Kit for constructing an orthotopic transplantation model of mouse liver cancer

Abstract

The present invention discloses a kit for constructing a mouse orthotopic liver cancer transplantation model, which comprises: H22 cell line; 1640 medium, FBS, P / S, PBS, D-Luciferin potassium salt, Matrigel; experimental equipment, including cell culture flasks, centrifuge tubes; surgical instruments, including surgical scissors, curved ophthalmic forceps, straight forceps, needle holder, suture needle with thread, "O" ring, cotton swab. Using this kit to construct a mouse orthotopic liver cancer transplantation model is simple in operation, time-saving, high in success rate, and reduces animal suffering.

Description

Technical Field

[0001] The present invention belongs to the field of clinical medicine, and particularly relates to a kit for constructing an orthotopic transplantation model of mouse liver cancer, its application, and the use of the constructed orthotopic transplantation model of mouse liver cancer. Background Art

[0002] Liver cancer is one of the major malignant tumors that seriously threaten human life and health, and the research on liver cancer has always been a hot topic in the field of medical research. Mouse liver cancer models provide effective ways for basic and clinical research on the occurrence, development, treatment, etc. of liver cancer. Currently, the main mouse liver cancer models include subcutaneous liver cancer models, mouse orthotopic models, etc. However, the mouse liver cancer orthotopic model is one of the essential main tools for studying the mechanisms of liver cancer growth, invasion, and metastasis. The orthotopic model of liver cancer not only maintains the original tumor characteristics but also preserves the original blood supply and accurate anatomical location of the liver, providing an accurate animal model basis for studying the biological properties of liver cancer and experimental intervention. The orthotopic mouse model is divided into carcinogen-induced, transplantable, gene-edited mouse liver cancer models, etc. However, the carcinogen-induced mouse model has disadvantages such as chemical toxicity, difficulty in controlling the dosage, and a relatively long cycle; gene-edited mice are costly, difficult to implement technically, and are less used in experiments. The orthotopic liver transplantation model has a relatively short modeling cycle and a high success rate and is currently widely used. However, due to the great difficulty in implementation, long time, the need for animal anesthesia and awakening, and the cumbersome operation, the orthotopic transplantation model of liver cancer has high requirements for the operator, experimental environment, and personnel technology and is only adopted by a few laboratories. Currently, the tumors of the widely used orthotopic transplantation model of liver cancer are generally human-derived liver cancer surgical specimens and mouse-derived specimens for transplantation. The mouse-derived tumor samples are mainly used. However, the orthotopic transplantation model has problems such as difficult tumor body fixation, excessive surgical blood loss, and long time.

[0003] There have been many reports on improving the construction method of the orthotopic transplantation model of mouse liver cancer. For example, patent document CN112262815A discloses a preparation method of an orthotopic transplantation liver cancer model in mice. After the mice are anesthetized, a midline abdominal incision is made to enter the abdomen, and a non-traumatic hemostatic forceps is used to block the first porta hepatis. At the left lobe of the liver, at 30 1 / 2Inject 0.05 ml of liver cancer cells into the curved needle, coagulate the puncture site with an electrocoagulation pen. After there is no active bleeding, release the hepatic hilar occlusion clamp and close the abdomen. This method overcomes the defects such as the outflow of tumor cells caused by blood flow, non-localized implantation, and low tumor formation rate, and reduces the death caused by hemorrhagic shock due to excessive bleeding at the puncture site and the peritoneal implantation of tumors. CN113875689A discloses a method for establishing a nude mouse orthotopic liver cancer transplantation model. Cut the solid tumor prepared in the subcutaneous transplantation tumor model into tumor tissue blocks with a size of about 2 mm×2 mm×1 mm for standby. Then anesthetize the nude mouse, place it in the supine position, disinfect the skin, make a transverse laparotomy about 5-10 mm below the xiphoid process along the midline of the abdomen to the right side of the animal, with an opening length of about 1 cm, expose the liver, and use the "sandwich method" to take out the tumor tissue. The "sandwich method" is to directly sew the tumor tissue block on the inner side of the right lobe of the liver with a surgical suture: first pass a suture needle with a suture through the tumor tissue, then penetrate from the inner side to the outer side of the right lobe of the liver, then make a surgical knot, gently tighten it, and cut the surgical suture. This method has the advantages of less bleeding during the experiment, less harm to animals, short operation time, and low difficulty.

[0004] In the solutions of these prior arts, there are generally operation procedures such as surgical suture fixation and bioadhesive fixation. However, due to the close color of the surgical suture and the liver during the operation, the suture is difficult and time-consuming, and requires an operator with rich experience. Moreover, the hepatic capsule is relatively thin, and there are disadvantages such as the failure of suture due to the pulling of the liver parenchyma by the suture. The bioadhesive is an organic substance, and there are interferences of foreign substances in the method of fixing the tumor mass. During the use of the bioadhesive, the wound surface needs to be kept dry, and it should be as uniform and slow as possible, otherwise it is easy to form large clumps, easy to adhere to surrounding organs, and it is relatively difficult to observe the postoperative anatomical position and tumor. Summary of the Invention

[0005] In view of the important clinical research value of the nude mouse orthotopic liver cancer transplantation model, in order to overcome the above deficiencies existing in the prior art, the inventor provided a new method for constructing a nude mouse orthotopic liver cancer transplantation model through multiple experiments, making the modeling operation simpler, reducing the pain of animals, and significantly improving the success rate, thus greatly saving the scientific research cost. Specifically, the present invention includes the following technical solutions.

[0006] A kit for constructing an orthotopic transplantation model of mouse liver cancer, characterized by comprising: H22 cell line; biochemical reagents, including 1640 medium, FBS (fetal bovine serum), P / S (penicillin / streptomycin mixed solution, i.e., P / S double antibody), PBS (balanced salt solution PBS, phosphate buffered saline), D-Luciferin potassium salt (D-luciferin potassium salt) such as XenoLight D-Luciferin potassium Salt (PerkinElmer), Matrigel; experimental equipment, including cell culture flasks, centrifuge tubes; surgical instruments, including surgical scissors, suture needles with thread, "O"-rings.

[0007] Preferably, the above-mentioned H22 cell line is H22-LUC cells (H22-luc, mouse liver cancer cells - luciferase labeled).

[0008] In one embodiment, the culture and use conditions of the above-mentioned H22 cell line can be: H22-luc cells are resuspended in complete medium (89% 1640 medium, 10% FBS, 1% P / S), placed in a carbon dioxide incubator at 37 °C and 5% CO2 concentration for 24 h. When the cultured cells are in the logarithmic growth phase (when the color of the medium changes from red to dark yellow), the cell density reaches 80 - 100%, and subculture is carried out at a ratio of 1:2 or 1:3. The culture solution is collected, centrifuged, the supernatant is discarded, washed twice with PBS and then resuspended with PBS, and the cell concentration is adjusted to about 2×10 7 / mL, and 0.1 mL is injected into each mouse.

[0009] The above-mentioned mice can be BALB / c mice.

[0010] The above-mentioned "O"-ring is a plastic "O"-ring with an inner diameter of about 5 mm, which is used to cover the incision buried in the mouse liver, for Matrigel to be added until it completely solidifies, to limit the flow range of the added Matrigel, and then it is removed.

[0011] For example, the "O"-ring can be selected from the following groups: ① Dongtai Pengkai Co., Ltd., product number 7*1; ② Yuanmeng Industry (Shenzhen) Co., Ltd., product number 7*1; ③ Wenzhou Nazhi Fastener Co., Ltd., product number 7*1.

[0012] The above-mentioned Matrigel is a commonly used Matrigel in animal model construction, and can be selected from the following groups: ① Corning, product number 356234 / 354234; ② Beyotime Co., Ltd., product number C0386 / C0387.

[0013] The above suture needle with thread is a commonly used suture needle with thread (0#5) in clinical surgeries and the construction of animal models, and can be selected from the following groups: ① Ningbo Lingqiao Co., Ltd., product number 0#5; ② Johnson & Johnson Co., Ltd. of the United States, product number W580; ③ Shanghai Pudong Jinhuan Co., Ltd., product number 0#5.

[0014] Preferably, the above surgical instruments further include ophthalmic forceps (including curved forceps and straight forceps), needle holders, cotton swabs, etc.

[0015] The above kit may further include: povidone iodine, 75% alcohol, and gentamicin.

[0016] Optionally, the above kit may further include: an anesthetic for anesthetizing mice, selected from isoflurane or sodium pentobarbital. The anesthetic can be configured according to the suggestions of an anesthesiologist.

[0017] When the anesthetic is sodium pentobarbital anesthesia, the dose of sodium pentobarbital is approximately 30 - 50 mg / kg of mouse body weight, such as approximately 50 mg / kg of mouse body weight.

[0018] When the anesthetic is isoflurane gas, the administration of the anesthetic is to anesthetize the mice using a small animal anesthesia machine such as the Matrx VIP3000 type gas anesthesia machine. When the mice are under isoflurane gas inhalation anesthesia, the flow rate of the anesthetic is adjusted according to the specific situation of the mice during the specific operation process.

[0019] To reduce the probability of modeling failure, the above kit does not include liquid bio - glue, so as to prevent the operator from misusing it as a matrix glue with low fluidity, resulting in the accidental adhesion of the highly fluid bio - glue to other visceral tissues around the liver, which is not conducive to the incision healing of the mice and hinders tumor growth.

[0020] Furthermore, to reduce the probability of modeling failure, the above kit also does not include surgical sutures, so as to prevent the operator from mistakenly using them as sutures for the suture needle with thread (0#5). Because the surgical sutures are close in color to the liver, the suture is difficult, time - consuming, requires high operating skills, and there is a risk of suture failure due to the pulling of the liver parenchyma by the sutures.

[0021] The second aspect of the present invention provides the use of the above kit in the construction of a mouse orthotopic liver cancer transplantation model.

[0022] As a specific usage mode of the above kit, the construction of a mouse orthotopic liver cancer transplantation model is implemented by a method including the following steps:

[0023] 1) Culturing H22 cells: H22-luc cells were resuspended in complete medium consisting of 89% 1640 medium, 10% FBS, and 1% P / S. The cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2 for approximately 24 hours. When the cells reached the logarithmic growth phase (when the color of the culture medium changed from red to dark yellow) and the cell density reached 80-100%, a 1:2 or 1:3 subculture was performed.

[0024] 2) Collect the H22 cell culture medium, centrifuge, discard the supernatant, wash twice with PBS, and resuspend in PBS to adjust the cell concentration to approximately 2×10 7 / mL, mice were injected subcutaneously with cells;

[0025] 3) One week later, after the subcutaneous model was established, the mice were anesthetized, shaved, disinfected with iodine, the epidermis was incised, the subcutaneous tumor was removed, the tumor capsule was separated, and the tumor parenchyma was exposed. The tumor was immersed in PBS and trimmed into small tumor tissue pieces using ophthalmic forceps.

[0026] 4) The recipient mouse (another mouse) was shaved, anesthetized, and disinfected with iodine. The epidermis, peritoneum, and liver capsule were incised to expand the surgical field. The liver was then removed from the body using a cotton swab soaked in PBS. The liver capsule was carefully incised using ophthalmic curved scissors, gradually creating a 3 mm tunnel. Hemostasis was achieved by applying pressure with a dry cotton swab, and the tumor tissue was embedded in the liver tunnel.

[0027] 5) Place an O-ring over the incision where the liver was buried, add Matrigel, and after the Matrigel has completely solidified, carefully remove the O-ring with ophthalmic forceps. Once the Matrigel has solidified and bleeding has ceased, close the abdomen in layers using a suture needle and 0#5 sutures. Disinfect the incision, inject gentamicin, and keep the mouse warm until it recovers from anesthesia.

[0028] 6) D-Luciferin Potassium Salt (15 mg / ml) was injected intraperitoneally weekly. In vivo imaging using the IVIS IV system was performed to observe tumor growth and confirm the establishment of the orthotopic liver cancer transplantation model in mice.

[0029] Preferably, the above-mentioned mouse is a BALB / c mouse.

[0030] It should be understood that when expressing numerical characteristics in this article, the term "about" or "approximately" means that the number represented may have an error range or floating range of ±10%, ±8%, ±6%, ±4% or ±2%.

[0031] The present invention provides novel tools and methods for constructing a mouse orthotopic liver cancer transplantation labeling model, which can be used for the establishment of a mouse orthotopic liver cancer transplantation model and subsequent experiments on liver cancer mice. Compared with the prior art, the operation steps of modeling the orthotopic liver cancer transplantation model are simplified, the operation time is shortened, the pain of animals is reduced, the experimental work efficiency is not only improved, but also the success rate is increased, and the production cost of the mouse orthotopic liver cancer transplantation model can be greatly reduced, which has economic significance. Brief Description of the Drawings

[0032] Figure 1 Shows a schematic flow chart for constructing a mouse orthotopic liver cancer transplantation labeling model.

[0033] Figure 2 Shows a bioluminescence detection diagram of the mouse orthotopic liver cancer transplantation labeling model constructed by the present invention. Among them, the fluorescent luminescence parts in the photo are circled in blue. Detailed Embodiments

[0034] The mouse orthotopic transplantation labeled liver cancer model can be used for basic research on liver cancer drugs, drug research, pre-clinical drug clinical trials, etc., and is an important and commonly used experimental animal model. Therefore, it is of great significance to develop a modeling technology for orthotopic liver cancer transplantation models that is simple to operate, time-saving, biologically stable, and has a high success rate, including significant economic benefits.

[0035] As mentioned above, there are mainly three types of existing in-situ liver cancer mouse models: induced liver cancer models, transgenic mouse liver cancer models, and transplanted mouse liver cancer models. The induced liver cancer model refers to using chemical, physical or biological factors to act on experimental mice to induce the formation of liver cancer in mice. Among them, the most commonly used is chemical carcinogen induction, such as aflatoxin and diethylnitrosamine. This method has a long tumor formation time, with an average of 4 to 6 months, and sometimes even 1 year. The tumor formation rate is unstable and there are large individual differences.

[0036] The transgenic liver cancer model means that after a mouse receives an exogenous gene, the exogenous gene is stably integrated into the chromosomal genome and can be inherited by its offspring, causing it to form liver cancer. This method can obtain stable in-situ liver cancer mice, but the technical requirements are extremely high, and the cultivation of the first-generation transgenic liver cancer mice is extremely difficult.

[0037] The transplanted liver cancer model includes two transplantation methods: tumor mass inoculation and cell suspension injection. Its tumor formation time is short, and liver cancer growth can be seen in 1 week. Moreover, the technical requirements are relatively low, it is easy to implement, and the success rate is relatively high. It is a currently commonly used in-situ liver cancer model.

[0038] Based on years of clinical experience, the inventors have improved existing methods for establishing orthotopic transplantation-labeled liver cancer models in mice. Through repeated exploration and comparative experiments, they developed a modeling method using specific biochemical agents and surgical instruments. Using this method in BALB / c mice, imaging after two weeks confirmed successful orthotopic liver cancer suppression and growth (n=8), with a 100% success rate and 0% mortality rate.

[0039] In this article, the term "mouse orthotopic transplantation labeled liver cancer model" may also be referred to as "mouse orthotopic transplantation liver cancer model," "mouse orthotopic liver cancer transplantation model," "mouse orthotopic liver cancer model," "mouse orthotopic liver cancer model," "mouse liver cancer transplantation model," or "orthotopic mouse model." For simplicity, the term "mouse liver cancer model" may be shortened to "mouse liver cancer model." These terms have the same meaning and can be used interchangeably.

[0040] It is well known in the medical field that the in situ transplant model modeling has problems such as difficulty in tumor fixation, high blood loss during surgery, and long time. The current technical solutions include surgical suture fixation, biological glue fixation and other operational links. However, since the color of the liver and the sutures are close during the operation, surgical sutures are difficult to sew and take a long time, requiring a surgeon with rich experience; furthermore, the liver capsule is relatively thin, and there are disadvantages such as the liver parenchyma being pulled by the sutures, resulting in suture failure. The bioglue tumor fixation method has the problem of interference from foreign substances. During the use of bioglue, the wound surface needs to be kept dry. Bioglue is liquid and has strong fluidity, otherwise it is easy to form large lumps. However, mice are small in size and their livers are also small. During operation, they are prone to adhesion to surrounding organs, which is not conducive to the healing of the mouse incision, hinders tumor growth, interferes with experiments, and is difficult to operate.

[0041] As an improvement, the present invention abandons surgical suture and bio-glue fixation methods and instead uses Matrigel to fix the transplanted tumor. Matrigel is less fluid than bio-glue and is rich in nutrients, which promotes successful tumor transplantation. In conjunction with Matrigel's tumor fixation, an O-ring is used for auxiliary support. The O-ring limits the Matrigel's fluidity, preventing the bio-glue from accidentally adhering to other tissues. Furthermore, the O-ring is reusable and easy to use.

[0042] In the modeling implementation method of the present invention, conventional operations such as H22 cell injection, in vivo imaging, and tumor growth observation are well known to those skilled in the art.

[0043] In a specific embodiment, the kit for constructing an orthotopic transplantation model of mouse liver cancer provided by the present invention, in addition to the aforementioned H22 cell line, biochemical reagents, experimental equipment, and surgical instruments, may further include at least one of the following items: a carrying toolbox, the space of which is divided into defined spaces that can accommodate one or more containers and operating utensils. Such containers are, for example, medicine bottles, test tubes, and the like, and each container contains a separate component for the method of the present invention; an instruction manual, which can be written on bottles, test tubes, and the like, or on a separate piece of paper, or on the outside or inside of the container. For example, it is a paper piece with a download window for an operation demonstration video APP such as a QR code. The instruction manual can also be in a multimedia form, such as a USB flash drive, a network disk, etc.

[0044] With the kit composed of the above, the present invention has established an implementation technique for an orthotopic transplantation model of liver cancer by a simple method, implementing a more convenient modeling operation method, shortening the operation time, reducing the pain of animals, improving the experimental efficiency, increasing the success rate, and the constructed mouse model has biological stability.

[0045] The following further elaborates on the present invention with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0046] Embodiment

[0047] In this article, the addition amounts, contents, and concentrations of various substances are involved. Among them, unless otherwise specified, the percentage content refers to the mass percentage content (wt%) or the volume / weight percentage content (w / v%).

[0048] In the embodiments of this article, if no specific description is made for the operating temperature, the temperature generally refers to room temperature (10 - 40 °C).

[0049] Main materials and instruments:

[0050] Animals: BALB / c mice (6 weeks old, male)

[0051] Cell line: H22-luc cells (The H22 cells were purchased from the Institute of Zoology, Chinese Academy of Sciences; the H22-luc cells were constructed by GeneChem.)

[0052] Surgical instruments: surgical scissors, ophthalmic curved forceps, straight forceps, needle holder, suture needle with thread (Ningbo Lingqiao Co., Ltd., specification 0#5), plastic "O" ring with an inner diameter of 5 mm (Yuanmeng Industrial (Shenzhen) Co., Ltd.), Matrigel (Corning, catalog number 354234), cotton swabs

[0053] Experimental reagents and equipment: 1640 medium, FBS, P / S, PBS, XenoLight D-Luciferin Potassium Salt, culture flasks, centrifuge tubes.

[0054] Main instruments: CO2 incubator, autoclave, refrigerator, syringe, laminar flow hood, centrifuge, small animal in vivo imaging system, fluorescence inverted phase contrast microscope.

[0055] Example 1: Cell culture and injection

[0056] Take out H22-luc cells from the liquid nitrogen tank; resuscitate the cells in a water bath at 37°C; centrifuge, discard the cryopreservation solution, add an equal volume of complete medium (89% RPMI 1640 medium, 10% FBS, 1% P / S), mix well and centrifuge. Then resuspend with complete medium and place in a CO2 incubator for 24 h. When the cell density reaches 80-100%, passage at a ratio of 1:2.

[0057] Construction of orthotopic transplantation model: Collect the culture medium of H22-luc cells, centrifuge, discard the supernatant, wash twice with PBS and then resuspend with PBS. Count under a microscope and adjust the cell concentration to 2×10 7 / mL, and inject 0.1 mL into each mouse.

[0058] Example 2: Construction of orthotopic transplantation model

[0059] One week after injecting H22-luc cells into the mice, a subcutaneous model is formed. Anesthetize the mice with 50 mg / kg pentobarbital, shave the hair, disinfect with iodophor, incise the epidermis, take out the subcutaneous tumor, separate the tumor capsule, expose the tumor parenchyma, immerse the tumor body in PBS solution, and trim it into small tumor tissue blocks of 2 mm×2 mm×2 mm with ophthalmic forceps.

[0060] According to the Figure 1 shown modeling process, shave the recipient mice, anesthetize with 50 mg / kg pentobarbital, disinfect with iodophor, incise the epidermis, peritoneum, cut open the liver capsule, expand the surgical field, then use a cotton swab dipped in PBS to take out the liver outside the body, carefully cut open the liver capsule with ophthalmic curved scissors to gradually form a 3-mm tunnel, compress the bleeding with a dry cotton swab, and bury the tumor tissue block into the liver tunnel. Note: Keep the liver surface moist with a cotton ball dipped in PBS throughout the operation. Cover the incision of the liver buried with a moist PBS "O" ring, add matrix glue, add pre-cooled matrix glue after 3-5 min, wait for it to completely solidify, carefully remove the "O" ring with ophthalmic forceps, wait for the matrix glue to solidify and stop bleeding, suture the abdomen layer by layer with 0#8 suture, disinfect the incision, inject gentamicin subcutaneously to prevent infection, keep the mice warm, and wait for the anesthesia to wear off.

[0061] Example 3: Detection of orthotopic transplantation model

[0062] After the operation, the mice were intraperitoneally injected with XenoLight D-Luciferin Potassium Salt (15 mg / ml) every week, and the animals were imaged in vivo by the IVIS IV system to observe the growth of tumors. We modeled the BALB / c mice by the above steps, and imaging was performed 2 weeks later. See Figure 2 , which confirmed that the orthotopic liver cancer inhibition model was successful and active (n = 8), with a success rate of 100% and a mortality rate of 0%.

[0063] The above-established orthotopic liver cancer transplantation model can be used for basic research on liver cancer drugs, drug research, pre-clinical drug clinical trials, etc.

[0064] The above embodiments are for the convenience of those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily modify the embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A kit for constructing an orthotopic transplantation model of mouse liver cancer, characterized in that, Including: H22 cell line; biochemical reagents, including 1640 medium, FBS, P / S, PBS, D-Luciferin potassium salt, Matrigel; experimental equipment, including cell culture flasks, centrifuge tubes; surgical instruments, including surgical scissors, suture needles with thread, "O" rings, wherein the H22 cell line is H22-luc cells; the "O" ring is a plastic "O" ring with an inner diameter of 5 mm, and the kit does not include liquid bioadhesive.

2. The kit according to claim 1, characterized in that, The H22 cell line is used for subcutaneous injection into mice to form subcutaneous tumors.

3. The kit according to claim 1, wherein The mice are BALB / c mice.

4. The kit according to claim 1, wherein The surgical instruments further include curved ophthalmic forceps, straight forceps, needle holders, cotton swabs.

5. The kit according to claim 1, wherein The specification of the suture needle with thread is suture needle with thread 0#5.

6. The kit according to claim 1, wherein Also including: Iodophor, Gentamicin.

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