CN107490701B - Method for establishing systemic bone injury model based on local irradiation and analyzing pathological mechanism - Google Patents
Method for establishing systemic bone injury model based on local irradiation and analyzing pathological mechanism Download PDFInfo
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Abstract
The invention discloses a method for establishing a systemic bone injury model and analyzing a pathological mechanism based on local irradiation, which adopts local knee joint part irradiation to avoid high mortality and complex pathological mechanism of animals after the systemic irradiation; the injury of organs closely related to bone metabolism such as intestinal tracts, kidneys, gonads and the like by abdominal irradiation is also avoided; the pathological mechanism of the whole body radioactive bone injury caused by local irradiation is effectively clarified by singly damaging bone tissue organs by rays; the discovery of the pathological mechanism of the radioactive bone injury is beneficial to early preventive intervention of patients with clinical radiotherapy and is used as a model for new drug development and drug effect test.
Description
Technical Field
The invention relates to a local irradiation-based systemic bone injury model method and application of a pathological mechanism.
Background
Radiation bone injuries (Radiation bone injures) are a common type of Radiation injury to the irradiated in clinical malignant Radiation therapy. With the increasing incidence of malignant tumors and the wide application of radiotherapy in the treatment of malignant tumors, the incidence of radioactive bone injuries is increasing day by day, and as reported by Ogino I and the like, the incidence of necrotic fractures of patients undergoing radiotherapy due to uterine cancer is as high as 17%. Once radioactive fracture or osteonecrosis occurs, only palliative conservative treatment or operative resection of dead bones for bone grafting can be clinically adopted for treatment, and the effect is not ideal. Current models of radioactive bone injury are based on either total or local irradiation of the animal. The whole body irradiation affects all organs and tissues, and although the aim of rapid bone injury can be achieved, the death rate of animals is high, the etiology of the bone injury is complex, the single ray factor is not included, and the difference from the clinical radiotherapy mode is obvious. The local irradiation is usually abdominal irradiation, because the animal body is small in size and is difficult to effectively protect like a clinical patient, the intestinal tract, the kidney and the gonad of the abdomen are inevitably affected, and the intestine, the kidney and the gonad are closely related to bone metabolism, so that an abdominal irradiation model cannot well reflect the pathogenesis of general bone mass reduction of a clinical radiotherapy patient, in particular to the radioactive fracture phenomenon which appears in a non-radiotherapy area 5-10 years after the radiotherapy.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a systemic bone injury model method based on local irradiation and application of a pathological mechanism, so as to solve the defects in the prior art.
The technical scheme for solving the technical problems is as follows:
a method for establishing a systemic bone injury model and analyzing a pathological mechanism based on local irradiation comprises the following steps:
1) selecting an animal model: dividing a plurality of SD rats with the age of 4 months into an irradiated group and a non-irradiated group, fasting for 12h before irradiation, and freely feeding water; then ketamine is used for anesthesia, rats in the irradiated group are in a supine position, the four limbs and the head are fixed, the external marking and positioning range of the knee joint part of the right hind limb is within the external marking and positioning range, and a 137 Cs-gamma radiation device is used for completing irradiation with 10-50Gy dose in one step; the rest parts are shielded by lead plates; rats in the non-irradiated group were anesthetized with chloranthrone, but not irradiated; after irradiation, two groups of rats are bred conventionally, and the rat materials are sacrificed at 2 and 12 weeks respectively, wherein the rat in the irradiation group takes the femur and the tibia on the irradiation side, the femur, the tibia and the lumbar in the non-irradiation area; taking femur, tibia and lumbar vertebrae from the non-irradiated group;
2) phenotypic analysis of model bone injury: taking the femur, tibia and lumbar vertebrae of 12 weeks for carrying out Micro CT three-dimensional reconstruction bone structure analysis;
3) application of a model pathological mechanism: respectively taking rats in an irradiated group and rats in a non-irradiated group, and analyzing serum inflammatory factors for 2 weeks and 12 weeks; 2 and 12 weeks serum oxidation and antioxidant factor analysis; 2 and 12 weeks blood, bone marrow, thymus and spleen lymphocyte analysis; analyzing the differentiation function of the mesenchymal stem cells; and analyzing the differentiation capability of the bone marrow lymphocytes and the mesenchymal stem cells after co-culture.
Further, in the step 1), the concentration of ketamine is 0.2ml/100 g; the radiation device is Gamma cell 40, and the radiation dose is dose rate of 0.83 Gy/min; the irradiation range was 1cm × 1cm, and the thickness of the lead plate was 4 cm.
The invention has the beneficial effects that:
1. the scheme adopts local knee joint part irradiation, thereby avoiding high mortality rate and complex pathological mechanism of animals after whole body irradiation; also avoids the abdominal irradiation from damaging closely related organs of bone metabolism such as intestinal tract, kidney, gonad and the like.
2. The scheme effectively clarifies the pathological mechanism of the whole body radioactive bone injury caused by local irradiation by singly damaging bone tissues and organs through rays.
3. The discovery of the pathological mechanism of the radioactive bone injury is beneficial to early preventive intervention of clinical radiotherapy patients and is used as a model for new drug development and drug effect test.
Detailed Description
The principles and features of this invention are described below in conjunction with specific embodiments, which are set forth merely to illustrate the invention and are not intended to limit the scope of the invention.
1. Animal irradiation model: SD rats (4 months old). Fasted 12h before irradiation and free access to water. Ketamine (0.2ml/100g) is anesthetized, the supine position is taken, the four limbs and the head are fixed, the external marking and positioning range of the knee joint part of the right hind limb is adopted, and the 10-50Gy dose irradiation is completed once in a 137 Cs-Gamma radiation device (dose rate of 0.83Gy/min, Gamma cell 40). The range was 1cm × 1cm, and the remaining portion was shielded with a lead plate (lead plate thickness was 4 cm). Animals were sacrificed at 2, 12 weeks (irradiated side femur and tibia, non-irradiated femur, tibia and lumbar; control femur, tibia and lumbar) using a non-irradiated control group, anesthetized with chloranthron, but not irradiated, and reared conventionally.
2. Phenotypic analysis of model bone injury: 12-week-old femur, tibia and lumbar vertebra Micro CT three-dimensional reconstruction bone structure analysis has the following indexes:
tBMD:trabecular bone mineral density；
BS/BV:ratio of bone surface to the bone volume；
BV/TV:bone volume fraction,ratio of the segmented bone volume to the total volume of the regi on of the region of interest；
Tb/Th:trabecular thickness,mean thickness of trabeculae；
Tb.Sp:trabecular separation,mean distance between trabeculae；
Tb.N:trabecular number,measure of the average number of trabeculae per unit length；
Ct.Th:average cortical thickness；
OB.S/BS:osteoblast surface to bone surface。
3. analysis of model pathological mechanism
2 and 12 weeks serum inflammatory factor assay;
2 and 12 weeks serum oxidation and antioxidant factor analysis;
2 and 12 weeks blood, bone marrow, thymus and spleen lymphocyte analysis;
analyzing the differentiation function of the mesenchymal stem cells;
analyzing the differentiation capacity of the marrow lymphocytes and the mesenchymal stem cells after coculture;
wherein, the analysis methods are respectively as follows:
one, 2 and 12 week serum inflammatory factor assays including TNF α, IL-6, IFN γ, IL-1 α, IL-1 β, MCP-1, Rantes and MIP, reagents including 12strips, each coated with 8different antibodies against murine inflammatory factors, Biotin labeled antibody mix obtained 8 differential inflammation cytokines, 1X Diluent cells (1X dilution buffer), 5X Assay buffer (5X Assay wash), Substrate Solution and Stop reaction Solution (Solution);
the experimental method comprises the following steps:
adding 100 mul of serum to be detected into a 96-well plate with 12 enzyme immune strips, and incubating for 1 hour at room temperature;
removing liquid, adding 200 μ l of analytical cleaning solution, and cleaning for 3 times;
add 100. mu.l biotin-labeled antibody mixture to each well and incubate for 1 hour at room temperature;
repeating the step 2;
adding 100. mu.l of streptavidin-horseradish peroxidase conjugate, and incubating for 45 minutes;
repeating the step 2;
add 100. mu.l of substrate, incubate for 10-30 minutes at room temperature;
adding 50 μ l of stop solution, and testing by OD 450;
serum oxidation and antioxidant factor analysis for two, 2 and 12 weeks: superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), Catalase (CAT), Malondialdehyde (MDA) and total antioxidant capacity (T-AOC) (Nanjing institute of bioengineering);
the experimental method comprises the following steps: refer to the 5 kit instructions above.
Three, 2 and 12 week blood, bone marrow, thymus and spleen lymphocytes analysis: anti-rat CD3-FITC/CD45RA-PC7/CD161a-APC triple antibody (Beckman Coulter, England), anti-rat CD3-FITC/CD4-PC7/CD8-APC triple antibody (Beckman Coulter, England), OptiLyse C (Beckman Coulter, England);
the experimental method comprises the following steps:
taking 1ml of heparin anticoagulation tube for later use; washing bone marrow with PBS containing heparin for later use; grinding thymus and spleen with PBS containing heparin, and sieving with 100 μm sieve;
adding 5 μ l each of CD3-FITC/CD45RA-PC7/CD161a-APC or CD3-FITC/CD4-PC7/CD8-APC into 500 μ l of each group of samples in the above step, and incubating at room temperature for 20 min;
adding 100 mu l of OptiLyse C to each sample, and incubating for 10 minutes at room temperature;
5ml of PBS buffer solution containing 1% FBS is added into each sample, 250g of PBS buffer solution is centrifuged for 5 minutes, and supernatant is removed;
repeating the steps for three times;
500 μ l PBS containing 1% FBS was resuspended in a flow tube and analyzed by an up flow meter;
fourthly, analyzing the differentiation function of the mesenchymal stem cells of the control group, the irradiation side and the non-irradiation side of the irradiation group;
the experimental method comprises the following steps:
taking the corresponding thighbone and shinbone of each group, and washing out bone marrow by PBS containing heparin;
1:3, adding erythrocyte lysate, incubating for 10 minutes at room temperature, centrifuging for 10 minutes at 300g, and removing supernatant;
adding PBS 5ml containing 1% FBS, centrifuging for 5 minutes at 250g, and removing supernatant;
repeating the steps for three times;
adding 500 ul Trizol LS into the sample in the above step, and immediately sending the sample to Invitrogen company for whole transcriptome chip analysis;
fifthly, analyzing the differentiation capacity of the bone marrow lymphocytes after the bone marrow lymphocytes and the normal mesenchymal stem cells are co-cultured;
the experimental method comprises the following steps:
taking a femur and a tibia of a normal rat, precooling a PBS containing heparin to wash out bone marrow in an aseptic state, adding 20ml of 10% FBS DMEM culture medium, and completely inoculating into a 100mm culture dish;
after 3 days of culture, gently removing 10ml of culture solution, and adding another 10ml of fresh culture solution;
after culturing for 7 days, the solution is completely changed until the cells are 90% confluent;
taking the thighbone and the shinbone of each group of the model, washing the bone marrow with 5ml of precooled lymphocyte separation medium (Sigma) in an aseptic state, transferring the bone marrow into a 15ml centrifuge tube, and slowly adding 1ml of culture solution along the tube wall;
centrifuging at 500g for 20-30 minutes at 300-;
transferring the nebulous cells into a clean 15ml centrifuge tube, adding 5ml PBS, centrifuging for 5 minutes at 250g, and removing supernatant;
repeating the steps for 3 times;
10ml of 10% FBS 1640 culture medium was resuspended in cell culture, and the medium was changed every 3 days;
taking the cells in the step 3, and subculturing the cells to a Transwell 6-hole plate;
after 24 hours, the lymphocytes obtained in the step 8 are inoculated into a Transwell chamber and cultured together with the cells in the step 9 for 6 days;
after culturing for 6 days, removing the small chamber, and replacing the osteogenesis induction culture solution and the adipogenesis induction culture solution for each 6-hole plate respectively; the formula of the osteogenesis induction culture solution is as follows: 10% FBS DMEM (DMEM medium containing 10% fetal bovine serum), 50mg/ml Ascorbic acid (50mg/ml Ascorbic acid), 10mM b-glycerophosphate (10mM b-glycerophosphate); the formula of the adipogenic induction culture solution is as follows: 10% FBS DMEM (DMEM medium containing 10% fetal bovine serum), 10mg/ml Iinusulin (10mg/ml insulin), 1mM Dexamethasone (1mM Dexamethasone), 0.5mM 3-isobutyl-1-methylxanthine (0.5mM 3-isobutyl-1-methylxanthine);
after 2 weeks of osteogenic induction, fixing with 4% paraformaldehyde, and staining with alizarin red;
after 3 weeks of adipogenic induction, 4% paraformaldehyde was fixed and oil red O was stained;
the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A method for establishing a systemic bone injury model and analyzing a pathological mechanism based on local irradiation is characterized by comprising the following steps:
1) selecting an animal model: dividing a plurality of SD rats with the age of 4 months into an irradiated group and a non-irradiated group, fasting for 12h before irradiation, and freely feeding water; then ketamine is used for anesthesia, rats in the irradiated group are in a supine position, the four limbs and the head are fixed, the external marking and positioning range of the knee joint part of the right hind limb is within the external marking and positioning range, and a 137 Cs-gamma radiation device is used for completing irradiation with 10-50Gy dose in one step; the rest parts are shielded by lead plates; rats in the non-irradiated group were anesthetized with chloranthrone, but not irradiated; after irradiation, two groups of rats are bred conventionally, and the rat materials are sacrificed at 2 and 12 weeks respectively, wherein the rat in the irradiation group takes the femur and the tibia on the irradiation side, the femur, the tibia and the lumbar in the non-irradiation area; taking femur, tibia and lumbar vertebrae from the non-irradiated group;
2) phenotypic analysis of model bone injury: taking the femur, tibia and lumbar vertebrae of 12 weeks for carrying out Micro CT three-dimensional reconstruction bone structure analysis;
3) analyzing a model pathological mechanism: respectively taking rats in an irradiated group and rats in a non-irradiated group, and analyzing serum inflammatory factors for 2 weeks and 12 weeks; 2 and 12 weeks serum oxidation and antioxidant factor analysis; 2 and 12 weeks blood, bone marrow, thymus and spleen lymphocyte analysis; analyzing the differentiation function of the mesenchymal stem cells; and analyzing the differentiation capability of the bone marrow lymphocytes and the mesenchymal stem cells after co-culture.
2. The method for modeling and analyzing pathological mechanism of a systemic bone injury model based on local irradiation as claimed in claim 1, wherein: in the step 1), the concentration of ketamine is 0.2ml/100 g; the radiation device is Gamma cell 40, and the radiation dose is dose rate of 0.83 Gy/min; the irradiation range was 1cm × 1cm, and the thickness of the lead plate was 4 cm.
3. The method for modeling and analyzing pathological mechanism of a systemic bone injury model based on local irradiation as claimed in claim 1, wherein: the serum inflammatory factor assay for 2 weeks and 12 weeks in the above step 3) comprises the following factors, TNF α, IL-6, IFN γ, IL-1 α, IL-1 β, MCP-1, Rantes and MIP, and the reagents comprise 12 enzyme immunoassay strips each coated with 8different antibodies against murine inflammatory factors, a biotin-labeled antibody mixture against 8 inflammatory factors, streptavidin-horseradish peroxidase conjugate, 1X dilution buffer, 5X assay wash, substrate solution and stop reaction solution; the analysis steps are as follows:
1) adding 100 mul of serum to be detected into a 96-well plate with 12 enzyme immune strips, and incubating for 1 hour at room temperature;
2) removing liquid, adding 200 μ l of analytical cleaning solution, and cleaning for 3 times;
3) add 100. mu.l biotin-labeled antibody mixture to each well and incubate for 1 hour at room temperature;
4) repeating the step 2;
5) adding 100. mu.l of streptavidin-horseradish peroxidase conjugate, and incubating for 45 minutes;
6) repeating the step 2;
7) adding 100 mul of substrate solution, and incubating for 10-30 minutes at room temperature;
8) add 50. mu.l of stop solution and test at OD 450.
4. The method for modeling and analyzing pathological mechanism of a systemic bone injury model based on local irradiation as claimed in claim 1, wherein: the 2-week and 12-week blood, bone marrow, thymus and spleen lymphocyte analyses in the above step 3) included the following reagents: anti-rat CD3-FITC/CD45RA-PC7/CD161a-APC triple antibody, anti-rat CD3-FITC/CD4-PC7/CD8-APC triple antibody, OptiLyse C, the analytical steps are as follows:
1) taking 1ml of heparin anticoagulation tube for later use; washing bone marrow with PBS containing heparin for later use; grinding thymus and spleen with PBS containing heparin, and sieving with 100 μm sieve;
2) taking 500 ul of each group of samples obtained in the step 1), adding 5 ul of each of CD3-FITC/CD45RA-PC7/CD161a-APC or CD3-FITC/CD4-PC7/CD8-APC, and incubating for 20 minutes at room temperature;
3) adding 100 mu l of OptiLyse C to each group of samples, and incubating for 10 minutes at room temperature;
4) adding 5ml of PBS buffer solution containing 1% FBS into each group of samples, centrifuging for 5 minutes at 250g, and removing supernatant;
5) step 4) repeating for three times;
500 μ l PBS containing 1% FBS was resuspended in flow tubes and analyzed by up flow.
5. The method for modeling and analyzing pathological mechanism of a systemic bone injury model based on local irradiation as claimed in claim 1, wherein: the step of analyzing the differentiation function of the mesenchymal stem cells in the step 3) comprises the following steps:
1) taking the corresponding thighbone and shinbone of each group, and washing out bone marrow by PBS containing heparin;
2)1:3, adding erythrocyte lysate, incubating for 10 minutes at room temperature, centrifuging for 10 minutes at 300g, and removing supernatant;
3) adding PBS 5ml containing 1% FBS, centrifuging for 5 minutes at 250g, and removing supernatant;
4) step 3) repeating for three times;
5) the sample obtained in step 4) was immediately sent to Invitrogen for whole transcriptome chip analysis after adding 500. mu.l of Trizol LS.
6. The method for modeling and analyzing pathological mechanism of a systemic bone injury model based on local irradiation as claimed in claim 1, wherein: the differentiation capacity analysis steps of the co-cultured bone marrow lymphocytes and mesenchymal stem cells in the step 3) are as follows:
1) taking a femur and a tibia of a normal rat, precooling a PBS containing heparin to wash out bone marrow in an aseptic state, adding 20ml of 10% FBS DMEM culture medium, and completely inoculating into a 100mm culture dish;
2) after 3 days of culture, gently removing 10ml of culture solution, and adding another 10ml of fresh culture solution;
3) after culturing for 7 days, the solution is completely changed until the cells are 90% confluent;
4) taking each group of thighbone and shinbone of the model, washing bone marrow with 5ml of precooled lymphocyte separation liquid under an aseptic condition, transferring the bone marrow into a 15ml centrifuge tube, and slowly adding 1ml of culture solution along the tube wall;
5) centrifuging at 500g for 20-30 minutes at 300-;
6) transferring the nebulous cells into a clean 15ml centrifuge tube, adding 5ml PBS, centrifuging for 5 minutes at 250g, and removing supernatant;
7) step 6) repeating for 3 times;
8)10ml of 10% FBS 1640 culture medium was resuspended in cell culture, and the medium was changed every 3 days;
9) taking the cells in the step 3, and subculturing the cells to a Transwell 6-hole plate;
10) inoculating the lymphocytes obtained in the step 8) into a Transwell after 24 hours, and co-culturing the lymphocytes and the cells in the step 9) for 6 days;
11) after culturing for 6 days, removing the small chamber, and replacing an osteogenesis induction culture solution and a adipogenesis induction culture solution for each 6-pore plate respectively, wherein the osteogenesis induction culture solution comprises the following formula: 10% fetal bovine serum DMEM medium, 50mg/ml ascorbic acid, 10mM b-glycerophosphate; the formula of the adipogenic induction culture solution is as follows: 10% fetal bovine serum DMEM culture solution, 10mg/ml insulin, 1mM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine;
12) after 2 weeks of osteogenic induction, fixing with 4% paraformaldehyde, and staining with alizarin red;
13) after 3 weeks of adipogenic induction, 4% paraformaldehyde was fixed and stained with oil red O.
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