CN117814181A - Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain - Google Patents
Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain Download PDFInfo
- Publication number
- CN117814181A CN117814181A CN202311777915.9A CN202311777915A CN117814181A CN 117814181 A CN117814181 A CN 117814181A CN 202311777915 A CN202311777915 A CN 202311777915A CN 117814181 A CN117814181 A CN 117814181A
- Authority
- CN
- China
- Prior art keywords
- cells
- liver
- mice
- rfp
- injection
- 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
- 241000282414 Homo sapiens Species 0.000 title claims abstract description 23
- 210000004024 hepatic stellate cell Anatomy 0.000 title claims abstract description 21
- 238000010276 construction Methods 0.000 title claims abstract description 17
- 238000010172 mouse model Methods 0.000 title claims abstract description 11
- 206010019668 Hepatic fibrosis Diseases 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 82
- 241000699670 Mus sp. Species 0.000 claims abstract description 44
- 210000004185 liver Anatomy 0.000 claims abstract description 35
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 16
- 210000000952 spleen Anatomy 0.000 claims abstract description 13
- 238000010171 animal model Methods 0.000 claims abstract description 12
- 230000035755 proliferation Effects 0.000 claims abstract description 11
- 238000002474 experimental method Methods 0.000 claims abstract description 10
- 238000001727 in vivo Methods 0.000 claims abstract description 9
- 108060001084 Luciferase Proteins 0.000 claims abstract description 7
- 239000005089 Luciferase Substances 0.000 claims abstract description 7
- 108010054624 red fluorescent protein Proteins 0.000 claims abstract description 7
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 5
- 206010061598 Immunodeficiency Diseases 0.000 claims abstract description 4
- 208000029462 Immunodeficiency disease Diseases 0.000 claims abstract description 4
- 238000001815 biotherapy Methods 0.000 claims abstract description 4
- 238000002651 drug therapy Methods 0.000 claims abstract description 4
- 230000007813 immunodeficiency Effects 0.000 claims abstract description 4
- 208000019425 cirrhosis of liver Diseases 0.000 claims description 30
- 239000007924 injection Substances 0.000 claims description 28
- 238000002347 injection Methods 0.000 claims description 28
- QPNKYNYIKKVVQB-UHFFFAOYSA-N crotaleschenine Natural products O1C(=O)C(C)C(C)C(C)(O)C(=O)OCC2=CCN3C2C1CC3 QPNKYNYIKKVVQB-UHFFFAOYSA-N 0.000 claims description 20
- QVCMHGGNRFRMAD-XFGHUUIASA-N monocrotaline Chemical compound C1OC(=O)[C@](C)(O)[C@@](O)(C)[C@@H](C)C(=O)O[C@@H]2CCN3[C@@H]2C1=CC3 QVCMHGGNRFRMAD-XFGHUUIASA-N 0.000 claims description 20
- QVCMHGGNRFRMAD-UHFFFAOYSA-N monocrotaline Natural products C1OC(=O)C(C)(O)C(O)(C)C(C)C(=O)OC2CCN3C2C1=CC3 QVCMHGGNRFRMAD-UHFFFAOYSA-N 0.000 claims description 20
- 210000005228 liver tissue Anatomy 0.000 claims description 15
- 241000700605 Viruses Species 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 8
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000002285 corn oil Substances 0.000 claims description 7
- 235000005687 corn oil Nutrition 0.000 claims description 7
- 230000001575 pathological effect Effects 0.000 claims description 7
- 238000002054 transplantation Methods 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 6
- 239000007928 intraperitoneal injection Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 238000001890 transfection Methods 0.000 claims description 5
- 230000037396 body weight Effects 0.000 claims description 4
- 229950010131 puromycin Drugs 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 241000713666 Lentivirus Species 0.000 claims description 3
- 108700008625 Reporter Genes Proteins 0.000 claims description 3
- 230000006378 damage Effects 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000012737 fresh medium Substances 0.000 claims description 3
- 238000011503 in vivo imaging Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 238000005199 ultracentrifugation Methods 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 2
- 210000001130 astrocyte Anatomy 0.000 claims description 2
- 238000011156 evaluation Methods 0.000 claims description 2
- 238000010859 live-cell imaging Methods 0.000 claims description 2
- 230000007246 mechanism Effects 0.000 claims description 2
- 230000000144 pharmacologic effect Effects 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 239000012096 transfection reagent Substances 0.000 claims description 2
- 206010067125 Liver injury Diseases 0.000 abstract description 9
- 231100000753 hepatic injury Toxicity 0.000 abstract description 9
- 238000002513 implantation Methods 0.000 abstract description 2
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- 238000007912 intraperitoneal administration Methods 0.000 abstract 1
- 230000029918 bioluminescence Effects 0.000 description 6
- 238000005415 bioluminescence Methods 0.000 description 6
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000004761 fibrosis Effects 0.000 description 4
- UMFJAHHVKNCGLG-UHFFFAOYSA-N n-Nitrosodimethylamine Chemical compound CN(C)N=O UMFJAHHVKNCGLG-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- 229940096422 collagen type i Drugs 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002440 hepatic effect Effects 0.000 description 3
- 238000010874 in vitro model Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- WBNQDOYYEUMPFS-UHFFFAOYSA-N N-nitrosodiethylamine Chemical compound CCN(CC)N=O WBNQDOYYEUMPFS-UHFFFAOYSA-N 0.000 description 2
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 2
- 210000003995 blood forming stem cell Anatomy 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011563 immunodeficient animal model Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 206010019670 Hepatic function abnormal Diseases 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229960003471 retinol Drugs 0.000 description 1
- 235000020944 retinol Nutrition 0.000 description 1
- 239000011607 retinol Substances 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 1
- 229950004616 tribromoethanol Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Abstract
The invention belongs to the technical field of animal model construction, and provides a construction method and application of a hepatic fibrosis mouse model based on a human hepatic stellate cell strain, aiming at the problem of hepatic fibrosis animal model caused by lack of human hepatic stellate cells. The invention takes an immunodeficiency mouse as an animal model, uses luciferase and LX-2 marked by red fluorescent protein, and uses the MCT to be injected intraperitoneally to prepare a liver injury model of the mouse, and LX-II is injected into the spleen after liver injury2, transplanting the humanized LX-2 cells into NSG mice, and then injecting CCl into the intraperitoneal of the mice 4 . By this method, the implantation, distribution, proliferation and hepatic fibrosis of transplanted LX-2 in the liver were successfully studied. The model is suitable for in vivo experiments of therapeutic methods such as biological therapy and drug therapy of targeted hepatic stellate cells or fibroblasts.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a liver fibrosis mouse model.
Background
Liver fibrosis is characterized by excessive accumulation of fibrous connective tissue in the liver, resulting in impaired liver function. Hepatic stellate cells (hepatic stellate cells, HSCs) are considered to be major contributors to liver fibrosis. Following liver injury, HSCs migrate to the site of injury and become live, producing excessive extracellular matrix (extracellular matrix, ECM) components, ultimately leading to liver fibrosis.
Current in vivo studies in mice directed to liver fibrosis have relied primarily on exogenously added carbon tetrachloride (CCl 4 ) Chemical inducers such as Dimethylnitrosamine (DMNA), D-galactosamine (DGA) and Diethylnitrosamine (DEN) are used for constructing liver fibrosis models of mice. By CCl 4 The induced liver fibrosis model is simple and easy, but has higher death rate and CCl 4 The liver fibrosis state caused by isochemical induction is difficult to simulate the occurrence and development states of fibrosis diseases, and the mouse fibrosis model is difficult to simulate the liver fibrosis disease progress of human beings due to the difference of species of mice and human beings. LX-2 is a primary human hepatic stellate cell strain capable of serial passage by SV40 large T antigen treatment. Xu et al established a serial-passaged LX-2 cell line and provided a source of stably proliferating human hepatic stellate cells (Xu L, hui AY, albanis E, arthur MJ, O' Byrne SM, blancer WS, mukherjee P, friedman SL, eng FJ.Human hepatic stellate cell lines, LX-1and LX-2:new tools for analysis of hepatic fibrosis.Gut.2005Jan;54 (1): 142-51). LX-2 retains key characteristics of liver astrocyte cytokine signal, nerve gene expression, retinol metabolism and fibrosis, and is very suitable for useCulture-based studies of human liver fibrosis were performed in combination. Because of the difficulty in the field planting of humanized cells in other animal models, the survival rate is low, and the current research on humanized liver fibrosis models is mainly in vitro models, for example, patent document CN109337860a discloses a method for constructing a liver fibrosis 3D model in vitro, single cells of hepatic parenchyma cells, single cells of hepatic stellate cells and single cells of cumic cells are inoculated on a 3D culture medium for co-culture, and a liver fibrosis inducer is added into the obtained 3D co-cultured cells for induction to obtain the liver fibrosis 3D model. Although such in vitro models can be constructed based on humanized cells, in vitro models cannot mimic complex conditions in an organism, for example, changes in the levels of fluids in an organism, changes in metabolites and in vivo environments can result in changes in the state of cell viability.
Although it has been found that an immunodeficient animal model is more suitable for human cell transplantation, the inventors have found during the course of the present invention that the selection of an immunodeficient animal model alone does not actually result in the desired engraftment rate and survival rate, and that human hepatic stellate cells are present in an immunodeficient mouse,
thus, there is a need to construct a humanized liver fibrosis mouse model for better scientific research.
Disclosure of Invention
Aiming at the problems that a mouse with liver fibers of a murine origin cannot fully meet scientific research requirements and a liver fibrosis animal model caused by human hepatic stellate cells is lacking at present, the invention provides a construction method of the liver fibrosis mouse model based on a human hepatic stellate cell strain. The invention takes an immunodeficiency mouse as an animal model, uses LX-2 marked by Luciferase (LUC) and Red Fluorescent Protein (RFP) to inject MCT intraperitoneally to prepare a liver injury model of the mouse, and uses LX-2 injected from spleen after liver injury to transplant human LX-2 cells into NSG mice, and then performs intraperitoneally injection CCl of the mice 4 By this method, the implantation, distribution, proliferation and hepatic fibrosis of transplanted LX-2 in the liver were successfully studied.
The invention is realized by the following technical scheme:
a method for constructing a hepatic fibrosis mouse model based on a human hepatic stellate cell strain comprises the following steps:
(1) Constructing LX-2-LUC-RFP cells, screening puromycin-resistant cells, monoclonalizing the cells, and screening monoclonal cells FFLuc-RFP LX-2 with good cell state and high fluorescence intensity;
(2) Taking an immunodeficiency mouse as an animal model, injecting 180-220mg/kg of Monocrotaline (MCT) into the abdominal cavity within 16-24 hours before cell transplantation, preparing an injection by dissolving the monocrotaline in a medically available solvent, and injecting an equivalent solvent into a control group;
(3) Transplanting FFLuc-RFP LX-2 cells into NSG mice by spleen injection;
(4) Starting on day 10 after cell transplantation, intraperitoneal injections of 0.18-0.22mL/kg body weight CCl were given twice a week 4 ,CCl 4 The corn oil was mixed with the control to prepare an injection for 6 weeks, and the control was injected with an equal amount of corn oil.
Preferably the mice are heavy immunodeficient mice, more preferably NSG mice, which lack mature T, B and NK cells.
Preferably by intraperitoneal injection of MCT at 200mg/kg body weight. Preferably, the solvent that dissolves MCT is phosphate buffer.
Preferably, 0.2mL/kg body weight CCl is administered intraperitoneally 4 . Preferably CCl 4 Mixing with corn oil according to the following ratio of 1: (6-15) by volume ratio, more preferably 1:10 volume ratio.
The LX-2-LUC-RFP cells are constructed by a method comprising the steps of:
(1) Constructing a plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing a target gene for expressing a luciferase reporter gene and a red fluorescent protein;
(2) Respectively carrying out high-purity endotoxin-free extraction on the three plasmid vectors, transfecting 293T cells in a growing period together, collecting virus supernatant and concentrating;
(3) And adding viruses into the LX-2 cells for incubation to obtain the LX-2-LUC-RFP cells.
Further, the 293T cells were replaced with complete medium 6 hours after co-transfection, cell supernatants containing lentiviral-enriched particles were collected at 24 and 48 hours of culture, and virus supernatants were concentrated by ultracentrifugation.
Further, LX-2 cells were plated on 24 well plates 18-24 hours prior to lentivirus transfection, the culture just prepared was replaced with the original medium the next day, 5 μg/mL of transfection reagent was added, and virus suspension was added followed by incubation at 37 ℃ for 24 hours, after which fresh medium was replaced with virus containing medium.
Pathological sections are made of NSG mouse livers at different time points after the injection of the monocrotaline, and the damage condition of liver tissues after the MCT injection is observed.
Performing living imaging of the mice at different time points after the spleen of the mice is injected with FFLuc-RFP LX-2, and observing the proliferation condition of LX-2 cells; after the mice were sacrificed, pathological sections of liver tissues were prepared, and the distribution of LX-2 cells in liver tissues was observed.
In mouse CCl 4 Performing in-vivo imaging of mice at different time points after injection, and observing the distribution of LX-2 cells in liver tissues; after the mice were sacrificed, pathological sections of liver tissue were prepared, and liver fibrosis was observed.
The liver fibrosis animal model obtained by the method for constructing the liver fibrosis mouse model based on the human hepatic stellate cell strain is applied to research of liver fibrosis mechanism or drug effect evaluation or pharmacological experiment of drugs.
Preferably, the application is in vivo experiments of human origin of biotherapy or drug therapy targeting hepatic stellate cells or fibroblasts.
The invention has the following beneficial effects:
according to the invention, NSG mice are used for experiments, and an external stimulus is combined to construct a humanized liver fibrosis mouse model, so that the engraftment rate and the survival rate of human cells are improved, and the human liver fibrosis process is better simulated.
According to the invention, MCT is injected intraperitoneally to prepare a liver injury model of a mouse, after liver injury, LX-2 injected by spleen is easy to colonize in liver, and the colonization quantity of LX-2 in the liver can be obviously increased.
The invention can be stably planted in the liver of a mouse after the LX-2 cell strain is injected through the spleen and is positioned in CCl 4 Is rapidly proliferated under the stimulation of (a).
The model is suitable for in vivo experiments of therapeutic methods such as biological therapy and drug therapy of targeted hepatic stellate cells or fibroblasts.
Compared with the model constructed by using primary human hepatic stellate cells or hepatic fibroblasts, the invention has small difficulty and is suitable for popularization and application in laboratories.
Drawings
FIG. 1 is a plasmid map of example expression of Luciferase and RFP;
FIG. 2 is a fluorescence image (fluorescence microscope, 200X) of a monoclonal cell line of example FFLuc-RFP LX-2;
FIG. 3 is a flow chart showing the procedure of the procedure for the colonization and proliferation of example LX-2 in the liver of NSG mice;
FIG. 4 is a graph of HE staining of the liver injury model of the example (left graph, liver HE staining of normal NSG mice; right graph, liver HE staining of liver injured NSG mice, 100X);
FIG. 5 is a photograph of a living animal of example LX-2 cells in liver colonization (left: not colonized; middle: no MCT; right: MCT);
FIG. 6 shows the results of frozen sections of liver after liver colonization of example LX-2 (fluorescence microscope, 200X);
FIG. 7 is a schematic diagram of an embodiment CCl 4 After injection, LX-2 cells were imaged in vivo in animals with liver proliferation (left panel, 3 weeks; right panel, 6 weeks);
FIG. 8 is a schematic diagram of an embodiment CCl 4 3D imaging of proliferated FFLuc-RFP LX-2 colonization in liver 6 weeks after injection;
FIG. 9 is a schematic diagram of an embodiment CCl 4 Distribution of FFLuc-RFP LX-2 cells in liver tissue after 6 weeks of injection (left panel, non-transplanted liver tissue; right panel, transplanted liver tissue, arrow FFLuc-RFP LX-2, 100×);
FIG. 10 is a schematic diagram of an embodiment CCl 4 Sirius scarlet staining of mouse liver 6 weeks after injectionColor (Sirius red), HE staining, anti-human collagen type I staining (left side not injected CCl) 4 A group; right side injection CCl 4 Group, 100×).
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1: selection of animal models
And 6-8 weeks old NSG mice are selected to construct a human model. In order to ensure the health status of mice, the required feeding goods should be autoclaved and the feeding environment should reach SPF level, which is very advantageous for feeding immunodeficient mice. A high fat low protein feed can be used, while the NSG mice feed should contain more than or equal to 18% protein and more than or equal to 6% fat, and be sterilized by Co-60 irradiation.
2. Construction of LX-2-LUC-RFP cells
(1) Plasmid construction
Construction of plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro expressing Luciferase reporter gene and red fluorescent protein RFP, plasmid map is shown in FIG. 1.
(2) Virus package
Lentiviral plasmids and their auxiliary packaging original vector plasmids were prepared, and three plasmid vectors were highly purified, respectively, to ensure endotoxin-free extraction. Then, 293T cells were co-transfected and replaced with complete medium 6 hours after transfection. Cell supernatants containing enriched lentiviral particles were collected at 24 and 48 hours of incubation, respectively. Finally, the virus supernatant is concentrated by ultracentrifugation.
(3) Viral transfection plasmid
1X 10 will be taken 18-24 hours prior to lentivirus transfection 5 The LX-2 cells were plated on 24-well plates to ensure that each well was transfected withLX-2 cell number was about 1X 10 5 . The next day, the freshly prepared medium (1.5 mL) was replaced with the original medium, 5. Mu.g/mL polybrene was added, and the appropriate amount of virus suspension was added, followed by incubation at 37 ℃. After a continuous incubation time of 24 hours, the virus-containing medium was replaced with fresh medium.
(4) Screening of monoclonal cell lines
As the plasmid contains puromycin resistance gene, a proper amount of puromycin is added for screening to obtain drug-resistant LX-2 cells, and the cells are named as FFLuc-RFP LX-2, so that the subsequent FFLuc-RFP LX-2 cell pool with high brightness is formed. FFLuc-RFP LX-2 cell pool with good cell state is selected, cells are subjected to suspension treatment by PBS, and counted by a cell counter. 300 cells were taken, mixed well with 30mL of medium, and then added to 3 96-well plates, 100uL per well. The plates were placed in a cell incubator for cultivation. On the third day of culture, 100uL of fluid was supplied per well. After 10 days of culture, the growth and fluorescence of the cells were observed by using a fluorescence microscope. And selecting a monoclonal cell strain with good cell state and strongest fluorescence intensity for amplification. For cells with poor cell state and poor agglomeration effect, the proliferation effect of the cells may be negatively affected and even the proliferation failure may occur when the cells are stimulated by carbon tetrachloride. In addition, when selecting cell lines, preference should be given to monoclonal cell lines which have a strong fluorescent intensity and which are convenient to track using live imaging of small animals and staining of pathological sections with antibodies. Such a choice will help to improve the accuracy and reliability of the experiment.
3. Experimental procedure for LX-2 colonization and proliferation in the liver of NSG mice
(1) And establishing a liver injury model. 200mg/kg of Monocrotaline (MCT) was dissolved in Phosphate Buffered Saline (PBS) at pH 7.4 by intraperitoneal injection during 16-24 hours prior to cell transplantation. The control group was injected with an equal dose of PBS.
(2) Using 2X 10 5 The FFLuc-RFP LX-2 cells were suspended in 200uL of PBS for use. Subsequently, mice were anesthetized with tribromoethanol and usedIodophors disinfect the skin of mice. During the procedure, the back skin of the mice was gently lifted using sterilized forceps and an incision of about 1cm was made along the skin using sterilized scissors. Next, the endothelial tissue at the incision was grasped using another pair of sterilized forceps, and a 1cm incision was cut along the endothelium again using scissors so as to fully expose the spleen. Then, 200uL of the suspended FFLuc-RFP LX-2 cells were aspirated using a 1mL injection needle, and injected from the spleen site of the mouse. Immediately after injection, the sterile cotton ball was used to stop bleeding by compression. Subsequently, a suturing operation is performed using a sterile suture needle line, which first sutures the endothelium and then the skin. The mice can be injected with an appropriate amount of antibiotics, as desired. Finally, the mice were placed on a warm and sterilized pad and waited for awakening.
(3) To induce toxin-mediated liver fibrosis, 0.2mL/kg CCl was injected intraperitoneally twice a week, starting on day 10 after cell transplantation 4 And corn oil 1:10 mixture for 6 weeks. The control group received only corn oil.
(4) Six weeks later, in vivo imaging and pathology biopsy were performed to evaluate model effects.
4. LX-2 colonization in NSG mouse liver
(1) Liver injury model
NSG mouse liver sections were prepared by hematoxylin-eosin staining (HE) following MCT injection. As can be clearly seen from the slice images of figure 4, the liver tissue was significantly damaged after MCT injection.
(2) Liver field planting condition
To examine the status of LX-2 colonization in liver cells, mice spleen was imaged in vivo (using PerkinElmer IVIS Spectrum) in small animals on day 10 of FFLuc-RFP LX-2 injection, as shown in FIG. 5. The results showed that FFLuc-RFP LX-2 was transferred from the spleen to the liver and that MCT-injected mice had more liver colonization with FFLuc-RFP LX-2 than without MCT injection.
After the mice were sacrificed, their tissues were subjected to frozen section observation, see fig. 6. The results showed that FFLuc-RFP LX-2 cells transferred from the spleen to the liver.
5. Proliferation of LX-2 in the liver of NSG mice and liver fibrosis
Mouse CCl 4 Animals were imaged in vivo at weeks 3 and 6 after injection, as shown in fig. 7. The results showed that LX-2 cells proliferated in the liver. In the process of CCl 4 3 weeks after intraperitoneal injection, the bioluminescence intensity of the liver part of the mouse reached a peak value of 3×10 5 And (3) radius. As the injection time was prolonged, the peak of bioluminescence intensity increased to 1.33X10 by 6 weeks 6 And (3) radius. This result is derived from the fact that FFLuc-RFP LX-2 has a bioluminescence gene, and thus the bioluminescence intensity can reflect the proliferation status of FFLuc-RFP LX-2. Experimental data shows that in CCl 4 Under stimulation, FFLuc-RFP LX-2 proliferated significantly.
CCl 4 At the 6 th week after injection, observing the coronal plane, sagittal plane and cross section of the mouse, wherein the region with higher bioluminescence intensity is mainly distributed at the liver part of the mouse; further construction of three-dimensional images, the results were more pronounced, showing that the higher bioluminescence intensity remained concentrated in the mouse liver region, as shown in fig. 8.
After the mice were sacrificed, liver tissues were taken and observed for anti-RFP immunohistochemical staining, and the results are shown in fig. 9. Compared to the control group, the experimental group exhibited numerous areas of tan-colored clusters, as indicated by the arrows on the right in fig. 9, which were FFLuc-RFP LX-2 stained with anti-RFP antibodies, characterized by regular distribution along fibrous tissue, but not within hepatic lobules.
HE staining was then performed, and liver fiber-related Sirius red (Sirius red) and anti-human collagen type I were stained, and the results are shown in FIG. 10. HE staining experiments showed that, via CCl 4 After injection treatment, mice had developed liver fibrosis. The fibrosis-related staining, such as Sirius red staining and anti-human collagen type I staining, was positive compared to the control, with a clear distribution of fibrous tissue.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (10)
1. The method for constructing the hepatic fibrosis mouse model based on the human hepatic stellate cell strain is characterized by comprising the following steps of:
(1) Constructing LX-2-LUC-RFP cells, screening puromycin-resistant cells, monoclonalizing the cells, and screening monoclonal cells FFLuc-RFP LX-2 with good cell state and high fluorescence intensity;
(2) Taking an immunodeficiency mouse as an animal model, injecting 180-220mg/kg of monocrotaline into the abdominal cavity within 16-24 hours before cell transplantation, preparing injection by dissolving the monocrotaline into a pharmaceutically available solvent, and injecting the same amount of solvent into a control group;
(3) Transplanting FFLuc-RFP LX-2 cells into mice by spleen injection;
(4) Starting on day 10 after cell transplantation, intraperitoneal injections of 0.18-0.22. 0.22mL/kg body weight CCl were given twice a week 4 ,CCl 4 The corn oil was mixed with the control to prepare an injection for 6 weeks, and the control was injected with an equal amount of corn oil.
2. Construction method according to claim 1, characterized in that the immunodeficient mice are NSG mice.
3. Construction method according to claim 1, wherein the LX-2-LUC-RFP cells are constructed by a method comprising the steps of:
(1) Constructing a plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing a target gene for expressing a luciferase reporter gene and a red fluorescent protein;
(2) Respectively carrying out high-purity endotoxin-free extraction on the three plasmid vectors, transfecting 293T cells in a growing period together, collecting virus supernatant and concentrating;
(3) And adding viruses into the LX-2 cells for incubation to obtain the LX-2-LUC-RFP cells.
4. Construction method according to claim 3, characterized in that the 293T cells are co-transfected and then replaced with complete medium 6 hours later, and the cell supernatants containing the lentiviral particles enriched are collected at 24 and 48 hours of culture, respectively, and the virus supernatant is concentrated by ultracentrifugation.
5. Construction method according to claim 3, characterized in that LX-2 cells are plated on 24 well plates 18-24 hours before lentivirus transfection, the culture just prepared the next day is replaced with original medium, transfection reagent is added and virus suspension is added, followed by incubation at 37 ℃ and after incubation for 24 hours the virus containing medium is replaced with fresh medium.
6. The construction method of claim 1, wherein pathological sections are made of the liver of the mice at different time points after injection of the monocrotaline, and the damage condition of liver tissues after MCT injection is observed.
7. Construction method according to claim 1, characterized in that live imaging of mice is performed at different time points after injection of FFLuc-RFP LX-2 into their spleen, and the proliferation of LX-2 cells is observed; after the mice were sacrificed, pathological sections of liver tissues were prepared, and the distribution of LX-2 cells in liver tissues was observed.
8. Construction method according to claim 1, characterized in that in the mouse CCl 4 Performing in-vivo imaging of mice at different time points after injection, and observing the distribution of LX-2 cells in liver tissues; after the mice were sacrificed, pathological sections of liver tissue were prepared, and liver fibrosis was observed.
9. Use of a liver fibrosis animal model obtained by the method for constructing a human liver astrocyte-based liver fibrosis mouse model according to any one of claims 1-8 for studying liver fibrosis mechanism or for drug efficacy evaluation or pharmacological experiment.
10. The use according to claim 9, wherein the use is in vivo in a human-derived experiment of biotherapy or drug therapy targeting hepatic stellate cells or fibroblasts.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311777915.9A CN117814181A (en) | 2023-12-22 | 2023-12-22 | Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311777915.9A CN117814181A (en) | 2023-12-22 | 2023-12-22 | Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117814181A true CN117814181A (en) | 2024-04-05 |
Family
ID=90522205
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311777915.9A Pending CN117814181A (en) | 2023-12-22 | 2023-12-22 | Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117814181A (en) |
-
2023
- 2023-12-22 CN CN202311777915.9A patent/CN117814181A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Quarta et al. | Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss | |
CN102388127B (en) | Lung tissue engineered | |
CN1812800B (en) | Muscle derived cells(mdc) for promoting and enhancing nerve repair and regeneration | |
Geuze et al. | Luciferase labeling for multipotent stromal cell tracking in spinal fusion versus ectopic bone tissue engineering in mice and rats | |
US20060247195A1 (en) | Method of altering cell properties by administering rna | |
CN104884611A (en) | NPRCP, PFDNC and uses thereof | |
CN1860222A (en) | Stem cells for clinical and commercial uses | |
CN104324053B (en) | A kind of dog stem cell secretion factor reparation liquid of quick healing dog wound tissue | |
KR102597594B1 (en) | A composition for bio transplanting of organoid | |
Lim et al. | Development of a model of sacrocaudal spinal cord injury in cloned Yucatan minipigs for cellular transplantation research | |
Talbot et al. | A novel orthotopic implantation technique for osteosarcoma produces spontaneous metastases and illustrates dose-dependent efficacy of B7-H3-CAR T cells | |
Connor et al. | Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis | |
EP0748374A1 (en) | Method for preparing clonogenic fibroblasts, method for gene transfection of fibroblasts and gene-transfected fibroblasts so obtained | |
DE69432856T2 (en) | SUPPLY OF GENE PRODUCTS BY MEANS OF MESANGIUM CELLS | |
Liu et al. | Nestin-expressing stem cells from the hair follicle can differentiate into motor neurons and reduce muscle atrophy after transplantation to injured nerves | |
CN106659560B (en) | The side group stem cell in gonad source | |
CN117814181A (en) | Construction method and application of hepatic fibrosis mouse model based on human hepatic stellate cell strain | |
JPWO2004045666A1 (en) | How to regenerate an organ | |
Laflamme et al. | Bone marrow chimeras to study neuroinflammation | |
WO2023143637A1 (en) | Construction and use of practical brain-like microorgan | |
CN100487754C (en) | Human angioma rat animal model and its configuration method | |
KR20160115522A (en) | Producing method of rheumatoid arthritis animal model, AbartaRA, via transplanting rheumatoid arthritis synovium | |
Chen et al. | Optical tissue window: a novel model for optimizing engraftment of intestinal stem cell organoids | |
Zhong et al. | Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope | |
Kuehn | Kainic acid-induced lumbar spinal cord damage leads to coordination deficits for the examination of cellular replacement therapies |
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 |