CN107537098B - Method for establishing mouse acute radiation duodenitis model - Google Patents
Method for establishing mouse acute radiation duodenitis model Download PDFInfo
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- CN107537098B CN107537098B CN201610465613.1A CN201610465613A CN107537098B CN 107537098 B CN107537098 B CN 107537098B CN 201610465613 A CN201610465613 A CN 201610465613A CN 107537098 B CN107537098 B CN 107537098B
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Abstract
The invention belongs to the technical field of radiology, and particularly relates to a method for establishing a mouse acute radiation duodenitis model, which comprises the steps of irradiating a mouse with 8Gy gamma rays, wherein the irradiated part is an abdomen, and performing duodenum pathological analysis and detecting the protein expression level of apoptosis-related factor BCL-2 and inflammatory-related factor TGF- β 12 weeks after irradiation.
Description
Technical Field
The invention belongs to the technical field of radiology, and particularly relates to a method for establishing a mouse acute radiation duodenitis model.
Background
The animal model of human diseases is an animal experimental object with human disease simulation performance, and the use of the animal model is a very important experimental means in modern biomedical science research, which is helpful for more conveniently and effectively understanding the rules of occurrence and development of human diseases, thereby providing an experimental basis for the research of prevention and treatment measures.
Radiation Enteritis (RE) is a common intestinal complication caused by radiation therapy of malignant tumors of the pelvic cavity and abdominal cavity, and no effective clinical treatment means exists at present. Cervical cancer is the second most common tumor that occurs in women worldwide, second only to breast cancer, and is the first to female malignant tumor in developing countries, seriously threatening the life safety of women. Radiation therapy is a common means of treating cervical cancer. However, inflammatory symptoms have been reported in the intestinal tract of 70% of patients with pelvic radiotherapy, which severely affect the quality of life of the patients. The serious near and far radioactive adverse reactions severely limit the exertion of clinical curative effects. Once radiation enteritis occurs, the treatment is troublesome. At present, no effective clinical treatment means for radioactive intestinal injury still exists, and the problem is a difficult problem in the medical field. Therefore, the method has important clinical and theoretical values for the research on the prevention and treatment of the radiation enteritis.
BCL-2 is an important apoptosis inhibitor, which can inhibit apoptosis mediated by a plurality of apoptosis inducing factors such as rays, chemotherapeutic drugs and other factors, and has strong anti-apoptosis effect, therefore, the expression level of BCL-2 can reflect the apoptosis condition of cells to a certain extent, helper T (helper, Th) cells have important effect in organisms and can induce other immune cells to be transformed into effector cells, Th cells have at least two subgroups, Th1 cells and Th2 cell subgroups, which can secrete different cytokines, the representative cytokines of Th1 cells are IL-12, TGF- β and the like, and can regulate cellular immune response, TGF- β is a strong fibroblast division factor, can promote the division and proliferation and the differentiation of fibroblasts, promote the gene transcription and the protein synthesis of extracellular matrixes such as collagen, mucin and the like, and can prevent the degradation of matrix protein by inhibiting the synthesis of protease, and the ionizing effect of radiation- β in tissues of mice can be clinically proved to be expressed on the level of tissues of mice.
Disclosure of Invention
The invention aims to provide a method for establishing a mouse acute radiation duodenitis model, which is used for research on prevention and treatment of duodenitis induced by clinical radiotherapy.
The technical scheme of the invention is as follows: a method for establishing a mouse acute radiation duodenitis model comprises the following steps:
(1) quarantining the mice, randomly grouping the mice by adopting a random number table after one week, and dividing the mice into a control group and a dosage group;
(2) anaesthetizing two groups of mice of a control group and a dose group, and carrying out whole abdominal irradiation on the mice of the dose group, wherein the single absorption dose is 8 Gy; control mice were not irradiated;
(3) continuously raising for two weeks after irradiation, killing all mice by dissection, and taking duodenum;
(4) cleaning duodenal tissues with normal saline, fixing with 40g/L formaldehyde solution, dehydrating the fixed 1cm of duodenal ethanol, embedding in paraffin, slicing, HE staining, and observing pathological morphological changes under a mirror;
(5) weighing duodenal tissues, diluting the duodenal tissues with 1 × PBS according to a proportion, grinding the tissues by a tissue homogenizer to prepare homogenate, centrifuging the homogenate at 12000rpm for 10min, collecting supernatant, carrying out ELISA analysis, setting 3 parallel samples in each group, detecting OD values at 450nm on an enzyme-linked immunosorbent assay (ELISA) instrument, drawing a standard curve according to a standard substance, and obtaining a formula to obtain corresponding concentrations of BCL-2 and TGF- β 1 in the detected samples.
Further, the method for establishing the mouse acute radiation duodenitis model comprises the step (1) of 10 mice in the control group, 14 mice in the dose group and half of males and females.
Further, according to the method for establishing the mouse acute radiation duodenitis model, in the step (2), the anesthetized mouse adopts a mode of intraperitoneal injection of 1% sodium pentobarbital, and the dosage is 0.01mg/g of body weight.
Further, in the method for establishing the acute radiation duodenitis model of the mouse as described above, in the step (2), the source skin distance for irradiating the whole abdomen of the dose group mouse is 80 cm.
Further, the method for establishing the mouse acute radiation duodenitis model comprises the steps of (4) shrinking, shortening or falling off villi of the duodenum tissue part of the dose group mouse; the intestinal mucosa epithelium is extensively necrosed and stripped, the intestinal cavity is filled with necrosed and stripped tissues, and more apoptotic cells can be seen in the glandular fossa.
Further, according to the method for establishing the mouse acute radiation duodenitis model, in the step (5), after 8Gy ionizing radiation acts on the mice in the dose group, the expression of BCL-2 in the duodenal tissue is remarkably reduced, and the expression of TGF- β 1 is remarkably increased.
The invention has the following beneficial effects: the invention provides a method for establishing a mouse acute radiation duodenitis model, which can be used for researching the prevention and treatment of human radiation duodenitis induced by clinical radiotherapy. The irradiation mode in the invention is different from other related researches, adopts an irradiation mode closer to clinical radiotherapy, and establishes a radioactive duodenitis model through full-abdomen irradiation (the irradiation mode of cervical cancer patients is that a source is implanted into a body and the whole abdomen is irradiated), the source skin distance is 80cm (the irradiation source skin distance outside the clinical radiotherapy is 80cm or 1m), thereby providing an experimental basis for the prevention and treatment research of clinical radioactive intestinal injury.
Drawings
FIG. 1 is a flow chart of the method for establishing a mouse acute radiation duodenitis model according to the present invention;
FIG. 2 is a graph showing the results of the analysis of the duodenal tissue case of the control group;
FIG. 3 is a graph showing the results of a case analysis of duodenal tissues in a dose group;
FIG. 4 shows the concentration of BCL-2 in the control and dose groups of the duodenal tissue ELISA assay;
FIG. 5 shows the concentration of TGF- β 1 in the duodenum tissue ELISA assay control and dose groups.
Detailed Description
The invention is described in detail below with reference to the figures and examples.
The invention provides a specific method for establishing a mouse acute radiation enteritis model and identification.
The SPF-grade Kunming mouse is purchased from China food and drug testing research institute, is 6-8 weeks old and has the weight of 26-28 g. Control group 10, 8Gy dose group 14, male and female halves.
The cobalt-60 remote therapy machine is provided by a radiotherapy department in a hospital affiliated to the Chinese radiation protection institute, and has the following types: GWGP80, manufacturer: equipment manufacturers of the institute of nuclear power research and design, china.
The ELISA kit of the invention is purchased from the company R & D in the United states.
As shown in figure 1, the method for establishing the mouse acute radiation duodenitis model provided by the invention comprises the following steps:
1. animal quarantine grouping
And (3) quarantine is carried out on the newly purchased SPF-grade Kunming mice, and the quarantine period is one week. The mice in the quarantine period are bred and managed in a GLP center animal laboratory of China institute of radiation protection. The breeding conditions are 21 +/-2 ℃ and 60% of humidity. The staff regularly observed the animal vital signs. One week later animals were randomized into groups using a random number table. Control group 10, 8Gy dose group 14, male and female halves.
2. Irradiation
The two groups of mice of the control group and the 8Gy dose group are both transported to a radiotherapy department of a hospital affiliated to the Chinese radiation protection research institute, and the two groups of mice are anesthetized by 1% sodium pentobarbital in the radiotherapy department in an intraperitoneal injection mode. The dose was 0.01mg/g body weight. The mice in the 8Gy dose group are irradiated in a machine room of a cobalt-60 remote therapy machine in the radiotherapy department, the irradiation field is full abdomen (combination of sternal process and xiphoid process to pubic bone), and the source skin distance is 80 cm. The dose rate was 0.678Gy/min, and the single-absorbed dose was 8 Gy. The control group was not irradiated.
Previous studies have established a model of radioactive duodenitis by fixing irradiation fields at the site of the duodenum with a source-skin distance of 4 m. The scheme of the invention mainly establishes the radioactive duodenitis model through whole abdominal irradiation (the irradiation mode of cervical cancer patients is that the source is implanted into the body, the whole abdomen is irradiated), the source skin distance is 80cm (the irradiation source skin distance outside the clinical radiotherapy is 80cm or 1m in most cases). The mode is closer to the irradiation mode of clinical radiotherapy, and provides an experimental basis for the prevention and treatment research of clinical radioactive intestinal injury. In addition, the time for killing the animals in the technical scheme of the invention is 2 weeks after irradiation, which is the high incidence time of acute radiation enteritis after clinical radiotherapy.
3. Observing and taking materials
The irradiated mice are continuously raised in a GLP center animal laboratory of China institute of radiation protection, and the raising and management conditions are the same. Two weeks later, all mice were killed by dissection, and the duodenum was taken.
4. Pathological analysis
Washing duodenal tissues with normal saline, fixing with 40g/L formaldehyde solution, dehydrating fixed 1cm of duodenal ethanol, embedding in paraffin, slicing, HE staining, and observing pathological morphological changes under a mirror. The results are shown in FIGS. 2 and 3. FIG. 2 shows the control group and FIG. 3 shows the 8Gy dose group. From fig. 3 it can be seen that part of the intestinal section villi atrophies, shortens or sloughs; the intestinal mucosa epithelium is extensively necrosed and stripped, the intestinal cavity is filled with necrosed and stripped tissues, and more apoptotic cells can be seen in the glandular fossa.
5. ELISA assay
Weighing duodenal tissues, diluting with 1 × PBS according to the proportion of adding 10ml of PBS to 1g of body weight, grinding by using a tissue homogenizer to prepare homogenate (the operation is carried out on ice), centrifuging at 12000rpm for 10min, collecting supernatant, strictly operating according to the specification of an ELISA kit, arranging 3 parallel samples in each group, detecting OD values at 450nm on an enzyme-linked immunosorbent assay, drawing a standard curve according to the standard samples and obtaining a formula to obtain corresponding concentrations of BCL-2 and TGF- β in the detected samples, and obtaining results shown in figures 4 and 5.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations.
Claims (5)
1. A method for establishing a mouse acute radiation duodenitis model comprises the following steps:
(1) quarantining the mice, randomly grouping the mice by adopting a random number table after one week, and dividing the mice into a control group and a dosage group;
(2) anaesthetizing two groups of mice of a control group and a dose group, and performing whole abdominal irradiation on the mice of the dose group, wherein the source skin distance is 80cm, and the single absorption dose is 8 Gy; control mice were not irradiated;
(3) continuously raising for two weeks after irradiation, killing all mice by dissection, and taking duodenum;
(4) cleaning duodenal tissues with normal saline, fixing with 40g/L formaldehyde solution, dehydrating the fixed 1cm of duodenal ethanol, embedding in paraffin, slicing, HE staining, and observing pathological morphological changes under a mirror;
(5) weighing duodenal tissues, diluting the duodenal tissues with 1 × PBS according to a proportion, grinding the tissues by a tissue homogenizer to prepare homogenate, centrifuging the homogenate at 12000rpm for 10min, collecting supernatant, carrying out ELISA analysis, setting 3 parallel samples in each group, detecting OD values at 450nm on an enzyme-linked immunosorbent assay (ELISA) instrument, drawing a standard curve according to a standard substance, and obtaining a formula to obtain corresponding concentrations of BCL-2 and TGF- β 1 in the detected samples.
2. The method of establishing a model of acute radiation duodenitis in a mouse of claim 1, wherein: in the step (1), 10 mice in a control group, 14 mice in a dose group and male and female halves respectively.
3. The method of establishing a model of acute radiation duodenitis in a mouse of claim 1, wherein: in the step (2), the anesthetized mice adopt a mode of intraperitoneal injection of 1% sodium pentobarbital, and the dosage is 0.01mg/g body weight.
4. The method of establishing a model of acute radiation duodenitis in a mouse of claim 1, wherein: in the step (4), villi in the duodenum tissue part of the dose group mice shrink, shorten or fall off; the intestinal mucosa epithelium is extensively necrosed and stripped, the intestinal cavity is filled with necrosed and stripped tissues, and more apoptotic cells can be seen in the glandular fossa.
5. The method for modeling acute radiation duodenitis of a mouse of claim 1, wherein in step (5), after 8Gy ionizing radiation exposure of the mice in the dose group, the expression of BCL-2 in the duodenal tissue is significantly down-regulated, and the expression of TGF- β 1 is significantly up-regulated.
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