A non-invasive cervical cancer orthotopic model modeling method of mice

Abstract

The present application relates to in situ model modeling method, especially for a kind of non-invasive cervical cancer in situ model modeling method of mouse, including selecting several 8 weeks old BALB / c-nude mouse, all mice are divided into non-invasive group and operation group, the age, weight of two groups of mice are compared, and there is no statistically significant difference;Non-invasive group is widened with the cotton stick of diameter 3mm and wipes dry vagina, the tumor block with luciferase fluorescence is immersed in modified hydrogel, after heating and making modified hydrogel solidification, the tissue glue mixed with hard gypsum in modified hydrogel is dropped on the surface of modified hydrogel and quickly inserted into vagina, when there is obstruction, stop deepening, pause 30s and wait for tissue glue to be completely dry, when tumor block is fixed in cervix, it is modeling completion;Operation group first opens abdominal cavity, and tumor tissue of same size is sutured on cervix.This kind of non-invasive cervical cancer in situ model modeling method of mouse, using non-invasive method to replace operation method to establish non-invasive cervical cancer in situ model, can reduce the pain of mouse, reduce experimental difficulty.

Description

Technical Field

[0001] The invention relates to an in situ model building method, in particular to a non-invasive in situ model building method for cervical cancer in mice. Background Art

[0002] The main morphological characteristics of nude mice are: hairless, naked and without thymus. Because nude mice have no thymus, they lack immune response and are immunodeficient animals, making them suitable for preparing in situ tumor models.

[0003] The commonly used method for establishing an orthotopic cervical cancer model is surgery. After the mouse is anesthetized, the abdominal cavity is opened and a 2-3 mm 3 The tumor is sutured to the cervix with fine sutures, and the abdominal cavity is closed with sutures. This modeling method is traumatic and causes significant pain to the animal, affecting its welfare. It also requires a certain amount of time to recover after surgery.

[0004] Directly inserting the tumor mass into the mouse vagina often results in the tumor mass being difficult to fix and easily slipping off, making it difficult to form a model. Furthermore, due to the fewer blood vessels in the vaginal environment, the tumor grows more slowly. Summary of the Invention

[0005] The main purpose of the present invention is to provide a method for modeling a non-invasive in situ cervical cancer model in mice, so as to solve the problems of low production efficiency and easy wear of the dental and maxillary models during fixation proposed in the related art.

[0006] To achieve the above object, according to one aspect of the present invention, a method for establishing a non-invasive in situ model of cervical cancer in mice is provided, comprising the following steps:

[0007] Step 1: 8-week-old BALB / c-nude mice were selected and divided into a non-invasive group and a surgical group. No statistically significant differences were found in age and weight between the two groups.

[0008] Step 2: In the non-invasive group, a 3mm diameter cotton swab was used to widen and dry the vagina. The tumor mass with luciferase fluorescence was immersed in the modified hydrogel. After heating to solidify the modified hydrogel, tissue glue mixed with hard gypsum was dripped onto the surface of the modified hydrogel and quickly inserted into the vagina. When a blockage was felt, the insertion was stopped and paused for 30 seconds to allow the tissue glue to completely dry. When the tumor mass was fixed in the cervix, the model was completed.

[0009] Step 3: The surgical team first opens the abdominal cavity and sutures tumor tissue of the same size to the cervix.

[0010] Furthermore, the body weight of the BALB / c-nude mice was 24±0.56 g.

[0011] Furthermore, after Step 3, in vivo imaging is used for evaluation to compare the tumor formation rates and conditions of the two methods. If fluorescence is clearly visible in the cervix of the mouse, the model is successfully constructed.

[0012] Furthermore, after Step 3, prime8 statistical software was used for analysis, and P < 0.05 was considered statistically significant.

[0013] Furthermore, the modified hydrogel is made from the following raw materials in parts by weight:

[0014]

[0015] This formula can accelerate tumor mass growth, promote blood vessel connections, and increase the rate of cervical cancer invasion by increasing estrogen.

[0016] Furthermore, the in vivo imaging method is specifically as follows:

[0017] Furthermore, a working solution at a dose of 10 μl / g was intraperitoneally injected into nude mice, anesthetized with isoflurane 10-15 minutes later, and then in vivo imaging observation was performed using an in vivo imaging device.

[0018] Furthermore, the working fluid is made of the following raw materials in parts by weight:

[0019] 70-90 portions of DPBS;

[0020] 3-6 copies of mCherry.

[0021] The mCherry working solution prepared with DPBS can be directly fused to the target protein after injection, which effectively ensures accurate imaging of the target cells.

[0022] Furthermore, the concentration of DPBS is 15-17 mg / ml.

[0023] Furthermore, the working solution was prepared as follows: after selecting DPBS (buffered saline solution), mCherry (mCherry fluorescent protein sample) was slowly added and gently stirred at 3-4°C to mix, ultrasonically vibrated for 1-3 minutes, and then refrigerated for later use.

[0024] Before modeling, mice were injected with 1-2 μl / g of prednisone or methylprednisolone. Prednisone or methylprednisolone can reduce the mouse's rejection of transplanted xenogeneic cells, allowing the model to adhere more stably.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In this method for modeling a non-invasive in situ model of cervical cancer in mice, a non-invasive method is used instead of a surgical method to establish a non-invasive in situ model of cervical cancer, which can reduce the pain of mice, reduce the difficulty of experiments, and improve the speed of experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the growth rate of cervical cancer in mice in the non-invasive group according to a preferred embodiment of the present invention;

[0028] Figure 2 This is a graph showing the growth rate of cervical cancer in the surgical group according to a preferred embodiment of the present invention;

[0029] Figure 3 This is a graph showing the fluorescence area monitoring results of in vivo imaging in a preferred embodiment of the present invention;

[0030] Figure 4 This is a comparison diagram of cervical cancer tumor mass in a preferred embodiment of the present invention;

[0031] Figure 5 This is a normal cervix image in a preferred embodiment of the present invention;

[0032] Figure 6 This is a cervical image of the non-invasive group in the preferred embodiment of the present invention;

[0033] Figure 7 This is a cervical image of the surgical group in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0035] In this embodiment, the in vivo imaging device is manufactured by Talon Corporation, model ABL-X6, and the tissue glue is manufactured by 3M Corporation, product number 1469SB.

[0036] See also Figure 1-Figure 2 As shown, the purpose of this embodiment is to provide a method for establishing a non-invasive in situ model of cervical cancer in mice, comprising:

[0037] Ten 8-week-old BALB / c-nude mice weighing (24±0.56) g were selected and divided into a non-invasive group and a surgical group. There was no statistically significant difference in age and weight between the two groups (P>0.05), indicating that the two groups were comparable. Before modeling, 1.5 μl / g of prednisone or methylprednisolone was injected.

[0038] To prepare the modified hydrogel: 50 μl of hydrogel was added to 10 ng of VEGF (vascular endothelial growth factor), 10 ng of FGF9 (fibroblast growth factor 9), 10 ng of EGF (epidermal growth factor), and 20 ng of E2 (estradiol). This formulation accelerated tumor growth, promoted vascular connections, and, by increasing estrogen levels, promoted the invasion of cervical cancer. Next, 100 mg of anhydrite (CaSO4·2H2O) was mixed with 100 ml of tissue glue to increase its water resistance, preventing the tumor from slipping out of the vagina.

[0039] (1) Non-invasive group: First, use a 3mm diameter cotton swab to widen and dry the vagina, immerse the tumor mass with luciferase fluorescence into the modified hydrogel, heat the modified hydrogel to solidify it, and then drop tissue glue mixed with hard gypsum in the modified hydrogel on the surface of the hydrogel and quickly insert it into the vagina. When there is a sense of obstruction, stop going deeper and pause for 30 seconds until the tissue glue is completely dry. When the tumor mass is fixed at the cervix, the model is completed;

[0040] (2) Surgical group: The abdominal cavity was first opened according to the traditional method, and the tumor tissue of the same size was sutured to the cervix.

[0041] In order to verify the effect of the non-invasive in situ model of cervical cancer in mice, the inventors inoculated cervical cancer tumor tissue blocks into the cervix of mice by non-invasive and surgical methods on day 0 of the experiment. The tumor block size was 2mm. 3 On the fourth day after tumor inoculation, in vivo imaging was used to monitor the size of cervical cancer tumors. Figure 1 , Figure 2 , Figure 3 As shown. The fluorescence area was monitored by in vivo imaging ( Figure 3 ) found that the growth rate of cervical cancer in mice in the non-invasive group ( Figure 1 ) faster than the growth rate of cervical cancer in the surgical group ( Figure 2 ), and there was a significant difference (p<0.05)

[0042] In vivo imaging to monitor fluorescence area: First, a dose of 10 μl / g of the working solution was injected intraperitoneally into nude mice. After 10 minutes, isoflurane anesthesia was performed and in vivo imaging was performed using an in vivo imaging device. After preparing 3 ml of 15 mg / ml DPBS with the working solution, 0.18 mg of mCherry was slowly added and gently stirred at 3-4°C. After ultrasonic oscillation for 2 minutes, the mixture was refrigerated for later use.

[0043] At the end of the experiment, the mice in each group were euthanized, and the tumors of the mice in each group were removed and weighed. Figure 4As shown, 8 of 10 mice in the noninvasive group developed tumors, a tumor formation rate of 80%; 5 of 10 mice in the surgical group developed tumors, a tumor formation rate of 50%. Among the mice with tumors, the mass of cervical cancer tumors in the noninvasive group was significantly greater than that in the surgical group (p < 0.05). Therefore, the autopsy results indicate that the noninvasive group had a higher cervical cancer formation rate and tumor growth rate than the surgical group. Figure 5 Normal cervix, Figure 6 For the non-invasive group, Figure 7 Cervix of the surgical group.

[0044] Judging from the results, the non-invasive in situ model technology for cervical cancer in mice has a significantly better modeling effect than the surgical method. At the same time, it reduces the pain of mice, reduces the difficulty of the experiment, and improves the speed of the experiment.

[0045] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for establishing a non-invasive in situ cervical cancer model in mice, comprising the following steps: Step 1: 8-week-old BALB / c-nude mice were selected and divided into a non-invasive group and a surgical group. No statistically significant differences were found in age and weight between the two groups. Step 2: In the non-invasive group, a 3mm diameter cotton swab was used to widen and dry the vagina. The tumor mass with luciferase fluorescence was immersed in the modified hydrogel. After heating to solidify the modified hydrogel, tissue glue mixed with hard gypsum was dripped onto the surface of the modified hydrogel and quickly inserted into the vagina. When a blockage was felt, the insertion was stopped and paused for 30 seconds to allow the tissue glue to completely dry. When the tumor mass was fixed in the cervix, the model was completed. Step 3: The surgical team first opens the abdominal cavity and sutures the tumor tissue of the same size to the cervix; After Step 3, in vivo imaging was used to evaluate the tumor formation rate and tumor formation status of the two groups. When fluorescence was clearly visible in the cervix of the mouse, the model was successfully established. The in vivo imaging shooting method is specifically as follows: The working solution (10µl / g) was intraperitoneally injected into nude mice. After 10-15 minutes, the mice were anesthetized with isoflurane and then observed using an in vivo imaging system. The working fluid is made from the following raw materials in parts by weight: 70-90 portions of DPBS; 3-6 copies of mCherry.

2. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: The body weight of the BALB / c-nude mice was 24±0.56 g.

3. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: After Step 3, prime8 statistical software was used for analysis. P The difference was statistically significant when the value was <0.

05.

4. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: The modified hydrogel is made from the following raw materials in parts by weight: Hydrogel 500,000-501,000 parts; VEGF 0.008-0.012 parts; FGF9 0.008-0.012 copies; EGF 0.008-0.012 parts; E2 0.016-0.027 parts.

5. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: The concentration of the DPBS is 15-17 mg / ml.

6. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: The working solution is prepared as follows: after selecting DPBS solution, mCherry is slowly added and gently stirred at 3-4° C., ultrasonically vibrated for 1-3 minutes, and then refrigerated for use.

7. The method for establishing a non-invasive in situ model of cervical cancer in mice according to claim 1, wherein: The mice were injected with prednisone or methylprednisolone 1-2 μl / g before modeling.

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