CN108504624A - A kind of new opplication of immunodeficient mouse model - Google Patents
A kind of new opplication of immunodeficient mouse model Download PDFInfo
- Publication number
- CN108504624A CN108504624A CN201810306457.3A CN201810306457A CN108504624A CN 108504624 A CN108504624 A CN 108504624A CN 201810306457 A CN201810306457 A CN 201810306457A CN 108504624 A CN108504624 A CN 108504624A
- Authority
- CN
- China
- Prior art keywords
- cell
- mouse
- people
- human
- fah
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0679—Cells of the gastro-intestinal tract
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The present invention provides a kind of new opplication of immunodeficient mouse model, the NOD/SCID/IL2rg‑/‑/Fah‑/‑Mouse can be used for expanding human body cell in vivo.The NOD/SCID/IL2rg of the present invention‑/‑/Fah‑/‑Mouse immune defect level is high, hepar damnification induction is controllable, and rejection is not generated after transplanting human liver cell, can be used as internal amplification and the tracing in vivo model of human liver cell.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of new opplication of immunodeficient mouse model, more particularly to a kind of
NOD/SCID/IL2rg-/-/Fah-/-Mouse model expands in vivo and the application of tracing in vivo human liver cell.
Background technology
Liver is the first target organs of the maximum most important biochemical reaction internal organs of human body and drug induced injury.It has been found that close
1000 kinds of drugs are related with hepatic injury, and hepatic injury is the most common single reason for causing drug to be removed from market.In the U.S., half
Above acute hepatic failure is caused by drug, and most common drug is paracetamol (Paracetamol), i.e. paracetamol
Or acetaminophen (AAP).The Annual occurence rate of AAP correlation acute hepatic failures rises to 51% in 2003 from 28% in 1998.
In the acute hepatic failure case caused by non-AAP, antimicrobial especially anti-tubercular drug is main liver injury medicament.Although such as
This, the incidence and seriousness of drug induced hepatic injury are still underestimated significantly.According to estimates in every 100 patients for receiving drug therapy
There are about 1 to occur drug induced hepatic injury during being hospitalized.
Currently, drug needs the internal verification by experimental animal, but wherein only have before entering clinical experimental stage
8% drug can be applied to human body diseases treatment by clinical test.And drug candidate is in three phase of human clinical trial
Mortality is more than 50%, has greatly aggravated fund input and the risk of new drug development, reduces the patent medicine probability of drug.It causes
A low major reason of clinical test percent of pass is, the preclinical animal studies of inhuman kind can not Accurate Prediction drug in people
The intermediate product of internal metabolic pathway and metabolite and metabolic process acts on the latent lesion of body.To human body
In drug with notable drug induced hepatic injury, about 50% drug shows hypotoxicity or nontoxicity in zoopery.
In the phase ii clinical trial of antiviral drugs Fialuridine (Fialuridine, FIAU), there are 7 to occur sternly in 15 patients
The drug induced hepatic injury of weight, wherein there is 5 patients still dead after receiving Liver Transplantation for Treatment.The bad clinical is caused to test thing
The reason of part is a kind of albumen --- mankind's equilibrative nucleoside transport body 1 for expressing and have notable racial difference in mitochondria
(hENT1, Human Equilibrative Nucleotide Transporter 1) induces the toxicity generated very to FIAU
It is sensitive.Therefore, species specificity is one of key factor needed to be considered in new drug preclinical study.
Currently, researcher is mainly by vitro culture human body liver cell aids drug in the metabolic process of human body, assessment
Damaging action of the drug metabolism to liver.However, liver cell in vitro toxicity assessment models can not accurate recreation drug it is multiple in vivo
Miscellaneous metabolic process can not also simulate the disposition of drug access that dependence is organized in organs of living beings.Therefore it provides a kind of live body mould
Type carries out amplification human primary hepatocyte in vivo and is of great significance to metabolism of the aids drug in organs of living beings.
The method that CN 102460162A disclose amplification human liver cell in vivo, the method includes transplanting human liver cell
To immune deficiency (Rag-/-Defect, B6 mouse backgrounds) and Fah defects mouse, the mouse also has IL-1R defects or is given
IL-1R antagonists, and the liver cell is allowed to expand, however, the mouse model of the invention needs IL-1R defects or needs IL-
1R antagonists, immunity is stronger, and rejection is easy to happen after transplanting human liver cell.
Therefore, a kind of mouse that immune deficiency degree is high progress human liver cell transplanting is developed, rejection does not occur, in life
Object field of medicaments is of great significance.
Invention content
In view of the deficiencies of the prior art, the present invention provides a kind of new opplication of immunodeficient mouse model, the NOD/
SCID/IL2rg-/-/Fah-/-Mouse immune defect level is high, does not generate rejection after Transplanting Human cell, can be used for people liver
The internal amplification of cell and tracing in vivo.
For this purpose, the present invention provides following technical scheme：
In a first aspect, the present invention provides a kind of NOD/SCID/IL2rg-/-/Fah-/-Mouse model expand in vivo and/or
The application of tracer human body cell.
In the present invention, NOD/SCID/IL2rg-/-/Fah-/-Mouse model is in the higher mouse mould of existing immune deficiency degree
Type (NOD/SCID/IL2rg-/-) on the basis of, Fah genes are further knocked out by gene targeting and are obtained.With NOD/SCID/
IL2rg-/-It compares, NOD/Rag-/-/IL2rg-/-/Fah-/-Mouse immune defect level higher, the repulsive interaction to human body cell
Less.NOD/SCID/IL2rg-/-Mouse has the advantage that：
(1) complement deficiency：Complement system is the innate immune system of a polyprotein ingredient, can help antibody and gulp down
Phagocyte destroys and removes the pathogen of body, and the compound that complement protein is formed can drill on heterogenous cell film to split
Solution invasion cell, NOD/SCID/IL2rg-/-Mouse lacks functionality C5, and complement protein cannot generate；
(2) NK Cells Depletions：NK cells belong to cytotoxic cell in innate immune system, are not having the case where antibody
It can rapidly react to the cell being infected down, it is often more important that, NK cells are most important obstruction human hematopoietics
Cell is transplanted to the factor of mouse, NOD/SCID/IL2rg-/-The NK Cells Depletions of mouse largely promote human cell
Heterograft；
(3) macrophage and antigen presenting cell variation and afunction：Macrophage and antigen presenting cell are to swallow up
The important immunocyte of pathogen, antigen are derived by pathogen on the surface of antigen presenting cell, and antigen is suitable
Play an important roll in terms of the immune response of answering property, NOD/SCID/IL2rg-/-The macrophage of mouse remains many jejune
The characteristic of macrophage, function are very weak；
The above attribute makes with NOD/SCID/IL2rg-/-The mouse of genetic background is the different of idealization
Kind transplantation model, in conjunction with Fah-/-Missing, the new mouse of generation can be preferably applied to the isocellular xenogenesis of human liver and move
It plants.
In the present invention, NOD/SCID/IL2rg-/-/Fah-/-Mouse with NOD, SCID, IL2rg defect and Fah in addition to lacking
Outside falling into, the gene defect of any other type is not needed, there is very high immune deficiency degree, be that a kind of excellent cell is different
Kind transplantation model；Meanwhile NOD/SCID/IL2rg-/-/Fah-/-Mouse only need to be in NOD/SCID/IL2rg-/-Fah is knocked out on mouse
Gene can be prepared, and preparation method is simple.
Preferably, the mouse model is Fah gene expression deficient mices.
Preferably, the mouse model lacks any one in functional T cell, B cell or NK cells or at least two
The combination of kind.
Preferably, the human body cell includes any one in human liver cell, people's gastric cells, HK cells or human pneumonocyte
Kind or at least two combination, preferably human liver cell.
Preferably, the human body cell is marked with luciferase.
In the present invention, human body cell is marked by using luciferase, it can be with vivo observation human body cell in Mice Body
Migration path, realize tracing in vivo.
Second aspect, the present invention provide a kind of method of internal amplification human body cell, include the following steps：
(1) people's Primary somatic cells are implanted into NOD/SCID/IL2rg-/-/Fah-/-In Mice Body；
(2) step (1) described mouse is raised.
Preferably, a concentration of the 10 of step (1) people's Primary somatic cells4-106A/kg, such as can be 104A/kg,
105A/kg or 106A/kg, preferably 105A/kg.
Preferably, the organ of step (1) described transplanting is spleen.
Preferably, the time of step (2) described raising is 1-8 months, such as can be 1 month, 2 months, 3 months, 4
The moon, 5 months, 6 months, 7 months or 8 months, preferably 2-6 months.
Preferably, the drinking-water of step (2) described raising is added with nitisinone.
Preferably, a concentration of 5-10mg/L of the nitisinone, for example, can be 5mg/L, 5.2mg/L, 5.5mg/L,
5.8mg/L、6mg/L、6.2mg/L、6.5mg/L、6.8mg/L、7mg/L、7.2mg/L、7.5mg/L、7.8mg/L、8mg/L、
8.2mg/L, 8.5mg/L, 8.8mg/L, 9mg/L, 9.2mg/L, 9.5mg/L, 9.8mg/L or 10mg/L, preferably 7.5mg/L.
In the present invention, NOD/SCID/IL2rg-/-/Fah-/-Mouse is a kind of T cell, B cell and NK cell major defects
With Fah deficient mices, raise in SPF environment, drinking-water need to add nitisinone (Nitisinone, NTBC), and NTBC is removed
After pin, hepar damnification gradually occurs for NSIF mouse, in dead in 3-5 weeks.
Preferably, further include the steps that in-vitro separation people's Primary somatic cells before step (1).
Preferably, the in-vitro separation people Primary somatic cells use two step perfusion of clostridiopetidase A.
The third aspect, the present invention provide a kind of method of tracing in vivo human body cell, include the following steps：
(1 ') co-cultures people's Primary somatic cells and luciferase slow virus, obtains the primary body of people for being marked with luciferase
Cell；
Step (1 ') people's Primary somatic cells are implanted into NOD/SCID/IL2rg by (2 ')-/-/Fah-/-In Mice Body, and
It is raised；
(3 ') injected fluorescein zymolyte carries out tracing in vivo detection.
Preferably, the preparation method of step (1 ') the luciferase slow virus includes the following steps：
(1 ") is by the mixed liquor of plasmid pWPXLD-LUC-2A-GFP, psPAX.2 and pMD2G and polyetherimide mixing；
(2 ") it is added in mammalian cell, virus is collected after culture.
In the present invention, the bis- mark live bodies of pWPXLD-LUC-2A-GFP are transfected into mammalian cell using polyetherimide (PEI)
It is imaged plasmid, packaging plasmid is psPAX.2 and pMD2G.
Preferably, the mass ratio of step (1 ") described pWPXLD-LUC-2A-GFP, psPAX.2 and pMD2G is (1-5):
(1-5):1, such as can be 1:1:1、1:2:1、1:3:1、1:4:1、1:5:1、2:1:1、2:2:1、2:3:1、2:4:1、2:5:
1、3:1:1、3:2:1、3:3:1、3:4:1、3:5:1、4:1:1、4:2:1、4:3:1、4:4:1、4:5:1、5:1:1、5:2:1、5:
3:1、5:4:1 or 5:5:1, preferably 2:2:1.
Preferably, the volume ratio of step (the 1 ") mixed liquor and the polyetherimide is (1-5):1, such as can be
1:1、2:1、3:1、4:1 or 5:1, preferably 3:1.
Preferably, step (the 2 ") mammalian cell is 293T cells.
Preferably, a concentration of the 10 of step (2 ') people's Primary somatic cells4-106A/kg, such as can be 104A/
kg、105A/kg or 106A/kg, preferably 105A/kg.
Preferably, the organ of step (the 2 ') transplanting is spleen.
Preferably, the time of step (the 2 ') raising is 1-8 months, for example, can be 1 month, 2 months, 3 months, 4
A month, 5 months, 6 months, 7 months or 8 months, preferably 2-6 months.
Preferably, the drinking-water of step (the 2 ') raising is added with nitisinone.
Preferably, a concentration of 5-10mg/L of the nitisinone, for example, can be 5mg/L, 5.2mg/L, 5.5mg/L,
5.8mg/L、6mg/L、6.2mg/L、6.5mg/L、6.8mg/L、7mg/L、7.2mg/L、7.5mg/L、7.8mg/L、8mg/L、
8.2mg/L, 8.5mg/L, 8.8mg/L, 9mg/L, 9.2mg/L, 9.5mg/L, 9.8mg/L or 10mg/L, preferably 7.5mg/L.
Preferably, a concentration of 100-200mg/kg of step (the 3 ') luciferase substrate, such as can be 100mg/
kg、110mg/kg、120mg/kg、130mg/kg、140mg/kg、150mg/kg、160mg/kg、170mg/kg、180mg/kg、
190mg/kg or 200mg/kg, preferably 150mg/kg.
Fourth aspect, the present invention provide a kind of method expanding human body cell in vivo as described in second aspect and/or such as the
The method of the three aspect tracing in vivo human body cells is probing into application of the small-molecule drug in body metabolism approach.
Compared with prior art, the present invention has the advantages that：
(1) NOD/SCID/IL2rg of the invention-/-/Fah-/-Mouse immune defect level is high, hepar damnification induction is controllable,
Rejection is not generated after transplanting human liver cell, can be used as the internal amplification model of human liver cell；
(2) after transplanting human liver cell, by feeding 7.5mg/L nitisinones, NOD/SCID/IL2rg is improved-/-/
Fah-/-The existence ratio of mouse, human albumin, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease content and mouse liver in mice serum
The expression quantity of middle people Alu genes is significantly increased；
(3) after transplanting human liver cell, NOD/SCID/IL2rg-/-/Fah-/-The quantity of human liver cell is notable in mouse liver
It improves；
(4) by NOD/SCID/IL2rg-/-/Fah-/-Mouse transplants the human liver cell for being marked with luciferase, realizes
Tracer of the human liver cell in Mice Body, to probe into metabolic pathway of the small-molecule drug in people's liver provides model.
Description of the drawings
Fig. 1 is the survivorship curve for transplanting the NSIF mouse after human liver cell；
Fig. 2 be transplant human liver cell after mice serum in human albumin content；
Fig. 3 be transplant human liver cell after mice serum in glutamic-pyruvic transaminase content；
Fig. 4 be transplant human liver cell after mice serum in glutamic-oxalacetic transaminease content；
Expression of Fig. 5 behaviour Alu genes in transplanting mouse liver；
Fig. 6 is the fluidic cell figure for the ratio that human liver cell accounts for mouse liver cell；
Fig. 7 is morphology colored graph of the human liver cell in mouse liver；
Fig. 8 is that immunohistochemical staining shows human liver cell specific markers FAH；
Fig. 9 is that immunohistochemical staining shows human liver cell specific markers CK-8/18；
Figure 10 is that immunohistochemical staining shows human liver cell specific markers human albumin；
Figure 11 is that luciferase reporter gene marks human liver cell and flow cytometer detection；
Figure 12 is that living imaging shows that the human liver cell of luciferase label specifically moves to mouse liver；
Figure 13 is after transplanting human liver cell, and human albumin secretion's amount is positively correlated with fluorescence intensity in mice serum；
Figure 14 is that testing result is metabolized in debrisoquine body.
Specific implementation mode
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, according to technology or condition described in document in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
Amplification in 1 human primary hepatocyte body of embodiment
(1) people's primary cell in-vitro separation
The primary liver parenchyma tissue of people of fresh donor is detached using two step perfusion of clostridiopetidase A, specific method is：Sterile item
PBS under part by fresh liver tissue first using addition bis- (the 2- amino-ethyls ether) tetraacethyls (EGTA) of 0.4mM ethylene glycol is slow
15 minutes (flow velocity 5mL/min) is perfused in fliud flushing；Then using addition 0.1mg/mL clostridiopetidase A IV and 2mM Ca2+PBS buffering
Liquid continues that 15 minutes (flow velocity 2mL/min) is perfused；After digestion fully, liver cell is gently shaken with ophthalmic tweezers, discharges single liver
Dirty cell does not digest complete tissue and continues digestion 20 minutes using 0.1mg/mL clostridiopetidase As IV, obtains a large amount of liver cells；It will be upper
It states cell and adds the DMEM culture mediums of 10% fetal calf serum with 50g rotating speeds centrifugation 3 times, every time 3 minutes；It is used for after cell count
Mouse is transplanted.
(2)NOD/SCID/IL2rg-/-/Fah-/-(NSIF) Mouse feeder
NSIF mouse are a kind of T cell, B cell and NK cells major defect and Fah deficient mices, are raised in SPF
In environment, drinking-water addition 7.5mg/L nitisinones (Nitisinone, NTBC), after NTBC revocations, liver gradually occurs for NSIF mouse
Dirty damage, in dead in 3-5 weeks.
(3) human liver cell transplants NSIF mouse
It transplants human liver cell the last week, does not add NTBC in mouse drinking-water, the human liver cell of separation is implanted into 24
Mouse specially injects 10 into the mouse spleen of anesthesia5-107The human liver cell (Trypan Blue exclusion dead cell) of a work,
Weight loss drinks water after weight recovery to NTBC drinking-water is restored when 20% one week and does not add NTBC after transplanting.
After mouse is rebuild, as shown in Figure 1, the weight gain of mouse, survival rate improves.
Eyeball of mouse blood sampling about 100mL, after being placed at room temperature for 2h, 1000g centrifuges 5min, careful collection upper serum, detection
The content of human albumin (hALB), glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminease (AST) in serum.
As shown in Fig. 2, human albumin content is gradually increasing in 12 weeks in serum；As shown in Figure 3-4, glutamic-pyruvic transaminase and
Glutamic-oxalacetic transaminease content is significantly improved compared with control group mice.
As shown in figure 5, by detecting expression of the Alu genes in transplanting mouse liver, the peripheral blood of mouse is found
Immune system is rebuild in order.
Embodiment 2 transplants the mouse liver detection after human liver cell
(1) flow cytometer detection
Take mouse liver tissue be added antibody blocking receptor, under the conditions of being protected from light be added fluorescent labeled antibody HLA-A/B/C and
MHC-I carries out flow cytometer detection after label afterwards twice with PBS buffer solution flushing.
The results are shown in Figure 6, and after stopping addition NTBC, the ratio of human liver cell obviously rises in mouse liver cell.
(2) H＆E is dyed
Mouse liver internal organs are placed in neutral formic acid solution and are fixed overnight, are dehydrated using the ethanol solution of increasing concen-trations,
Waxdip is handled after bleach in dimethylbenzene, and embedding forms the suitable wax stone of size；Tissue 4-6 μm is cut into slicer to cut
After piece, dewaxing and hydration process are carried out；Slice after aquation is rinsed through hematoxylin dyeing, flowing water flushing, eosin stains, flowing water
Afterwards, using dehydration bleach, neutral gum mounting dries；Photographic analysis under microscope.
The results are shown in Figure 7, and human liver cell is distributed in the liver of mouse, keeps normal cellular morphology.
(3) immunofluorescence
Mouse liver tissue, which is used, to be dyed identical step with H＆E and is fixed, is dehydrated, embeds, is sliced, dewaxes and aquation
Afterwards, antigen retrieval is carried out with citrate solution as needed, removes endogenous peroxydase；PBS is carried out after cleaning twice
BSA is closed, and is incubated at room temperature 30min, washes away extra confining liquid, and the primary antibody diluted is added dropwise, and is incubated at room temperature 1h；PBS cleanings 3
Time, add fluorescent marker secondary antibody, be incubated at room temperature 1h, PBS is cleaned 3 times, dropwise addition neutral gum mounting, photographic analysis under microscope.
As a result as seen in figs. 8-10, human liver cell specific markers FAH, CK-8/18 and the white egg of people are expressed in mouse liver
In vain.
3 human primary hepatocyte tracing in vivo of embodiment
(1) Tissue Culture Dish is pre-coated
Using the pre-coated culture dish of 0.1% gelatin (Gelatin) or culture plate, specially：1% gelatin of 1mL is dissolved in
It in 10mL DMEM culture mediums, is added in 60mm culture dishes, gently shaking makes liquid cover entire ware bottom, is placed in 37 DEG C of trainings
4h in case is supported, culture dish is taken out, sucks extra supernatant, DMEM culture mediums are added and clean 2 times, it is spare.
(2) human primary hepatocyte culture
Donor liver cell is detached, adjustment cell concentration is 5 × 105/cm2, it is inoculated in the coated 60mm culture dishes of Gelatin
In, culture medium is the DMEM culture mediums for adding the dual anti-+ 10nM/mL hepatocyte growth factor (HGF) of 10%FBS+1%；It is placed in 5%
CO2After cultivating 4h in 37 DEG C of incubators, the non-attached cell in fresh culture removal part is replaced；Replace fresh cultured within every 2 days
Base.
Human primary hepatocyte cannot pass on, in vitro culture about 14 days.Utilize the slow-virus transfection of expressing luciferase gene
Liver cell carries out the fluorescent marker of human primary hepatocyte.
(3) luciferase slow virus is packed
By 293T cells with 2 × 105The density of a mL passes in the culture dish of 100mm, is trained with the DMEM containing 10%FBS
Nutrient solution culture, in 5%CO under 37 degree2In incubator overnight, make growth to 80% degree of converging；By plasmid pWPXLD-LUC-2A-
GFP (10 μ g), psPAX.2 (10 μ g) and pMD2G (5 μ g) are uniformly mixed with serum-free without dual anti-a-MEM, and room temperature is static
5min is added the polyetherimide (PEI) of mixed liquor one third volume, 20min is stored at room temperature after mixing；293T is added dropwise
In Tissue Culture Dish, it is gently mixed uniformly, in 5%CO at 37 DEG C26h is cultivated in incubator；DMEM culture solutions are changed to continue to train
It supports, observes green fluorescence expression after transfection for 24 hours, hour collection is viral after 24,48 and 72.
(4) luciferase label human liver cell transplanting NSIF mouse and tracing in vivo
NSIF mouse peritoneals are injected 1% amobarbital and are anaesthetized, and fixed mouse is in prone position, in the middle part of back side, left side
It between midaxillary line and posterior axillary line, takes 5mm notch into abdomen, appears spleen, and pull out outside notch, with insulin needle syringe needle in pole on spleen
Inserting needle, depth about 1.5cm are slowly injected into luciferase label human liver cell (5 × 105)；Visible injection during being slowly injected into
The spleen envelope at position becomes bletilla swelling, extracts syringe needle, and a moment is clamped at pinprick, checks no bleeding, spleen is put back to original position,
Whole layer suture stomach wall, postoperative continuation are raised under the conditions of SPF, daily close observation；10min before living imaging, to mouse peritoneal
150mg/kg luciferase substrates are injected, then are anaesthetized with isoflurane, is placed in IVIS Spectrum operation consoles and carries out active inspection
It surveys.
As shown in figure 11, the infection proportion of the human liver cell of luciferase label reaches 53.6%.
As shown in figure 12, the human liver cell of luciferase label specifically moves to mouse liver portion.
As shown in figure 13, after transplanting human liver cell, the secretory volume Yu fluorescence intensity of human albumin are at positive in mice serum
It closes.
The in vivo functionality of 4 people's primary cell of embodiment is verified
After transplanting 8 to 12 week of human liver cell, is taken orally into NSIF mouse or vena gastrica injects 2mg/kg debrisoquines (DEB),
Blood sample, centrifugal separation plasma are acquired after 1,2,4 and 8h；Pass through HPLC system quantitative analysis 4-OH DEB.
As shown in figure 14, content of the specific metabolic drug debrisoquine in humanization hepatic model reaches after injecting 2 hours
To peak, it is reduced to minimum after injecting 8 hours, illustrates that humanization hepatic model has stronger metabolic energy to debrisoquine
Power.
In conclusion the NOD/SCID/IL2rg of the present invention-/-/Fah-/-Mouse immune defect level is high, hepar damnification lures
It leads controllably, does not generate rejection after transplanting human liver cell, can be used as the internal amplification model of human liver cell；Transplant human liver cell
Afterwards, by feeding nitisinone, NOD/SCID/IL2rg is improved-/-/Fah-/-The existence ratio of mouse, people is white in mice serum
The expression quantity of people's Alu genes is significantly increased in albumen, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease content and mouse liver, mouse
The quantity of human liver cell significantly improves in liver；To NOD/SCID/IL2rg-/-/Fah-/-Mouse transplanting is marked with luciferase
Human liver cell realizes tracer of the human liver cell in Mice Body, to probe into metabolic pathway of the small-molecule drug in people's liver
Provide model.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.
Claims (10)
1. a kind of NOD/SCID/IL2rg-/-/Fah-/-Mouse model expands in vivo and/or the application of tracer human body cell.
2. application according to claim 1, which is characterized in that the mouse model is Fah gene expression deficient mices；
Preferably, the mouse model lacks any one in functional T cell, B cell or NK cells or at least two
Combination.
3. application according to claim 1 or 2, which is characterized in that the human body cell includes that human liver cell, people's stomach are thin
In born of the same parents, HK cells or human pneumonocyte any one or at least two combination, preferably human liver cell；
Preferably, the human body cell is marked with luciferase.
4. a kind of method of internal amplification human body cell, which is characterized in that include the following steps：
(1) people's Primary somatic cells are implanted into NOD/SCID/IL2rg-/-/Fah-/-In Mice Body；
(2) step (1) described mouse is raised；
Preferably, a concentration of the 10 of step (1) people's Primary somatic cells4-106A/kg, preferably 5 × 105A/kg；
Preferably, the organ of step (1) described transplanting is spleen；
Preferably, the time of step (2) described raising is 1-8 months, preferably 2-6 months；
Preferably, the drinking-water of step (2) described raising is added with nitisinone；
Preferably, a concentration of 5-10mg/L of the nitisinone, preferably 7.5mg/L.
5. according to the method described in claim 4, it is characterized in that, further including the primary body of in-vitro separation people before step (1)
The step of cell；
Preferably, the in-vitro separation people Primary somatic cells use two step perfusion of clostridiopetidase A.
6. a kind of method of tracing in vivo human body cell, which is characterized in that include the following steps：
(1 ') co-cultures people's Primary somatic cells and luciferase slow virus, and the primary body of people for obtaining being marked with luciferase is thin
Born of the same parents；
Step (1 ') people's Primary somatic cells are implanted into NOD/SCID/IL2rg by (2 ')-/-/Fah-/-In Mice Body, and carry out
Raising；
(3 ') injected fluorescein zymolyte carries out tracing in vivo detection.
7. according to the method described in claim 6, it is characterized in that, the preparation method of step (1 ') the luciferase slow virus
Include the following steps：
(1 ") is by the mixed liquor of plasmid pWPXLD-LUC-2A-GFP, psPAX.2 and pMD2G and polyetherimide mixing；
(2 ") it is added in mammalian cell, virus is collected after culture；
Preferably, the mass ratio of step (1 ") described pWPXLD-LUC-2A-GFP, psPAX.2 and pMD2G is (1-5):(1-5):
1, preferably 2:2:1；
Preferably, the volume ratio of step (the 1 ") mixed liquor and the polyetherimide is (1-5):1, preferably 3:1；
Preferably, step (the 2 ") mammalian cell is 293T cells.
8. the method described according to claim 6 or 7, which is characterized in that step (2 ') people's Primary somatic cells it is a concentration of
104-106A/kg, preferably 5 × 105A/kg；
Preferably, the organ of step (the 2 ') transplanting is spleen；
Preferably, the time of step (the 2 ') raising is 1-8 months, preferably 2-6 months；
Preferably, the drinking-water of step (the 2 ') raising is added with nitisinone；
Preferably, a concentration of 5-10mg/L of the nitisinone, preferably 7.5mg/L.
9. according to claim 6-8 any one of them methods, which is characterized in that step (the 3 ') luciferase substrate it is dense
Degree is 100-200mg/kg, preferably 150mg/kg.
10. a kind of method expanding human body cell in vivo as described in claim 4 or 5 and/or such as any one of claim 6-9 institutes
The method for stating tracing in vivo human body cell is probing into application of the small-molecule drug in body metabolism approach.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810306457.3A CN108504624A (en) | 2018-04-08 | 2018-04-08 | A kind of new opplication of immunodeficient mouse model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810306457.3A CN108504624A (en) | 2018-04-08 | 2018-04-08 | A kind of new opplication of immunodeficient mouse model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108504624A true CN108504624A (en) | 2018-09-07 |
Family
ID=63380926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810306457.3A Pending CN108504624A (en) | 2018-04-08 | 2018-04-08 | A kind of new opplication of immunodeficient mouse model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108504624A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111696A1 (en) * | 2020-11-30 | 2022-06-02 | 江苏大学 | Maintenance of programmed chronic liver injury of fah gene-deficient animals and application thereof in preparation of heterologous liver models |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809156A (en) * | 2007-06-05 | 2010-08-18 | 俄勒冈健康科学大学 | The method of expanding human hepatocytes in vivo |
| CN102281758A (en) * | 2009-01-16 | 2011-12-14 | 公益财团法人实验动物中央研究所 | Mouse having human hepatocytes transplanted therein |
| CN102550486A (en) * | 2012-01-10 | 2012-07-11 | 浙江大学 | Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice |
| CN106661593A (en) * | 2016-02-25 | 2017-05-10 | 深圳市体内生物医药科技有限公司 | Immunodeficient mice, manufacturing method thereof and application |
| CN107137425A (en) * | 2017-05-22 | 2017-09-08 | 深圳市体内生物医药科技有限公司 | A kind of circulating tumor cell mouse model, its construction method and application |
-
2018
- 2018-04-08 CN CN201810306457.3A patent/CN108504624A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809156A (en) * | 2007-06-05 | 2010-08-18 | 俄勒冈健康科学大学 | The method of expanding human hepatocytes in vivo |
| CN102281758A (en) * | 2009-01-16 | 2011-12-14 | 公益财团法人实验动物中央研究所 | Mouse having human hepatocytes transplanted therein |
| CN102550486A (en) * | 2012-01-10 | 2012-07-11 | 浙江大学 | Method for establishing xenogeneic graft-versus-host disease model for NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice |
| CN106661593A (en) * | 2016-02-25 | 2017-05-10 | 深圳市体内生物医药科技有限公司 | Immunodeficient mice, manufacturing method thereof and application |
| CN107119076A (en) * | 2016-02-25 | 2017-09-01 | 深圳市体内生物医药科技有限公司 | A kind of immunodeficient mouse model, its preparation method and application |
| CN107137425A (en) * | 2017-05-22 | 2017-09-08 | 深圳市体内生物医药科技有限公司 | A kind of circulating tumor cell mouse model, its construction method and application |
Non-Patent Citations (6)
| Title |
|---|
| AZUMA H ET AL.: "Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice", 《NATURE BIOTECHNOLOGY》 * |
| EVA RAMBOER ET AL.: "Strategies for immortalization of primary hepatocytes", 《JOURNAL OF HEPATOLOGY》 * |
| LI F ET AL.: "Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology", 《SCIENTIFIC REPORTS》 * |
| ZHOU QJ ET AL.: "Humanized NOD-SCID IL2rg-/- mice as a preclinical model for cancer research and its potential use for individualized cancer therapies", 《CANCER LETTERS》 * |
| 房敬超等: "人脐带组织间充质干细胞对小鼠肿瘤的趋化作用", 《山东医药》 * |
| 苏宝亮等: "人成体肝细胞在Fah-/-Nod/Scid 小鼠肝脏中的显著增殖", 《中国科学：生命科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022111696A1 (en) * | 2020-11-30 | 2022-06-02 | 江苏大学 | Maintenance of programmed chronic liver injury of fah gene-deficient animals and application thereof in preparation of heterologous liver models |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Haldi et al. | Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish | |
| Kunzi-Rapp et al. | Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model | |
| CN101827933B (en) | Selective cell therapy for the treatment of renal failure | |
| KR100760367B1 (en) | In ovo activation of an avian egg in the shell | |
| CN103212071B (en) | Stem cell fusion model of carcinogenesis | |
| CN110402853B (en) | Detection model for tumor killing ability of immune cells and construction method and application thereof | |
| CN108779440A (en) | Improved adult liver progenitor prepared product | |
| CN101842105A (en) | Progenitor cells from urine and methods for using the same | |
| US6093872A (en) | Extended human hematopoiesis in a heterologous host | |
| USRE39337E1 (en) | Nude mouse model for human neoplastic disease | |
| CN102715132A (en) | Porcine reproductive and respiratory syndrome virus receptor CD163 knock-out swine and cultivation method thereof | |
| CN103509751B (en) | Co-culturing method of human primary hepatocytes and liver nonparenchymal cells | |
| Sanford et al. | Clonal analysis of variant cell lines transformed to malignant cells in tissue culture | |
| Ohshita et al. | Propagation of human hepatocytes in uPA/SCID mice: producing chimeric mice with humanized liver | |
| Naito et al. | In vivo selection of human renal cell carcinoma cells with high metastatic potential in nude mice | |
| Uloza et al. | Effect of laryngeal squamous cell carcinoma tissue implantation on the chick embryo chorioallantoic membrane: morphometric measurements and vascularity | |
| Chen et al. | Human liver cancer organoids: Biological applications, current challenges, and prospects in hepatoma therapy | |
| CN114717190A (en) | Human breast malignant phylliform tumor cell line BPT0713 and application thereof | |
| CN108504624A (en) | A kind of new opplication of immunodeficient mouse model | |
| CN109136273A (en) | Prepare the method and its application of the rat of immune deficiency | |
| Albani et al. | Development of a cell line from Echinococcus granulosus germinal layer | |
| US20240108590A1 (en) | Maintenance of programmed chronic liver injury of fah gene-deficient animals and application thereof in preparation of heterologous liver models | |
| CN107460170B (en) | Establishment and application of human pituitary adenoma cell line | |
| Heydarian et al. | Tissue models for Neisseria gonorrhoeae research—from 2D to 3D | |
| Squartini et al. | Reciprocal interference between mouse mammary tumour virus and leukaemia virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190314 Address after: 410007 402 houses of Zhongnan headquarters base 688, two, 688 Wan Li South Road, Yuhua District, Changsha, Hunan Applicant after: Hunan Zhao Tai Biological Medicine Co., Ltd. Address before: 518102 5th Floor, Five Blocks, Hongwan Industrial Zone, Gushi Community, Xixiang Street, Baoan District, Shenzhen City, Guangdong Province Applicant before: Shenzhen in vivo biological medicine technology Co., Ltd. |
|
| TA01 | Transfer of patent application right |