CN101020066A - In-situ transplantation process of constructing human liver cancer mouse model - Google Patents
In-situ transplantation process of constructing human liver cancer mouse model Download PDFInfo
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Abstract
The present invention discloses in-situ transplantation process of constructing human liver cancer mouse model. The process includes using Kunming sucking mouse as the tumor carrying acceptor, and injecting the mouse liver lobes with human liver cancer cell suspension to in-situ transplanted tumor carrying model of human liver cancer cell. The process is simple and high in successful rate. The process makes it possible to monitor the mutual effect between liver cancer cell and mouse immune system under normal mouse body immune environment, simulate the whole process of immunological tolerance and antitumor immunity in human body and provide ideal operation platform for the immunobiological medicine screening and pharmacological research.
Description
Technical field
A kind of animal hepatocarcinoma Preparation of model method involved in the present invention especially relates to a kind of being based upon under the physiological condition that common mice has normal immunologic function, makes up the method for human liver cancer animal model through xenogenesis orthotopic transplantation.
Background technology
(Primary Hepatocellular Carcinoma PHC) is one of grade malignancy and mortality rate are the highest in the world malignant tumor to primary hepatoma; Setting up ideal human liver cancer animal model is the important foundation of studying hepatocarcinoma, screening antitumor drug and carry out the experimental treatment of hepatocarcinoma.In research previously, once there was multiple animal hepatocarcinoma lotus tumor model to be invented, but because all multifactor in human-animal's difference and the human liver cancer forming process, setting up one, to be reflected in the animal model that has the human liver cancer biological nature in the animal livers fully be a problem that has challenge.
The transplanted hepatoma model is meant the model that liver cancer tissue piece or hepatoma cell strain is inoculated in laboratory animal (Mus commonly used) and sets up, and comprises that mainly (between Mus and the Mus) of the same race transplant and two kinds of xenogenesis (between people and the nude mice) transplanting.The tumor source can be people's hepatocarcinoma specimen or a hepatoma cell strain spontaneous, that bring out, excision.According to the receptor difference, the transplanted hepatoma model can be divided into intact animal's transplanted hepatoma model and immunodeficiency zoografting liver cancer model; The former receptor mostly is mice and rat, and latter receptor mostly is nude mice, nude rat; Can be with the different tissues of tumour transplatation in animal, in liver, position such as also implantable subcutaneous, abdominal cavity.
1, transplantability Mus-Hepar Mus cancer model
Intact animal's transplanted hepatoma model mostly is outside the spontaneous liver---liver homotransplantation Hepar Mus cancer model, this model Walker2256 cell strains that derive from rat liver cancer that adopt more, be implanted into the liver of rat by different approaches, thereby form the rat implantation liver cancer model.Laboratory animal is generally selected male Wistar rat for use, also useful SD male rat.And the transplantability rat liver cancer model is far fewer than transplantability rat liver cancer model, nineteen eighty-two, Zunyi Medical College set up China the 1st strain mouse bearing liver cancer model H615 with 615 inbred mouses, H615 hepatocarcinoma is 615 to be the strain of mice homogenic type tumor, also can only transplant in 615 mice bodies.
This type of model property is stable, manufacture method is simple, and success rate height (＞95%), cycle are short, and (7～10d), natural life span was 3～4 weeks, oncobiology stability of characteristics and homogeneous is the secretion of hepatocarcinoma and high concentration alpha-fetoprotein AFP; Be widely used in experiment of hepatocarcinoma imaging diagnosis and hepatocarcinoma topical therapeutic at present.But because this kind model generally can only be used for the tumour transplatation of (Mus) between allogenic animal, compare, certain limitation is arranged with nude mice people hepatocarcinoma transplantation model.
2, transplantability nude mice-people's liver cancer model
With human hepatoma cell strain or liver cancer tissue piece, be grafted directly in the nude mouse of no immunologic function and set up the transplanted hepatoma model, belong to people-Mus xenotransplantation.Mainly containing three kinds at present: people-nude mouse xenotransplantation liver cancer model, people-nude rat xenotransplantation liver cancer model and people's hepatocarcinoma nude mice be subcutaneous-the former shift value tumor of liver model.
Since nude mouse was transplanted successfully, China introduced nude mouse in 1979, had set up 5 strain nude mouse people liver cancer tissue model LTNM 1～5 and 1 strain nude mouse human hepatoma cell strain model LCNM1 from Shimosato reported first people liver cancer tissue in 1976.These models all keep the feature of former people's hepatocarcinoma form and function (secretion human a-fetoprotein).Nineteen eighty-two encourages formal plan academician etc. by soup and sets up China's the first human hepatocellular nude mouse transplantation model, makes that studying human liver cancer on one's body animal becomes possibility, and nude mice people hepatocarcinoma transplantation model has obtained significant progress in China since then.
This class model has higher xenotransplantation success rate, and incubation period is short, but most of rate of transform is low.Simultaneously, because nude mice lacks the immune system based on the T cell, raising and requirement of experiment condition height, life-span weak point, low to experiment processing factor toleration, especially transplant the cell as hepatocarcinoma, growth is rapidly in animal body, promptly produce a large amount of tuberosity and lethal bloody ascites of shifting in short time, make nude mice band tumor life span of short duration and influence experimentation, be unsuitable for the dynamic observation carried out in the drug targeting treatment, thereby limited extensive use this liver cancer model.
From above research as can be seen, existing people's liver cancer murine model mainly is to utilize nude mouse, the nude rat athymism, lack the such immunodeficiency animal of T lymphocyte, the transplanting of people's hepatocarcinoma of striding species of carrying out and the structure of model, though guaranteed the success rate of transplanting, but can not real simulated hepatocarcinoma take place and evolution in body to the cellullar immunologic response situation of tumor, cause the research of the anti-liver tumor immunne response of human body to pause, from having limited the research and the assessment of oncobiology and antineoplastic immune medication effect to a great extent in experiment in vitro always.Therefore, be based upon Mus-people's hepatocarcinoma transplantability model and become pressing in contemporary biologic pharmacological science and the tumour immunity research with normal immunologic function.
Summary of the invention
At the situation of prior art, the purpose of this invention is to provide the method that a kind of orthotopic transplantation makes up human liver cancer mouse model.
The present invention utilizes the alpha-fetoprotein (alpha-fetaprotein of high expressed in the neonatal rat liver, AFP) can suppress the biologic activity of antitumor immunity of organism and the principle of orthotopic transplantation, utilize Kunming kind neonatal rat first, set up people's liver cancer mouse orthotopic transplantation model with normal immunologic function.
A kind of orthotopic transplantation that the present invention relates to makes up the method for human liver cancer mouse model, and step is:
[1] human hepatoma cell strain is cultivated: the hepatoma cell strain routine of the choosing cultivation of going down to posterity, and standby;
[2] preparation of the cell suspension of fluorescent dye DiI labelling: choose one of human hepatoma cell strain of above-mentioned exponential phase, under 37 ℃～38 ℃, incubate 30min～60min with 20 μ g/ml living body fluorescent dyestuff DiI altogether, afterwards, 0.25% aseptic trypsinization is with the basic culture solution collecting cell and adjust concentration of cell suspension to 0.5 * 10
6Individual/ml～1.0 * 10
9Individual/ml;
[3] orthotopic transplantation makes up human liver cancer mouse model: select Kunming kind three age in days neonatal rats for use, after the skin of abdomen sterilization, utilize aseptic 30-gauge 50-μ l glass microsyringe, human liver cancer cell suspension with 10 μ l～above-mentioned DiI labelling of 50 μ l, directly transplanting is gone in the Hepar Mus leaf, sets up people's liver cancer mouse orthotopic transplantation model;
[4] model is identified.
Above-mentioned orthotopic transplantation makes up in the method for human liver cancer mouse model, preferred HepG-2 (the alph-fetoprotein positive of the described human hepatoma cell strain of step [1], purchase in Shanghai Inst. of Cytobiology, Chinese Academy of Sciences), Bel-7402 (alph-fetoprotein positive, purchase in Shandong Academy of Medical Sciences), SK-Hep-1 (alpha-fetoprotein feminine gender, one of purchase in Shanghai Inst. of Cytobiology, Chinese Academy of Sciences), the cell strain liquid nitrogen is preserved, the cultivation of going down to posterity of laboratory routine.
Described human hepatoma cell strain cultural method is: hyclone concentration is 10% cell culture fluid, 37 ℃ of CO
2The constant incubator cultivation of going down to posterity, wherein human hepatoma cell strain HepG-2, SK-Hep-1 select the DMEM-H basic culture solution for use; Bel-7402 selects the RPMI1640 basic culture solution for use.
Above-mentioned orthotopic transplantation makes up in the method for human liver cancer mouse model, and the method for the described living body fluorescent dyestuff of step [2] DiI labelling human hepatoma carcinoma cell is:
Before living body fluorescent dyestuff DiI uses with anhydrous alcohol solution to 2.0mg/ml, after the disposable filter filtration sterilization of 0.22 μ m, room temperature is airtight to keep in Dark Place, standby;
10% serum cell culture fluid is diluted to above-mentioned fluorescent dye DiI the final concentration of 20 μ g/ml, in 37 ℃ of incubators, incubate 50min～60min with the adherent human liver cancer cell of exponential phase altogether, every 10min the fluorescence dye liquor is shaken up once, hepatoma carcinoma cell is promptly by the complete labelling of fluorescent dye DiI.
Fluorescent dye DiI (1,1 ' '-Dioctadecyl-3,3,3 ' ', 3 ' '-tetramethylindocarbocyanineperchlorate) be a kind of lipotropy carbon cyanine fluorescent dye, easy embedding biomass membrane is interior and do the lateral diffusion motion in film, perhaps enters kytoplasm by pinocytosis, thus the whole cell of labelling.DiI is easy to use, to the active somatic cell avirulence, and not from the cell transfer of labelling to unlabelled cell, and fluorescence decay is slow, therefore, DiI is a kind of reliable fluorescent dye.Honig in 1986 etc. at first introduce DiI neural research, have successfully shown neuronic cell space of In vitro culture and projection.After this, DiI is widely used in the growth research of nervous pathway.But Soriano etc. at first find also labelling hepatocyte of DiI, pick out transplanted hepatocyte from liver parenchyma.Domestic less use fluorescent dye DiI labelling liver cancer cell carries out tracer study.
Above-mentioned orthotopic transplantation makes up in the method for human liver cancer mouse model, and the preparation method of the cell suspension of the described fluorescent dye DiI of step [2] labelling is:
The cell of the fluorescent dye DiI labelling behind the collection trypsinization, the centrifugal 3min of 1000r/min repeats 3 times, all uses 37 ℃ of pre-temperature basic culture solution resuspended at every turn; Through counting, after survival rate surpassed 80%, adjusting cell concentration with basic culture solution was 1.0 * 10
8Individual/ml; After fluorescence microscope exciting light 549nm observes down, determines that cell is by successful labelling, stand-by.
Above-mentioned orthotopic transplantation makes up in the method for human liver cancer mouse model, and the method that the described directly transplanting human liver cancer cell of step [3] suspension is gone in the Hepar Mus leaf is:
Clamp the neonatal rat cervical region with forefinger and middle finger, to fix the head of neonatal rat, nameless and little finger of toe is fixed mouse tail, and this moment, mouse web portion can expose to the open air fully, as seen is positioned at the bolarious liver area that is of mice thorax below;
Aseptic 30-gauge 50-μ l glass microsyringe is drawn the fluorescently-labeled human liver cancer cell suspension 30 μ l of step [2], the pin angle of going into 30 degree is thrust in the Mouse Liver leaf, go into pin degree of depth 2mm～2.5mm, human liver cancer cell is gone in the mice lobe of the liver by the external directly transplanting of mice, inject time be 2min/ only～3min/ only, go out behind the pin with the rapid flicking of cotton ball soaked in alcohol injection site 1min～2min, afterwards, give back female Mus, raise in a usual manner.
Above-mentioned orthotopic transplantation makes up in the method for human liver cancer mouse model, and the described model of step [4] is identified and adopted following identification of means:
(1) human liver cancer cell in the intravital transfer case of mice-----fluorescent dye DiI labelling technique in conjunction with the living imaging system;
(2) qualitative in the tumor-bearing mice liver of human liver cancer cell, location spike-----living body fluorescent dyestuff DiI labelling human hepatoma carcinoma cell, tumor-bearing mice hepatic tissue frozen section, fluorescence microscope; This method can directly be distinguished the hepatoma carcinoma cell and the receptor hepatocyte of transplanting, spike intuitively distribution and the survival condition of liver transplantation cancerous cell in Mouse Liver;
(3) tumor-bearing mice hepatopathy variation of science-----paraffin section HE dyeing is observed; This method is the optimal parameter of present clinical diagnosis hepatocarcinoma, can reflect the hepatic lesions state intuitively.；
(4) tumor-bearing mice serum alpha-fetoprotein (AFP) is expressed evaluation----the quantitative ELISA detection that changes; The Serum AFP positive is confirmed as diagnosis and finds the gold index of primary hepatocarcinoma (HCC).
Beneficial effect of the present invention:
Problem at existing rat liver cancer model existence, through investigation and experiment are groped for a long time, the applicant recognizes, common Kunming mouse to human similar aspect hereditism, pathology, the biology numerous characteristics, and adaptive faculty is very strong, low price and have higher breeding potential and survival rate, oneself be widely used research in fields such as pharmacology, toxicology, and the production and the calibrating of medicine, biological product.Martin Olsson etc. showed to 27 results of study that are the expression characteristic of the liver alpha-fetoprotein of mice carries out not of the same race that mice liver in the postnatal regular period still had the AFP of higher level to express in 1977; Studies show that AFP keeps the hepatocarcinoma malignancy, escape one of molecular basis of immune system attack, is the significant albumen of high expressed in mice embryonic period and the hepatocarcinoma generating process.So infer that the AFP of higher level expresses the survival that has the human liver cancer cell that is beneficial to the mouse liver transplanting in the liver of nascent neonatal rat, according to the orthotopic transplantation principle, has made up human hepatocellular carcinoma mice orthotopic transplantation model, it has the following advantages:
(1) manufacturing process is simple, the success rate height, and the animal low price obtains easily, the energy wide popularization and application;
(2) receptor of the Kunming kind neonatal rat of 3 ages in days as the lotus tumor selected in this experiment for use, this moment, mouse skin was transparent, liver weight/weight ratio is bigger, need not implement under the operating prerequisite, improved the accuracy rate of lotus tumor injection, reduced in the past because anesthesia and operation to the damage of mice body, have guaranteed laboratory animal healthy physiological status during the lotus tumor;
(3) the living imaging system in conjunction with the spike of living body fluorescent dyestuff DiI labelling technique be implanted into the intravital human liver cancer cell of mice, xenogenesis orthotopic transplantation of the present invention is positioned, qualitative investigation, directly perceived, easy, operation easily, domestic correlational study is few;
(4) implantation rate and the nude mice of hepatoma carcinoma cell in mice is approaching, and trophophase is short, it is big to bear the cancer ascites capacity, tolerance is strong, and band tumor life span prolongs more than 1 times, simultaneously than nude mice, immune development along with mice, not only can carry out real-time monitored, and can study the interaction of hepatoma carcinoma cell and mouse immune system, very truly the overall process of immunologic tolerance and antineoplastic immune in the simulating human body the growth of hepatoma carcinoma cell;
(5) simple with a kind of enforcement, the method that success rate is high, under normal immunity of organism environment, structure is striden the mice orthotopic transplantation people liver cancer model of species, for the screening of immune bio-pharmaceutical and the research of pharmacology provide even more ideal operating platform, so far still do not have relevant report, have the novelty in the theory and practice.
Description of drawings
Fig. 1. mouse-borne tumor the 7th day, the liver outside drawing,
Tumor-bearing mice abdominal cavity anatomic observation, and the tangible Lycoperdon polymorphum Vitt tumor nodule of appearance one place on the lobe of the liver (about 3cm * 2cm);
Fig. 2. human liver cancer cell is in the intravital transfer case of tumor-bearing mice,
The binding fluorescent dyes DiI of living imaging system labelling technique, the intravital human liver cancer cell of spike mice transfer case;
Fig. 3. after the lotus tumor, the occupy-place situation (* 40) of living body fluorescent dyestuff DiI spike human liver cancer cell in Mouse Liver,
A B: lotus tumor 60h frozen section shows the occupy-place situation of hepatoma carcinoma cell in liver,
C D: lotus tumor 5d, frozen section show the occupy-place situation of hepatoma carcinoma cell in liver,
E F: lotus tumor 14d frozen section shows the occupy-place situation of hepatoma carcinoma cell in liver;
Fig. 4. the inspection of tumor-bearing mice liver histopathology,
A. 5 days tumor tissues paraffin sections of lotus tumor, H-E coloration result (* 40),
B. 14 days tumor tissues paraffin sections of lotus tumor, H-E coloration result (* 40),
1.1 the hypochromatosis of tumor center position (* 400),
1.2 cellular morphology is streak (* 400),
1.3 borderline tumor is many times of nucleus (* 400) significantly;
Fig. 5 .ELISA detects the tumor-bearing mice serum alpha-fetoprotein to be changed,
Testing result shows that lotus tumor mice serum alpha-fetoprotein on the 3rd level obviously raises.
The specific embodiment
Below by implementing illustration, the present invention is described in detail further, it should be understood that described embodiment is only used for explanation rather than restriction the present invention.
The preparation of embodiment 1:DiI fluorochrome label human liver cancer cell HepG-2 suspension
1, test equipment
Fluorescence microscope (NIKON ECLIPSE TE2000-U); Cell culture incubator (SANYO, 2300TC); 0.22 μ m needle-based filter (U.S. MILLIPORE);
2, test reagent
DMEM-H liquid basic culture solution (HyClone); Top grade hyclone (HyClone); Fluorescent dye DiI (AnaSpec, Inc.); 0.25% trypsin SINGAMA) etc.
3, test method
AFP positive human hepatoma cell strain HepG-2 purchases in Shanghai Inst. of Cytobiology, Chinese Academy of Sciences, and liquid nitrogen is preserved, DMEM-H basic culture solution, the cultivation of going down to posterity of 10% hyclone, 37 ℃ of 5%CO2 constant incubators; 0.25% aseptic trypsinization goes down to posterity.
Anhydrous alcohol solution fluorescent dye DiI to 2.0mg/ml; 0.22 after the disposable filter filtration sterilization of μ m, room temperature is airtight to keep in Dark Place; Dilute the final concentration of fluorescent dye to 20 μ g/ml before using with 10% hyclone DMEM basic culture solution, and incubate 45min altogether in adherent 37 ℃ of incubators of human liver cancer cell, once every the 10min mixing.0.25% aseptic trypsinization, the human liver cancer cell behind the collection labelling, centrifugal 3 times of 1000r/min * 3min all uses 37 ℃ of pre-temperature basic culture solution resuspended at every turn; Through the trypan blue dyeing counting, after survival rate surpassed 80%, it was 1.0 * 10 that basic culture solution is adjusted cell concentration
8Individual/ml; After fluorescence microscope exciting light 549nm observes down, determines that cell is by successful labelling, stand-by.
Embodiment 2: the structure of orthotopic transplantation human liver cancer cell HepG-2 mouse model
1, test material
30-gauge 50-μ l glass microsyringe (the Shanghai glad glass apparatus of height factory); The human liver cancer cell HepG-2 suspension of DiI fluorochrome label, freezing microtome (MICROM HM 550); AO 820 microtomes (American OpticalCorporration); Paraffin (sangon) etc.;
2, experimental animal
Kind pregnant Mus in Kunming is purchased the Experimental Animal Center in Shandong university, raises in SPF clean laboratory animal room endoadaptation, and treats its production, and taking out and giving birth to back 3 age in days neonatal rats is lotus tumor receptor, and male and female all have, and average weight is 1.9g.
3, test method
1) get 3 age in days Kunming kind neonatal rats, skin of abdomen disinfects in alcohol.
2) clamp the mice cervical region with forefinger and middle finger, to fix the head of neonatal rat, firmly want light, in case mice is suffocated, nameless and little finger of toe is fixed mouse tail, make the slight palintrope of mice health, this moment, mouse web portion can expose to the open air fully, and the major part that as seen is positioned at mice thorax below is bolarious liver area.
3) get aseptic 30-gauge 50-μ l glass microsyringe and draw fluorescently-labeled HepG-2 cell suspension 30 μ 1, the pin angle of going into 30 degree is thrust in the Mouse Liver leaf, goes into pin degree of depth 2mm, and this moment, mouse liver was tender, bundle is worn easily, and causes the loss of cell suspension.
4) the human liver cancer cell suspension is injected in the mice lobe of the liver slowly, be 2min/ inject time, go out behind the pin with the rapid flicking of aseptic cotton ball soaked in alcohol injection site 1min, to prevent oozing out of cell suspension, if the injection position accurate in locating is in lobe of the liver, neonatal rat can acutely not struggle, and essentially no blood oozes out.
5) whole operation process must be guaranteed operator not directly with hands touching neonatal rat skin, to guarantee the identification of young Mus after female Mus is to the lotus tumor.
6) give back female Mus nurture, guarantee the cleaning of feeding environment, observe the survival condition of lotus tumor neonatal rat at any time.
7) behind the lotus tumor 60h, every 48h, living imaging detects once;
The tail vein is got blood, and 4 ℃ leave standstill, the next day separation of serum ,-80 ℃ of preservations are stand-by;
Get tumor-bearing mice with the intraperitoneal anesthesia of 10g/L pentobarbital sodium, anatomic observation;
Get to organize in cancer and cancer week and cook frozen section, observe down in fluorescence microscope exciting light 549nm;
Portion of tissue is fixed with the 40g/L neutral formalin, dehydration, paraffin embedding, section, hematoxylin-eosin (HE) dyeing, routine pathology histological observation under the light microscopic.
4, result of the test
1) during the lotus tumor, growth of tumor does not make significant difference to the survival condition of tumor-bearing mice, no natural death phenomenon.
2) anatomic observation, visible mice lobe of the liver have 1～2 significantly tuberosity (see figure 1) of likeness in form Semen Phaseoli, and be clear with the perienchyma interface, is canescence; The tumor surrounding tissue is normal, tumor formation rate nearly 100%.
3) living imaging shows human liver cancer cell position in Mouse Liver, does not have spontaneous transfer and disappears phenomenon generation (see figure 2).
4) the painted positive cell of DiI shows red fluorescence under exciting light 549nm, and cell is evenly painted, can not distinguish karyon and kytoplasm, and the frozen section result shows that the occupy-place of tumor cell in liver is more stable, and fluorescence intensity is not seen and obviously weakened (see figure 3).
5) the H-E check pathological section shows, tumor group is woven with complete thin film bag quilt, and boundary is clearly demarcated between the normal liver tissue, and cellular morphology is irregular, be strip, tumor nodule is arranged, and 7d begins after the lotus tumor, occurs caseous necrosis in the middle of the tumor, growth along with mice, necrotic zone does not have obvious increase, and its growth behavior is similar to hepatocarcinoma, is suitable for people's hepatocarcinoma relevant medical science and biological experiment Journal of Sex Research (see figure 4).
6) behind the lotus tumor 3d, obvious rising (see figure 5) appears in the alpha-fetoprotein level of mice serum, and the routine pathology that meets in people's hepatocarcinoma generating process is checked index and characteristics (detection sees embodiment 3 for details).
Test confirms the structure people liver cancer mouse orthotopic transplantation model that the method for the invention can be successful.
This modelling utilizes the immunosuppressive action of AFP in the animal body on normal immune system, avoided the generation of immunologic rejection substantially, has reappeared the biological characteristics of hepatoma carcinoma cell in liver ideally.
Embodiment 3:ELISA detects tumor-bearing mice serum alpha-fetoprotein concentration change
1. test material
Tumor-bearing mice serum; The alpha-fetoprotein enzyme exempt from test kit (BioCheck, Inc); Alpha-fetoprotein antibody (LABVISON)
2. test method
(1) preparation work cleaning mixture: concentrated cleaning solution 20ml/ bottle, use with 1: 20 dilution back of distilled water;
(2) application of sample: establish blank 1 hole, each 2 hole of positive and negative contrast add each 50 μ l of positive and negative contrast and 50 μ l samples to be tested respectively in respective aperture;
(3) enzyme-added: except that the blank hole, every hole adds 1 (50 μ l) enzyme labelling thing, and vibration is sealed plate hole with cellophane adhesive tape gently;
(4) incubation: 37 ℃ of water-bath 30min;
(5) washing: button removes liquid in the hole, fills with each hole with cleaning mixture, leaves standstill 20s, removes cleaning mixture, pats dry in absorbent paper after repeating 4 times;
(6) colour developing: every hole adds each 1 of substrate solution A, B, behind the shrouding that vibrates gently, puts 37 ℃ of water-baths 15 minutes;
(7) result judges: every hole adds 1 of stop buffer, detects (wavelength 450nm) each hole OD value (blank well zeroing) with microplate reader.
3. result of the test
Detect after the lotus tumor 3d mice serum alpha-fetoprotein expression (see figure 6) that obviously raises.
Embodiment 4: the structure of orthotopic transplantation human liver cancer cell SK-Hep-1 mouse model
1, test material
30-gauge 50-μ l glass microsyringe (the Shanghai glad glass apparatus of height factory); DiI fluorochrome label SK-Hep-1 hepatoma carcinoma cell suspension (0.5 * 10
6Individual/ml)
2, experimental animal
Kind pregnant Mus in Kunming is purchased the Experimental Animal Center in Shandong university, raises in SPF clean laboratory animal room endoadaptation, treats its production.
3, test method
1) get the Kunming kind neonatal rat of 3 ages in days, skin of abdomen disinfects in alcohol.
2) clamp the mice cervical region with forefinger and middle finger, to fix the head of neonatal rat, firmly want light, in case mice is suffocated, nameless and little finger of toe is fixed mouse tail, makes the slight palintrope of mice health, and this moment, mouse web portion can expose to the open air fully, as seen be positioned at most of liver area of mice thorax below, be kermesinus.
3) get aseptic 30-gauge 50-μ l glass microsyringe and draw DiI fluorochrome label people hepatocarcinoma SK-Hep-1 cell suspension 20 μ l, the pin angle of going into 30 degree is thrust, directly penetrate the mice lobe of the liver from the skin outside, going into pin degree of depth 2.5ml gets final product, this moment, mouse liver was tender, bundle is worn easily, and causes the loss of tumor cell suspension.
4) be injected into the people's hepatocarcinoma SK-Hep-1 cell suspension behind the fluorochrome label in the Mouse Liver slowly, be 2min/ inject time, go out behind the pin with the rapid flicking of aseptic cotton ball soaked in alcohol injection site 1min, to prevent oozing out of cell suspension, annotate: if the injection position accurate in locating is in lobe of the liver, neonatal rat can acutely not struggle, and essentially no blood oozes out.
5) give back female Mus nurture, guarantee the cleaning of feeding environment.
6) whole operation process please be guaranteed operator not directly with hands touching neonatal rat skin, to guarantee the identification of young Mus after female Mus is to the lotus tumor.
7) behind the lotus tumor 60h, every 48h, living imaging detects once;
The tail vein is got blood, and 4 ℃ leave standstill, the next day separation of serum ,-80 ℃ of preservations are stand-by;
Get tumor-bearing mice with the intraperitoneal anesthesia of 10g/L pentobarbital sodium, anatomic observation;
Get to organize in cancer and cancer week and cook frozen section, observe down in fluorescence microscope exciting light 549nm;
Portion of tissue is fixed with several neutral formalin of 40g, dehydration, paraffin embedding, section, hematoxylin-eosin (HE) dyeing, routine pathology histological observation under the light microscopic.
4, result of the test
Behind the lotus tumor 48h, anatomic observation, because carcinoma cell concentration is low excessively, lotus tumor success rate obviously is reduced to 0.5%, the mice of lotus tumor success, basal conditions is with embodiment 2.
Embodiment 5: the structure of orthotopic transplantation people hepatocarcinoma Bel-7402 mouse model
1, test material
30-gauge 50-μ l glass microsyringe (the Shanghai glad glass apparatus of height factory); The human liver cancer cell Bel-7402 suspension (1.0 * 10 of DiI fluorochrome label
7Individual/ml);
2, experimental animal
Kind pregnant Mus in Kunming is purchased the Experimental Animal Center in Shandong university, raises in SPF clean laboratory animal room endoadaptation, and taking out and giving birth to back 3d mice is lotus tumor receptor, and male and female all have, and average weight is 1.9g.
3, test method
1) get 3 age in days Kunming kind neonatal rats, skin of abdomen disinfects in alcohol.
2) clamp the mice cervical region with forefinger and middle finger, to fix the head of neonatal rat, firmly want light, in case mice is suffocated, nameless and little finger of toe is fixed mouse tail, make the slight palintrope of mice health, this moment, mouse web portion can expose to the open air fully, and the major part that as seen is positioned at mice thorax below is bolarious liver area.
3) get aseptic 30-gauge 50-μ l glass microsyringe and draw HepG-2 cell suspension 10 μ l, the needle angle with 30 degree directly penetrates the mice lobe of the liver from the skin outside, going into pin degree of depth 2ml gets final product, this moment, mouse liver was tender, and bundle is worn easily, and caused the loss of cell suspension.
4) cell suspension is injected in the Mouse Liver slowly, inject time be 2min/ only, go out behind the pin with the rapid flicking of aseptic cotton ball soaked in alcohol injection site 1min, to prevent oozing out of cell suspension, if the injection position accurate in locating is in lobe of the liver, neonatal rat can acutely not struggle, and essentially no blood oozes out.
5) whole operation process please be guaranteed operator not directly with hands touching neonatal rat skin, to guarantee the identification of young Mus after female Mus is to the lotus tumor.
6) give back female Mus nurture, guarantee the cleaning of feeding environment, observe the survival condition of neonatal rat at any time.
7) behind the lotus tumor 60h, every 48h, living imaging detects once;
The tail vein is got blood, and 4 ℃ leave standstill, the next day separation of serum ,-80 ℃ of preservations are stand-by;
Get tumor-bearing mice with the intraperitoneal anesthesia of 10g/L pentobarbital sodium, anatomic observation;
Get visible cancer of naked eyes and cancer week tissue freezing section, observe down in fluorescence microscope exciting light 549nm;
Portion of tissue is fixed with the 40g/L neutral formalin, dehydration, paraffin embedding, section, hematoxylin-eosin (HE) dyeing, routine pathology histological observation under the light microscopic.
4, result of the test
Because carcinoma cell suspension volume is low excessively, lotus tumor success rate obviously is reduced to 1%, and the mice basal conditions of lotus tumor success is with embodiment 2.
Claims (6)
1, a kind of orthotopic transplantation makes up the method for human liver cancer mouse model, and step is:
[1] cultivation of human hepatoma cell strain: the hepatoma cell strain routine of the choosing cultivation of going down to posterity, standby;
[2] preparation of the human liver cancer cell suspension of fluorescent dye DiI labelling: choose one of human hepatoma cell strain of above-mentioned exponential phase, under 37 ℃～38 ℃, incubate 30min～60min with 20 μ g/ml living body fluorescent dyestuff DiI altogether, afterwards, 0.25% aseptic trypsinization is with the basic culture solution collecting cell and adjust concentration of cell suspension to 0.5 * 10
6Individual/ml～1.0 * 10
9Individual/ml;
[3] orthotopic transplantation makes up human liver cancer mouse model: select Kunming kind three age in days neonatal rats for use, after the skin of abdomen sterilization, utilize aseptic 30-gauge50-μ l glass microsyringe, human liver cancer cell suspension with 10 μ l～above-mentioned DiI labelling of 50 μ l, directly transplanting is gone in the neonatal rat lobe of the liver, sets up people's liver cancer mouse orthotopic transplantation model;
[4] model is identified.
2, orthotopic transplantation makes up the method for human liver cancer mouse model according to claim 1, it is characterized in that the described human liver cancer cell of step [1] is: one of HepG-2, Bel-7402, SK-Hep-1;
Cultural method is: hyclone concentration is 10% cell culture fluid, 37 ℃ of CO
2The constant incubator cultivation of going down to posterity, wherein human hepatoma cell strain HepG-2, SK-Hep-1 select the DMEM-H basic culture solution for use; Bel-7402 selects the RPMI1640 basic culture solution for use.
3, orthotopic transplantation makes up the method for human liver cancer mouse model according to claim 1, it is characterized in that the method for the described living body fluorescent dyestuff of step [2] DiI labelling human hepatoma carcinoma cell is:
Before living body fluorescent dyestuff DiI uses with anhydrous alcohol solution to 2.0mg/ml, after the disposable filter filtration sterilization of 0.22 μ m, room temperature is airtight to keep in Dark Place, standby;
10% serum culture fluid is diluted to above-mentioned fluorescent dye DiI the final concentration of 20 μ g/ml, in 37 ℃ of incubators, incubate 45min with the adherent human liver cancer cell of exponential phase altogether, every 10min the fluorescence dye liquor is shaken up once, hepatoma carcinoma cell is promptly by the complete labelling of fluorescent dye DiI.
4, orthotopic transplantation makes up the method for human liver cancer mouse model according to claim 1, it is characterized in that the preparation method of the cell suspension of the described fluorescent dye DiI of step [2] labelling is:
The cell of the fluorescent dye DiI labelling behind the collection trypsinization, the centrifugal 3min of 1000r/min repeats 3 times, all uses 37 ℃ of pre-temperature basic culture solution resuspended at every turn; Through counting, after survival rate surpassed 80%, adjusting cell concentration with basic culture solution was 1.0 * 10
8Individual/ml; After fluorescence microscope exciting light 549nm observes down, determines that cell is by successful labelling, stand-by.
5, orthotopic transplantation makes up the method for human liver cancer mouse model according to claim 1, it is characterized in that the method that the described directly transplanting human liver cancer cell of step [3] suspension is gone into the neonatal rat lobe of the liver is:
Clamp the neonatal rat cervical region with forefinger and middle finger, to fix the head of neonatal rat, nameless and little finger of toe is fixed mouse tail, and this moment, mouse web portion can expose to the open air fully, as seen is positioned at the bolarious liver area that is of mice thorax below;
Aseptic 30-gauge 50-μ l glass microsyringe is drawn the fluorescently-labeled human liver cancer cell suspension 30 μ l of step [2], the pin angle of going into 30 degree is thrust in the Mouse Liver leaf, go into pin degree of depth 2mm～2.5mm, human liver cancer cell is gone in the mice lobe of the liver by the external directly transplanting of mice, inject time be 2min/ only～3min/ only, go out behind the pin with the rapid flicking of cotton ball soaked in alcohol injection site 1min～2min, give back female Mus afterwards, raise in a usual manner.
6, orthotopic transplantation makes up the method for human liver cancer mouse model according to claim 1, it is characterized in that the described model of step [4] is identified the following means that adopt:
(1) human liver cancer cell in the intravital transfer case of mice-----fluorescent dye DiI labelling technique in conjunction with the living imaging system;
(2) qualitative in the tumor-bearing mice liver of human liver cancer cell, location spike-----living body fluorescent dyestuff DiI labelling human hepatoma carcinoma cell, frozen section, fluorescence microscope;
(3) tumor-bearing mice hepatopathy variation of science-----paraffin section HE dyeing is observed;
(4) the tumor-bearing mice serum alpha-fetoprotein is expressed evaluation----the quantitative ELISA mensuration that changes.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102408756A (en) * | 2011-08-05 | 2012-04-11 | 中国人民解放军第四军医大学 | Method for simply preparing DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) microscopic particles and marking neurons |
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN110163195A (en) * | 2018-02-14 | 2019-08-23 | 中国医药大学附设医院 | Liver cancer divides group's prediction model, its forecasting system and liver cancer to divide group's judgment method |
| CN110876359A (en) * | 2019-06-17 | 2020-03-13 | 郑州大学第一附属医院 | Novel mouse in-situ pancreatic cancer model and establishment method thereof |
| CN117643283A (en) * | 2023-12-18 | 2024-03-05 | 广州铂晋生物科技有限公司 | Construction method and application of in-situ liver cancer eradication postoperative recurrence model |
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- 2007-01-19 CN CNA2007100132481A patent/CN101020066A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN102465113B (en) * | 2010-11-12 | 2015-04-08 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN102408756A (en) * | 2011-08-05 | 2012-04-11 | 中国人民解放军第四军医大学 | Method for simply preparing DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) microscopic particles and marking neurons |
| CN102408756B (en) * | 2011-08-05 | 2013-06-19 | 中国人民解放军第四军医大学 | Method for simply preparing DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) microscopic particles and marking neurons |
| CN110163195A (en) * | 2018-02-14 | 2019-08-23 | 中国医药大学附设医院 | Liver cancer divides group's prediction model, its forecasting system and liver cancer to divide group's judgment method |
| CN110876359A (en) * | 2019-06-17 | 2020-03-13 | 郑州大学第一附属医院 | Novel mouse in-situ pancreatic cancer model and establishment method thereof |
| CN117643283A (en) * | 2023-12-18 | 2024-03-05 | 广州铂晋生物科技有限公司 | Construction method and application of in-situ liver cancer eradication postoperative recurrence model |
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