Method for establishing osteoclast model based on combination of blue light irradiation and RANKL and application thereof
By transfecting ChR2 gene in RAW264.7 cells and combining blue light irradiation and RANKL treatment, an osteoclast model with short induction cycle, low RANKL dosage and stable osteoclast differentiation was established, solving the problems of high research costs and long test cycles in the prior art, and is suitable for the research and drug screening of osteoporosis diseases.
Patent Information
- Application Number
- CN202411824363.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-03-11
AI Technical Summary
In the prior art, the osteoclast model has a long induction cycle, large RANKL usage, and poor stability of differentiated osteoclasts, resulting in a long trial cycle and high research cost for osteoporosis disease research.
By lentiviral transfection of ChR2 gene into RAW264.7 cells, C-RAW264.7 cells stably expressing ChR2 protein were screened, and combined with 460nm blue light irradiation and RANKL treatment, C-RAW264.7 cells were induced to differentiate into mature osteoclast models.
The osteoclast model has achieved the effect of short induction cycle, low RANKL dosage, and stable osteoclast differentiation, which helps to reduce the cost and time of osteoporosis disease research and is suitable for screening drugs that regulate Ca2+ concentration in osteoclasts.
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Figure CN119592632A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell biology and biomedicine technology, and in particular to a method for establishing an osteoclast model based on blue light irradiation combined with RANKL and an application thereof. Background Art
[0002] Osteoporosis is a metabolic bone disease that causes bone loss and destruction of bone microstructure with aging, leading to increased bone brittleness and susceptibility to fractures. Its harmful effects include bone pain, shortened height, increased risk of fractures, spinal deformity, and decreased respiratory function, which seriously affect the quality of life of patients. Given the core role of osteoclasts in bone remodeling, that is, they absorb old bone and form bone resorption pits at the original site to provide osteoblasts with bone formation space and maintain bone integrity, the overactivation of osteoclasts is closely related to the onset of osteoporosis.
[0003] In order to deeply study the differentiation mechanism of osteoclasts, it is particularly important to establish relevant osteoclast models. Through the model, the formation process of osteoclasts can be simulated, the key factors and signal pathways of their differentiation can be explored, and new targets and strategies can be provided for the treatment of osteoporosis. At the same time, the model also helps to evaluate the efficacy and safety of drugs and provide a scientific basis for clinical medication. Therefore, the establishment of an osteoclast model is of great significance for the research and treatment of osteoporosis.
[0004] At present, there are mainly two types of osteoclast models induced in vitro: one is an osteoclast model (OCs-M) of mouse mononuclear macrophage leukemia cells (RAW264.7) induced by RANKL or LPS reagents, with an induction period of 5-6 days and an induction reagent concentration of RANKL or LPS 50-100ng / mL; the other is an osteoclast model of mouse bone marrow-derived macrophages (BMMs) induced by RANKL and M-CSF, with an induction reagent concentration of RANKL 50-100ng / mL, M-CSF 25-50ng / mL, and a period of 7-8 days. Both of the above methods have inevitable disadvantages, such as long induction period, large amount of RANKL, poor stability of differentiated osteoclasts, resulting in long experimental periods for researchers to conduct osteoporosis disease research and high research costs. Therefore, establishing an osteoclast model with a short induction period, low RANKL dosage and stable osteoclast differentiation is a major challenge in current research. Summary of the invention
[0005] Based on the above problems, the present invention provides a method for establishing an osteoclast model based on blue light irradiation combined with RANKL and its application.
[0006] The present invention first transfects the ChR2 (rhodopsin channel protein 2) gene into RAW264.7 cells through lentivirus, screens out C-RAW264.7 cells that stably express the ChR2 protein, and then treats the C-RAW264.7 cells with RANKL, and combines with 460nm blue light irradiation to obtain a mature osteoclast model (C-OCs-M) after 3-4 days of treatment. The transfected C-RAW264.7 can stably express the ChR2 protein. After 460nm blue light treatment, the influx of calcium ions assists RANKL in activating the NFATC1 signaling pathway and promoting osteoclast differentiation. This cell model has the characteristics of a short induction cycle and stable and reliable indicators of osteoclasts, and is expected to be used in screening osteoclasts that have been successfully induced by Ca 2+ Pathway inhibits osteoclast differentiation in drug experiments.
[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:
[0008] A method for establishing an osteoclast model based on blue light irradiation combined with RANKL:
[0009] Step 1: Obtain C-RAW264.7 cells stably expressing ChR2 protein: RAW264.7 cells are inoculated into a T25 cell culture flask, and when the growth fusion rate reaches 30% to 50%, a lentivirus carrying the ChR2 gene is added to infect the RAW264.7 cells, and puromycin is used to select cells stably expressing ChR2, thereby obtaining C-RAW264.7 cells;
[0010] Step 2: Inoculate C-RAW264.7 cells: Inoculate C-RAW264.7 cells into a 96-well plate at a seeding density of 1000 cells / well and incubate in a cell culture incubator for 24 h;
[0011] Step 3: Induce osteoclast differentiation: After 24 h, replace the complete culture medium, irradiate the C-RAW264.7 cells and add RANKL until a large number of osteoclasts are observed to terminate the induction.
[0012] Preferably, the lentivirus carrying the ChR2 gene in step 1 is pLenti-EF1-EGFP-P2A-Puro-CMV-hChR2.
[0013] Preferably, the complete culture medium replaced in step 3 is α-MEM complete culture medium, and the α-MEM complete culture medium contains 10% fetal bovine serum, 1% penicillin and streptomycin.
[0014] Preferably, the C-RAW264.7 cell irradiation conditions in step 3 are: blue light 460nm (0.4W cm -2 ) Irradiation for 0 to 5 minutes.
[0015] Preferably, the RANKL induction concentration in step 3 is 0 to 50 ng / mL.
[0016] Preferably, in step 3, the C-RAW264.7 cells are subjected to culture medium replacement, irradiation and RANKL addition once a day for 3 to 4 days.
[0017] The osteoclast model of the present invention is established based on blue light irradiation combined with RANKL, and can be applied to the study of the pathogenesis of osteoporosis or in the field of anti-osteoporosis drug screening.
[0018] Preferably, the application of the pathogenesis of osteoporosis is the application of studying the mechanism of inhibiting osteoclast differentiation in vitro.
[0019] Preferably, the application of the anti-osteoporosis drug screening is to screen for drugs that reduce Ca2+ in osteoclasts. 2 + Application of concentration drugs.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] (1) The present invention provides a new osteoclast model and a method for constructing the same. The ChR2 gene is transfected into RAW264.7 cells by lentivirus to obtain C-RAW264.7 cells that stably express the ChR2 protein. The C-RAW264.7 cells are induced to differentiate into osteoclasts by combining blue light irradiation and RANKL. The obtained osteoclast model is verified to have a large number of osteoclasts that are differentiated and mature, and can express osteoclast marker genes and proteins.
[0022] (2) The C-RAW264.7 osteoclast model of the present invention has a shorter induction time, a lower RANKL induction concentration, and a larger number of differentiated osteoclasts than the RAW264.7 osteoclast model of the prior art, and is a low-cost and high-efficiency osteoclast differentiation osteoclast model, which is expected to be applied to the study of the pathogenesis of osteoporosis, especially to the screening of osteoclasts that regulate Ca2+ in osteoclasts. 2+ Concentration of drug applications in research. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Characterization diagram of the successful stably transfected ChR2 protein in C-RAW264.7 cells: Figure 1 (ab) are the expression of ChR2 protein in RAW264.7 and C-RAW264.7 cells and its quantitative analysis statistics; Figure 1 (cd) are Ca2+ in RAW264.7 and C-RAW264.7 cells 2+Fluorescence images and statistical graphs of quantitative analysis of fluorescence intensity, scale bar 50 μm.
[0024] Figure 2 Data of screening conditions for inducing osteoclast differentiation in vitro: Figure 2 (a) is a TRAP (tartrate-resistant acid phosphatase) staining image. Figure 2 (b) is the statistical diagram of the number of mature osteoclasts (n≥3), with a scale bar of 100 μm.
[0025] Figure 3 Comparison data of osteoclast numbers in C-OCs-M and OCs-M: Figure 3 (a) is the TRAP staining image, Figure 3 (b) Statistical graph of the number of mature osteoclasts in the Vehicle group (n≥3), with a scale bar of 100 μm.
[0026] Figure 4 The intracellular Ca2+ in osteoclasts of C-OCs-M and OCs-M 2+ Concentration comparison analysis chart.
[0027] Figure 5 Comparison data of osteoclast bone resorption function in C-OCs-M and OCs-M: Figure 5 (a) is the fluorescence staining image of F-actin ring and cell nucleus of each group of cells. Figure 5 (b) is a statistical diagram of the number of F-actin rings in each group of cells. Figure 5 (c) Analysis of F-actin fusion of osteoclasts in each group.
[0028] Figure 6 Comparative data of osteoclast marker gene and protein expression in C-OCs-M and OCs-M: Figure 6 (a) is the expression bands of NFATc1 and c-Fos proteins in each group of cells. Figure 6 (b) is the quantitative statistical diagram of NFATc1 and c-Fos protein expression in each group of cells. Figure 6 (c) is the statistical graph of NFATc1 and c-Fos mRNA expression levels in each group of cells.
[0029] Figure 7 The data of the effect of alendronate (ALN) on the osteoclast differentiation of C-OCs-M and OCs-M are shown in the figure: Figure 7 (a) is the TRAP staining image, Figure 7 (b) is the statistical diagram of osteoclast numbers (n≥3), with a scale of 100 μm. Figure 7 (c) is the Ca in osteoclasts 2+ Concentration statistics graph. DETAILED DESCRIPTION
[0030] The present invention is described in detail below with reference to the embodiments.
[0031] Example 1: Obtaining C-RAW264.7 cells that stably express ChR2 protein
[0032] Experimental method: RAW264.7 cells were inoculated into T25 cell culture flasks. When the growth fusion rate reached 30% to 50%, lentivirus carrying or not carrying ChR2 gene (pLenti-EF1-EGFP-P2A-Puro-CMV-hChR2) was added to infect RAW264.7 cells. Puromycin was used to select cells stably expressing ChR2 to obtain C-RAW264.7 cells. Then, the expression of ChR2 protein in RAW264.7 and C-RAW264.7 cells was observed by WB experiment. In addition, the expression of ChR2 protein in RAW264.7 and C-RAW264.7 cells was detected by blue light 460nm (0.4W cm -2 ) illuminated RAW264.7 and C-RAW264.7 cells for 3 min, and the intracellular Ca2+ in the two cells was observed using a fluorescence microscope. 2 + Concentration.
[0033] Experimental results: WB results are as follows Figure 1 As shown in (ab), compared with RAW264.7 cells, ChR2 protein was highly expressed in C-RAW264.7 cells, indicating that ChR2 gene was successfully transferred and stably expressed the target protein; the results of fluorescence microscopy observation of RAW264.7 and C-RAW264.7 cells were as follows Figure 1 (c), after irradiation with blue light, the fluorescence intensity of calcium ions in C-RAW264.7 cells was greater than that in RAW264.7 cells; after statistical analysis, Figure 1 (d) The intracellular calcium concentration of C-RAW264.7 cells was significantly higher than that of RAW264.7 cells, indicating that the ChR2 protein expressed in C-RAW264.7 cells stably exerted its calcium channel function. 2+ The concentration detection results show that C-RAW264.7 cells stably express ChR2 protein and function normally.
[0034] Example 2: Method of osteoclast model—screening of conditions for inducing osteoclast differentiation in vitro
[0035] Experimental method: C-RAW264.7 cells within 20 generations were cultured in DMEM complete medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin) at 37°C and 5% CO2. When the cells grew to 70% of the cell density, C-RAW264.7 cells were seeded into 96-well plates at a density of 1000 / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. Blue light 460nm (0.4W cm -2 ) were irradiated for 0 min, 1 min, 3 min, and 5 min. The concentrations of RANKL induction were 0 ng / mL, 5 ng / mL, 25 ng / mL, and 50 ng / mL. On the second day after cell plating, the culture medium was replaced with α-MEM complete culture medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin). After light treatment, different concentrations of RANKL were added to the well plate. After continuous light and RANKL treatment for 3-4 days, the cell culture medium was discarded, and the cells at the bottom of the well were fixed with 4% paraformaldehyde and TRAP staining was performed according to the instructions of the TRAP staining kit. After the staining, photos were taken with a microscope, and the images were used to count osteoclasts using Image J (cell nuclei ≥ 3 were counted as one osteoclast).
[0036] The experimental results are as follows Figure 2 As shown in (ab), in the absence of blue light treatment, the number of osteoclasts increased with the increase of RANKL concentration; when the cells were treated with blue light for 1 min, the number of osteoclasts induced by RANKL 25 ng / mL was close to that induced by 50 ng / mL, indicating that blue light irradiation may activate the Ca2+ pathway in C-RAW264.7 cells. 2+ channel, accelerating the process of osteoclast differentiation; when blue light was irradiated for 3 minutes, the number of osteoclasts in each RANKL group reached a peak, among which the number of osteoclasts in the RANKL 25ng / mL group was the largest; it decreased in the RANKL 50ng / mL group, which may be that osteoclasts differentiated and matured too quickly under high RANKL concentrations, and the osteoclast survival time was short; when blue light was irradiated for 5 minutes, the number of RANKL was the largest at 25ng / mL, but the number of osteoclasts in each RANKL group showed a downward trend compared with the group irradiated for 3 minutes, which may be due to the long irradiation time, causing certain damage to C-RAW264.7 cells. Therefore, the optimal induction time for blue light irradiation is 460nm (0.4W cm -2 ) irradiation treatment for 3 minutes.
[0037] From the perspective of RANKL, when RANKL is not added, blue light irradiation will not affect the generation of osteoclasts, indicating that RANKL is still a necessary condition for osteoclast differentiation; when RANKL is 5ng / mL, the number of osteoclasts is small, but at a concentration of 25ng / mL, the number of osteoclasts begins to increase. After reaching 50ng / mL, the number of osteoclasts decreases. This may be due to the over-maturation of osteoclasts and their short survival time. Therefore, the optimal RANKL induction concentration is 25ng / mL ( Figure 2 ab).
[0038] In summary, C-RAW264.7 cells were exposed to blue light at 460 nm (0.4 W cm -2 ) irradiated for 3 min, and the best osteoclast differentiation condition was RANKL 25 ng / mL.
[0039] Example 3: Comparison of the number of osteoclasts generated by the C-RAW264.7 osteoclast model (C-OCs-M) and the RAW264.7 osteoclast model (OCs-M)
[0040] Experimental method: The common osteoclast model in current research is OCs-M, which is an osteoclast model differentiated from RAW264.7 cells stimulated by RANKL for 5-6 days. In order to compare the difference between the C-OCs-M of the present invention and the OCs-M commonly used in the prior art, the present invention designed a TRAP staining comparison experiment for observation. C-RAW264.7 or RAW264.7 cells within 20 generations were cultured with DMEM complete medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin) at 37°C and 5% CO2. When the cells grew to 70% of the cell density, C-RAW264.7 or RAW264.7 cells were inoculated into 96-well plates at a density of 1000 / well and incubated in a 37°C, 5% CO2 cell culture incubator overnight until the cells adhered to the wall. Both cell models were set up as control group (without RANKL) and vehicle group (with RANKL). The culture medium of cells was replaced with α-MEM complete medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin) on the second day of plating. The C-RAW264.7 group was illuminated at 460nm (0.4W cm -2 ) After 3 minutes of irradiation, RANKL was added to a final concentration of 25 ng / mL, and continuous illumination and RANKL treatment was performed for 3-4 days; RANKL was added to the RAW264.7 group to a final concentration of 50 ng / mL, and the culture medium was replaced every other day. The induction was terminated after 5-6 days. After the induction time was over, the cells in the two groups of cell models were fixed for TRAP staining and the number of osteoclasts was counted.
[0041] Experimental results: The induced contrast results are as follows Figure 3As shown in (a), in C-RAW264.7 or RAW264.7 cells, a large number of osteoclasts were generated in the model group compared with the blank group, indicating that both osteoclast models were successfully induced. Figure 3 (a) TRAP staining shows that compared with OCs-M, the osteoclasts in C-OCs-M are larger in size and the number of osteoclasts is greater under the same magnification; Figure 3 From the statistical results of (b), it can be seen that the number of osteoclasts in C-OCs-M is greater than that in OCs-M. The height of the error bar shows that the number of osteoclasts generated in C-OCs-M is relatively stable. This shows that the C-OCs-M of the present invention induces osteoclasts in a shorter time than the OCs-M in the prior art, requires a lower concentration of the inducing agent RANKL, and the number of osteoclasts generated is more stable, reflecting the good high-efficiency and low-cost stable model performance of C-OCs-M.
[0042] Example 4: Intracellular Ca2+ in osteoclasts of C-OCs-M and OCs-M 2+ Concentration comparison
[0043] Experimental method: The cell treatment method was as described in Example 3. After induction, the treated cells were transferred to a microcentrifuge tube and 400 μL Fluo-3 / AM was added to a final concentration of 5 μM / mL. The cells were incubated at 37°C in the dark for 30 minutes and then rinsed twice with PBS. 1×10 4 The mean fluorescence intensity of randomly selected cells for each sample was measured by flow cytometry at an emission wavelength of 516 nm and an alternating excitation wavelength of 494 nm. Each experiment for each treatment group was performed in triplicate.
[0044] Experimental results: According to the flow cytometry test results, Figure 4 As shown, compared with the uninduced Control group, the intracellular Ca 2+ The concentration increased significantly, indicating that RANKL induction caused the influx of cellular calcium ions. Compared with OCs-M, the intracellular calcium ion concentration of C-OCs-M was higher before and after induction, indicating that blue light treatment of C-RAW264.7 cells opened calcium ion channels and promoted the influx of calcium ions in C-RAW264.7 cells. This may be related to the fact that C-OCs-M induced more osteoclasts than OCs-M, had a shorter induction time, and required a lower RANKL concentration.
[0045] Example 5: Comparison of bone resorption function between C-OCs-M and OCs-M
[0046] Experimental method: The cell treatment method was as described in Example 3. After induction, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde, then washed 3 times with PBS, permeabilized with 0.1% Triton X-100 and incubated with Alexa Fluor488-phalloidin for 60 minutes. After washing with PBS, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes and photographed under a fluorescence microscope.
[0047] Experimental results: The cytoskeleton (F-actin ring) of osteoclasts is the key to their bone resorption function. After incubation with Alexa Fluor488-phalloidin, it appears red under a fluorescence microscope, and the nucleus appears blue under a fluorescence microscope after being stained with DAPI. Figure 5 (a) In the two cell models, compared with the Control group, the Vehicle group had more large red F-actin rings, indicating that both osteoclast models were successfully induced. Further observation in the Vehicle group showed that the volume of the osteoclast F-actin ring induced by C-OCs-M was larger than that of OCs-M. Combined with the quantitative analysis data Figure 5 (bc) It can be seen that the number of osteoclast F-actin rings and cell fusion rate of C-OCs-M are significantly higher than those of OCs-M.
[0048] Example 6: Comparison of the expression of signature genes and proteins between C-OCs-M and OCs-M
[0049] Experimental method: The cell treatment method was as described in Example 3. After induction, the cells were washed with PBS and the cell lysate was collected for WB experiment and RT-PCR experiment to detect the mRNA and protein expression levels of NFATc1 and c-Fos, the marker genes of osteoclast differentiation, in each group of cells.
[0050] Experimental results: According to WB experimental data Figure 6 (ab) It can be seen that compared with the Control group, the protein expression levels of NFATc1 and c-Fos in the Vehicle group were upregulated. In the Vehicle group, the protein expression levels of NFATc1 and c-Fos in C-OCs-M were higher than those in OCs-M, indicating that the degree of C-RAW264.7 differentiation into osteoclasts in C-OCs-M was higher. This result is consistent with the results of RT-PCR experiments. Figure 6 (c) Consistently, therefore, the mRNA and protein expression levels of the osteoclast marker genes NFATc1 and c-Fos in C-OCs-M were higher than those in the OCs-M group.
[0051] Example 7: Effect of ALN on osteoclast differentiation of C-OCs-M and OCs-M
[0052] Experimental method: ALN is a common anti-osteoporosis drug in clinical practice, which can inhibit the formation of osteoclasts. The present invention uses ALN to treat two osteoclast models, C-OCs-M and OCs-M, and compares and observes the application effect of C-OCs-M in drug screening. The cell treatment method is as described in Example 3. After the induction, the Control group, Vehilce group and ALN group are set up. The ALN group treats the two cell models with 10μM ALN for 24h. The drug administration time ends, the cells are collected and fixed, and TRAP staining is performed and the number of osteoclasts is counted under an optical microscope; the three groups of cells of the Control group, Vehilce group and ALN group treated in the same way are collected and collected into centrifuge tubes, and the Ca in the cells is labeled with Fluo-3 / AM 2+ Flow cytometry was used to detect the Ca 2+ concentration.
[0053] Experimental results: According to the results of TRAP staining experiment, Figure 7 As shown in (a), in both cell models, compared with the Control group, a large number of large multinucleated osteoclasts appeared in the Vehicle group. After ALN treatment, the number of multinucleated osteoclasts decreased; Figure 7 (b) is a statistical analysis of the number of osteoclasts in each group after TRAP staining. According to the results, it can be seen that in the two cell models, the number of osteoclasts in the Vehicle group increased sharply compared with the Control group. The number of osteoclasts in the Vehicle group of C-OCs-M was more than that in the Vehicle group of OCs-M, indicating that the C-OCs-M model was more stable. In addition, after 24 hours of ALN treatment of C-OCs-M and OCs-M, the number of osteoclasts in the ALN group of the two cell models decreased, indicating that ALN exerted its efficacy normally and inhibited the differentiation of osteoclasts. In order to further observe the Ca 2+ Fluo-3 / AM was used to label the Ca 2+ Flow cytometry results showed Figure 7 (c) In the two cell models, the intracellular Ca in the Vehicle group after induction was higher than that in the Control group. 2+ The concentration of C-OCs-M in the Vehicle group was increased. 2+ The concentration was higher than that of the OCs-M group, indicating that after blue light irradiation, the ChR2 calcium ion channel on the surface of the C-RAW264.7 cell membrane opened, promoting osteoclast differentiation. In addition, after ALN treatment, the intracellular Ca 2+The decrease in concentration indicates that osteoclast differentiation is inhibited by ALN. Therefore, combined with the results of TRAP staining and calcium ion concentration detection, it can be seen that the osteoclast differentiation of C-OCs-M can be inhibited by ALN, and the calcium ion concentration in osteoclasts is reduced, which further indicates that C-OCs-M can be used in the osteoclast model for screening drugs that inhibit osteoclast differentiation.
[0054] In summary, the C-OCs-M established in the present invention can open calcium ion channels under blue light irradiation, promote RANKL-induced C-RAW264.7 cells to differentiate into mature osteoclasts, and comparative experiments verify that C-OCs-M has a shorter induction time than OCs-M, requires a lower RANKL concentration, and induces more osteoclasts, indicating that C-OCs-M has the advantages of efficient and low-cost modeling; in addition, the results of the two cell models treated with ALN also fully demonstrate that C-OCs-M can be used in experiments for in vitro screening of anti-osteoporosis drugs, providing a more convenient and low-cost modeling method for anti-osteoporosis research.
[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.
Claims
1. A method for establishing an osteoclast model based on blue light irradiation combined with RANKL, the specific steps comprising the following: Step 1: Obtain C-RAW264.7 cells stably expressing ChR2 protein: RAW264.7 cells are inoculated into a T25 cell culture flask, and when the growth fusion rate reaches 30% to 50%, a lentivirus carrying the ChR2 gene is added to infect the RAW264.7 cells, and puromycin is used to select cells stably expressing ChR2, thereby obtaining C-RAW264.7 cells; Step 2: Inoculate C-RAW264.7 cells: Inoculate C-RAW264.7 cells into a 96-well plate at a seeding density of 1000 cells / well and incubate in a cell culture incubator for 24 h; Step 3: Induce osteoclast differentiation: After 24 h, replace the complete culture medium, irradiate the C-RAW264.7 cells and add RANKL until a large number of osteoclasts are observed to terminate the induction.
2. The method for establishing an osteoclast model according to claim 1, characterized in that: The lentivirus carrying the ChR2 gene in step 1 is pLenti-EF1-EGFP-P2A-Puro-CMV-hChR2.
3. The method for establishing an osteoclast model according to claim 1, characterized in that: The complete culture medium replaced in step 3 is α-MEM complete culture medium, which contains 10% fetal bovine serum, 1% penicillin and streptomycin.
4. The method for establishing an osteoclast model according to claim 1, characterized in that: The irradiation conditions of the C-RAW264.7 cells in step 3 are: blue light 460nm (0.4W cm -2 ) Irradiation for 0 to 5 minutes.
5. The method for establishing an osteoclast model according to claim 1, characterized in that: The induction concentration of RANKL in step 3 is 0 to 50 ng / mL.
6. The method for establishing an osteoclast model according to claim 1, characterized in that: In step 3, the C-RAW264.7 cells were subjected to culture medium replacement, irradiation and RANKL addition once a day for 3 to 4 days.
7. Application of an osteoclast model established based on blue light irradiation combined with RANKL as claimed in any one of claims 1 to 6 in studying the pathogenesis of osteoporosis or in screening anti-osteoporosis drugs.
8. Use according to claim 7, characterized in that The application of the pathogenesis of osteoporosis is to study the mechanism of inhibiting osteoclast differentiation in vitro.
9. The use according to claim 7, characterized in that The application of anti-osteoporosis drug screening is to screen for drugs that reduce Ca in osteoclasts 2 + Application of concentration drugs.