Construction method of animal model for spontaneous metastasis of popliteal space lymph nodes on foot pads of cholangiocarcinoma

Abstract

The invention provides a construction method of a bile duct cancer foot pad popliteal fossa lymph node spontaneous metastasis animal model, and relates to the technical field of animal models.The construction method comprises the steps that bile duct cancer cell lines (QBC939, HuCCT1) and tumor-related fibroblasts (CAFs) are co-injected and inoculated to a foot pad of an immunodeficient animal; the animal model for spontaneous metastasis of popliteal fossa lymph nodes on the foot pad of the bile duct cancer is successfully constructed. The model construction operation process is simple, anesthetic dissection of the mouse is not needed, and the success rate is high. Besides, the dynamic process of bile duct cancer lymph node metastasis is simulated in an animal body, the pathological and physiological characteristics of bile duct cancer lymph node metastasis are better met, and an important animal model is provided for fundamental research of bile duct cancer lymph node metastasis.

Description

Technical Field

[0001] The invention relates to the technical field of constructing animal models, and in particular to a method for constructing an animal model of spontaneous metastasis of popliteal lymph nodes in the foot pad of cholangiocarcinoma. Background Art

[0002] Cholangiocarcinoma (CCA) is the second most common primary liver malignancy after hepatocellular carcinoma (HCC). CCA lacks typical clinical symptoms in the early stage and is usually diagnosed when the disease has entered the middle and late stages, which greatly affects the treatment options and leads to poor prognosis. Since lymphatic metastasis is the most common metastasis of cholangiocarcinoma, it can be used as an independent risk factor for recurrence and prognosis of cholangiocarcinoma. Therefore, it is of great theoretical significance and clinical value to clarify the molecular mechanism of lymphatic metastasis of cholangiocarcinoma and explore the key molecules for early diagnosis and targeted treatment of cholangiocarcinoma.

[0003] At present, several large-sample studies have found that in patients with cholangiocarcinoma, regardless of whether R0 resection is achieved, lymph node metastasis is an independent risk factor for poor prognosis. A clinical study at Nagoya University in Japan found that after surgical resection, the 5-year survival rate of patients with lymph node-negative cholangiocarcinoma was 67.1%, while the 5-year survival rate of patients with lymph node-positive cholangiocarcinoma was only 22.1%. Therefore, it is of great clinical significance to clarify the mechanism of lymph node metastasis in cholangiocarcinoma. Constructing a reliable animal model that can be directly used to study spontaneous lymph node metastasis in cholangiocarcinoma will provide an important theoretical basis for the study of lymph node metastasis in cholangiocarcinoma.

[0004] Spontaneous lymphatic metastasis of cholangiocarcinoma is an extremely complex process, including: (1) lymphangiogenesis in the tumor microenvironment; (2) spread of tumor cells from the primary tumor site to the lymphatic system; (3) tumor cells are transported to local lymph nodes through lymphatic vessels; (3) tumor cells colonize in lymph nodes and change the local microenvironment; (4) tumor cells grow in lymph nodes to form metastases; (5) tumor cells metastasize to more distant lymph nodes through lymphatic vessels. Each of these steps is crucial. In the early stage, a research group directly micro-injected tumor cells HuCCT1 into the inguinal lymph nodes of immunodeficient mice to directly construct lymph node metastases, which provided a certain theoretical basis for the study of cholangiocarcinoma cells in the lymph node microenvironment. However, this model also has many shortcomings: (1) Spontaneous lymph node metastasis of cholangiocarcinoma is a dynamic process. This model directly injects tumor cells into the lymph nodes and cannot reflect the dynamic process of spontaneous tumor metastasis; (2) The operation process is very difficult and the procedure is very complicated. It requires anesthesia and dissection of mice, which can easily lead to the death of immunodeficient mice; (3) The success rate is low. If the number of injected tumor cells is too small, it is difficult to form a tumor focus. If the number of injected tumor cells is too large, the entire lymph node will be occupied by growing tumor cells and the entire lymph node structure will be destroyed, making it difficult to simulate the microenvironment of cholangiocarcinoma lymph node metastasis. Summary of the invention

[0005] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide an animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma and a method for constructing the same. By co-injecting cholangiocarcinoma cell lines (QBC939, HuCCT1) and tumor-associated fibroblasts (CAFs) into the footpad of immunodeficient animals, an animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma was successfully constructed, providing an important animal model for basic research on lymph node metastasis of cholangiocarcinoma.

[0006] The specific scheme adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for constructing an animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma, comprising the following four stages: Phase I: Cholangiocarcinoma cells and tumor fibroblasts were co-injected into the footpads of immunodeficient animals; Phase II: Two weeks after the first footpad injection of Phase I, the second footpad injection was performed. This time, bile duct cancer cells and tumor fibroblasts were co-injected into the same site. Phase III: After the second injection, decide whether to perform the third injection based on the in situ tumor formation. If further injection is required, refer to the injection steps of Phase II. Phase 4: Dynamic in vivo imaging will be performed starting from the fourth week to observe popliteal lymph node metastasis; if popliteal lymph node metastasis is observed by in vivo imaging, the experiment can be terminated according to the specific experimental design requirements.

[0008] As a further optimization of the above construction method, the first stage specifically includes the following steps: Step 1: Isolation, culture and immortalization of tumor-associated fibroblasts (CAFs); Step 2: In vitro culture of cholangiocarcinoma cell line QBC939 / HuCCT1; Step 3: co-injection of cholangiocarcinoma cell CAFs and tumor fibroblasts QBC939 / HuCCT1; wherein the ratio of the number of inoculated cells of QBC939 / HuCCT1 and CAFs is 10:1; and the cell suspension used during inoculation is PBS buffer; Step 4: Press the injection point for 1 minute and apply Fuaile medical glue to the puncture point.

[0009] Furthermore, in step 3, the number of cells inoculated: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells; inoculation site: the left posterior foot pad of each nude mouse; total injection volume: 50uL; cell suspension used: PBS buffer;

[0010] As a further optimization of the above construction method, the second stage specifically includes the following steps: Step 1: co-injection of cholangiocarcinoma cell CAFs and tumor fibroblasts QBC939 / HuCCT1; the ratio of the number of inoculated cells of QBC939 / HuCCT1 and CAFs was 10:1; the cell suspension used during inoculation was PBS and matrix gel at a volume ratio of 1:1; Step 2: Press the injection point for 1 minute after injection and apply Fuaile medical glue to the puncture point. Step 3. 7 days after the second cell inoculation, observe the tumor formation in the footpad of nude mice every 2 days and measure the size of the tumor in situ on the footpad. If drug treatment is required, administer the drug by intraperitoneal injection at a dose of 50 mg / kg per day.

[0011] Furthermore, in step 2, the number of cells inoculated: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells; the inoculation site is the first inoculation site; the total injection volume: 50uL; the final system: 50ul, including 25ul PBS and 25ul matrix gel.

[0012] In a second aspect, the present invention provides an animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma constructed by the above method.

[0013] Beneficial effects: The present invention successfully constructed an animal model of spontaneous metastasis of cholangiocarcinoma footpad popliteal lymph nodes by co-injecting cholangiocarcinoma cell lines (QBC939, HuCCT1) and tumor-associated fibroblasts (CAFs) into the footpads of immunodeficient animals. The model construction operation process is simple, no anesthesia is required for dissecting mice, and the success rate is high. In addition, the present invention simulates the dynamic process of cholangiocarcinoma lymph node metastasis in animals, which is more in line with the pathophysiological characteristics of cholangiocarcinoma lymph node metastasis, and provides an important animal model for basic research on cholangiocarcinoma lymph node metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A schematic diagram of the construction of a novel animal model of spontaneous metastasis of popliteal lymph nodes in the foot pad of cholangiocarcinoma provided by the present invention;

[0015] Figure 2 The result diagram of the gross anatomical evaluation of popliteal lymph node metastasis using a small animal in vivo imaging device provided by the present invention;

[0016] Figure 3 This is a diagram showing the results of HE staining and immunohistochemical staining of paraffin sections of popliteal lymph node tissue provided by the present invention. DETAILED DESCRIPTION

[0017] This invention is based on the previous research of this research group. The tumor-associated fibroblasts (CAFs) isolated from cholangiocarcinoma tumor tissue with lymph node metastasis can promote tumor progression and lymph node metastasis, and successfully constructed a new animal model of spontaneous lymph node metastasis of cholangiocarcinoma. By co-injecting cholangiocarcinoma cell lines (QBC939, HuCCT1) and tumor-associated fibroblasts (CAFs) into the footpads of immunodeficient animals, an animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma was successfully constructed, simulating the dynamic process of cholangiocarcinoma lymph node metastasis in animals, and providing an important scientific basis for the basic research of cholangiocarcinoma lymph node metastasis.

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0019] Based on previous work, we invented a new animal model of spontaneous lymph node metastasis of cholangiocarcinoma and its construction method, which includes the following four stages.

[0020] Phase 1:

[0021] Isolation and culture of tumor-associated fibroblasts (CAFs)

[0022] 1. Experimental Preparation

[0023] Reagents: tissue digestion solution (culture medium + 0.1% type I collagenase), DMEM culture medium, PBS.

[0024] Consumables: 6-well plate, 15ml / 50ml centrifuge tube, sterile tweezers / scissors, 10cm culture dish, 70μm filter, Pasteur tube.

[0025] Thaw the tissue digestion solution in a 37°C water bath (store in a -30°C refrigerator).

[0026] 2. Tissue Isolation

[0027] (1) Remove the burnt tissue and fat tissue, cut the tissue into small pieces (5mm 3 optimal).

[0028] (2) Add 3 ml of preheated digestion solution and transfer the tissue and digestion solution to a 15 ml centrifuge tube.

[0029] (3) Digest at 37°C and 35 rpm for 20 min (three digestions in total: the digestion time for tumor tissue and normal bile duct is 20 min, 15 min, and 10 min, respectively; the lymph node tissue is soft, and the digestion time is 10 min, 8 min, and 5 min, respectively). The digestion time can be appropriately extended for the last time to ensure complete digestion.

[0030] (4) Use a Pasteur tube to blow the tissue vigorously for at least 20 times. Let the 15 ml tube stand for 5 minutes. After the large pieces of tissue have settled, aspirate the supernatant into another 15 ml centrifuge tube.

[0031] (5) Add 1-2 ml of culture medium, centrifuge at 250 g, 4°C for 5 min, discard the supernatant, add 1 ml of culture medium to resuspend the precipitate, and store on ice after labeling.

[0032] (6) Add 3 ml of preheated digestion solution to resuspend the remaining tissue from the first digestion.

[0033] (7) Repeat steps 3-6.

[0034] (8) Filter through a 70 μm filter, collect all the precipitates in a 50 ml tube, add appropriate amount of culture medium, and centrifuge at 4°C, 250 g for 5 min.

[0035] (9) If a large number of red blood cells are present in the precipitate, they need to be lysed. The steps are as follows: A. Add 4 ml of red blood cell lysis buffer, lyse at room temperature for 2 minutes, and centrifuge at 4°C, 250g for 5 minutes (different lysis buffers have different usages, please refer to the instructions for details); B. Discard the supernatant, resuspend with appropriate amount of culture medium, and centrifuge at 4°C, 250g for 5 min.

[0036] (10) Discard the supernatant, take an appropriate amount of cells and plate them into a 6-well plate, adding 2 ml of culture medium to each well.

[0037] (11) Culture in a 37°C incubator. When a large number of long strips of fibroblasts appear on the bottom, change the medium every other day. When the wells are full, change the medium again. Collect the supernatant after 48 hours and subculture at a ratio of 1:2.

[0038] Immortalization of tumor-associated fibroblasts (CAFs)

[0039] It is difficult to permanently propagate primary CAFs cells. Generally, after the 7th to 8th generation, the cells will age. The establishment of this model requires a large number of CAFs. Therefore, we propagated the isolated CAFs to the 3rd to 4th generation and sent them to Guangzhou Yuexing Biotechnology Co., Ltd. for immortalization.

[0040] Culture and preparation of three in vitro cholangiocarcinoma cell lines

[0041] 1. Cultivate cholangiocarcinoma cell lines (QBC939, HuCCT1) labeled with luciferase in vitro; QBC939: DMEM complete medium, HuCCT1: RPMI-1640 complete medium, CAFs: DMEM complete medium. When the cell density reaches 90%, discard the old culture medium, and gently wash the cells 3 times with PBS. After trypsin digestion of the cells, terminate with culture medium, and transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 5min.

[0042] 2. After centrifugation, carefully pour off the supernatant. Resuspend the cells in PBS buffer and count them to adjust the cell density. Transfer the cell suspension to an EP tube and place it on ice at the same time; the planned number of cells to be inoculated in nude mice: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells (10:1). Inoculation site: This experiment was the left posterior foot pad of each nude mouse; total injection volume: 50uL.

[0043] 3. After fixing the nude mouse, use a pipette to blow and evenly distribute the cells. Then, inject 50 μL of cell suspension into the left posterior foot pad and press the injection point for 1 minute. To prevent the cell suspension from leaking out, apply "Fuaile Medical Glue" to the puncture point (this operation step is extremely important).

[0044] Fuaile Medical Glue: [Ingredients]: This product is made of α-octyl cyanoacrylate and α-butyl cyanoacrylate in a certain proportion and then put into a glass ampoule. It can be used with glue suction bottle, glue stick, glue spray bottle and nozzle according to clinical needs. [Indications]: It is used for sealing the edge of surgical incision close to the skin surface, including the sealing of minimally invasive interventional surgery puncture, and the sealing of wounds after complete debridement. It cannot be used for the closure of the sub-surface layer of the skin; when other methods are ineffective, it can be used for spraying the wound surface under direct vision or laparoscopic debridement in vivo, and sealing / hemostasis of bleeding on the wound surface of organs and tissues; it is used for embolization treatment of gastric varicose veins.

[0045] Phase 2:

[0046] Two weeks after the first footpad injection, the second footpad injection is performed. The specific steps are as follows:

[0047] 1. Cultivate cholangiocarcinoma cell lines (QBC939, HuCCT1) in vitro; QBC939: DMEM complete medium, HuCCT1: RPMI-1640 complete medium, CAFs: DMEM complete medium. When the cell density reaches 90%, discard the old culture medium, and gently wash the cells 3 times with PBS. After trypsin digestion of the cells, terminate with culture medium, and transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 1000rpm for 5min.

[0048] 2. After centrifugation, carefully pour off the supernatant. Resuspend the cells in PBS buffer and count them to adjust the cell density. Transfer the cell suspension to an EP tube and place it on ice at the same time; the planned number of cells to be inoculated in nude mice: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells (10:1). Inoculation site: the first inoculation site; total injection volume: 50uL. Final system: 50ul, including 25ulPBS and 25ulMatrigel; PBS:Matrigel = 1:1.

[0049] 3. After fixing the nude mouse, use a pipette to blow and evenly distribute the cells. Then, inject 50 μL of cell suspension into the left posterior foot pad and press the injection point for 1 minute. To prevent the cell suspension from leaking out, continue to apply "Fuaile Medical Glue" to the puncture point.

[0050] 4. 7 days after the second cell inoculation, observe the tumor formation in the footpad of nude mice every 2 days and measure the size of the tumor in situ on the footpad. If drug treatment is required, administer the drug by intraperitoneal injection at a dose of 50 mg / kg per day.

[0051] Phase 3:

[0052] In situ footpad tumor formation is a prerequisite for the successful establishment of this animal model. However, the subcutaneous tissue in the mouse footpad is dense and the space is limited. It is often difficult to form an in situ tumor with a single injection of tumor cells. Therefore, repeated footpad injections are required to ensure successful in situ tumor formation. Therefore, after the second injection, the decision on whether to perform a third injection is made based on the in situ tumor formation. For specific steps, refer to the second stage.

[0053] Phase 4:

[0054] Dynamic live imaging was performed from the 4th week to observe the metastasis of the popliteal lymph nodes. Generally, popliteal lymph node metastasis can be seen in the 6th to 8th week after the first footpad injection. When popliteal lymph node metastasis was observed by live imaging, the experiment was terminated according to the specific experimental design requirements. Nude mice were killed by cervical dislocation, and tumors and other tissues were fixed and embedded in formalin.

[0055] The above construction method was first created by our research team and has the following characteristics:

[0056] 1. There is no research on the construction of an animal model of spontaneous metastasis of popliteal lymph nodes in the foot pad of cholangiocarcinoma.

[0057] 2. This study used co-injection of cholangiocarcinoma cells and tumor fibroblasts (CAFs), which is simple to operate and does not require anesthesia of dissecting mice, making it easy to operate.

[0058] 3. In situ footpad tumor formation is a prerequisite for the successful construction of this animal model. However, the subcutaneous tissue in the mouse footpad is dense and the space is limited. It is often difficult to form a tumor in situ with a single injection of tumor cells. Therefore, this study used multiple injections to ensure the success rate of the model construction.

[0059] 4. During the first footpad injection, the cells were resuspended in PBS and then injected into the left posterior footpad. To prevent the leakage of the cell suspension, the clinically used "Folair Medical Glue" was used for the first time to construct the model and smear the puncture point to prevent the mouse from squeezing out the cell suspension from the puncture point during activity.

[0060] 5. During the second and third footpad injections, use PBS:matrigel = 1:1 to resuspend the cells to ensure the success rate of in situ tumor formation in the footpad.

[0061] Figure 1 A shows a schematic diagram of the construction of a novel animal model of spontaneous metastasis of popliteal lymph nodes in the foot pad of cholangiocarcinoma provided by the present invention. Figure 1 B shows the specific operation steps for constructing the model, including needle insertion, injection, application of medical glue, drying and sealing, etc. Figure 2 The present invention provides a method for evaluating popliteal lymph node metastasis by using a small animal in vivo imaging device through gross dissection. Figure 2A shows the in vivo imaging and gross anatomy of mice with popliteal lymph node metastasis. Figure 2 B shows the in vivo imaging and gross anatomy of a mouse with non-metastatic popliteal lymph nodes; Figure 3 The invention provides HE staining and immunohistochemical staining of paraffin sections of popliteal lymph node tissue.

[0062] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A method for constructing an animal model of spontaneous metastasis of popliteal lymph nodes in cholangiocarcinoma. Features: It includes the following four stages: Phase I: Cholangiocarcinoma cells and tumor fibroblasts were co-injected into the footpads of immunodeficient animals; Phase II: Two weeks after the first footpad injection of Phase I, the second footpad injection was performed. This time, bile duct cancer cells and tumor fibroblasts were co-injected into the same site. Phase III: After the second injection, decide whether to perform the third injection based on the in situ tumor formation. If further injection is required, refer to the injection steps of Phase II. Phase 4: Dynamic in vivo imaging will be performed starting from the fourth week to observe popliteal lymph node metastasis; if popliteal lymph node metastasis is observed by in vivo imaging, the experiment can be terminated according to the specific experimental design requirements.

2. The construction method according to claim 1, It is characterized in that The first stage specifically includes the following steps: Step 1: Isolation, culture and immortalization of tumor-associated fibroblasts (CAFs); Step 2: In vitro culture of cholangiocarcinoma cell line QBC939 / HuCCT1; Step 3: co-injection of cholangiocarcinoma cell CAFs and tumor fibroblasts QBC939 / HuCCT1; wherein the ratio of the number of inoculated cells of QBC939 / HuCCT1 and CAFs is 10:1; and the cell suspension used during inoculation is PBS buffer; Step 4: Press the injection point for 1 minute after injection and apply Fuaile medical glue to the puncture point.

3. The construction method according to claim 2, It is characterized in that In step 3, the number of cells inoculated: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells; inoculation site: the left posterior foot pad of each nude mouse; total injection volume: 50uL; cell suspension used: PBS buffer.

4. The construction method according to claim 3, It is characterized in that The second stage specifically includes the following steps: Step 1: co-injection of cholangiocarcinoma cell CAFs and tumor fibroblasts QBC939 / HuCCT1; the ratio of the number of inoculated cells of QBC939 / HuCCT1 and CAFs was 10:1; the cell suspension used during inoculation was PBS and matrix gel at a volume ratio of 1:1; Step 2: Press the injection point for 1 minute after injection and apply Fuaile medical glue to the puncture point. Step 3. 7 days after the second cell inoculation, observe the tumor formation in the footpad of nude mice every 2 days and measure the size of the tumor in situ on the footpad. If drug treatment is required, administer the drug by intraperitoneal injection at a dose of 50 mg / kg per day.

5. The construction method according to claim 4, It is characterized in that In step 2, the number of cells inoculated: QBC939 / HuCCT1: 2 million cells, CAFs: 200,000 cells; the inoculation site is the first inoculation site; the total injection volume: 50uL; the final system: 50ul, including 25ul PBS and 25ul matrix gel.

6. An animal model of spontaneous metastasis of popliteal lymph nodes in the footpad of cholangiocarcinoma constructed by the construction method described in any one of claims 1 to 5.

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