CN110999865B - Construction method and application of osteoporosis mouse model caused by secondary hyperthyroidism - Google Patents
Construction method and application of osteoporosis mouse model caused by secondary hyperthyroidism Download PDFInfo
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Abstract
The invention relates to a construction method and application of a mouse model for osteoporosis caused by secondary hyperthyroidism, wherein the construction method comprises the following steps: the parathyroid gland tissue from a secondary hyperthyroidism patient is pretreated and then transplanted into a mouse body, and a secondary hyperthyroidism-induced osteoporosis mouse model is constructed. The construction method of the secondary hyperthyroidism-induced osteoporosis mouse model lays a good foundation for development of diseases, screening of treatment drugs and scientific evaluation of curative effects, by adopting the method, the severity and treatment conditions of the onset of the mouse can be judged scientifically, quickly and accurately, and a convenient, quick and accurate judgment method is successfully constructed for the secondary hyperthyroidism-induced osteoporosis drug screening model. The construction method has high success rate and high clinical relevance.
Description
Technical Field
The invention belongs to the technical field of biological medical treatment, and particularly relates to a construction method and application of a mouse model for osteoporosis caused by secondary hyperthyroidism.
Background
Parathyroid hormone (PTH) is secreted by parathyroid chief cells, is an important calcium-phosphorus homeostasis regulatory factor consisting of 84 amino acids, can regulate expression and secretion of PTH according to changes in blood calcium concentration, and plays an important role in maintaining the balance of calcium and phosphorus in the blood and bone metabolism. Intermittent administration of low dose PTH promotes osteoblast maturation and differentiation, mediates osteogenic processes, and thereby promotes increased bone mass; continuous administration of high doses of PTH promotes osteoclast maturation, thereby mediating the osteoclast process and inducing bone loss. PTH is used clinically to treat osteoporosis in menopausal women; there are data indicating that daily administration of PTH can effectively improve the level of osteoporosis in menopausal women, a phenomenon suggesting that the rhythmic administration of PTH can promote osteogenesis. Hyperparathyroidism (hyperthyroidism) is a series of syndromes caused by excessive secretion of PTH by parathyroid gland, which can lead to bone pain, osteoporosis/fractures, hypercalcemia, etc.; that is, sustained high levels of PTH can induce a range of symptoms of bone metabolism disorders.
Previously, the understanding of osteoporosis has been limited to calcium deficiency and insufficient calcium absorption; therefore, the treatment of osteoporosis often falls short of calcium supplementation and the administration of related supplements that promote calcium absorption. In recent years, it has become recognized that excessive secretion of PTH can also lead to osteoporosis; however, the clinical screening still has the preference phenomenon of the chief complaint, namely, the hyperthyroidism patients are often concerned about the excessive secretion of PTH, and the osteoporosis accompanied by the same is easy to ignore.
At present, how to construct an animal model of osteoporosis caused by secondary hyperthyroidism is not disclosed in the prior art, and the animal model is crucial to the research work of osteoporosis diseases caused by secondary hyperthyroidism, so that the construction of the animal model of osteoporosis caused by secondary hyperthyroidism is very significant.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a construction method and application of a mouse model for osteoporosis caused by secondary hyperthyroidism.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a method for constructing a mouse model of osteoporosis induced by secondary hyperthyroidism, which comprises the following steps: the paraungual gland tissue from a secondary hyperthyroidism patient is pretreated and then transplanted into a mouse body, and a mouse model of osteoporosis caused by the secondary hyperthyroidism is constructed.
The construction method of the secondary hyperthyroidism-induced osteoporosis mouse model lays a good foundation for development of diseases, screening of treatment drugs and scientific evaluation of curative effects, by adopting the method, the severity and treatment conditions of the onset of the mouse can be judged scientifically, quickly and accurately, and a convenient, quick and accurate judgment method is successfully constructed for the secondary hyperthyroidism-induced osteoporosis drug screening model. The construction method has high success rate and high clinical relevance.
Preferably, the pretreatment mode is as follows: the tissue culture is carried out after the paraungual gland tissue from the secondary hyperthyroidism patient is sheared.
Preferably, the tissue is cut into pieces of 0.5-2mm3Of tissue mass, e.g. 0.5mm3、0.8mm3、1mm3、1.2mm3、1.5mm3Or 2mm3And the other specific point values in the range can be selected, and are not described in detail herein.
Preferably, the incubation refers to incubating the tissue in a medium containing serum and a diabody.
Preferably, the medium containing serum and double antibody is RPMI1640 medium containing 10% FBS and 1% double antibody.
Preferably, the incubation time is 3-7 days, for example, 3 days, 4 days, 5 days, 6 days, 7 days, etc., and any other specific point value within the range can be selected, which is not described herein.
Preferably, the incubation is at 37 ℃, 5% CO2In an incubator.
Preferably, the transplanting mode is as follows: the mouse is opened in the triangular area of the back, the opening is widened to form a pocket for containing the tissue block, and the tissue block is placed in the pocket and then is sewn.
Preferably, the mouse is opened in the triangular region of the back using a scalpel, the opening is extended with forceps and widened to form a pocket for holding the tissue mass, and 2-6 tissue masses (e.g., 2, 3, 4, 5 or 6) are placed in the pocket and the wound is sutured and disinfected.
As a preferred technical scheme, the construction method of the secondary hyperthyroidism-initiated osteoporosis mouse model comprises the following steps:
(1) placing the paraungual gland tissue of patient with secondary hyperthyroidism in precooled D-Hanks solution, stripping off blood vessel tissue, and cutting into pieces of 0.5-2mm3The tissue mass of (a);
(2) placing the tissue block obtained in the step (1) in a culture medium containing serum and double antibodies, and culturing for 3-7 days at 37 ℃ in a 5% CO2 incubator;
(3) and (3) opening the back triangular area of the mouse by using a scalpel, extending into the opening by using forceps and widening to form a pocket for containing the tissue block, placing 2-6 tissue blocks obtained in the step (2) in the pocket and then suturing, disinfecting wounds and constructing the osteoporosis mouse model caused by secondary hyperthyroidism.
The invention relates to a method for constructing a mouse model for secondary osteoporosis caused by hyperthyroidism, which comprises the following steps of: collecting blood samples of the mice 4 weeks after the transplantation operation is finished, respectively detecting serum PTH or blood calcium level, and comparing the blood samples with mice of a control group (a sham operation group, which only performs an operation process and does not perform the transplantation operation); scanning and reconstructing through micro-CT, respectively analyzing the change of parenchymal bone and cancellous bone, representing the bone metabolism condition by using parameters such as the number of trabeculae, bone density, trabecular bone gap and the like, and comparing with a control group mouse (a false operation group, only performing an operation process and not performing transplantation operation); the expression of alkaline phosphatase, an osteoblast marker, and tartrate-resistant acid phosphatase, an osteoclast marker were statistically analyzed by immunostaining, and the ratio of osteoblast number to bone volume (Ob/TV) and the ratio of osteoclast number to bone volume (Oc/TV) were also statistically analyzed.
If the serum PTH and the blood calcium of the mice in the transplant group are obviously higher than those of the control group, the tibia bone mass thickness of the mice in the transplant group is obviously reduced, the bone density and the bone body volume density are obviously reduced, the osteoblast marker alkaline phosphatase expression of the mice in the transplant group is increased, the osteoclast marker tartaric acid phosphatase expression is reduced, the ratio (Ob/TV) of the number of osteoblasts to the bone body volume is increased, and the ratio (Oc/TV) of the number of osteoclasts to the bone body volume is reduced, so that the success of the establishment of the hyperthyroidism nude mouse model can be proved.
In a second aspect, the invention provides an application of the construction method of the mouse model of osteoporosis caused by secondary hyperthyroidism in preparation of a medicament for treating osteoporosis caused by secondary hyperthyroidism.
Compared with the prior art, the invention has the following beneficial effects:
the construction method of the secondary hyperthyroidism-induced osteoporosis mouse model lays a good foundation for development of diseases, screening of treatment drugs and scientific evaluation of curative effects, by adopting the method, the severity and treatment conditions of the onset of the mouse can be judged scientifically, quickly and accurately, and a convenient, quick and accurate judgment method is successfully constructed for the secondary hyperthyroidism-induced osteoporosis drug screening model. The construction method has high success rate and high clinical relevance.
Drawings
FIG. 1 is a statistical graph of the serum PTH levels of the control and transplanted mice in example 4;
FIG. 2 is a structure diagram of the femur of a mouse in the control group and the transplant group in example 4;
FIG. 3 is a tibial scanning reconstruction diagram of mice from the control group and the transplant group in example 4;
FIG. 4 is a statistical graph of bone density (BMD) of mice in the control group and the transplanted group in example 4;
FIG. 5 is a statistical view of the body volume density (BV/TV) of mice in the control group and the transplanted group in example 4;
FIG. 6 is a statistical graph showing the ratio of the number of osteoblasts to the volume of bone (Ob/TV) in the control group and the transplanted group of mice in example 4;
FIG. 7 is a graph showing a statistical graph of the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the mice of the control group and the transplanted group in example 4;
FIG. 8 is a statistical graph of the serum PTH levels of the control and transplanted mice in example 5;
FIG. 9 is a statistical graph of bone density (BMD) of mice in the control group and the transplanted group in example 5;
FIG. 10 is a statistical view of the body volume density (BV/TV) of mice in the control group and the transplanted group in example 5;
FIG. 11 is a statistical graph showing the ratio of the number of osteoblasts to the volume of bone (Ob/TV) in the control group and the transplanted group of mice in example 5;
FIG. 12 is a graph showing a statistical graph of the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the mice of the control group and the transplanted group in example 5;
FIG. 13 is a statistical graph showing the serum PTH levels of the control and transplanted mice in example 6;
FIG. 14 is a statistical graph of bone density (BMD) of mice in the control group and the transplanted group in example 6;
FIG. 15 is a statistical view of the body volume density (BV/TV) of mice in the control group and the transplanted group in example 6;
FIG. 16 is a statistical graph showing the ratio of the number of osteoblasts to the volume of bone (Ob/TV) in the control group and the transplanted group of mice in example 6;
FIG. 17 is a graph showing a statistical graph of the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the mice of the control group and the transplanted group in example 6.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.
The test animals referred to in the following examples were male CD-1 nude mice, purchased from the center of medical laboratory animals of Guangdong province, aged 6-8 weeks, and had a body weight of 20-25 g.
The paraungual gland tissues of the secondary hyperthyroidism patients related to the following examples are derived from the secondary hyperthyroidism patients in the first human hospital of Shenzhen city; all organizations obtain informed consent signed by patients; the experiment obtained by the inventor is approved by ethical review of medical ethical committee of the first people hospital in Shenzhen city.
Example 1
The embodiment provides a method for constructing a mouse model of osteoporosis caused by secondary hyperthyroidism, which comprises the following steps:
(1) placing the paraungual gland tissue of patient with secondary hyperthyroidism in pre-cooled D-Hanks solution, removing blood vessel tissue with fine forceps and operation scissors, and cutting into pieces of 1mm3The tissue mass of (a);
(2) adding culture medium (RPMI1640+ 10% FBS + 1% double antibody) into a 6-well plate, transferring the tissue block obtained in the step (1), and culturing in a 5% CO2 incubator at 37 ℃ for 5 days;
(3) and (3) opening the back triangular area of the mouse by using a scalpel, extending into the opening by using forceps and widening to form a pocket for containing the tissue block, placing the 4 tissue blocks obtained in the step (2) in the pocket, suturing, and sterilizing the wound to construct the osteoporosis mouse model caused by the secondary hyperthyroidism.
Example 2
The embodiment provides a method for constructing a mouse model of osteoporosis caused by secondary hyperthyroidism, which comprises the following steps:
(1) placing paraungual gland tissue from secondary hyperthyroidism patient in pre-cooled D-Hanks solution, stripping off blood vessel tissue with fine forceps and operation scissors, and cutting into pieces of 1.5mm3The tissue mass of (a);
(2) adding culture medium (RPMI1640+ 10% FBS + 1% double antibody) into a 6-well plate, transferring the tissue block obtained in the step (1), and culturing for 7 days at 37 ℃ in a 5% CO2 culture box;
(3) and (3) opening the back triangular area of the mouse by using a scalpel, extending the opening into the triangular area by using forceps, widening the opening to form a pocket for containing the tissue block, placing the 3 tissue blocks obtained in the step (2) in the pocket, sewing, and disinfecting the wound to construct the osteoporosis mouse model caused by the secondary hyperthyroidism.
Example 3
The embodiment provides a method for constructing a mouse model of osteoporosis caused by secondary hyperthyroidism, which comprises the following steps:
(1) placing the paraungual gland tissue of patient with secondary hyperthyroidism in pre-cooled D-Hanks solution, removing blood vessel tissue with fine forceps and operation scissors, and cutting into pieces of 0.5mm3The tissue mass of (a);
(2) adding culture medium (RPMI1640+ 10% FBS + 1% double antibody) into a 6-well plate, then transferring the tissue block obtained in the step (1) into a culture box at 37 ℃ and in a 5% CO2 culture box for 3 days;
(3) and (3) opening the back triangular area of the mouse by using a scalpel, extending into the opening by using forceps and widening to form a pocket for containing the tissue block, placing 5 tissue blocks obtained in the step (2) in the pocket, suturing, and sterilizing the wound to construct the osteoporosis mouse model caused by the secondary hyperthyroidism.
Example 4
This example demonstrates that the mouse model of osteoporosis induced by secondary hyperthyroidism referred to in example 1 was successfully constructed, and the contents include the following:
(1) collecting blood samples of mice 4 weeks after the transplantation operation, detecting the serum PTH level, and comparing the blood samples with mice of a control group (a sham operation group, which only performs the operation process and does not perform the transplantation operation);
the results are shown in FIG. 1: serum PTH levels were significantly higher in the transplanted mice relative to the control group.
(2) Carrying out Mciro-CT scanning, reconstructing a mouse bone tissue sample, analyzing bone metabolism indexes including parameters such as bone density, bone volume density and bone structure, and comparing the bone metabolism indexes with a control group mouse (a false operation group, only carries out an operation process and does not carry out transplantation operation);
the structure of the femur of the mouse is shown in fig. 2: the transplanted mice showed significant osteoporosis relative to the control group.
The tibial scanning reconstruction of the mice is shown in figure 3: the tibial bone mass of the control group of mice is larger, and the bone mass of the transplanted group of mice is obviously thinner.
The bone density (BMD) statistical profile of the mice is shown in fig. 4: bone density (BMD) was significantly reduced in the transplanted mice relative to the control group.
A bone volume density (BV/TV) histogram of mice is shown in FIG. 5: compared with the control group, the body volume density (BV/TV) of the mice in the transplanted group is obviously reduced.
(3) By using immunostaining technique, the ratio of osteoblast number to bone volume (Ob/TV) and the ratio of osteoclast number to bone volume (Oc/TV) were counted and compared with control mice (sham group, which were only operated without transplantation).
A statistical plot of the ratio of osteoblast number to bone volume (Ob/TV) for the mice is shown in FIG. 6: the ratio of osteoblast number to bone volume (Ob/TV) was significantly reduced in the transplanted mice relative to the control group.
A statistical plot of the osteoclast number to bone volume ratio (Oc/TV) in mice is shown in FIG. 7: the ratio of osteoclast number to bone volume (Oc/TV) was significantly increased in the mice of the transplanted group relative to the control group.
The results show that the mouse model of osteoporosis caused by secondary hyperthyroidism is successfully constructed.
Example 5
This example demonstrates that the mouse model of osteoporosis induced by secondary hyperthyroidism referred to in example 2 was successfully constructed, and the contents include the following:
(1) collecting blood samples of mice 4 weeks after the transplantation operation is finished, detecting the serum PTH level, and comparing the blood samples with mice of a control group (a sham operation group, which only performs an operation process and does not perform the transplantation operation);
the results are shown in FIG. 8: serum PTH levels were significantly higher in the transplanted mice relative to the control group.
(2) Carrying out Mciro-CT scanning, reconstructing a mouse bone tissue sample, analyzing bone metabolism indexes including bone density and bone volume density, and comparing with a control group mouse (a pseudo operation group, only carrying out an operation process and not carrying out transplantation operation);
the bone density (BMD) statistical profile of the mice is shown in fig. 9: bone density (BMD) was significantly reduced in the transplanted mice relative to the control group.
A bone volume density (BV/TV) histogram of mice is shown in FIG. 10: compared with the control group, the body volume density (BV/TV) of the mice in the transplanted group is obviously reduced.
(3) Using immunostaining, the ratios of osteoblast to bone volume (Ob/TV) and osteoclast to bone volume (Oc/TV) were counted and compared with control mice (sham, which were treated only with the surgical procedure and were not transplanted).
A statistical plot of the ratio of osteoblast number to bone volume (Ob/TV) for the mice is shown in FIG. 11: the osteoblast count to bone volume ratio (Ob/TV) was significantly reduced in the transplanted mice relative to the control group.
A statistical plot of the osteoclast number to bone volume ratio (Oc/TV) in mice is shown in FIG. 12: the ratio of osteoclast number to bone volume (Oc/TV) was significantly increased in the mice of the transplanted group relative to the control group.
The results show that the mouse model of osteoporosis caused by secondary hyperthyroidism is successfully constructed.
Example 6
This example demonstrates that the mouse model of osteoporosis induced by secondary hyperthyroidism referred to in example 3 was successfully constructed, and the contents include the following:
(1) collecting blood samples of mice 4 weeks after the transplantation operation, detecting the serum PTH level, and comparing the blood samples with mice of a control group (a sham operation group, which only performs the operation process and does not perform the transplantation operation);
the results are shown in FIG. 13: serum PTH levels were significantly higher in the transplanted mice relative to the control group.
(2) Carrying out Mciro-CT scanning, reconstructing a mouse bone tissue sample, analyzing bone metabolism indexes including bone density and bone volume density, and comparing with a control group mouse (a pseudo operation group, only carrying out an operation process and not carrying out transplantation operation);
the bone density (BMD) statistical profile of the mice is shown in fig. 14: bone density (BMD) was significantly reduced in the transplanted mice relative to the control group.
Bone volume density (BV/TV) statistics for mice are shown in FIG. 15: compared with the control group, the body volume density (BV/TV) of the mice in the transplanted group is obviously reduced.
(3) By using immunostaining technique, the ratio of osteoblast number to bone volume (Ob/TV) and the ratio of osteoclast number to bone volume (Oc/TV) were counted and compared with control mice (sham group, which were only operated without transplantation).
A statistical plot of the ratio of osteoblast number to bone volume (Ob/TV) for the mice is shown in FIG. 16: the osteoblast count to bone volume ratio (Ob/TV) was significantly reduced in the transplanted mice relative to the control group.
A statistical plot of the osteoclast number to bone volume ratio (Oc/TV) in mice is shown in FIG. 17: the ratio of osteoclast number to bone volume (Oc/TV) was significantly increased in the mice of the transplanted group relative to the control group.
The results show that the mouse model of osteoporosis caused by secondary hyperthyroidism is successfully constructed.
The applicant states that the invention is illustrated by the above examples to describe the construction method and application of the mouse model of osteoporosis induced by secondary hyperthyroidism, but the invention is not limited by the above examples, i.e. it does not mean that the invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (6)
1. A construction method of a mouse model of osteoporosis caused by secondary hyperthyroidism is characterized by comprising the following steps: shearing paraungual gland tissue from a secondary hyperthyroidism patient, placing the sheared paraungual gland tissue in a culture medium containing serum and double antibodies, culturing for 3-7 days at 37 ℃ in a 5% CO2 incubator, opening a triangular area on the back of a mouse, widening the opening to form a pocket for containing a tissue block, placing the cultured tissue block in the pocket, and suturing to construct an osteoporosis mouse model caused by the secondary hyperthyroidism.
2. The method for constructing a mouse model of osteoporosis induced by secondary hyperthyroidism in accordance with claim 1, wherein said tissue is minced to 0.5-2mm3The tissue mass of (1).
3. The method for constructing a mouse model of osteoporosis induced by secondary hyperthyroidism according to claim 1, wherein said medium containing serum and double antibody is RPMI1640 medium containing 10% FBS and 1% double antibody.
4. The method for constructing a mouse model of osteoporosis induced by secondary hyperthyroidism of claim 1, wherein said opening is made in the triangular area of the back of the mouse, the opening is widened to form a pocket for holding the tissue mass, and then the tissue mass is placed in the pocket and then sutured, specifically comprising: the mouse back triangle area is opened with a scalpel, the opening is extended into the mouse back triangle area and widened with forceps to form a pocket for containing tissue blocks, and 2-6 tissue blocks are placed in the pocket and then the wound is sutured and disinfected.
5. The method of constructing a mouse model of osteoporosis secondary to hyperthyroidism in claim 1, comprising:
(1) placing paraungual gland tissue from secondary paraungual hyperthyroidism patient in precooled D-Hanks liquid, peeling off blood vessel tissue, and cutting into pieces of 0.5-2mm3The tissue mass of (a);
(2) placing the tissue block obtained in the step (1) in a culture medium containing serum and double antibodies, and culturing for 3-7 days at 37 ℃ in a 5% CO2 incubator;
(3) and (3) opening the back triangular area of the mouse by using a scalpel, extending into the opening by using forceps and widening to form a pocket for containing the tissue block, placing 2-6 tissue blocks obtained in the step (2) in the pocket and then suturing, disinfecting wounds and constructing the osteoporosis mouse model caused by secondary hyperthyroidism.
6. Use of the method of any one of claims 1-5 for the preparation of a medicament for the treatment of a secondary hyperthyroidism-induced osteoporosis mouse model.
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