CN115088675A - Method for constructing novel testis injury model caused by ionizing radiation - 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
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention relates to a method for constructing a novel testis injury model caused by ionizing radiation, which comprises the following steps: placing a sample rack of the radiation instrument at the height of 50cm, drawing a circle with the diameter of 50cm by taking the vertical projection of a radiation source as the center, and fixing the position of a circular turntable; selecting a male C57BL/6 mouse with the age of 7-8 weeks, putting the mouse into a circular turntable, and performing whole-body radiation exposure, wherein the irradiation dose is 6Gy, and the dose rate is 130 cGy/min; after 8w of radiation, the testis can be obviously damaged, the vital signs are kept good, and the construction of a testis damage model is completed. The model construction method is simple to operate, accurate in radiation dose, consistent in radiation action part, wide and uniform in radiation action range, and can reduce the intra-group difference caused by complex radiation source control to the greatest extent.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for constructing a novel testis injury model caused by ionizing radiation.
Background
The ionizing radiation technology is widely applied in the modern production and living process, and how to prevent the ionizing radiation from damaging normal tissues is a difficult problem to be solved when the ionizing radiation technology is applied efficiently. The radiation-sensitive tissues of the hematopoietic system and the digestive system have been studied more deeply in the protection against radiation damage, but the protection against radiation damage of the reproductive system is still under basic research.
In order to develop a method for efficiently preventing the damage of radiation to the testis and the reproductive system, a great deal of related researches are carried out at home and abroad one after another. A large number of reproductive system radiation damage models are modeled by deep fixed therapeutic radiometers, testicular damage can be caused by the series of devices, but because the devices are large in size relative to mice, control of the acting positions, the irradiation dose, the dose rate and the like of radiation exposure of the mice has more influencing factors which are difficult to control, precision of all aspects needs to be improved, operation of the devices is relatively complex, and difficulty is brought to experimental operation.
In addition, the radiation in production, life, scientific application and medical use hardly has the condition of only targeting on the testis to cause injury, and the testis injury is common due to the irradiation of the whole body or the pelvic cavity, which can cause the adverse effect of the whole body of the organism. To do this, we first need to screen a whole-body radiation dose that can cause testicular damage without life threatening, in order to develop a long-term effect of testicular tissue. Meanwhile, radiation exposure may excite the autoimmune and defense mechanism, the protective compensation mechanism and the accumulation of testicular damage of the mouse testicle, so that the damage degree and the characterization of the testicle of the mouse are different in different time periods after the radiation exposure, after the radiation dose capable of effectively damaging the testicle is screened and determined, the testicular damage condition in different time periods after the radiation is further observed, and an efficient testicular damage model is provided for follow-up multi-dimensional thinking.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the defects existing in the existing testicular injury model establishment, the invention provides a novel method for establishing a testicular injury model caused by ionizing radiation, the model establishing method is simple to operate, accurate in radiation dose, consistent in radiation action part, wide and uniform in radiation action range, and can reduce the intra-group difference caused by the complex radiation source control to the greatest extent.
The technical scheme is as follows: a method for constructing a novel testis injury model caused by ionizing radiation comprises the following steps:
1) placing a sample rack of the radiation instrument at a height of 50cm, drawing a circle with the diameter of 50cm by taking the vertical projection of a radiation source as a center, and fixing the position of a circular turntable;
2) selecting a male C57BL/6 mouse with the age of 7-8 weeks, putting the mouse into a circular turntable, and performing whole-body radiation exposure, wherein the irradiation dose is 6Gy, and the dose rate is 130 cGy/min;
3) after 8w of radiation, the testis can be obviously damaged, and the vital signs are kept good, so that the construction of a testis damage model is completed.
The body weight of the mice in the step 2) is (22 +/-2) g, and the mice are not subjected to any injury treatment before being subjected to the radiation treatment.
Has the advantages that: the invention provides a method for constructing a novel testis injury model caused by ionizing radiation, which has the following beneficial effects:
1. the model construction operation of the invention is simple, the radiation dose is accurate, the radiation action parts are consistent, the radiation action range is wide and uniform, and the intra-group difference caused by the complicated control of the radiation source can be reduced to the maximum extent;
2. the model of the invention can cause testicular injury and ensure the life health of mice, and provides an effective model for the relevant research of testicular radiation injury protection.
Drawings
Fig. 1 is a plot of testis-related indices in stage one ([ p ] 0.05vs 0Gy, [ p ] 0.001vs 0 Gy).
Fig. 2 is an example of a typical diagram of a teratospermia at stage one.
Fig. 3 is a plot of the sperm mass parameters in stage one ([ p ] p <0.05vs 0Gy, [ p ] p <0.01vs 0Gy, [ p ] p <0.001vs 0 Gy). FIG. 4 is a graph showing HE staining of testis tissue of the unirradiated, 2Gy, 4Gy and 6Gy groups at stage one after irradiation of 8w (magnification 400 times).
Fig. 5 is a parameter statistic for seminiferous tubules in stage one ([ p ] p <0.05vs 0Gy, [ p ] p <0.001vs 0 Gy). A is a raw tubule area statistical graph, and B is a raw tubule diameter statistical graph.
FIG. 6 shows testis in stage oneLesion model testis PLZF and SOX9 immunohistochemical staining patterns. A is testis tissue PLZF + Staining pattern (400 times magnification), B is testis tissue SOX9 + Staining pattern (400-fold magnification).
FIG. 7 is stage one testicular injury model testis PLZF + And SOX9 + Statistical plot (. dot.p)<0.01vs 0 Gy). A is PLZF + Staining statistical chart, B is SOX9 + Staining statistical chart.
Fig. 8 is a plot of testis-associated indices in stage two (×) p < 0.001.
Fig. 9 is a graph of the sperm mass parameters in stage two ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001).
FIG. 10 is the epididymis HE staining pattern in stage two (100-fold magnification).
FIG. 11 is a graph of the HE staining of testis at radiation dose of 6Gy, exposure time of 24h, 7d, 6w, and 8w in stage two (400-fold magnification).
Fig. 12 is the seminiferous tubule associated parameter statistics in stage two (× p < 0.001). A is a raw tubule area statistical graph, and B is a raw tubule diameter statistical graph.
FIG. 13 is a graph of stage two testis lesion model testis PLZF and SOX9 immunohistochemical staining. A is testis tissue PLZF + Staining pattern (400 times magnification), B is testis tissue SOX9 + Staining pattern (400-fold magnification).
FIG. 14 is stage two testis injury model testis PLZF + And SOX9 + Statistical plot (. p)<0.05，***p<0.001). A is PLZF + Staining statistical chart, B is SOX9 + Staining statistical chart.
FIG. 15 is a graph of testis BrdU and TUNEL staining in the stage two testis injury model. A is staining pattern of testis tissue BrdU (400 times magnification), and B is staining pattern of testis tissue TUNEL (400 times magnification).
FIG. 16 is stage two testis injury model testis BrdU + And TUNEL + Statistical plot (. about.. p)<0.01，***p<0.001). A is BrdU + Staining statistical chart, B is TUNEL + Staining statistical chart.
Detailed Description
The present invention is described in further detail below by way of specific embodiments, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
The mouse X-Ray whole body irradiation source in this embodiment was an X-RAD 225 radiometer available from Precision X-Ray, USA. X-Rad 225 is a small animal applicator that can provide targeted radiation. The fixing device for mouse irradiation is a plastic circular turntable with the diameter of 50cm, 11 independent small spaces are provided, and only 1 mouse can be accommodated in each space and placed in a normal position. A circular shape with the diameter of 50cm is drawn on a sample placing frame of the radiation instrument by taking a radiation source as a center, and a mouse fixing device is placed in the range, so that the accurate irradiation of the mice with the same dose and the same body position is ensured, and the intra-group difference caused by the complexity of the operation and control of the radiation source is reduced to the maximum extent. The radiation dose rate was 130cGy/min, and the radiation meter was calibrated by a skilled technician before use.
The materials and reagents used in the present embodiment are commercially available unless otherwise specified.
In order to detect and verify testicular damage, detect testicular volume and testicular organ coefficients, and observe visual morphology of the testicles, sperm quality parameters are used to detect sperm quality produced by the testicles, HE staining is used to observe morphology and histopathological structure of the testicles and epididymis, and PLZF and SOX9 are used for testis immunohistochemical staining to mark spermatogonial cells and supporting cells respectively. The specific method and process are as follows:
(1) sperm quality parameter
Preparation of sperm suspension: putting the right intact epididymis into 1mL physiological saline preheated at 37 ℃, cutting into multiple sections with scissors, incubating in a water bath kettle at 37 ℃ for 20min, and fully dissociating sperm from the epididymis. Detecting sperm survival rate, sperm motility rate, sperm density and sperm malformation rate.
(2) Testicular organ coefficient and volume measurements
Testis organ coefficient: weighing bilateral intact testis, wherein the testis organ coefficient (%). is bilateral testis weight (g)/body weight (g) × 100%; unilateral testicular volume: the left testis was submerged in 1mL of saline in the 2.5mL syringe at the 1mL scale, and the volume of saline higher than the 1mL scale, i.e., the volume of the left testis, was measured with a 100uL micropipette. After the volume is measured, the testis tissues are photographed and stored.
(3) Image analysis and morphological parameter quantification of HE staining and immunohistochemical staining
1) Measuring the circumference and area of the seminiferous tubule: taking about 10 pictures of one sample without repetition under a 10 × 20 microscope field; after the seminiferous tubules are circled out one by using Image Pro Plus, the perimeter and the area of each seminiferous tubule are measured, and 50 seminiferous tubules are randomly measured and calculated for each sample, and the average value is calculated.
2)PLZF + 、SOX9 + 、BrdU + 、TUNEL + Cell counting: under a 10X 20 microscope field, 50 seminiferous tubules were randomly estimated for each sample, and the average number of positive cells per seminiferous tubule was calculated.
The optimal irradiation dose and the optimal irradiation time in the testicular injury model construction method are determined through the following stages.
Stage one, determination of optimal irradiation dose in a testis injury model construction method:
20 male SPF-grade C57BL/6 mice 7-8 weeks old, with the weight of 22g +/-2 g, purchased from Schleksideda laboratory animals Co., Ltd, Hunan, certificate number (SCXK (Hunan) 2019-. C57BL/6 mice were randomly divided into 4 groups: non-irradiated group, 2Gy group, 4Gy group, 6Gy group (different dose irradiation groups are all x-ray mice whole body exposure).
8w after radiation exposure, mice were harvested for bilateral testis and epididymis: weighing the cervical vertebra, dislocating and killing the mouse, taking out the left and right testicles and epididymis, fixing the left epididymis for 20 hours at 4 ℃ by adopting 4% paraformaldehyde, and using the right epididymis for preparing sperm suspension; the left testis was fixed in modified Davidson's fixative at 4 deg.C for 20h, and the right testis was snap frozen in liquid nitrogen and stored at-80 deg.C for RNA and protein extraction.
The testis weighing and sperm mass changes for different radiation doses are shown in figures 1, 2, and 3. The testis coefficient and the testis volume of mice in 4Gy and 6Gy irradiation groups are obviously reduced. After 4Gy and 6Gy irradiation, the mouse epididymis has more abnormal sperms, such as double-headed sperms, double-tailed sperms, indefinite head sperms, folded tail and the like. No moving sperm is seen under the 6Gy irradiation rear mirror, and the number of sperm in epididymis is obviously reduced. Both 4Gy and 6Gy radiation exposure doses resulted in testicular size reduction and sperm quality reduction, with 6Gy being the most significant effect.
The damage to testicular tissue caused by different radiation doses is shown in figures 4 and 5. The diameter of the seminiferous tubule of the mice in the 4Gy and 6Gy irradiation groups is obviously reduced, the wall of the seminiferous tubule becomes thin and vacuole appears, the cell arrangement is disordered, and the area and the diameter of the seminiferous tubule are obviously reduced. Thus, 4Gy and 6Gy radiation exposure can significantly damage testicular tissue structure.
The changes in germ cell numbers induced by different radiation doses are shown in figures 6 and 7. The number of the mouse PLZF immunoreactive positive spermatogonial stem cells is not obviously changed 8w after 2Gy, 4Gy and 6Gy irradiation. The supporting cells in the seminiferous tubules are marked by SOX9, and 8w after irradiation of 4Gy and 6Gy, the supporting cells in the seminiferous tubules are distributed more densely, the number of the supporting cells is increased, and the number of the supporting cells in the seminiferous tubules of mice exposed to the radiation dose of 6Gy is obviously increased.
In different radiation dose models, 4Gy and 6Gy ionizing radiation doses acting on a mouse can cause testicle damage and sperm quality reduction of the mouse, but the 6Gy radiation dose damage effect is obviously stronger than the 4Gy radiation dose. 6Gy can cause obvious damage to testis and does not affect the life health of mice.
In conclusion, the optimal irradiation dose of the testicular injury model construction method of the present invention is 6 Gy.
Stage two, determination of optimal irradiation time in the testicular injury model construction method:
male SPF grade C57BL/6 mice at 7-8 weeks were randomly divided into 24h, 7d, 6w, 8w after irradiation and non-irradiated groups corresponding to each group, 3/group, 24 in total; the total dose of the X-ray irradiation is 6Gy, and the dose rate is 130 cGy/min; collecting relevant indexes of testicle and epididymis detection at each time point after irradiation. 2h before material drawing, the mouse is injected with 100mg/kg BrdU drug reagent in the abdominal cavity. Weighing, dislocating cervical vertebra, killing mouse, taking out left and right testis and epididymis, and collecting materials and treating.
The change in testicular and sperm quality at different times after radiation exposure is shown in figures 8, 9, 10, and 11. After 6Gy irradiation, the testis is obviously reduced by 6w and 8w, the visceral organ index and the testis volume are obviously reduced, the sperm teratospermia rate is obviously increased, the sperm motility rate and the sperm density are obviously reduced, and no or only a small amount of sperms exist in the epididymis.
The 6w and 8w induced testicular structural damage after ionizing radiation is shown in figure 12. In a mouse testis injury model at different time after 6Gy x-ray radiation, the seminiferous tubules of 6w and 8w mice testis after radiation become smaller obviously, irregular seminiferous tubules increase, the lumen becomes larger, the seminiferous epithelium becomes thinner obviously, a large number of vacuoles exist, and the pipe diameter and the area are both reduced obviously.
The changes in germ cell numbers at 24h and 7d after ionizing radiation are evident in fig. 13, 14. 24h and 7d after 6Gy irradiation, the number of spermatogonial stem cells is obviously reduced, and the number of supporting cells is obviously increased.
In conclusion, in the testis injury model construction method, spermatogonial stem cell reduction is taken as a main characteristic in 7d after radiation, and 8w after radiation is mainly expressed as testis tissue structure defect. Therefore, the optimal irradiation time of the testis injury model construction method is 8 w.
In order to further explore and verify the damage of the testis caused by radiation, immunohistochemistry technology is applied to testis tissues treated in the second stage to detect the proliferation and apoptosis of germ cells after ionizing radiation, and the detection results are shown in fig. 15 and 16. BrdU can label cells in proliferative state, TUNEL staining to label apoptotic cells. After 6Gy irradiation for 24h and 7d, the number of proliferated cells is obviously reduced, and apoptotic cells are obviously increased.
Example 1
The embodiment provides a method for constructing a novel testis injury model caused by ionizing radiation, which comprises the following steps:
1) placing a sample rack of the radiation instrument at the height of 50cm, drawing a circle with the diameter of 50cm by taking the vertical projection of a radiation source as the center, and fixing the position of a circular turntable;
2) selecting a male C57BL/6 mouse with the age of 7-8 weeks, wherein the weight of the mouse is (22 +/-2) g, no damage treatment is performed before the mouse is subjected to radiation treatment, putting the mouse into a circular turntable, and performing whole-body radiation exposure, wherein the irradiation dose is 6Gy, and the dose rate is 130 cGy/min;
3) after 8w of radiation, the testis can be obviously damaged, and the vital signs are kept good, so that the construction of a testis damage model is completed.
The technology of the invention simply and accurately controls the radiation dose on the premise of ensuring the consistency of the X-ray whole body radiation exposure dose, the exposure position and the range of the mouse, probes the influence of radiation on the testis land, and consists of two stages of X-ray whole body irradiation different doses and testis damage conditions at each time after radiation exposure. The effective radiation dose can cause obvious damage to the testis and dose not threatening the life of the mouse, and the dynamic change of the testis at different time after the damage dose is acted can be observed at different time after radiation exposure, thereby providing wide thinking space and research foundation for the research of the testis radiation damage mechanism.
While the embodiments of the present invention have been described in detail, those skilled in the art will appreciate that the various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Claims (2)
1. A method for constructing a novel model of testis injury caused by ionizing radiation is characterized by comprising the following steps of:
1) placing a sample rack of the radiation instrument at a height of 50cm, drawing a circle with the diameter of 50cm by taking the vertical projection of a radiation source as a center, and fixing the position of a circular turntable;
2) selecting a male C57BL/6 mouse with the age of 7-8 weeks, putting the mouse into a circular turntable, and performing whole-body radiation exposure, wherein the irradiation dose is 6Gy, and the dose rate is 130 cGy/min;
3) after 8w of radiation, the testis can be obviously damaged, the vital signs are kept good, and the construction of a testis damage model is completed.
2. The method for constructing the novel model of testicular injury caused by ionizing radiation according to claim 1, wherein: the body weight of the mice in the step 2) is (22 +/-2) g, and the mice are not subjected to any injury treatment before being subjected to radiation treatment.
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