CN103828763B - A kind of liver cancer patient source xenograft tumor mouse model and construction method thereof - Google Patents
A kind of liver cancer patient source xenograft tumor mouse model and construction method thereof Download PDFInfo
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Abstract
The invention provides a kind of liver cancer patient source xenograft tumor mouse model and method for building up thereof.Described liver cancer patient source xenograft tumor mouse model comprises the mouse that the inoculation of forelimb shoulder dorsal sc has the liver cancer tissue block processed.Its method for building up comprises gets fresh HCC tissue, processes it, is inoculated in by the liver cancer tissue block after process without knurl mouse forelimb shoulder dorsal sc, conventional raising, obtains liver cancer patient source xenograft tumor mouse model.Process of establishing of the present invention is simple, becomes knurl incidence to reach 40%-50%, can be used for liver-cancer medicine screening and liver cancer Mechanism Study.To have manufacturing process simple for the liver cancer patient source xenograft tumor models set up by the present invention, and the caused lethality of operation is low, and success rate is high, be easy to promote; Can large quantities of making and the good advantage of synchronism, be applicable to drug screening and experimental study.
Description
Technical field
The present invention relates to a kind of liver cancer patient source xenograft tumor mouse model and construction method thereof.
Background technology
Primary carcinoma of liver is the malignant tumour ranking the whole world the 5th, China the 3rd.Estimate according to the World Health Organization, before the year two thousand thirty, liver cancer all will keep sustainable growth, and its case fatality rate is in second especially.At present, one of operation primary treatments being still liver cancer, but liver cancer patient more than 60% first medical time entered middle and advanced stage and with transfer, recurrence, thus lose the chance of operative treatment.
Animal model for tumour is the Important Platform carrying out tumour related mechanism and drug screening research.At present, the method for preclinical oncology animal model many employings tumor cell line suspension modeling, it has tumor cell line and easily obtains and test advantages such as easily repeating.But utilize the tumor model of Establishment of Cell Line to there is many defects.Tumour cell is built the process being in vitro and has been departed from natural tumor microenvironment, causes its gene to change; Selection pressure and through digestive ferment process in Process of in vitro, destroys tumor tissues and cell is heterogeneous and original structure, and the animal model that a strain cell-line is set up only can reflect a cancer patient; Tumour formed by cell-line lacks primary tumor and to be correlated with microenvironment component.Finally cause tumor biological behavior and natural quality irreversibility to change, the serious mankind of obstruction are to tumour mechanism and the research for the treatment of response, and it has become the bottleneck affecting therapeutic efficacy for hepatic carcinoma and improve, urgently to be resolved hurrily.
Excision liver cancer tissue is directly planted in immunodeficient mouse body by liver cancer patient source xenograft tumor mouse model, farthest simulate human internal environment, retain the specific gene of tumour, molecule and histology thereof heterogeneous, etiology of liver cancer, pathogenesis, evolution and treatment can be widely used in.Compared with the animal model for tumour set up with traditional human carcinoma cell line, humanized's animal model for tumour is owing to decreasing extracorporeal treatment step, the protistology characteristic sum histologic characteristics of clinical tumor tissue can be kept better, farthest save the microenvironment of tumour, comprise tumour cell, the lymphocyte of infiltration, fibroblast, extracellular matrix and vascular system; And humanized's Transplanted tumor model comes from different patients and does not cultivate through artificial, can reflect the genetic diversity of patient.Therefore, humanized's Transplanted tumor model will more adequately predict the clinical test results of medicine, for tumor individual therapy provides more reasonably conceptual design, is a kind of ideal Gene expression.Jackson Lab (The Jackson Laboratory) of famous American is with every thousands of dollars sell at competitive tumor patient source xenograft tumor mouse model.French industrial portion subsidizes the amount of money up to 5,400,000 Euros to this model plan.Therefore, tumor patient source xenograft tumor models oneself define a huge market.At present, tumor patient source xenograft tumor mouse model, as a kind of novel tumor model, mainly concentrates on breast cancer and lung cancer research, and its reason is that liver cancer patient source xenograft tumor mouse model success rate is extremely low.At Chin J Cancer Res, Yan M etc. report that building liver cancer patient source xenograft tumor mouse model method is in 2013: liver cancer tissue is divided into 2mm
3after fritter, directly transplanting is below the omoplate of mouse.The method plantation success rate is only 20.83%.
Therefore, in order to adapt to clinical and scientific research needs, need a kind of new liver cancer patient source xenograft tumor mouse model construction method badly, the method significantly can improve model plantation success rate, constructed model is applicable to drug screening and experimental study, also have manufacturing process simple, the caused lethality of operation is low simultaneously, and success rate is high, be easy to the feature promoted.
Summary of the invention
First object of the present invention is the deficiency overcoming existing liver cancer model, provides a kind of animal model that highly can reduce human liver cancer genetics information and biological property, to meet the right demand of medical market.
Another object of the present invention is to provide a kind of construction method of liver cancer patient source xenograft tumor models, significantly improves model plantation success rate.
In order to achieve the above object, the invention provides a kind of liver cancer patient source xenograft tumor mouse model, it is characterized in that, comprise the mouse that the inoculation of forelimb shoulder dorsal sc has the liver cancer tissue block processed.
Preferably, described mouse is non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse.
Present invention also offers the method for building up of above-mentioned liver cancer patient source xenograft tumor mouse model, it is characterized in that, concrete steps comprise: get fresh HCC tissue, it is processed, liver cancer tissue block after process is inoculated in without knurl mouse forelimb shoulder dorsal sc, conventional raising, obtains liver cancer patient source xenograft tumor mouse model.
Preferably, the processing method of described fresh HCC tissue comprises: the test tube that fresh HCC tissue is put into containing culture fluid is moved to super-clean bench, for subsequent use; Taking-up fresh HCC tissue, rinses 2-3 time with culture fluid, is divided into volume to be 8-32mm
3liver cancer tissue block, with aseptic Incubating Solution at 1-10 DEG C of submergence liver cancer tissue block 30-90 minute, obtain process after liver cancer tissue block.
More preferably, described culture fluid is DMEM culture fluid, its preparation method is: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
More preferably, the preparation method of described aseptic Incubating Solution is: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and the Matrigel glue mixing of 50 parts by volume, add epidermal growth factor (EGF), fibroblast growth factor (bFGF), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 5-20ng/ml, the final concentration of fibroblast growth factor is 5-20ng/ml, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
Compared with prior art, the invention has the beneficial effects as follows:
Patient's liver cancer tissue is directly seeded in the forelimb shoulder dorsal sc of experiment mice by the present invention, retain tumor microenvironment, correlated characteristic in the patient verily reduced corresponding to it, whole model process of establishing is simple, become knurl incidence to reach 40%-50%, can be used for liver-cancer medicine screening and liver cancer Mechanism Study.To have manufacturing process simple for the liver cancer patient source xenograft tumor models set up by the present invention, and the caused lethality of operation is low, and success rate is high, be easy to promote; Can large quantities of making and the good advantage of synchronism, be applicable to drug screening and experimental study.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Test method in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Same life is divided into A, B, C, D tetra-groups 3-4 week, 120 male non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse, and often organizing quantity is 30.
Embodiment 1
A kind of liver cancer patient source xenograft tumor mouse model takes on dorsal sc inoculation by forelimb has the mouse of the liver cancer tissue block processed to form, and its method for building up is:
(1) culture fluid is prepared: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) aseptic Incubating Solution is prepared: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and Matrigel glue (the BD company of 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgery excisions is inserted in the test tube containing the culture fluid of (1) middle preparation in steps, put into ice and transfer to super-clean bench rapidly, for subsequent use;
(4) aseptically the culture fluid of preparation in tumor tissues step (1) is rinsed 2 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 2 × 2mm
3fritter, the aseptic Incubating Solution prepared by step (2) 4 DEG C infiltrate 30 minutes, for subsequent use;
(5) select A group totally 30 male, 3 week age, weight 18-22g, non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse (NOD/SCID).The liver cancer tissue of each patient gets three fritters, and be seeded in the forelimb shoulder dorsal sc of 3 mouse respectively, concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 2 × 2mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, and tissue sends into forelimb shoulder dorsal sc the most at last, and compressing wound is to without hemorrhage;
(6) under SPF level environment, routine is raised 60 days, complete and set up liver cancer patient source xenograft tumor models, and be No. 1-10 (be more than or equal to 1 in 3 mouse corresponding to each numbering and become knurl, namely assert this numbering model modeling success) to pattern number.
A group mouse inoculation observes mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value with vernier caliper measurement transplantable tumor, by volume formula V=1/2 (LW
2) calculate transplantable tumor average external volume.Transplant naked eyes after about 4 weeks and be partially formed tumor nodule as seen, within the 10th week, tumour is hemispherical, and average tumor volume is about 1.59cm
3.Dissect mouse, transplantable tumor tissue is in flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, confirms that pathological is hepatocellular carcinoma after H & E dyes, consistent with corresponding patient's pathological.Through above-mentioned steps, have 2 to become knurls in corresponding 3 mouse of No. 2 models, No. 4 models have 2 to become knurls, and No. 8 and No. 9 models have 1 to become knurl respectively.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 2
A kind of liver cancer patient source xenograft tumor mouse model takes on dorsal sc inoculation by forelimb has the mouse of the liver cancer tissue block processed to form, and its method for building up is:
(1) culture fluid is prepared: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) aseptic Incubating Solution is prepared: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and Matrigel glue (the BD company of 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgery excisions is inserted in the test tube containing the culture fluid of (1) middle preparation in steps, put into ice and transfer to super-clean bench rapidly, for subsequent use;
(4) aseptically the culture fluid of preparation in tumor tissues step (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 2 × 2mm
3fritter, the aseptic Incubating Solution prepared by step (2) 4 DEG C infiltrate 40 minutes, for subsequent use;
(5) select B group totally 30 male, 3 week age, weight 18-22g, non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse (NOD/SCID).The liver cancer tissue of each patient gets three fritters, and be seeded in the forelimb shoulder dorsal sc of 3 mouse respectively, concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 2 × 2mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, and tissue sends into forelimb shoulder dorsal sc the most at last, and compressing wound is to without hemorrhage;
(6) under SPF level environment, routine is raised 60 days, complete and set up liver cancer patient source xenograft tumor models, and be No. 1-10 (be more than or equal to 1 in 3 mouse corresponding to each numbering and become knurl, namely assert this numbering model modeling success) to pattern number.
B group mouse inoculation observes mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value with vernier caliper measurement transplantable tumor, by volume formula V=1/2 (LW
2) calculate transplantable tumor average external volume.Transplant naked eyes after about 4 weeks and be partially formed tumor nodule as seen, within the 11st week, tumour is hemispherical, and average tumor volume is about 1.67cm
3.Dissect mouse, transplantable tumor tissue is in flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, confirms that pathological is hepatocellular carcinoma after H & E dyes, consistent with corresponding patient's pathological.Through above-mentioned steps, have 2 to become knurl in No. 1 corresponding 3 mouse of model, No. 2 models have 2 to become knurl, and No. 6 models have 2 to become knurl, and No. 9 models have 1 to become knurl.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 3
A kind of liver cancer patient source xenograft tumor mouse model takes on dorsal sc inoculation by forelimb has the mouse of the liver cancer tissue block processed to form, and its method for building up is:
(1) culture fluid is prepared: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) aseptic Incubating Solution is prepared: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and Matrigel glue (the BD company of 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgery excisions is inserted in the test tube containing the culture fluid of (1) middle preparation in steps, put into ice and transfer to super-clean bench rapidly, for subsequent use;
(4) aseptically the culture fluid of preparation in tumor tissues step (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 3 × 3mm
3fritter, the aseptic Incubating Solution prepared by step (2) 4 DEG C infiltrate 60 minutes, for subsequent use;
(5) select C group totally 30 male, 3 week age, weight 18-22g, non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse (NOD/SCID).The liver cancer tissue of each patient gets three fritters, and be seeded in the forelimb shoulder dorsal sc of 3 mouse respectively, concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 3 × 3mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, and tissue sends into forelimb shoulder dorsal sc the most at last, and compressing wound is to without hemorrhage;
(6) under SPF level environment, routine is raised 60 days, complete and set up liver cancer patient source xenograft tumor models, and be No. 1-10 (be more than or equal to 1 in 3 mouse corresponding to each numbering and become knurl, namely assert this numbering model modeling success) to pattern number.
C group mouse inoculation observes mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value with vernier caliper measurement transplantable tumor, by volume formula V=1/2 (LW
2) calculate transplantable tumor average external volume.Transplant naked eyes after about 5 weeks and be partially formed tumor nodule as seen, within the 10th week, tumour is hemispherical, and average tumor volume is about 1.36cm
3.Dissect mouse, transplantable tumor tissue is in flesh of fish sample substantially, and blood is for abundant, and central authorities are visible downright bad, confirms that pathological is hepatocellular carcinoma after H & E dyes, consistent with corresponding patient's pathological.Through above-mentioned steps, have 2 to become knurl in No. 2 corresponding 3 mouse of model, No. 5 models have 3 to become knurl, and No. 7 models have 1 to become knurl, and No. 10 models have 2 to become knurl.Finally set up 4 models, the tumor formation rate of mouse is 40%.
Embodiment 4
A kind of liver cancer patient source xenograft tumor mouse model takes on dorsal sc inoculation by forelimb has the mouse of the liver cancer tissue block processed to form, and its method for building up is:
(1) culture fluid is prepared: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, add penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains culture fluid.
(2) aseptic Incubating Solution is prepared: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and Matrigel glue (the BD company of 50 parts by volume; Article No.: 356234) mix, add epidermal growth factor (EGF) (Gibco company; Article No.: PHG0314), fibroblast growth factor (bFGF) (Gibco company; Article No.: PHG0264), penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 10ng/ml, the final concentration of fibroblast growth factor is 10ng/ml, the final concentration of penicillin is 100U/ml, and the final concentration of streptomycin is 100U/ml, obtains aseptic Incubating Solution.
(3) respectively the fresh HCC tissue (in vitro 5 minutes) of 10 corrective surgery excisions is inserted in the test tube containing the culture fluid of (1) middle preparation in steps, put into ice and transfer to super-clean bench rapidly, for subsequent use;
(4) aseptically the culture fluid of preparation in tumor tissues step (1) is rinsed 3 times; Remove slough, select euangiotic liver cancer tissue to be divided into 2 × 4 × 4mm
3fritter, the aseptic Incubating Solution prepared by step (2) 4 DEG C infiltrate 90 minutes, for subsequent use;
(5) select D group totally 30 male, 3 week age, weight 18-22g, non-obese patients with type Ⅰ DM severe combined immunodeficiency mouse (NOD/SCID).The liver cancer tissue of each patient gets three fritters, and be seeded in the forelimb shoulder dorsal sc of 3 mouse respectively, concrete inoculation method is: at mouse web portion preserved skin, then with iodophor disinfection.With No. 20 trochars by total amount 2 × 4 × 4mm
3liver cancer tissue thrust in belly midaxillary line place, syringe needle traveling is between skin and peritonaeum, and tissue sends into forelimb shoulder dorsal sc the most at last, and compressing wound is to without hemorrhage;
(6) under SPF level environment, routine is raised 60 days, complete and set up liver cancer patient source xenograft tumor models, and be No. 1-10 (be more than or equal to 1 in 3 mouse corresponding to each numbering and become knurl, namely assert this numbering model modeling success) to pattern number.
D group mouse inoculation observes mouse general activity and nutritional status etc. after transplanting.Within every three days, grow (L) short (W) footpath mean value with vernier caliper measurement transplantable tumor, by volume formula V=1/2 (LW
2) calculate transplantable tumor average external volume.Transplant naked eyes after about 4 weeks and be partially formed tumor nodule as seen, within the 10th week, tumour is hemispherical, and average tumor volume is about 1.71cm
3.Dissect mouse, transplantable tumor tissue is in flesh of fish sample substantially, and blood is for abundant, and central necrosis is obvious, confirms that pathological is hepatocellular carcinoma after H & E dyes, consistent with corresponding patient's pathological.Through above-mentioned steps, have 3 to become knurls in corresponding 3 mouse of No. 1 model, No. 3, No. 4 and No. 5 models have 2 to become knurls, and No. 10 models have 3 to become knurls.Finally set up 5 models, the tumor formation rate of mouse is 50%.
Claims (2)
1. a processing method for fresh HCC tissue, is characterized in that, comprising: the test tube that fresh HCC tissue is put into containing culture fluid is moved to super-clean bench, for subsequent use; Taking-up fresh HCC tissue, rinses 2-3 time with culture fluid, is divided into volume to be 8-32mm
3liver cancer tissue block, with aseptic Incubating Solution at 1-10 DEG C of submergence liver cancer tissue block 30-90 minute, obtain process after liver cancer tissue block; Described culture fluid is DMEM culture fluid, and its preparation method is: mixed with the hyclone of 10 parts by volume by the DMEM of 90 parts by volume, adds penicillin and streptomycin, obtain DMEM culture fluid, wherein, the final concentration of penicillin is 50-200U/ml, and the final concentration of streptomycin is 50-200U/ml.
2. the processing method of fresh HCC tissue as claimed in claim 1, it is characterized in that, the preparation method of described aseptic Incubating Solution is: by the DMEM of 45 parts by volume, the hyclone of 5 parts by volume and the Matrigel glue mixing of 50 parts by volume, add epidermal growth factor, fibroblast growth factor, penicillin and streptomycin, obtain aseptic Incubating Solution, wherein, the final concentration of epidermal growth factor is 5-20ng/ml, the final concentration of fibroblast growth factor is 5-20ng/ml, the final concentration of penicillin is 50-200U/ml, the final concentration of streptomycin is 50-200U/ml.
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