell 50be respectively 24.1 μm of ol/L and 335.2 μm ol/L; The IC of nude mice A549/nude and A549/DDP/nude 50be respectively 23.8 μm of ol/L and 321.4 μm ol/L, calculate the drug resistance multiple of primary cell and nude mice model oncocyte, drug resistance multiple=mdr cell IC 50/ parental cell IC 50, be respectively 13.9 and 13.5, show A549/DDP transplanted tumor cell and primary cell to the drug resistance multiple of DDP without significant difference, illustrate that nude mice model cancer drug resistance is stablized;
The sxemiquantitative of d, Mdr In Lung Cancer transplanted tumor in nude mice multidrug resistance gene mdr1 and Multidrug Resistance associated Protein Gene mrp Messenger RNA mRNA, the relative expression quantity that Transcription-Polymerase Chain formula amplified reaction RT-PCR analyzes multidrug resistance gene mdr1 and Multidrug Resistance associated Protein Gene mrp in A549/DDP cell transplanted tumor in nude mice is respectively: 2.079 ± 0.071,3.503 ± 0.044, compared with A549 transplanted tumor in nude mice, in A549/DDP transplanted tumor in nude mice, the relative expression quantity of the mRNA of multidrug resistance gene and Multidrug Resistance associated Protein Gene all significantly raises p < 0.01; Prompting expression of drug resistance genes in nude mice drug resistance transplanted tumor is higher, maintains its drug resistance;
E, Mdr In Lung Cancer transplanted tumor in nude mice Mdr-p P-gp and multidrug-associated protein MRP express, and Mdr-p P-gp expresses in cell membrane and cytoplasm, and multidrug-associated protein MRP expresses in cell membrane; A549/DDP transplanted tumor Mdr-p P-gp and multidrug-associated protein MRP expresses in strong positive, visible a large amount of larger brown yellow granule; Statistical result shows that A549/DDP transplanted tumor Mdr-p P-gp and multidrug-associated protein MRP expression are all significantly higher than A549 transplanted tumor group p < 0.05.
Advantage of the present invention is: Pulmonary carcinoma nude mice Transplanted tumor model drug resistance is stablized, shallow being convenient to of tumor table is observed, and embody complete biological characteristics, namely there is organizational structure and the functional character of human lung adenocarcinoma, it is comparatively ideal multidrug resistance of tumor model, the multidrug resistance reversing adenocarcinoma of lung for research provides same human body source, the easy good laboratory animal system repeated, and may be used for the research of adenocarcinoma of lung resistance mechanism and chemotherapeutics screening.
Accompanying drawing explanation
Fig. 1 is flow chart of the present invention;
Fig. 2 is A549/DDP transplanted tumor in nude mice tissue morphology observation figure (hematoxylin-eosin staining, light Microscopic observation);
Fig. 3 is Messenger RNA (mRNA) the semi-quantitative expressed figure of nude mice model tumor tissue Multi-drug resist-ance-1 (mdr1);
Fig. 4 is Messenger RNA (mRNA) the semi-quantitative expressed figure of nude mice model tumor tissue Multidrug Resistance associated Protein Gene (mrp); In figure, M is scale, and 1 is A549 transplanted tumor, and 2 is A549/DDP transplanted tumor.
Fig. 5 is that A549/DDP transplanted tumor Mdr-p (P-gp) expresses figure (immunohistochemical method, light Microscopic observation);
Fig. 6 is that A549 transplanted tumor Mdr-p (P-gp) expresses figure (immunohistochemical method, light Microscopic observation);
Fig. 7 is that A549/DDP transplanted tumor multidrug-associated protein (MRP) expresses figure (immunohistochemical method, light Microscopic observation);
Fig. 8 is that A549 transplanted tumor multidrug-associated protein (MRP) expresses figure (immunohistochemical method, light Microscopic observation).
Detailed description of the invention
A kind of foundation of multidrug resistance lung cancer animal models and detection method:
1. the method for building up of lung cancer in nude mice multidrug resistance model:
A, first to carry out between cell culture and Mus inoculation and go down to posterity.Human A459 lung cancer cell line and drug-resistant cell strain A549/DDP, be placed in the RPMI-1640 culture fluid containing 10% hyclone, at 37 DEG C, 5%CO 2cultivate under condition.In A549/DDP cell culture fluid, the maintenance concentration of cisplatin (DDP) is 6 μm of ol/L.Routine passage is cultivated, and the cell of trophophase of taking the logarithm is for subsequent use;
Between b, Mus, inoculation is gone down to posterity, Modling model.Female inbred lines nude mouse (BALB/C-nu/nu), in 4 ~ 5 week age, body weight 15g ~ 17g, raises, free intake water and food under the qualified environment of no-special pathogen (SPF) level animal, and each experiment is all carried out in aseptic superclean bench.Under aseptic condition, trophophase tumor cell (A549 and A549/DDP) of taking the logarithm, prepares cell suspension, and concentration is 5 × 10 7individual/ml, nude mice right oxter inoculation 0.2ml/ only, sets up first generation human A549 cell lines and persister A549/DDP Nude Mouse Model.
Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, A549, A549/DDP tumor block taken respectively, is placed in glass grinding device, add RPMI-160 culture medium (sampling proves not pollute), prepare tumor cell suspension, carry out cell counting, nude mice right oxter inoculation 0.2ml/ only, complete in 30min, set up second filial generation nude mice model.
Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, A549, A549/DDP tumor block taken respectively, is placed in glass grinding device, add RPMI-160 culture medium (sampling proves not pollute), prepare tumor cell suspension, carry out cell counting, nude mice right oxter inoculation 0.2ml/ only, complete in 30min, set up third generation nude mice model.
3 generations (6/generation) are passaged between Mus.Moulding property detection is carried out by the 4th generation nude mice A549 transplanted tumor group and A549/DDP transplanted tumor group.
2. the detection method of lung cancer in nude mice multidrug resistance model:
A, Mdr In Lung Cancer transplanted tumor in nude mice upgrowth situation are observed, 1st ~ 3 generation nude mice all after transplanting, go out tumor about 15 ± 6 days (i.e. 9-21 days), can 5mm be grown to about 3 weeks diameters.4th generation A549/DDP Xenografts in nude mice success rate is 100%, and average latency is (7 ± 1.35) sky, (i.e. 5.65-8.35 days); Average every day, the speed of growth was (102.23 ± 31.66) mm3.Two groups of tumor volumes extend in time and increase gradually.
Table 1 is that the growing state of A549 cell and A549/DDP cell transplanted tumor in nude mice compares (mean ± standard deviation)
Group Tumor formation rate (%) Incubation period (d) The speed of growth (mm 3）
A549 group 100 10±1.01 74.5480±16.5622
A549/DDP group 100 7±1.35 102.2317±31.6571
B, Mdr In Lung Cancer transplanted tumor in nude mice morphological observation, perusal: observation the 4th generation transplanted tumor is shown in that tumor body is in local nodal-like growth, smooth surface is not easily movable.Regard sb. as an outsider when collecting transplanted tumor and see in yellow-white, have angiogenic growth to distribute, with surrounding tissue sharpness of border, without adhesion.Section pinkiness, central authorities are even hemorrhagic necrosis or liquefaction.Light microscopy checking: transplanted tumor PD adenocarcinoma feature in presenting under mirror.A549/DDP transplanted tumor is then many in solid lumps.Ovalize or spindle shape, the large engrain of core, in moderate heteromorphism, misaligned, kernel is obvious.Oncocyte is in invading profit growth, downright bad less.Interstitial has more blood capillary to be formed.
C, Mdr In Lung Cancer transplanted tumor in nude mice Resistance detection, get nude mice drug resistance transplanted tumor, and screen cloth method isolated cell carries out IC to primary cell and transplanted tumor cell 50detect, the IC of primary A549 cell and A549/DDP cell 50be respectively 24.1 μm of ol/L and 335.2 μm ol/L; The IC of nude mice A549/nude and A549/DDP/nude 50be respectively 23.8 μm of ol/L and 321.4 μm ol/L.Calculate the drug resistance multiple (drug resistance multiple=mdr cell IC of primary cell and nude mice model oncocyte 50/ parental cell IC 50), be respectively 13.9 and 13.5, show A549/DDP transplanted tumor cell and primary cell to the drug resistance multiple of DDP without significant difference.Illustrate that nude mice model cancer drug resistance is stablized.
Table 2 is the comparison (mean ± standard deviation) of A549/DDP and A549/DDP/nude cell to DDP drug resistance multiple
Cell IC 50（μmol/L） Drug resistance multiple
Wild A549 24.1±2.2
Wild A549/DDP 335.2±41.4 13.91
A549/nude 23.8±2.4
A549/DDP/nude 321.4±42.5 13.5
D, the sxemiquantitative of Mdr In Lung Cancer transplanted tumor in nude mice Multi-drug resist-ance-1 (mdr1) and Multidrug Resistance associated Protein Gene (mrp) Messenger RNA (mRNA), the relative expression quantity that Transcription-Polymerase Chain formula amplified reaction (RT-PCR) analyzes Multi-drug resist-ance-1 (mdr1) and Multidrug Resistance associated Protein Gene (mrp) in A549/DDP cell transplanted tumor in nude mice is respectively: (2.079 ± 0.071), (3.503 ± 0.044), compared with A549 transplanted tumor in nude mice, in A549/DDP transplanted tumor in nude mice the relative expression quantity of mdr1 and mrp mRNA all significantly raise ( p < 0.01).Prompting expression of drug resistance genes in nude mice drug resistance transplanted tumor is higher, maintains its drug resistance.
Table 3 be A549 nude mice and A549/DDP nude mice gene relative expression quantity comparison ( mean ± standard deviation)
Group mdr1 mrp
A549 transplanted tumor in nude mice group 1.222±0.029 1.635±0.006
A549/DDP transplanted tumor in nude mice group 2.079±0.071 3.503±0.044
E, Mdr In Lung Cancer transplanted tumor in nude mice Mdr-p (P-gp) and multidrug-associated protein (MRP) are expressed, and Mdr-p (P-gp) is expressed in cell membrane and cytoplasm, and multidrug-associated protein (MRP) is expressed in cell membrane.A549/DDP transplanted tumor Mdr-p (P-gp) and multidrug-associated protein (MRP) are expressed in strong positive, visible a large amount of larger brown yellow granule.Statistical result show A549/DDP transplanted tumor Mdr-p (P-gp) and multidrug-associated protein (MRP) expression be all significantly higher than A549 transplanted tumor group ( p < 0.05).
Table 4 is the comparison (mean ± standard deviation) of the average optical density value of transplanted tumor in nude mice P-gp and MRP protein expression
Group P-gp MRP
A549 transplanted tumor in nude mice 84057.79±15846.98 69268.83±14038.53
A549/DDP transplanted tumor in nude mice 123281.09±43051.92 101821.93±15744.64

Claims (2)

1. a method for building up for multidrug resistance lung cancer animal models, is characterized in that:
A, first to carry out between cell culture and Mus inoculation and go down to posterity, human A459 lung cancer cell line and drug-resistant cell strain A549/DDP, be placed in the RPMI-1640 culture fluid containing 10% hyclone, at 37 DEG C, 5%CO 2cultivate under condition; In A549/DDP cell culture fluid, the maintenance concentration of cisplatin DDP is 6 μm of ol/L; Routine passage is cultivated, and the cell of trophophase of taking the logarithm is for subsequent use;
Between b, Mus, inoculation is gone down to posterity, Modling model, female inbred lines nude mouse, 4 ~ 5 week age, body weight 15g ~ 17g, raise under the qualified environment of no-special pathogen SPF level animal, free intake water and food, each experiment is all carried out in aseptic superclean bench, under aseptic condition, take the logarithm trophophase tumor cell A549 and A549/DDP, prepares cell suspension, and concentration is 5 × 10 7individual/ml, nude mice right oxter inoculation 0.2ml/ only, sets up first generation human A549 cell lines and persister A549/DDP Nude Mouse Model; Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, and A549, A549/DDP tumor block taken respectively, be placed in glass grinding device, add RPMI-160 culture medium, sampling proves not pollute, and prepares tumor cell suspension, carry out cell counting, nude mice right oxter inoculation 0.2ml/ only, completes in 30min, sets up second filial generation nude mice model; Treat that nude mice by subcutaneous enclosed mass grows to about 100mm 3time, de-cervical approach puts to death nude mice, under aseptic condition, and A549, A549/DDP tumor block taken respectively, be placed in glass grinding device, add RPMI-160 culture medium, sampling proves not pollute, and prepares tumor cell suspension, carry out cell counting, nude mice right oxter inoculation 0.2ml/ only, completes in 30min, sets up third generation nude mice model; Be passaged to for 3 generations, per generation 6 between Mus; Moulding property detection is carried out by the 4th generation nude mice A549 transplanted tumor group and A549/DDP transplanted tumor group.
2. a detection method for the lung cancer in nude mice multidrug resistance model set up according to the method for building up of multidrug resistance lung cancer animal models according to claim 1, is characterized in that:
A, Mdr In Lung Cancer transplanted tumor in nude mice upgrowth situation are observed, 1st ~ 3 generation nude mice all in transplanting after 15 ± 6 days, namely within about 9-21 days, tumor is gone out, 5mm can be grown to about 3 weeks diameters, 4th generation A549/DDP Xenografts in nude mice success rate is 100%, average latency is 7 ± 1.35 days, i.e. 5.65-8.35 days; Average every day, the speed of growth was 102.23 ± 31.66mm3, and two groups of tumor volumes extend in time and increase gradually;
B, Mdr In Lung Cancer transplanted tumor in nude mice morphological observation, perusal: observation the 4th generation transplanted tumor is shown in that tumor body is in local nodal-like growth, smooth surface, not easily movable, regard sb. as an outsider when collecting transplanted tumor and see in yellow-white, angiogenic growth is had to distribute, with surrounding tissue sharpness of border, without adhesion, section pinkiness, central authorities are even hemorrhagic necrosis or liquefaction, light microscopy checking: transplanted tumor PD adenocarcinoma feature in presenting under mirror, A549/DDP transplanted tumor is then many in solid lumps, ovalize or spindle shape, the large engrain of core, in moderate heteromorphism, misaligned, kernel is obvious, oncocyte is in invading profit growth, downright bad less, interstitial has more blood capillary to be formed,
C, Mdr In Lung Cancer transplanted tumor in nude mice Resistance detection, get nude mice drug resistance transplanted tumor, and screen cloth method isolated cell carries out IC to primary cell and transplanted tumor cell 50detect, the IC of primary A549 cell and A549/DDP cell 50be respectively 24.1 μm of ol/L and 335.2 μm ol/L; The IC of nude mice A549/nude and A549/DDP/nude 50be respectively 23.8 μm of ol/L and 321.4 μm ol/L, calculate the drug resistance multiple of primary cell and nude mice model oncocyte, drug resistance multiple=mdr cell IC 50/ parental cell IC 50, be respectively 13.9 and 13.5, show A549/DDP transplanted tumor cell and primary cell to the drug resistance multiple of DDP without significant difference, illustrate that nude mice model cancer drug resistance is stablized;
The sxemiquantitative of d, Mdr In Lung Cancer transplanted tumor in nude mice multidrug resistance gene mdr1 and Multidrug Resistance associated Protein Gene mrp Messenger RNA mRNA, the relative expression quantity that Transcription-Polymerase Chain formula amplified reaction RT-PCR analyzes multidrug resistance gene mdr1 and Multidrug Resistance associated Protein Gene mrp in A549/DDP cell transplanted tumor in nude mice is respectively: 2.079 ± 0.071,3.503 ± 0.044, compared with A549 transplanted tumor in nude mice, in A549/DDP transplanted tumor in nude mice, the relative expression quantity of the mRNA of multidrug resistance gene and Multidrug Resistance associated Protein Gene all significantly raises p < 0.01; Prompting expression of drug resistance genes in nude mice drug resistance transplanted tumor is higher, maintains its drug resistance;
E, Mdr In Lung Cancer transplanted tumor in nude mice Mdr-p P-gp and multidrug-associated protein MRP express, and Mdr-p P-gp expresses in cell membrane and cytoplasm, and multidrug-associated protein MRP expresses in cell membrane; A549/DDP transplanted tumor Mdr-p P-gp and multidrug-associated protein MRP expresses in strong positive, visible a large amount of larger brown yellow granule; Statistical result shows that A549/DDP transplanted tumor Mdr-p P-gp and multidrug-associated protein MRP expression are all significantly higher than A549 transplanted tumor group p < 0.05.
CN201310369232.XA 2013-08-22 2013-08-22 Establishment and detection method of multidrug resistance lung cancer nude mouse transplantation tumor model Pending CN104414771A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112080471A (en) * 2020-08-26 2020-12-15 北京科途医学科技有限公司 Cisplatin-resistant lung cancer organoid and culture method, culture medium and application thereof
CN112863601A (en) * 2021-01-15 2021-05-28 广州微远基因科技有限公司 Pathogenic microorganism drug-resistant gene attribution model and establishing method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112080471A (en) * 2020-08-26 2020-12-15 北京科途医学科技有限公司 Cisplatin-resistant lung cancer organoid and culture method, culture medium and application thereof
CN112863601A (en) * 2021-01-15 2021-05-28 广州微远基因科技有限公司 Pathogenic microorganism drug-resistant gene attribution model and establishing method and application thereof

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Application publication date: 20150318