CN110999865A - Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism - Google Patents
Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism Download PDFInfo
- Publication number
- CN110999865A CN110999865A CN202010014282.6A CN202010014282A CN110999865A CN 110999865 A CN110999865 A CN 110999865A CN 202010014282 A CN202010014282 A CN 202010014282A CN 110999865 A CN110999865 A CN 110999865A
- Authority
- CN
- China
- Prior art keywords
- tissue
- mouse model
- secondary hyperparathyroidism
- osteoporosis
- constructing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000001132 Osteoporosis Diseases 0.000 title claims abstract description 56
- 238000010172 mouse model Methods 0.000 title claims abstract description 40
- 238000010276 construction Methods 0.000 title claims abstract description 15
- 208000005770 Secondary Hyperparathyroidism Diseases 0.000 title claims abstract 20
- 241000699670 Mus sp. Species 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 36
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 21
- 210000002966 serum Anatomy 0.000 claims description 17
- 238000002054 transplantation Methods 0.000 claims description 16
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 5
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 210000002990 parathyroid gland Anatomy 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000000849 parathyroid Effects 0.000 claims 2
- CLVOYFRAZKMSPF-UHFFFAOYSA-N n,n-dibutyl-4-chlorobenzenesulfonamide Chemical compound CCCCN(CCCC)S(=O)(=O)C1=CC=C(Cl)C=C1 CLVOYFRAZKMSPF-UHFFFAOYSA-N 0.000 claims 1
- 238000007781 pre-processing Methods 0.000 claims 1
- 238000002203 pretreatment Methods 0.000 claims 1
- 230000002792 vascular Effects 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 238000007877 drug screening Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000011156 evaluation Methods 0.000 abstract description 3
- 229940126585 therapeutic drug Drugs 0.000 abstract 1
- 210000000988 bone and bone Anatomy 0.000 description 45
- 206010053260 Secondary hyperthyroidism Diseases 0.000 description 37
- 210000001519 tissue Anatomy 0.000 description 35
- 102000003982 Parathyroid hormone Human genes 0.000 description 24
- 108090000445 Parathyroid hormone Proteins 0.000 description 24
- 239000000199 parathyroid hormone Substances 0.000 description 24
- 229960001319 parathyroid hormone Drugs 0.000 description 24
- 210000002997 osteoclast Anatomy 0.000 description 18
- 210000000963 osteoblast Anatomy 0.000 description 17
- 230000037182 bone density Effects 0.000 description 14
- 210000004369 blood Anatomy 0.000 description 12
- 239000008280 blood Substances 0.000 description 12
- 230000008569 process Effects 0.000 description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 229910052791 calcium Inorganic materials 0.000 description 7
- 230000004097 bone metabolism Effects 0.000 description 6
- 210000004907 gland Anatomy 0.000 description 6
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 206010020850 Hyperthyroidism Diseases 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 210000004204 blood vessel Anatomy 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000012744 immunostaining Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002591 computed tomography Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 208000018083 Bone metabolism disease Diseases 0.000 description 1
- 206010006002 Bone pain Diseases 0.000 description 1
- 206010006956 Calcium deficiency Diseases 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 201000002980 Hyperparathyroidism Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 1
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 1
- ZQBZAOZWBKABNC-UHFFFAOYSA-N [P].[Ca] Chemical compound [P].[Ca] ZQBZAOZWBKABNC-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000010603 microCT Methods 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- 210000002655 parathyroid chief cell Anatomy 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000001020 rhythmical effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Diabetes (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Endocrinology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及一种继发性甲旁亢引发的骨质疏松小鼠模型的构建方法及其应用，所述构建方法包括：将来源于继发性甲旁亢病人的甲旁腺组织进行预处理，再将其移植入小鼠体内，构建继发性甲旁亢引发的骨质疏松小鼠模型。本发明所涉及的继发性甲旁亢引发的骨质疏松小鼠模型的构建方法为疾病的发展、治疗药物的筛选及疗效的科学评价打下了较好的构建模型基础，采用该方法可以较好地科学、快速、准确地判断出小鼠发病的严重程度及治疗情况，也为治疗继发性甲旁亢引发的骨质疏松的药物筛选模型成功构建了一种便捷、快速、准确的判断方法。该构建方法具有很高的成功率，且具有很高的临床相关性。
The invention relates to a method for constructing a mouse model of osteoporosis caused by secondary hyperparathyroidism and its application. , and then transplanted into mice to construct a secondary hyperparathyroidism-induced osteoporosis mouse model. The method for constructing a mouse model of osteoporosis caused by secondary hyperparathyroidism involved in the present invention lays a good foundation for constructing the model for the development of the disease, the screening of therapeutic drugs and the scientific evaluation of the curative effect. It can scientifically, quickly and accurately judge the severity of the disease in mice and the treatment situation, and also successfully constructed a convenient, rapid and accurate judgment for the drug screening model for the treatment of osteoporosis caused by secondary hyperparathyroidism. method. This construction method has a 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 current clinical screening still has the main symptom preference phenomenon, namely, hyperthyroidism patients are often concerned about the excessive secretion of PTH, and the osteoporosis accompanied by the PTH 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 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.
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 osteoblast marker alkaline phosphatase and osteoclast marker tartrate-resistant acid phosphatase 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 statistical graph showing the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the control group and the transplanted group of mice 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 statistical graph showing the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the control group and the transplanted group of mice in example 5;
FIG. 13 is a statistical graph of 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 statistical graph showing the ratio of the number of osteoclasts to the volume of bone (Oc/TV) in the control group and the transplanted group of mice 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 limitations 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 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 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 into the opening by using forceps and widening to form a pocket for containing the tissue block, placing the 3 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 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, transferring the tissue block obtained in the step (1), and culturing in a 5% CO2 incubator at 37 ℃ 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 pseudo 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, 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. 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) 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. 11: 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. 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.
A bone volume density (BV/TV) histogram of mice is 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 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. 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 (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010014282.6A CN110999865B (en) | 2020-01-07 | 2020-01-07 | Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010014282.6A CN110999865B (en) | 2020-01-07 | 2020-01-07 | Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110999865A true CN110999865A (en) | 2020-04-14 |
| CN110999865B CN110999865B (en) | 2022-06-17 |
Family
ID=70120502
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010014282.6A Active CN110999865B (en) | 2020-01-07 | 2020-01-07 | Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110999865B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050060764A1 (en) * | 2003-09-17 | 2005-03-17 | Susan Gregory | Mouse model for bone metabolism |
| US20110092949A1 (en) * | 2008-04-25 | 2011-04-21 | Wang Taylor G | Immunoisolation patch system for cellular transplantation |
| CN102051344A (en) * | 2009-10-30 | 2011-05-11 | 上海交通大学医学院附属瑞金医院 | Human osteosarcoma cell line and mouse in-vivo transplantation model |
| JP2012235715A (en) * | 2011-05-10 | 2012-12-06 | Hamamatsu Univ School Of Medicine | Aortal aneurysm model animal |
| US20130324505A1 (en) * | 2011-01-07 | 2013-12-05 | Beth Israel Deaconess Medical Center, Inc. | Assays and methods of treatment relating to vitamin d insufficiency |
| CN106619719A (en) * | 2016-12-27 | 2017-05-10 | 中国科学院广州生物医药与健康研究院 | Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells |
| CN108753831A (en) * | 2018-06-01 | 2018-11-06 | 郑州大学第附属医院 | Utilize the immunodeficient mouse model constructed by NK/T lymphoma cell strains |
| CN109022362A (en) * | 2018-08-02 | 2018-12-18 | 武汉大学 | A kind of method for building up of hyperleucocyte acute leukemia PDX model |
| CN109090039A (en) * | 2018-09-07 | 2018-12-28 | 广州长峰生物技术有限公司 | The method for building up of humanized's Tumor Xenograft Models through in vitro culture |
-
2020
- 2020-01-07 CN CN202010014282.6A patent/CN110999865B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050060764A1 (en) * | 2003-09-17 | 2005-03-17 | Susan Gregory | Mouse model for bone metabolism |
| US20110092949A1 (en) * | 2008-04-25 | 2011-04-21 | Wang Taylor G | Immunoisolation patch system for cellular transplantation |
| CN102051344A (en) * | 2009-10-30 | 2011-05-11 | 上海交通大学医学院附属瑞金医院 | Human osteosarcoma cell line and mouse in-vivo transplantation model |
| US20130324505A1 (en) * | 2011-01-07 | 2013-12-05 | Beth Israel Deaconess Medical Center, Inc. | Assays and methods of treatment relating to vitamin d insufficiency |
| JP2012235715A (en) * | 2011-05-10 | 2012-12-06 | Hamamatsu Univ School Of Medicine | Aortal aneurysm model animal |
| CN106619719A (en) * | 2016-12-27 | 2017-05-10 | 中国科学院广州生物医药与健康研究院 | Model and method for detecting inhibiting effect of chimeric antigen receptor (CAR) T cells on hepatoma cells |
| CN108753831A (en) * | 2018-06-01 | 2018-11-06 | 郑州大学第附属医院 | Utilize the immunodeficient mouse model constructed by NK/T lymphoma cell strains |
| CN109022362A (en) * | 2018-08-02 | 2018-12-18 | 武汉大学 | A kind of method for building up of hyperleucocyte acute leukemia PDX model |
| CN109090039A (en) * | 2018-09-07 | 2018-12-28 | 广州长峰生物技术有限公司 | The method for building up of humanized's Tumor Xenograft Models through in vitro culture |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110999865B (en) | 2022-06-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Huang et al. | High-purity weight-bearing magnesium screw: translational application in the healing of femoral neck fracture | |
| Cao et al. | The use of autologous enriched bone marrow MSCs to enhance osteoporotic bone defect repair in long-term estrogen deficient goats | |
| CN101868245B (en) | Synthetic peptide containing bone forming peptide 1(BFP 1) for stimulating osteoblast differentiation, and pharmaceutical composition comprising synthetic peptide | |
| Nagata et al. | Influence of the proportion of particulate autogenous bone graft/platelet-rich plasma on bone healing in critical-size defects: an immunohistochemical analysis in rat calvaria | |
| Fang et al. | Vascular restoration through local delivery of angiogenic factors stimulates bone regeneration in critical size defects | |
| AU2019222935B2 (en) | Enhancement of osteogenic potential of bone grafts | |
| Seon et al. | Peptide derived from stromal cell-derived factor 1δ enhances the in vitro expression of osteogenic proteins via bone marrow stromal cell differentiation and promotes bone formation in in vivo models | |
| CN110999865A (en) | Construction method and application of a mouse model of osteoporosis induced by secondary hyperparathyroidism | |
| Xiao et al. | Porcine platelet lysates exert the efficacy of chondroregeneration and SMAD2-mediated anti-chondrofibrosis on knee osteoarthritis | |
| Lin et al. | Serum N‑terminal telopeptide of type I collagen as an early marker of fracture nonunion in rabbits | |
| CN103919908B (en) | Medicine of a kind of prevention and therapy disuse osteoporosis and preparation method thereof | |
| CN107937334A (en) | A kind of cell factor extract and its application | |
| RU2370227C1 (en) | Method of treating multi-splintered and multiple fractures of long tubular bones | |
| CN117618424B (en) | Application of dihydroberberine in inhibiting osteoclast differentiation | |
| Tschon et al. | In vivo efficacy of an injectable piezoelectric nanocomposite hydrogel and low-intensity pulsed ultrasound in two preclinical models of osteoarthritis | |
| Zhang et al. | Mechanism of low-intensity pulse ultrasound combined with Rhodiola to promote bone formation in spinal fusion | |
| CN111214763A (en) | Rhythmic light stimulation mode system and application thereof | |
| Yu et al. | BMSCs Combined with Grafted igament Promote ACL Healing by Regulating NF-kB Signaling Pathway The mechanism of action of the combination | |
| Kikuchi et al. | Development of a nasal mucosa-removal model for evaluating cell therapy | |
| 菊地瞬 | Development of a nasal mucosa-removal model for evaluating cell therapy | |
| Lazishvili et al. | Experimental assessment of biological potential of collagen membranes in reconstruction of full-thickness hyaline cartilage defects | |
| Bethel et al. | The properties of inducible membranes in animals and humans | |
| Jassem | Evaluation the Effect of local Application of Red Clover Oil (Trifolium pratense) Covered by Hemostatic Gelatin Sponge on Bone Healing in Rats by histological and Immunohistochemical Study on (P1NP) | |
| CN118987039A (en) | Application of pulp stem cell exosome in preparation of bone injury repair preparation | |
| TWI649085B (en) | A pharmaceutical composition for increasing skeletal density and the using method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |