A method for constructing a mouse colorectal carcinoma in situ model

Abstract

The present invention relates to a method for constructing a mouse colorectal carcinoma in situ model, comprising the following steps: fixing an anesthetized mouse under a stereomicroscope, pulling out the mouse's colon and rectum under light, injecting colorectal cancer cells into the colorectal submucosal membrane, pressing to stop bleeding after the injection, returning the colon and rectum to the abdominal cavity and waiting for the mouse to wake up, thereby constructing a mouse colorectal carcinoma in situ model. The animal model obtained by the present invention has significantly reduced mortality and infection rates during and after surgery, and truly achieves in situ implantation. After the tumor cells are injected into the target organ, there is no metastasis phenomenon at all. Based on this, the carcinoma in situ model, especially when used as a colorectal cancer metastasis model, obtains more accurate and reliable results, has higher reference value, and has advantages that cannot be matched by existing technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of animal experimental models, and in particular to a method for constructing a mouse colorectal carcinoma in situ model. Background Art

[0002] Colon cancer is a malignant tumor of worldwide concern. Distant metastasis and invasive growth are the main causes of death from colorectal cancer. Therefore, research on the pathogenesis and metastasis of colorectal cancer is crucial. Establishing an animal model of colorectal cancer metastasis that is easily observed and whose biological behaviors more closely resemble clinical manifestations is crucial for understanding the growth, progression, and metastasis of colorectal cancer and for evaluating the efficacy of various treatment options.

[0003] Currently, commonly used methods for establishing colon cancer models include spontaneous and in situ induced colon cancer models and transplanted colon cancer models. Depending on the location, these can be further divided into subcutaneous transplantation, liver transplantation, and orthotopic transplantation. Clinical observations have shown that tumor metastasis is often organ-specific rather than randomly distributed, and certain tumor cells have a particular tendency to metastasize to certain organs, thus proposing the "seed and soil theory." Although subcutaneous transplanted tumors can maintain the tissue structure and growth characteristics of the original tumor, they are separated from the microenvironment of their tissue and organ of origin and are encapsulated by a fibrous membrane (pseudocapsule), often growing in a localized manner. Their biological characteristics, especially metastatic characteristics, are difficult to express. Orthotopic transplantation is the inoculation of human tumors into the host organ tissue corresponding to the primary tumor site, allowing them to obtain a microenvironment similar to that of human tumors, which is more suitable for tumor growth and metastasis. The orthotopic transplantation model effectively simulates the growth and metastasis process of clinical tumors and is an ideal model for tumor prevention and treatment and anti-metastasis research.

[0004] However, due to their larger size, more visible body parts, and ease of manipulation, existing technologies primarily target rats for orthotopic tumor model development. Compared to rats, mice offer the following advantages as animal tumor models for discovering gene function, exploring cellular processes, studying human disease mechanisms, and accelerating drug development and efficacy evaluation: ① They are one of the smallest mammals (20-25 g) with a short generation cycle; ② They are evolutionarily close to humans (60-75 million years old); ③ Their placental formation and early embryonic development are similar to those of humans; ④ Their tissue, organ structure, and cellular function are similar to those of humans; ⑤ They possess advanced neural activity; ⑥ The mouse genome has been sequenced; ⑦ 99% of human genes are found in mice, with a high homology of 78.5%; ⑧ 93% of the genome regions have the same gene order as humans; ⑨ The technology for genome engineering is mature. Given these advantages, there is an urgent need to develop methods for developing orthotopic colorectal cancer models in mice. Existing methods for establishing mouse colorectal carcinoma in situ models include orthotopic implantation via the mesocecal triangle, cecal subserosal cell injection, spleen injection, rectal mucosal injection, and subcutaneous tumor formation. However, these methods require surgical incision to expose the cecum and access the mesentery. This surgery carries a high risk of infection, requires suturing each mouse, and is time-consuming and cumbersome. Subcutaneous tumor formation requires a long operation cycle, as the tumor is not initially generated in the animal, significantly different from natural occurrence. Furthermore, surgical incision to expose the cecum is required, which carries a high risk of infection, is cumbersome, and takes a long time, limiting the number of mice treated at one time. Splenic injection requires surgical incision, which carries a high risk of infection, is cumbersome, and is prone to bleeding. Furthermore, this method only simulates colorectal cancer metastasis, not tumor development. Its metastasis mechanism differs from normal clinical occurrence, and its significance remains questionable, as it does not fully represent clinical metastasis. Due to the small size of mice and the thin anal intestinal wall, the needle easily pierces the intestinal wall during injection under naked eye observation, making it impossible to clearly observe the depth of needle insertion and the injected cells. This makes in situ injection of the colon wall surgery more difficult. This can lead to tumor inoculation failure due to the inability to accurately judge the depth of needle penetration, or multiple extra-intestinal implants due to leakage. Therefore, it is necessary to find a new method for preparing a mouse colorectal carcinoma in situ model. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for constructing a mouse colorectal carcinoma in situ model. The method uses a stereomicroscope to operate and inject mice, and the depth of the needle and the injected cells can be clearly observed. The operation can be precisely controlled without the need for laparotomy, causing little harm to the mice, and not easily infected. The mice can then move normally after waking up.

[0006] The first object of the present invention is to provide a method for constructing a mouse colorectal carcinoma in situ model, comprising the following steps:

[0007] The anesthetized mouse was fixed under a stereomicroscope, the mouse colon and rectum were pulled out under light, and colorectal cancer cells were injected submucosally into the colorectal mucosa. After the injection, pressure was pressed to stop bleeding, and the colorectum was returned to the abdominal cavity until the mouse woke up, thereby constructing the mouse colorectal carcinoma in situ model.

[0008] Furthermore, the mice were anesthetized by intraperitoneal injection of chloral hydrate.

[0009] Furthermore, the mice are C57 mice of 6-8 weeks old and weighing 20-25 g.

[0010] Furthermore, the colorectal cancer cells are injected in the form of a cell suspension.

[0011] Furthermore, the cell suspension is prepared by the following method: colorectal cancer cells are inoculated into RPMI-1640 culture medium containing fetal bovine serum, and cultured, and then digested with trypsin to prepare a cell suspension.

[0012] Furthermore, the concentration of injected colorectal cancer cells was (2.5±0.5)×10 7 cells / mL.

[0013] Furthermore, colorectal cancer cells were injected 0.2-0.5 mm below the colorectal mucosa.

[0014] Furthermore, the mouse colorectal carcinoma in situ model is an animal model for studying colorectal cancer metastasis.

[0015] The second object of the present invention is to provide a mouse colorectal carcinoma in situ model obtained by the above-mentioned construction method.

[0016] The third object of the present invention is to provide the use of the above-mentioned mouse colorectal carcinoma in situ model in screening colorectal cancer drugs.

[0017] Furthermore, the colorectal cancer drug is a drug for preventing or treating colorectal cancer metastasis.

[0018] By means of the above solution, the present invention has at least the following advantages:

[0019] The present invention provides a method for constructing a mouse colorectal carcinoma in situ model under a stereomicroscope using the anal prolapse method. Compared to traditional model construction, the present invention does not require laparotomy, significantly reducing the risk of infection and harm to experimental animals. The operation method is fast and convenient, and large numbers of mice can be injected in a short period of time. Based on experimental results, true in situ implantation was achieved with a high tumor formation rate and uniform tumors. After injection of tumor cells into the target organ, there was no metastasis. Based on this, the carcinoma in situ model prepared by the present invention, especially when used as a colorectal cancer metastasis model, produces more accurate and reliable results, has a higher reference value, and has advantages that are unmatched by existing technologies.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0022] Figure 1 Schematic diagram of the device for establishing an orthotopic C57 mouse colorectal cancer model under a stereomicroscope;

[0023] Figure 2 This is a top view of the operation of establishing an orthotopic model of colorectal cancer in C57 mice under a stereomicroscope;

[0024] Figure 3 This is a tumor peeling picture of a C57 mouse bearing a tumor 15 days after modeling using the present invention;

[0025] Figure 4 This is a tumor peeling picture of a C57 mouse bearing a tumor 21 days after modeling using the present invention;

[0026] Figure 5 Graph showing the survival curves corresponding to the six groups in Example 2. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0028] Example 1 Construction of an orthotopic colorectal cancer model in C57 mice under a stereomicroscope

[0029] (1) The experimental animals were 40 male C57 mice, weighing 20-25 g each and aged 6-8 weeks.

[0030] (2) Preparation of MC38.WT cell suspension

[0031] For routine cell culture, MC38.WT cells were inoculated in RPMI-1640 medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% CO2. Cells in the exponential growth phase were obtained and digested with 0.25% trypsin containing 0.02% EDTA. The cells were mechanically pipetted into a cell suspension, filtered through a 200-mesh sieve, and centrifuged at 800 rpm for 3 minutes. The supernatant was discarded and the cells were washed twice with PBS. The cell pellet was resuspended in an appropriate amount of physiological saline. Cell viability was determined by trypan blue staining to be >95%, and the cell concentration was adjusted to (2.5±0.5)×10 7 / ml, and store in ice bath for later use.

[0032] (3) Turn on the stereo microscope, adjust the microscope brightness to an appropriate brightness, take the anesthetized C57 mouse and observe it under the microscope and adjust the position until the mouse's anus is exactly in the center of the field of view (such as Figure 1 ).

[0033] (4) Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.05 ml / g), placed in a supine position, and their limbs were fixed non-invasively on the operating table with tape (eg Figure 2 Under anesthesia, the mouse's anus and colorectum are relaxed. One operator uses curved, toothless forceps to gently pull out a 5mm section of the mouse's colorectum from the anus, tightening it until it is taut. Another operator holds a syringe, inserts the needle into the colorectal mucosa (0.2-0.5mm), and pushes the syringe to slowly inject 20-50ul of tumor cell suspension into the submucosal area for each mouse. Once the injection is complete, gently withdraw the needle and press the injection site with a cotton swab to stop bleeding and prevent the tumor cell fluid from leaking out of the body. After pressing for 1-2 minutes, gently return the prolapsed colorectum to the abdominal cavity.

[0034] (5) After all injections are completed, place the mice in a supine position, allow them to wake up in a warm environment, and allow them to eat and drink freely. Observe for 21 consecutive days and you will see the tumor protruding from the anus.

[0035] (6) Results: Forty C57 mice were injected with tumor cell suspension under a stereomicroscope to prepare an orthotopic colon cancer model. The tumor formation rate was 100% (40 / 40) and the injection concentration was (2.5±0.5)×10 7 Tumor cells / ml, 20-50ul tumor cell suspension was injected into each mouse. During the entire experimental process (21 days), all tumor-bearing mice grew well, no intestinal obstruction occurred, and no mice died. Ten days after the operation, a small hard nodule could be seen at the anus of the mouse, which gradually increased in size. By the end of the experiment, a clear mass (such as Figure 3-4 ).

[0036] As shown in the image, the orthotopic mouse colon cancer model, created by injecting a tumor cell suspension under a stereomicroscope, shows that the tumor lesions are easily exfoliated, with a clear demarcation from the surrounding tissue. The surface of the isolated tumor is smooth, with multiple nodular protrusions, an overall pink color, and a firm texture. Localized ulceration and erosion are visible on the tumor lesions. The cross-section is off-white, with visible necrosis and a rich blood supply. Microscopically, the orthotopic mouse colorectal tumor cells exhibit significant atypia, with large, darkly stained nuclei, unequal cell size, irregular morphology, and pathological mitotic figures, retaining the characteristics of the original inoculated cancer cells.

[0037] Example 2 Comparison of the effects of different methods on colorectal carcinoma in situ in mice

[0038] 1. Experiment

[0039] A total of 120 male C57 mice were used for the experiment, weighing 20-25g each and aged 6-8 weeks. They were randomly divided into 6 groups. Group 1 consisted of 20 C57 mice injected with orthotopic colon cancer under a stereomicroscope; Group 2 consisted of 20 mice injected directly into the rectal mucosa (i.e., non-stereomicroscopic injection); Group 3 consisted of 20 mice injected with subcutaneous tumors; Group 4 consisted of 20 mice injected with cells through the cecum subserosal membrane; Group 5 consisted of 20 mice injected with spleen; and Group 6 consisted of 20 mice injected with orthotopic colon cancer established through the cecal mesenteric triangle. Each group was injected with MC38.WT cells at a concentration of (2.5±0.5)×10 7 Each mouse was injected with 20-50 μl of tumor cell suspension.

[0040] The specific steps are as follows:

[0041] (1) Group 1: The operation steps are the same as those in Example 1.

[0042] (2) Group 2: Fix the mouse in a mouse holder, and the assistant fully exposes the anus. Insert the needle into the two side walls and the back wall of the mouse anus and slowly inject the tumor cell suspension.

[0043] (3) Group 3: Digest adherent cells into single cell suspension and count them. 7 MC38.WT colon cancer cells (1000 tumor cells / ml) were suspended in 200 μl of saline, mixed thoroughly by pipetting, and then inoculated subcutaneously into the scapula of nude mice. Two weeks later, the subcutaneous tumor reached approximately 1 cm in diameter. The skin was disinfected and the tumor removed, and the surrounding fibrous connective tissue was trimmed. Fresh tissue surrounding the tumor was removed and cut into small pieces approximately 1 mm in diameter using a razor blade. The tissue was placed in saline on ice for orthotopic tumor implantation. The time from tumor explantation to implantation should not exceed 3 hours.

[0044] The MC38.WT tumor implanted subcutaneously in mice was removed, and the fresh tumor tissue outside the tumor was cut into small pieces of 1 mm in size and placed on ice for later use. C57 mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.05 ml / g), the abdominal cavity was opened through a midline incision in the lower abdomen, and the cecum was found from the left lower abdomen and dragged out of the incision. Under a microscope, a sharp blade was used to scrape the serosal layer in the middle of the cecum for about 1 mm. The pre-prepared tumor tissue block was placed on the cecum scraped by the blade, and the surrounding serosal membrane was wrapped with 6-0 Prolene sutures. After stopping the bleeding and confirming that the implanted tissue block had not fallen out, the cecum was repositioned and the abdominal cavity was closed.

[0045] (4) Group 4: C57 mice were anesthetized with 10% chloral hydrate (0.05 ml / g) intraperitoneally. The abdominal cavity was opened by midline incision. The cecum was found from the left lower abdomen and pulled out of the incision. Under a microscope, a 30G fine needle was used to remove (2.5±0.5)×10 7 Inject 100 tumor cells / ml into the subserosa of the central cecum, a region with a rich blood supply. Inject slowly, within 30 seconds. After the injection, remove the needle and apply pressure with a small cotton ball for 1 minute. Once the injected liquid has been absorbed by the intestinal wall, reposition the cecum, avoid applying pressure, and close the abdominal cavity.

[0046] (5) Group 5: A 1.5 cm incision was made in the middle of the dorsal region, between the left mid-axillary line and the posterior axillary line, to expose the spleen. The needle was inserted along the longitudinal axis of the spleen at the upper pole of the spleen, and MC38.WT cell suspension (2.5 ± 0.5) × 10 7 Tumor cells / ml were detected, and then a sterilized cotton swab dipped in complex iodine was used to gently press the needle hole for a few seconds. After checking for bleeding, the spleen was put back in place and the abdominal wall was sutured throughout the thickness.

[0047] (6) Group 6: The operation steps were the same as those described in Patent No. 201410818207.X. The colon cancer cell suspension was injected into the cecal mesenteric triangle of mice anesthetized by intraperitoneal injection of 10% chloral hydrate (0.05 ml / g), so that the tumor cells were distributed and grew along the cecal axial direction along the mesenteric attachment of the cecal body, but the specific dosage of each substance was the same as above.

[0048] 2. Results

[0049] ① The average operation time for each of the 20 mice in group 1 using the orthotopic implantation method of mouse colon cancer injected under a body microscope was less than 1 minute. After 10 days, the tumor formation rate was 100%, the ectopic implantation rate was 0, there was no risk of surgical infection, and the size of the colorectal orthotopic tumors was consistent after 21 days.

[0050] ② The 20 mice in group 2 were injected directly into the rectal mucosa (i.e., injected under a non-stereoscopic microscope). The average operation time was less than 1 minute per mouse. After 10 days, the tumor formation rate was as low as 30%. 13 mice were implanted in the abdominal cavity without the risk of surgical infection. After 21 days, the sizes of the colorectal in situ tumors varied greatly. Among them, 2 colorectal in situ tumors were relatively large, and 5 tumors were extremely small.

[0051] ③ The average operation time for the 20 mice in group 3 using the subcutaneous tumor formation test method was more than 5 minutes per mouse. After 10 days, the tumor formation rate was as low as 50%. Liver implantation was more common in 7 mice, and 3 mice died of acute infection after surgery. After 21 days, the sizes of the colorectal in situ tumors varied greatly, among which 4 had larger colorectal in situ tumors and 6 had smaller tumors.

[0052] ④ The average operation time for 20 mice in Group 4 was about 15 minutes per mouse by subserosal cell injection in the mouse cecum. After 10 days, the tumor formation rate was as low as 65%. The tumor formation speed was fast but the survival period of the mice was short. Two mice had liver implants, and five mice died of acute sepsis after surgery. After 21 days, the sizes of the colorectal in situ tumors varied greatly. Among them, 4 had larger colorectal in situ tumors, 2 had slightly smaller tumors, and 2 were colorectal liver metastases.

[0053] ⑤ The average operation time for the 20 mice in Group 5 via spleen injection was about 10 minutes per mouse. After 10 days, the tumor formation rate was as low as 70%. The tumor formation speed was fast, but the survival period of the mice was extremely short. 14 mice were implanted in the liver, and 7 died of acute sepsis and bacteremia after surgery. After 21 days, the sizes of the colorectal in situ tumors varied greatly. Among them, there were 0 colorectal in situ tumors and all 14 were colorectal liver metastases.

[0054] ⑥ In group 6, the average operation time for the 20 mice with colon cancer orthotopic implantation established through the cecal mesotrioneal triangle was more than 10 minutes per mouse. After 10 days, the tumor formation rate was as low as 90%. There were 3 mice with a higher tumor formation rate and mild abdominal implantation. One mouse died of acute sepsis after surgery. After 21 days, the sizes of the colorectal orthotopic tumors varied greatly, among which 10 were larger and 8 were smaller.

[0055] The details are shown in the table below. The results of mouse survival rate are shown in Figure 5 .

[0056]

[0057]

[0058] 3. Conclusion

[0059] The experiment in group 1 used the method of in situ implantation of C57 mouse colon cancer injected under a body microscope to successfully induce in situ tumor formation of mouse colorectal cancer, which was superior to the experimental methods of control groups 2, 3, 4, 5, and 6, with a high success rate (p < 0.01) and statistically significant.

[0060] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse colorectal carcinoma in situ model, characterized in that: The construction method comprises the following steps: The anesthetized mouse is fixed under a stereomicroscope and its position is adjusted until the anus of the mouse is exactly in the center of the field of view. The anus and colorectum of the mouse are relaxed under anesthesia. Under light illumination, the colorectum of the mouse is pulled out from the anus of the mouse using curved toothless forceps. The length of the mouse colorectum is about 5 mm and it is tightened until it is taut. Colorectal cancer cells are injected 0.2-0.5 mm below the colorectal mucosa. After the injection, pressure is applied to stop bleeding. The colorectum is returned to the abdominal cavity and the mouse is allowed to wake up, thereby establishing the mouse colorectal carcinoma in situ model. The concentration of injected colorectal cancer cells was (2.5±0.5)×10 7 cells / mL; The mouse colorectal carcinoma in situ model is an animal model used to study colorectal cancer metastasis; The construction method utilizes the anal prolapse method to construct a mouse colorectal carcinoma in situ model under a stereomicroscope, without the need for laparotomy, greatly reducing the risk of infection and harm to experimental animals. The tumor formation rate is high, the in situ tumors are of uniform size, and in situ implantation is achieved. After the tumor cells are injected into the target organ, there is no metastasis at all.

2. The construction method according to claim 1, wherein: The mice were C57 mice aged 6-8 weeks and weighing 20-25 g.

3. The construction method according to claim 1, wherein: The colorectal cancer cells are injected in the form of a cell suspension.

4. The construction method according to claim 3, wherein: The cell suspension is obtained by inoculating colorectal cancer cells into RPMI-1640 culture medium containing fetal bovine serum and then digesting with trypsin.

5. Use of the mouse colorectal carcinoma in situ model obtained by the construction method according to any one of claims 1 to 4 in screening colorectal cancer drugs.

6. The use according to claim 5, characterized in that: The colorectal cancer drug is a drug for preventing or treating colorectal cancer metastasis.

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