CN113940310A - Method for establishing mouse gastric cancer model - 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
- A01K67/02—Breeding vertebrates
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses a method for establishing a mouse gastric cancer model, and belongs to the field of gastric cancer models. A method for establishing mouse gastric cancer model comprises collecting cultured and amplified product in microaerophilic environmentH.felisPreparing the helicobacter pylori to brain-heart immersion liquid, and preparing homogeneous bacterial liquid in a culture medium; then, gavage is carried out on fasted experimental mice by using freshly prepared homogeneous bacterial liquid, gavage is carried out for 1 time every 1 day, gavage is carried out for 5 times totally, finally, the experimental mice after gavage is fed by using drinking liquid contained in a light-resistant container, 1 time is recorded in 1 week of continuous feeding, 1 time is carried out every other week of feeding, 5 times is carried out totally, and the drinking liquid is liquid which is freshly prepared by using drinking water of the mice and contains 240ppm of N-methyl-N-nitrosourea; the primary mouse gastric cancer model is obtained, the survival rate of the mouse and the occurrence probability of gastric tumors are greatly improved, and the specificity of helicobacter pylori in a gastric mucosa local immune microenvironment is ensured, so that the method is more suitable for relevant immunological research.
Description
Technical Field
The invention relates to the field of gastric cancer models, in particular to a method for establishing a mouse gastric cancer model.
Background
Due to the lack of mature and stable mouse gastric cancer cell lines, the technical and ethical problems of constructing humanized mice by planting human gastric cancer cells exist, so that the development of gastric cancer immunity-related experimental research by using animal models is greatly limited. Inducing spontaneous gastric cancer in mice is the most feasible strategy.
However, the following problems exist in the process of inducing spontaneous gastric cancer of mice at present:
1. in the traditional experiment, Kunming mice are adopted, although gastric cancer can be induced, the gastric cancer incidence rate is low, a model with high stability and consistency is not convenient to obtain, the model is not suitable for immunological research, and the C57BL/6 mice capable of obtaining the model with high stability and consistency have extremely low spontaneous gastric cancer probability and can bring serious damage to other organs by inducing chemical carcinogens; however, mice infected with helicobacter pylori for a long time can only achieve chronic gastritis, the probability of canceration is extremely low, and the mice can not progress to gastric cancer almost for the whole life.
2. Although the commonly used human helicobacter pylori strain can cause transient gastritis in mice and can simulate the condition of human body infection to a certain extent, the long-term permanent planting is difficult, and malignant lesions at the later stage of chronic inflammation cannot be induced. Few clinically isolated strains of helicobacter pylori were utilized, which were CagA and VagA positive, but still species-specific.
3. Nitrosoguanidine is a strong carcinogenic chemical, not only can induce gastric mucosa to locally generate pathological changes, but also has certain carcinogenic effect on all organs of the whole body.
4. According to the reference and the pre-experimental results, the concentration of MNU, the feeding mode and the like all influence the final molding result to a great extent. Since the stomach structure of mice is different from that of humans, there is a "forestomach" portion without glandular structures, and the intragastric approach adopted by the prior art has a high probability of inducing squamous carcinoma in the forestomach, rather than the adenocarcinoma which is clinically common and is aggravated by glandular epithelium.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for establishing a mouse gastric cancer model.
In order to solve the above problems, the present invention adopts the following technical solutions.
A method for establishing a mouse gastric cancer model comprises the following steps:
a. fasting the experimental mice for 14-18 hours, preferably 16 hours;
b. collecting the culture amplified in a microaerophilic environmentH.felisHelicobacter pylori, preparing into homogeneous bacterial liquid in Brain Heart Infusion (BHI) liquid culture medium, and measuring OD600The value is between 1.8 and 2.2, preferably 2.0;
c. b, after the step b is finished, performing intragastric administration on the experimental mouse fasted in the step a by using the freshly prepared homogeneous bacterial liquid in the step b, and performing intragastric administration for 1 time every 1 day and 5 times in total;
d. preparing a drinking liquid containing 240ppm of N-methyl-N-nitrosourea with drinking water for the mice, putting the drinking liquid in a light-resistant container to feed the experimental mice after the intragastric administration in the step c, continuously feeding for 1 week for 1 time, and feeding for 1 time every other week for 5 times;
e. and d, after the step d is completed, confirming the colonization and canceration of the helicobacter pylori.
Furthermore, the experimental mice in the step a are all C57BL/6 female mice with healthy immune function and large size of 5-7 weeks. The weight range is preferably 20-25 g.
Further, the stomach filling amount of the experimental mouse in the step a is that the mouse is filled with 100-130 mul/mouse according to the weight of the mouse, the stomach filling is performed for 1 time every other day, and the stomach filling is not performed firstly on the first day.
Further, the microaerophilic environment in step b contains 6-12% oxygen and 5-8% carbon dioxide.
Further, the drinking liquid of the mice in the step d is prepared by using common high-temperature and high-pressure sterilized water with the concentration of 240 ppmMNU.
Further, in the process of establishing the mouse gastric cancer model, a treatment group and a control group can be set for effect comparison, wherein the treatment group and the control group are respectively as follows:
first, the treatment group is first passedH.felisInjecting helicobacter pylori into brain-heart infusion liquid culture medium to obtain homogeneous bacterial liquid, and performing gastric lavage for inflammation induction, wherein 2 months after inflammation induction, the mice enter into chronic gastritis stage, and then performing chemical induction with N-methyl-N-nitrosourea liquid to confirmH.felisHelicobacter pylori colonization and carcinogenesis;
② a treatment group, which is firstly chemically induced by N-methyl-N-nitrosourea liquid and is carried out every other weekH.felisInflammation induced by helicobacter pylori, confirmationH.felisHelicobacter pylori colonization and carcinogenesis;
processing group, only inducing by N-methyl-N-nitrosourea chemical liquid;
(ii) control group, onlyH.felisHelicobacter pylori induces inflammation.
Compared with the prior art, the invention has the advantages that:
(1) the scheme firstly passesH.felisAfter a stable chronic gastritis model is constructed, N-methyl-N-nitrosourea (MNU) is added for chemical induction, a mouse primary gastric cancer model is established, a Correa gastric cancer model which finally causes gastric cancer development due to helicobacter pylori infection and various chemical substance injuries in a human body is simulated, and compared with a gastric cancer tissue obtained by conventionally planting a tumor cell line, the model has higher heterogeneity, is closer to a real state and is more beneficial to related immunological research.
(2) According to the scheme, the mouse tumorigenic speed is accelerated by a combined induction mode of first inflammation induction and then chemical induction, the survival rate and the probability of gastric tumor tumorigenic during mouse experiments are greatly improved, the specificity of helicobacter pylori in a local immune microenvironment of a gastric mucosa is ensured, the conditions that diseases such as thymoma, intestinal cancer and genital organ tumor are caused by independent MNU chemical induction are avoided, and the conditions that chemical injury is firstly avoided, and then the normal planting of the helicobacter pylori is influenced by the acute injury caused by the strategy of bacterial infection to the gastric mucosa are avoided.
(3) The C57BL/6 mouse is adopted in the scheme, the problem that the probability of the spontaneous gastric cancer of the C57BL/6 mouse is extremely low is effectively solved, the probability of the spontaneous gastric cancer of the C57BL/6 mouse is improved, a model with high stability and consistency is obtained by utilizing the characteristic of high consistency of the genetic background of the mouse, the C57BL/6 mouse with complete immune function is adopted in the test, convenience is provided for carrying out immune-related experiments in the mouse body, rejection reaction caused by immune cell reinfusion can be avoided, and the obtained experimental result is more reliable.
(4) In the scheme, adoptH.felisThe strain has stronger affinity to mice, can be planted in the gastric mucosa of the mice for a long time, and can spontaneously induce malignant lesions such as adenoma, adenocarcinoma and the like after long-term infection.
(5) According to the invention, the N-methyl-N-nitrosourea liquid with the concentration of 240ppm is adopted to irrigate the stomach of the mouse, and 100-130 mul/mouse is irrigated in a mode of weighing 5ml/kg of the mouse, so that a better final molding result is obtained, and the establishment of a mouse gastric cancer model is facilitated.
(6) Only female mice were used in this protocol, as female pairs of miceH.felisThe sensitivity is higher, and the tumor formation rate of the gastric cancer is higher.
Drawings
FIG. 1 is a schematic diagram of the successful colonization of the gastric mucosa by H.pylori in the present invention;
FIG. 2 is a schematic diagram showing that gastritis is induced successfully by helicobacter pylori infection in the present invention (the left picture is a picture of normal gastric mucosa, the middle picture is a picture of acute gastritis occurring in gastric mucosa, and the right picture is a picture of chronic gastritis occurring in gastric mucosa);
FIG. 3 shows the mouse stomach cancer specimen (the left picture is the normal gastric mucosa picture, the middle picture is the picture of chronic gastritis of gastric mucosa, and the right picture is the canceration of stomach).
Detailed Description
Please refer to fig. 1-3, which shows a method for constructing a mouse gastric cancer model, wherein a C57BL/6 mouse with a robust immune function is selected for research; the method comprises the following steps:
a. fasting the experimental mice;
b. collecting the culture amplified in a microaerophilic environmentH.felisHelicobacter pylori, preparing into homogeneous bacterial liquid in Brain Heart Infusion (BHI) liquid culture medium, and measuring OD600The value is between 1.8 and 2.2;
c. b, after the step b is finished, performing intragastric administration on the experimental mouse fasted in the step a by using the freshly prepared homogeneous bacterial liquid in the step b, and performing intragastric administration for 1 time every 1 day and 5 times in total;
d. preparing a drinking liquid containing 240ppm of N-methyl-N-nitrosourea with drinking water for the mice, putting the drinking liquid in a light-resistant container to feed the experimental mice after the intragastric administration in the step c, continuously feeding for 1 week for 1 time, and feeding for 1 time every other week for 5 times;
e. and d, after the step d is completed, confirming the colonization and canceration of the helicobacter pylori.
In the step a, the experimental mice are C57BL/6 female mice 5-7 weeks old, the number of the experimental mice is 200-300, and the weight range of each experimental mouse is 20-25 g. The fasting time in step a ranges from 14 hours to 18 hours. And c, in the step a, the stomach filling amount of the experimental mouse is that the mouse is filled with 100-130 mul/mouse according to the weight of the mouse, the stomach filling is carried out for 1 time every other day, and the stomach filling is not carried out firstly on the first day. The microaerophilic environment in step c is an environment containing 6-12% oxygen and 5-8% carbon dioxide. And d, preparing the liquid for drinking by the mice in the step d by using common high-temperature and high-pressure sterilized water with the concentration of 240 ppmMNU.
The first embodiment is as follows: amplified by incubation in microaerophilic environmentH.felisSoaking helicobacter pylori in the liquid, homogenizing in culture medium, and measuring OD600Value at 2.0, then group: randomly selecting 50 mice of 6 weeks old, placing in a culture device with the temperature of 19 ℃ and the humidity of 42%, fasting for 16 hours, then performing gavage on the freshly prepared bacterial liquid for 1 time every 1 day, performing gavage on 100 mu l/mouse for 5 times in total, as shown in figure 1,H.felishelicobacter pylori successfully colonizes the gastric mucosa, two months later, as shown in figure 2,H.felishelicobacter pylori successfully induces gastritis by chemical induction of N-methyl-N-nitrosourea, preparing a drinking water containing 240ppm of N-methyl-N-nitrosourea with mouse drinking water, continuously feeding for 1 week for 1 time, feeding for 1 time every other week for 5 times for ten weeks, as shown in FIG. 3H.felisAnd the primary gastric cancer is successfully induced by combining the N-methyl-N-nitrosourea. Firstly, useH.felisAfter a stable chronic gastritis model is constructed, the survival rate of mice is higher through MNU chemical induction.
The second embodiment is as follows: advanced mobile object grouping: randomly selecting 50 mice of 6 weeks old, placing in culture equipment with temperature of 19 deg.C and humidity of 42%, and preparing drinking water containing N-methyl-N-nitrosourea with concentration of 240ppm from mouse drinking waterThe liquid is fed continuously for 1 week for 1 time, fed every other week for 1 time, 5 times, ten weeks for a total time, and cultured and amplified in microaerophilic environment after one week intervalH.felisSoaking helicobacter pylori in the liquid, homogenizing in culture medium, and measuring OD600At the value of 2.0, fasting is performed for 16 hours, then the freshly prepared bacterial liquid is perfused, and the perfusion is performed for 1 time every 1 day, 100 mul/mouse is perfused each time, and the perfusion is performed for 5 times in total, as shown in figure 3, byH.felisHelicobacter pylori and N-methyl-N-nitrosourea can be combined to successfully induce primary gastric cancer, but the strategy of firstly chemical injury and then bacterial infection is adopted, so that the condition that mice influence the normal colonization of the helicobacter pylori due to the acute injury of gastric mucosa is easily caused; moreover, MNU is very easy to induce diseases such as thymoma, intestinal cancer, reproductive organ tumor and the like, so that the survival rate of the mouse is low, the survival time of the mouse is shorter than that of the mouse in the first embodiment, and the experiment in subsequent related immunological research is not facilitated.
The third concrete embodiment: advanced mobile object grouping: randomly selecting 50 mice of 6 weeks old, placing in a culture device with the temperature of 19 ℃ and the humidity of 42%, preparing drinking water containing 240ppm N-methyl-N-nitrosourea with the drinking water of the mice, continuously feeding for 1 week for 1 time, feeding for 1 time every other week for 5 times and ten weeks, wherein other organs of the mice are seriously damaged along with the increase of time, and cannot be used as samples for subsequent detection.
Comparative example one: advanced mobile object grouping: randomly selecting 50 mice of 6 weeks old, placing in a culture device at 19 deg.C and 42% humidity, and culturing in microaerophilic environmentH.felisSoaking helicobacter pylori in the liquid, homogenizing in culture medium, and measuring OD600The value is 2.0, fasting is carried out for 16 hours, then fresh bacterial liquid is perfused into the stomach, the stomach is perfused for 1 time every 1 day, 100 mu l/mouse is perfused for each time, and the stomach is perfused for 5 times, as shown in figure 1-2, after helicobacter pylori is successfully fixed, gastritis is successfully induced, but the probability of inducing primary gastric cancer is extremely low.
Claims (7)
1. A method for establishing a mouse gastric cancer model is characterized by comprising the following steps: the method comprises the following steps:
a. fasting the experimental mice for 14-18 hours;
b. collecting the culture amplified in a microaerophilic environmentH.felisHelicobacter pylori, preparing into homogeneous bacterial liquid in brain-heart infusion liquid culture medium, and measuring OD600The value is between 1.8 and 2.2;
c. b, after the step b is finished, performing intragastric administration on the experimental mouse fasted in the step a by using the freshly prepared homogeneous bacterial liquid in the step b, and performing intragastric administration for 1 time every 1 day and 5 times in total;
d. preparing a drinking liquid containing 240ppm of N-methyl-N-nitrosourea with drinking water for the mice, putting the drinking liquid in a light-resistant container to feed the experimental mice after the intragastric administration in the step c, continuously feeding for 1 week for 1 time, and feeding for 1 time every other week for 5 times;
e. and d, after the step d is completed, confirming the colonization and canceration of the helicobacter pylori.
2. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: in the step a, the experimental mice are C57BL/6 mice with complete immune function and female mice with 5-7 weeks old, and the weight range is 20-25 g.
3. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: the fasting time of the experimental mice in the step a is 16 hours.
4. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: OD of the homogenized bacterial liquid in the step b600The value was 2.0.
5. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: and c, filling the stomach of the experimental mouse in the step c by 100-130 mul/mouse according to the weight of the mouse of 5ml/kg, filling the stomach every other day for 1 time, and not filling the stomach on the first day.
6. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: the microaerophilic environment in step b contains 6-12% oxygen and 5-8% carbon dioxide.
7. The method for establishing a mouse gastric cancer model according to claim 1, wherein the method comprises the following steps: and d, the drinking water for the mice in the step d is high-temperature and high-pressure sterilized water.
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