Construction method and application of mouse model of liver fibrosis based on human hepatic stellate cell line

Abstract

The present application belongs to the technical field of animal model construction, and aims at the problem of the lack of human hepatic stellate cells leading to the liver fibrosis animal model, and provides a construction method and application of a liver fibrosis mouse model based on a human hepatic stellate cell strain.The present application uses an immunodeficient mouse as an animal model, uses luciferase and red fluorescent protein labeled LX-2, and injects MCT into the abdominal cavity to make a liver injury model of the mouse, injects LX-2 into the spleen after liver injury, transplants human LX-2 cells into the NSG mouse, and then injects CCl4 into the abdominal cavity of the mouse.Through the method, the implantation, distribution, proliferation and liver fibrosis of the transplanted LX-2 in the liver are successfully studied.The model is suitable for human in-vivo experiments of biological treatment and drug treatment and other treatment methods targeting hepatic stellate cells or fibroblasts.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal model construction, and particularly relates to a method for constructing a liver fibrosis mouse model. BACKGROUND

[0002] Liver fibrosis is characterized by excessive accumulation of fibrous connective tissue in the liver, leading to impaired liver function. Hepatic stellate cells (HSCs) are considered to be the main contributors to liver fibrosis. After liver injury, HSCs migrate to the injury site and become activated, producing excessive extracellular matrix (ECM) components, ultimately leading to liver fibrosis.

[0003] Currently, the mouse in vivo studies on liver fibrosis mainly rely on the exogenous addition of chemical inducers such as carbon tetrachloride (CCl4), dimethyl nitrosamine (DMNA), D-galactosamine (DGA), diethyl nitrosamine (DEN) to construct mouse liver fibrosis model. Although the CCl4-induced liver fibrosis model is simple and easy to operate, the mortality rate is high, and the liver fibrosis state caused by CCl4 and other chemical inducers is difficult to simulate the state of the development of fibrosis disease, and due to the species difference between mice and humans, the mouse fibrosis model is difficult to simulate the progression of human liver fibrosis disease. LX-2 is a primary human hepatic stellate cell line that can be continuously passaged by SV40 large T antigen treatment. Xu et al. established a continuously passaged LX-2 cell line and provided a stable source of human hepatic stellate cells (Xu L, Hui AY, Albanis E, Arthur MJ, O'Byrne SM, Blaner WS, Mukherjee P, Friedman SL, Eng FJ. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut. 2005 Jan; 54(1): 142-51). LX-2 retains key features of hepatic stellate cells, such as cytokine signaling, neural gene expression, retinol metabolism, and fibrosis, making it ideal for culture-based research on human liver fibrosis. Due to the difficulty of humanized cells to colonize in other animal models and the low survival rate, 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, which co-cultures liver parenchymal cells, hepatic stellate cells and Kupffer cells on a 3D culture medium, and then adds liver fibrosis inducers to the obtained 3D co-cultured cells to induce a liver fibrosis 3D model. Although this in vitro model can be constructed based on humanized cells, the in vitro model cannot simulate the complex conditions in vivo, such as changes in the level of body fluids, metabolic products and changes in the in vivo environment, which can change the survival state of cells.

[0004] Although some studies have found that immunodeficient animal models are more suitable for human cell transplantation, the inventors have found in the research process of the present application that simply relying on immunodeficient animal models cannot achieve ideal colonization rate and survival rate results, and human hepatic stellate cells in immunodeficient mice,

[0005] Therefore, there is an urgent need to construct a humanized liver fibrosis mouse model for better scientific research. SUMMARY

[0006] In order to meet the research needs of the mouse liver fibrosis mouse, the present application provides a method for constructing a liver fibrosis mouse model based on a human hepatic stellate cell strain.

[0007] The present application is realized by the following technical solutions:

[0008] A method for constructing a liver fibrosis mouse model based on a human hepatic stellate cell strain, comprising the following steps:

[0009] (1) Constructing LX-2-LUC-RFP cells, screening cells resistant to puromycin, monoclonalizing the cells, and screening single clone cells FFLuc-RFP LX-2 with good cell state and high fluorescence intensity;

[0010] (2) Using immunodeficient mice as animal models, injecting 180-220 mg / kg body weight of MCT through the abdominal cavity within 16-24 hours before cell transplantation, dissolving MCT in a medically available solvent to prepare an injection, and injecting an equal amount of solvent into the control group;

[0011] (3) Transplanting FFLuc-RFP LX-2 cells into NSG mice through spleen injection;

[0012] (4) Starting on the 10th day after cell transplantation, injecting 0.18-0.22 mL / kg body weight of CCl4 through the abdominal cavity twice a week, mixing CCl4 with corn oil to prepare an injection, and continuing for 6 weeks, and injecting an equal amount of corn oil into the control group.

[0013] Preferably, the mouse is a severe immunodeficient mouse, more preferably an NSG mouse, and the NSG mouse lacks mature T, B, and NK cells.

[0014] Preferably, 200 mg / kg body weight of MCT is injected through the abdominal cavity. Preferably, the solvent for dissolving MCT is phosphate buffer.

[0015] Preferably, 0.2 mL / kg body weight of CCl4 is injected through the abdominal cavity. Preferably, CCl4 is mixed with corn oil at a volume ratio of 1:(6-15) to prepare an injection, more preferably at a volume ratio of 1:10.

[0016] The LX-2-LUC-RFP cell is constructed by using a method comprising the following steps:

[0017] (1) Constructing a plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing a target gene for expressing luciferase reporter gene and red fluorescent protein;

[0018] (2) High-purity endotoxin-free extraction is respectively performed on the three plasmid vectors, 293T cells in a growth period are co-transfected, virus supernatant is collected and concentrated treatment is performed;

[0019] (3) Virus incubation is added to the LX-2 cells, and the LX-2-LUC-RFP cell is obtained.

[0020] Further, 6 hours after co-transfection of the 293T cells, the complete culture medium is replaced, and at the 24 and 48 hour time points of culture, the cell supernatant containing enriched lentivirus particles is collected, and the virus supernatant is concentrated by ultracentrifugation.

[0021] Further, 18-24 hours before lentivirus transfection, the LX-2 cells are plated on a 24-well plate, the next day, the just prepared culture is replaced with the original culture medium, 5 μg / mL of transfection reagent is added, and the virus suspension is added, then incubated at 37°C, and after 24 hours of continuous culture, the virus-containing culture medium is replaced with fresh culture medium.

[0022] Pathological sections of the liver of the NSG mouse at different time points after injection of monocrotaline are prepared, and the damage of the liver tissue after MCT injection is observed.

[0023] The mouse is imaged in vivo at different time points after injection of FFLuc-RFP LX-2 in the mouse spleen, and the proliferation of the LX-2 cells is observed; after the mouse is killed, pathological sections of the liver tissue are prepared, and the distribution of the LX-2 cells in the liver tissue is observed.

[0024] The mouse is imaged in vivo at different time points after injection of CCl4 in the mouse, and the distribution of the LX-2 cells in the liver tissue is observed; after the mouse is killed, pathological sections of the liver tissue are prepared, and the fibrosis of the liver is observed.

[0025] The application of the liver fibrosis animal model obtained by the construction method of the liver fibrosis mouse model based on a human hepatic stellate cell strain in the research of the mechanism of liver fibrosis or in the pharmacodynamic evaluation or pharmacological experiment of a drug.

[0026] Preferably, the application is a human in-vivo experiment of biological treatment or drug treatment targeting hepatic stellate cells or fibroblasts.

[0027] The application has the following beneficial effects:

[0028] This invention uses NSG mice for experiments and combines them with external stimuli to construct a humanized liver fibrosis mouse model, thereby improving the colonization and survival rate of human cells and better simulating the process of human liver fibrosis.

[0029] This invention uses intraperitoneal injection of MCT to create a mouse liver injury model. After liver injury, LX-2 injected into the spleen is more likely to colonize in the liver, significantly increasing the number of LX-2 colonized in the liver.

[0030] This invention allows LX-2 cell lines to be stably colonized in mouse livers after injection into the spleen, and to rapidly proliferate under CCl4 stimulation.

[0031] This model is suitable for in vivo human trials of treatments such as biotherapy and drug therapy targeting hepatic stellate cells or fibroblasts.

[0032] Compared to using primary human hepatic stellate cells or hepatic fibroblasts to construct models, this invention is less difficult to implement and is suitable for widespread application in laboratories. Attached Figure Description

[0033] Figure 1 The plasmid diagram for the example shows Luciferase and RFP;

[0034] Figure 2 Fluorescence image of the FFLuc-RFP LX-2 monoclonal cell line in the example (fluorescence microscope, 200×);

[0035] Figure 3 This is a flowchart illustrating the colonization and proliferation process of LX-2 in the liver of NSG mice in this example.

[0036] Figure 4 HE staining images of the liver injury model in the example (left image, HE staining of the liver of normal NSG mice; right image, HE staining of the liver of NSG mice with liver injury, 100×).

[0037] Figure 5 This is an in vivo animal imaging image of LX-2 cells colonizing the liver in the example (left: no colonization; middle: no MCT; right: with MCT);

[0038] Figure 6 The results of frozen sections of the liver after LX-2 liver colonization (fluorescence microscope, 200×) are shown in Example 1.

[0039] Figure 7 The image shows in vivo animal imaging of LX-2 cell proliferation in the liver after CCl4 injection (left, 3 weeks). Figure 6 week);

[0040] Figure 8 Figure 6. 3D imaging of FFLUC-RFP LX-2 colonization in liver after CCl4 injection for 6 weeks, for example C;

[0041] Figure 9 Figure 7. Distribution of FFLUC-RFP LX-2 in liver tissue after CCl4 injection for 6 weeks, for example C. Left panel, non-colonized liver tissue; right panel, colonized liver tissue, arrows point to FFLUC-RFP LX-2, 100x;

[0042] Figure 10 Figure 8. Sirius red staining, HE staining and anti-human collagen type I staining of mouse liver after CCl4 injection for 6 weeks, for example C. Left panel, non-injected CCl4 group; right panel, injected CCl4 group, 100x. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with specific examples and drawings.

[0044] Unless otherwise specified, the specific techniques or conditions in the examples are in accordance with the techniques or conditions described in the literature or in accordance with the product specifications. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0045] Example 1: Selection of animal model

[0046] A 6-8 week old NSG mouse was selected to construct a human model. In order to ensure the health status of the mouse, the required feeding materials should be sterilized by high pressure sterilization, and the feeding environment should reach the SPF level, which is very beneficial for the feeding of immunodeficient mice. High-fat low-protein feed can be used, and the feed for NSG mice should contain protein ≥ 18% and fat content ≥ 6%, and be sterilized by Co-60 irradiation.

[0047] II. Construction of LX-2-LUC-RFP cells

[0048] (1) Plasmid construction

[0049] The plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro expressing luciferase reporter gene and red fluorescent protein RFP was constructed, and the plasmid map is shown in Figure 1 .

[0050] (2) Virus packaging

[0051] Prepare lentivirus plasmid and its auxiliary packaging element carrier plasmid, and purify the three plasmid carriers to ensure that there is no endotoxin in the extraction. Then, co-transfect 293T cells, and replace the complete culture medium 6 hours after transfection. At 24 and 48 hours of culture, collect the cell supernatant containing enriched lentivirus particles. Finally, concentrate the virus supernatant by ultracentrifugation.

[0052] (3) Virus transfection plasmid

[0053] 18-24 hours before lentivirus transfection, 1×10 5 LX-2 cells were plated in a 24-well plate to ensure that the number of LX-2 cells in each well was about 1×10 5 The next day, replace the original culture medium with the just prepared culture medium (1.5 mL), add 5 μg / mL of polybrene, and add an appropriate amount of virus suspension, and then incubate at 37°C. After 24 hours of continuous culture, replace the virus-containing culture medium with fresh culture medium.

[0054] (4) Screening of monoclonal cell lines

[0055] Since the plasmid contains a puromycin resistance gene, an appropriate amount of puromycin is added for screening to obtain drug-resistant LX-2 cells, which are named FFLuc-RFP LX-2 and constitute the subsequent high-brightness FFLuc-RFP LX-2 cell pool. Select a well-conditioned FFLuc-RFP LX-2 cell pool, suspend the cells with PBS, and count them using a cell counter. Take 300 cells, mix them evenly with 30 mL of culture medium, and then add them to 3 96-well plates, 100 uL per well. Place the plates in the cell culture incubator for culture. On the third day of culture, add 100 uL of liquid to each well. After 10 days of culture, observe the growth and fluorescence intensity of the cells using a fluorescence microscope. Select the single clone cell line with the best cell state and strongest fluorescence intensity for amplification. For cells with poor cell state and poor aggregation, their proliferation may be negatively affected when stimulated with carbon tetrachloride, or they may not be able to proliferate. In addition, when selecting cell lines, priority should be given to single clone cell lines with strong fluorescence brightness, which are easy to use for small animal live imaging and antibody staining for tracking when making pathological sections. Such selection will help improve the accuracy and reliability of the experiment.

[0056] III. Experimental steps for LX-2 colonization and proliferation in the liver of NSG mice

[0057] (1) Establish a liver injury model. Within 16-24 hours before cell transplantation, inject 200 mg / kg of MCT into the abdominal cavity. MCT is dissolved in PBS with a pH value of 7.4. The control group is injected with an equal dose of PBS.

[0058] (2) Use 2x10 5 FFLuc-RFP LX-2 cells, suspended in 200uL of PBS solution as standby. Then, anesthetize the mouse with tribromoethanol, and disinfect the skin of the mouse with iodophor. During the operation, use a disinfected forceps to gently lift the back skin of the mouse, and use a disinfected scissors to cut about 1cm incision along the skin. Then, use another disinfected forceps to pinch the endothelial tissue at the incision, and use the scissors again to cut a 1cm incision along the endothelium to fully expose the spleen. Then, use a 1mL injection needle to suck 200uL of suspended FFLuc-RFP LX-2 cells, and inject from the spleen of the mouse. After injection, immediately use a sterile cotton ball to compress the bleeding. Then, use a sterile suture needle to suture the endothelium first, and then the epidermis. As needed, inject the mouse with an appropriate amount of antibiotic. Finally, place the mouse on a warm and disinfected cushion, and wait for it to wake up.

[0059] (3) To induce toxin-mediated liver fibrosis, start injecting 0.2mL / kg CCl4 and corn oil 1:10 mixture into the abdominal cavity twice a week from the 10th day after cell transplantation, for 6 weeks. The control group only receives corn oil.

[0060] (4) After six weeks, perform small animal in vivo imaging and pathological biopsy to evaluate the model effect.

[0061] Four, the colonization of LX-2 in the liver of NSG mice

[0062] (1) Liver injury model

[0063] After MCT injection, prepare liver sections of NSG mice by hematoxylin-eosin staining (HE). From Figure 4 The slice image clearly shows that the liver tissue has been significantly damaged after MCT injection.

[0064] (2) Liver colonization

[0065] To detect the colonization of LX-2 in liver cells, on the 10th day after the mouse spleen injection of FFLuc-RFP LX-2, do small animal in vivo imaging (use PerkinElmer IVIS Spectrum), as Figure 5The results show that FFLuc-RFP LX-2 cells are transferred from the spleen to the liver. The mice injected with MCT have more FFLuc-RFP LX-2 cells in the liver than the mice without MCT injection.

[0066] After the mice are sacrificed, the tissues are observed by frozen sectioning, and the results are shown in Figure 6 The results show that FFLuc-RFP LX-2 cells are transferred from the spleen to the liver.

[0067] Five, proliferation of LX-2 in the liver of NSG mice and liver fibrosis

[0068] The mice are imaged in vivo at 3 weeks and 6 weeks after CCl4 injection, and the results are shown in Figure 7 The results show that LX-2 cells proliferate in the liver. After 3 weeks of CCl4 intraperitoneal injection, the bioluminescence intensity in the liver of the mice reaches a peak of 3 x 10 5 radiance. As the injection time is prolonged, the peak of bioluminescence intensity rises to 1.33 x 10 6 radiance at 6 weeks. This result is due to the fact that FFLuc-RFP LX-2 has a bioluminescence gene, so the bioluminescence intensity can reflect the proliferation of FFLuc-RFP LX-2. The experimental data show that FFLuc-RFP LX-2 proliferates significantly under the stimulation of CCl4.

[0069] At 6 weeks after CCl4 injection, the coronal, sagittal and transverse sections of the mice are observed, and the regions with high bioluminescence intensity are mainly distributed in the liver of the mice. Further construction of three-dimensional images shows that the regions with high bioluminescence intensity are still concentrated in the liver of the mice, as shown in Figure 8

[0070] After the mice are sacrificed, the liver tissues are taken and observed by anti-RFP immunohistochemical staining, and the results are shown in Figure 9 Compared with the control group, the experimental group shows many brownish clumps, as shown in Figure 9 the right arrow, which are FFLuc-RFP LX-2 stained by anti-RFP antibody, and the distribution characteristics are regular along the fibrous tissue, but not in the liver lobule.

[0071] Then HE staining, Sirius red staining related to liver fibrosis, anti-human collagen type I staining, and the results are shown in Figure 10 ​HE staining experiments showed that the mice had developed liver fibrosis after CCl4 injection treatment. Compared with the control group, fibrosis-related staining such as Sirius red staining and anti-human collagen type I staining were positive, and the distribution of fibrous tissue was clearly visible.

[0072] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application.

Claims

1. A method for constructing a mouse model of liver fibrosis based on a human hepatic stellate cell line, characterized by, The method comprises the following steps: (1) Constructing LX-2-LUC-RFP cells, screening cells resistant to puromycin, monocloning the cells, and screening single clone cells FFLuc-RFP LX-2 with good cell state and high fluorescence intensity; (2) Using immunodeficient mice as animal models, injecting 180-220 mg / kg of body weight of the hyacinth alkaloid into the abdominal cavity of the mice within 16-24 hours before cell transplantation, and preparing the injection by dissolving the hyacinth alkaloid in a medically usable solvent, and injecting the same amount of solvent into the control group; (3) Transplanting FFLuc-RFP LX-2 cells into the mice by means of spleen injection; (4) Starting from the 10th day after cell transplantation, injecting 0.18-0.22 mL / kg of body weight of CCl4 into the abdominal cavity of the mice twice a week, mixing CCl4 with corn oil to prepare an injection, and continuously injecting the same amount of corn oil into the control group for 6 weeks.

2. The construction method according to claim 1, characterized in that, The immunodeficient mice are NSG mice.

3. The construction method of claim 1, wherein, The LX-2-LUC-RFP cells are constructed by the method comprising the following steps: (1) Constructing a plasmid pCDH-CMV-fLuc-EF1-turboRFP-T2A-Puro containing a target gene for expressing luciferase reporter gene and red fluorescent protein, and preparing a lentivirus plasmid and a helper packaging element vector plasmid; (2) Extracting the above three kinds of plasmid vectors with high purity and endotoxin-free, and co-transfecting 293T cells in the growth phase, collecting virus supernatant, and performing concentration treatment; (3) Adding virus to LX-2 cells for incubation to obtain LX-2-LUC-RFP cells.

4. The construction method according to claim 3, characterized in that, The 293T cells are co-transfected for 6 hours, and then the complete culture medium is replaced, and the virus supernatant containing enriched lentivirus particles is collected at 24 and 48 hours of culture, and the virus supernatant is concentrated by ultracentrifugation.

5. The construction method according to claim 4, characterized in that, The LX-2 cells are plated on a 24-well plate 18-24 hours before lentivirus transfection, the original culture medium is replaced with the just prepared culture medium the next day, the transfection reagent is added, and the virus suspension is added, then incubated at 37°C, and the culture medium containing the virus is replaced with fresh culture medium after 24 hours of continuous culture.

6. The construction method of claim 1, wherein, The liver of the mouse at different time points after injection of the hyacinth alkaloid is made into a pathological section, and the damage of the liver tissue after injection of the hyacinth alkaloid is observed.

7. The construction method of claim 1, wherein The mouse is imaged in vivo at different time points after injection of FFLuc-RFP LX-2 into the spleen of the mouse, and the proliferation of the LX-2 cells is observed; after the mouse is killed, the liver tissue pathological section is made, and the distribution of the LX-2 cells in the liver tissue is observed.

8. The construction method of claim 1, wherein, The mouse is imaged in vivo at different time points after injection of CCl4, and the distribution of the LX-2 cells in the liver tissue is observed; after the mouse is killed, the liver tissue pathological section is made, and the liver fibrosis is observed.

9. The application of the liver fibrosis animal model obtained by the method for constructing the liver fibrosis mouse model based on the human hepatic stellate cell strain according to any one of claims 1-8 in the research of the mechanism of liver fibrosis or in the pharmacodynamic evaluation or pharmacological experiment of a drug.

10. Use according to claim 9, characterized in that, The application is a human in vivo experiment of biological therapy or drug therapy targeting hepatic stellate cells or fibroblasts.

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