CN115136930A - Mouse breast cancer pain model and establishment method thereof - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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Abstract
The invention discloses a mouse breast cancer pain model and an establishment method thereof, belonging to the field of medical basic research. The mouse breast cancer pain model is obtained by placing protein gel containing MADB106 cells at the periphery of mouse sciatic nerve and then culturing. The creation of the tumor pain model provides a useful tool for the research of the topic related to the tumor pain, in particular to the targeted antagonism of the pain-related pathway and the research and development of the related anti-tumor pain medicine. As the cell strain is derived from a rat breast cancer metastatic cancer tissue, the application of the cell strain to create a tumor pain model can be regarded as a breast cancer metastatic model, and the cell strain has important scientific research value and application potential.
Description
Technical Field
The invention belongs to the field of medical basic research, and particularly relates to a mouse breast cancer pain model and an establishment method thereof.
Background
Tumor pain is one of the high clinical complications in the process of treating tumors, and is also one of the main causes of suffering of patients with advanced tumors and causing the reduction of life quality. In pain patients, about 50% to 80% of pain is not effectively controlled for various reasons. The cause of tumor pain is the sensation caused by the transmission of information to the nerve center that the site of pain needs to be repaired or modulated. Because the change of local micro-environments before and after the occurrence of the tumor cannot be completely reproduced by in vitro experiments, including the interaction among multiple micro-environments such as tumor cells, immune cells, nerve cells and the like, an in vivo tumor model is still the best model for researching tumor pain. In recent years, although there are reports of establishment of tumor pain models in succession, in view of immunosuppression of an immune normal mouse on an exogenous graft, a general tumor model needs to be established on an immune deficient mouse, and the breeding environment of the immune deficient mouse limits monitoring of pain indexes, which further hinders development of related targeted drugs, so how to establish the tumor pain model of the immune normal mouse is one of the problems to be solved for researching tumor pain.
The MADB106 cell line is derived from rat breast cancer lung metastatic tumor tissue, has the advantages of high growth speed and easiness in culture, and reports of constructing a tumor pain model by using the MADB106 cell line are not searched so far.
Disclosure of Invention
The invention aims to provide a mouse breast cancer pain model and an establishment method thereof, overcomes the defect that the existing tumor transplantation model needs to be carried out in an immunodeficient mouse, provides application of the model in batch establishment of tumor pain models in an immune normal mouse, and provides a reliable in vivo model for clinical screening of tumor pain targeted drugs.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mouse breast cancer pain model is obtained by placing protein gel containing MADB106 cells around the sciatic nerve of a mouse and then culturing.
Further, the protein gel containing the MADB106 cells is prepared by mixing the MADB106 cells and rat tail collagen.
Further, the protein gel containing the MADB106 cells is prepared by the following steps:
step 1, to 100mL of 10 × EBSS solution, 2.45g NaHCO was added 3 7.5mL of 1M NaOH, adjusting the pH value to 7.45, filtering for sterilization, fully and uniformly mixing the rat tail collagen and the obtained solution according to the volume ratio of 2;
step 2, after counting the MADB106 cells, digesting and centrifuging, and preparing the number of the cells to be 1 multiplied by 10 7 Mixing the cells with 1.25mL of the mixed solution to prepare an MADB106 cell suspension;
Further, the mice are BALB/c mice, female, clean grade, weight 15-20g, and basal pain threshold 0.98-1.16 g.
The method for establishing the model comprises the following steps:
step 1, preparing a protein gel containing MADB106 cells;
and 2, placing the protein gel prepared in the step 1 around sciatic nerves of the mice, and feeding the mice in an SPF environment to obtain the breast cancer pain model of the mice.
Further, the feeding time was 40 days.
Specifically, the method comprises the following steps: anaesthetizing the mouse, placing on an operating table capable of maintaining an anaesthesia state, shaving hair and preparing skin at the back leg by using a hair shaving machine, locally disinfecting an alcohol cotton ball at the operation position, obliquely cutting and incising the skin at the hip by using an operating knife, separating muscle gaps in a blunt manner by using a microscope under an operating microscope, exposing sciatic nerves, placing protein gel mixed with MADB106 cells on the periphery of the sciatic nerves, closing an incision, suturing fascia and skin of the mouse layer by layer, and coating anti-infective medicine on the sutured part to prevent infection. And (5) continuously feeding the mice after the operation for 40 days to establish a tumor pain model.
The cell gel is placed without contacting the skin or other tissues, and after the placement is finished, gelatin sponge is needed to cover the incision on the lower layer of the muscle before the incision is closed, otherwise, the cell gel is lost, the number of cells is different, and the molding failure is easy to cause.
The rat tail collagen should be preserved at 0 deg.C or below before mixing with MADB106 cells, mixed with MADB106 cells, divided into 25 μ L cell-containing gel, and put at 37 deg.C, 5% CO 2 Was allowed to clot and 2mL of complete medium was added after clotting, otherwise cell viability was affected.
The creation of the tumor pain model provides a useful tool for the research of the topic related to the tumor pain, in particular to the targeted antagonism of the pain-related pathway and the research and development of the related anti-tumor pain medicine. Because the cell strain is derived from a rat breast cancer metastatic cancer tissue, the cell strain is applied to create a tumor pain model which can be regarded as a breast cancer metastatic model, and has important scientific research value and application potential.
Drawings
FIG. 1 is a schematic view of the molding of the present invention.
FIG. 2 is a diagram showing the preparation of a cell gel before modeling in the model of example 1.
FIG. 3 is the mechanical pain threshold of mice on days 0 and 40 of the model set-up of example 2.
FIG. 4 shows the total scratching, and licking time of mice on days 0 and 40 of the model set-up in example 2.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
The materials used in the following examples were as follows:
1. tumor strain: MADB106 rat breast cancer lung metastasis tumor cell strain provided by Shanghai cells of Chinese academy of sciences;
2. animals: BALB/c mice, female, clean grade, weight 15-20g, provided by the laboratory animal center of the university of southeast university school of medicine;
3. the instrument comprises: small animal anaesthesia machine (produced by revold), von Frey action needle set (produced by Danmic Global), animal test cage (produced by revold), small animal surgical dissecting mirror (produced by revold), fluorescence microscope (produced by Nikon), and cryomicrotome (Leica).
The rat tail collagen is prepared by the following steps:
1. cleaning rat tail, and soaking in 75% alcohol for 5 min;
2. holding the tail by hand, clamping the tip of the tail with hemostatic forceps, breaking the coccyx, and pulling out the tail tendon;
3. cutting the tendon and placing in a plate, and soaking in normal saline;
4. aspirate the tendon (0.5-1 g) with saline;
5. placing the tail tendon in a plate, shearing into pieces, and transferring to a triangular flask;
6. adding 0.1% acetic acid solution according to the proportion of 50mL of the tail tendon per gram;
7. shaking to disperse the tendon into acetic acid solution, and standing at 4 deg.C for one week;
8. centrifuging at 4000 rpm for 10-15 minutes;
9. the supernatant was aspirated, split-filled into small bottles, and stored at 4 ℃.
Example 1
1. To 100mL of a 10 × EBSS (sigma E-6132) solution, 2.45g NaHCO was added 3 7.5mL of 1M NaOH was adjusted to pH 7.45, and then sterilized by filtration through a 0.2 μ M filter. And (2) fully and uniformly mixing the rat tail collagen with the obtained solution according to the volume ratio of 2.
The MADB106 cells were counted, digested and centrifuged to prepare a number of 1X 10 7 The cells (2) were mixed with 1.25mL of the above mixture to prepare a MADB106 cell suspension.
25 mu L of cell suspension is sucked up and dropped into a 6cm culture dish at a constant speed, then the culture dish is placed in an incubator at 37 ℃ for 30 minutes, and 1-2mL of complete culture medium is added into the culture dish to maintain the cell activity after collagen forms gel.
2. Molding machine
Model group: anaesthetizing a mouse by using a gas anaesthesia device, placing the mouse on an operating table capable of maintaining an anaesthesia state with the abdomen facing downwards, shaving hair and preparing skin at the back leg by using a shaving machine, locally disinfecting an alcohol cotton ball at the operation position, cutting the skin at the hip part by beveling with an operating knife, separating a muscle gap under an operating microscope by using a micro forceps in a blunt manner, exposing a sciatic nerve, placing protein gel mixed with MADB106 cells at the periphery of the sciatic nerve, placing a layer of gelatin sponge at the lower layer of the muscle, closing an incision, suturing the fascia and the skin of the mouse layer by layer, and coating iodophor at the suturing part to prevent infection.
Control group: an equal volume of gel without MADB106 cells was placed around the mouse sciatic nerve and the rest of the procedure was performed in the same model group.
3. The model group and the control group obtained above were evaluated as follows:
the mechanical pain threshold and the total time to scratch, scratch and lick were measured with a Von Frey behavioral needle set and a stopwatch on day 5, 10, 15, 20, 25, 30, 35, 40 before and after surgery, respectively.
Mechanical pain threshold: a certain number of animals are selected, and are adapted to an overhead metal mesh animal test cage for 3 days, about 2 hours each day, and formal detection is carried out from the 4 th day. Using von Frey filaments (0.16 g-2.00 g) perpendicular to the area tested (plantar surface of hind paw) for each animal, the filaments were bent 1-2 seconds each time the same force was applied, 10 seconds between the two measurements, if the animals had paw withdrawal, paw shaking, licking etc., indicating that the animals felt pain, marked as "x"; if the paw of the animal is not changed, the animal does not feel pain, the mark is O, after the animal is measured for 6 times, the corresponding mechanical pain threshold value is obtained according to the corresponding scale, and the animal with the pain threshold value of 0.98-1.16g is screened for the experiment.
Measuring scratching, scratching and licking time: the screened animals are first adapted in an elevated metal mesh animal test cage for 3 days, about 2 hours per day, and formal detection is carried out from the 5 th day. The total time of scratching, and licking of the left or right limb by the animal per unit time was recorded using a stopwatch.
Statistical treatment: graphPad Prism 6 was used for data statistics and histogram generation.
As shown in fig. 3, compared with the basic threshold before the model, the mechanical pain threshold of the model side and the mechanical pain threshold of the control side on the left and right sides of the mouse are significantly different (P < 0.01) from the 15 th day on the model side, and then the mechanical pain threshold of the model side is continuously reduced while the mechanical pain threshold of the control side is not significantly changed, and the significant difference can be compared on the 15 th day, the 20 th day, the 25 th day, the 30 th day, the 35 th day and the 40 th day on the both sides. This indicates that mechanical hyperalgesia has occurred on day 10 on the right side of the mouse and a gradual and intensified trend.
As shown in FIG. 4, the mice showed statistical differences in the scratching, scratching and licking changes per unit time on day 40 of the model building operation (P < 0.01). This indicates spontaneous pain in the right lower limb of mice seeded with MADB106 cells.
Claims (6)
1. A mouse breast cancer pain model, comprising: the model is obtained by placing protein gel containing MADB106 cells around the sciatic nerve of a mouse and then culturing.
2. The mouse breast cancer pain model of claim 1, wherein: the protein gel containing the MADB106 cells is prepared by mixing the MADB106 cells and rat tail collagen.
3. The mouse breast cancer pain model of claim 2, wherein: the protein gel containing the MADB106 cells is prepared by the following steps:
step 1, to 100mL of 10 × EBSS solution, 2.45g NaHCO was added 3 7.5mL of 1M NaOH, adjusting the pH value to 7.45, filtering for sterilization, fully and uniformly mixing the rat tail collagen and the obtained solution according to the volume ratio of 2;
step 2, digesting and centrifuging the counted MADB106 cells to prepare the MADB cells with the number of 1 multiplied by 10 7 Mixing the cells with 1.25mL of the mixed solution to prepare an MADB106 cell suspension;
step 3, 25. Mu.L of cell suspension is taken and dropped into a 6cm culture dish, and then the culture dish is placed in an incubator at 37 ℃ for 30 minutes to form collagen into gel.
4. The mouse breast cancer pain model of claim 1, wherein: the mouse is a BALB/c mouse, and the basic pain threshold value is 0.98-1.16 g.
5. The method of establishing a mouse breast cancer pain model of claim 1, wherein: the method comprises the following steps:
step 1, preparing protein gel containing MADB106 cells;
and 2, placing the protein gel prepared in the step 1 around sciatic nerves of the mouse, and feeding the mouse in an SPF environment to obtain the mouse breast cancer pain model.
6. The method of establishing according to claim 5, wherein: the breeding time is 40 days.
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| CN202210950766.0A CN115136930A (en) | 2022-08-09 | 2022-08-09 | Mouse breast cancer pain model and establishment method thereof |
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| CN202210950766.0A CN115136930A (en) | 2022-08-09 | 2022-08-09 | Mouse breast cancer pain model and establishment method thereof |
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Application publication date: 20221004 |