Cell in-vitro calcification inducer, induction culture medium and application thereof, aortic valve calcification in-vitro induction model and modeling method of aortic valve calcification in-vitro induction model

Abstract

The invention belongs to the technical field of biological medicine research, and particularly relates to a cell in-vitro calcification inducer, an induction culture medium and application thereof, an aortic valve calcification in-vitro induction model and a modeling method thereof. The inducer provided by the invention is prepared from the following components in parts by weight: 2160.4 parts of beta-sodium glycerophosphate, 0.039 parts of dexamethasone, 50 parts of vitamin C and 34 parts of vitamin D. The invention further provides a culture medium formed by using the inducer and a method for modeling an aortic valve calcification in-vitro induction model by using the inducer. According to the method, osteogenic differentiation of the aortic valve interstitial cells can be stably induced within a short time, an efficient and repeatable standardized induction scheme is provided for in-vitro aortic valve calcification models and drug screening, and the method has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical research technology, specifically relating to an in vitro cell calcification inducing agent, an induction culture medium and its uses, and an in vitro aortic valve calcification induction model and its modeling method. Background Technology

[0002] Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the elderly, characterized by abnormal osteogenic differentiation of valvular interstitial cells (VICs) leading to fibrotic calcification and remodeling of the valve tissue. Currently, there are no effective drug interventions, and patients ultimately require aortic valve replacement surgery. Therefore, there is an urgent need to establish reliable in vitro research models to elucidate the disease mechanism and screen for therapeutic drugs.

[0003] In the prior art, those skilled in the art commonly use osteogenic medium (OM) to simulate the pathological environment of CAVD in vitro to construct a calcification model of aortic valve interstitial cells (VICs). The model typically uses high-viscosity endothelial cells (HVICs) as seed cells, and induction is achieved by adding osteogenic stimulating factors to the basal culture medium. Typical components of existing inducing agents include: sodium β-glycerophosphate or inorganic phosphate (5-10 mM), ascorbic acid (25 μg / mL to 100 μg / mL), dexamethasone (10-100 nM), and optionally insulin (10⁻⁻¹). 7 mM) was used as a supplementary factor, with an induction period of 7-21 days (see: Cell Death and Disease (2023) 14:108; Adv. Sci. 2024, 11, 2307319). This model allowed for the observation of gradual osteogenic phenotypic transformation in VICs, with identifying indicators including: alizarin red staining showing calcium salt deposition nodules, increased alkaline phosphatase (ALP) activity, upregulation of osteogenic markers RUNX2 and OPN expression, and pathological features such as Warburg effect metabolic reprogramming.

[0004] The drawback of existing calcification inducers is their slow induction rate, typically requiring 7-21 days, which significantly impacts the efficiency of drug screening. Therefore, improving the formulation of calcification inducers to increase the induction rate and shorten the induction period remains a pressing issue in this field. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides an in vitro cell calcification inducing agent, an induction culture medium and its uses, and an in vitro aortic valve calcification induction model and its modeling method.

[0006] An in vitro calcification inducer for cells comprises the following components in parts by weight: Sodium β-glycerophosphate 1080.2-2160.4 parts Dexamethasone 0.0039-0.039 parts Vitamin C 25-100 servings Vitamin D3 1-10 servings.

[0007] Preferably, the components include the following parts by weight: 2160.4 parts of sodium β-glycerophosphate Dexamethasone 0.039 parts, Vitamin C 50 servings Vitamin D3 (4 servings).

[0008] Preferably, it further includes at least one of the following components in parts by weight: Transforming growth factor β 0.001-0.01 parts, Alternatively, 444-1110 parts of calcium chloride.

[0009] The present invention also provides an in vitro calcification induction culture medium, comprising a basal culture medium and the above-mentioned in vitro calcification inducer, wherein the final concentration of the in vitro calcification inducer in the culture medium is: β-glycerophosphate sodium 5.0-10.0 mmol / L Dexamethasone 10.0-100.0 nmol / L Vitamin C 25.0-100.0 mg / L Vitamin D3 1.0-10.0 μg / mL.

[0010] The present invention also provides the above-mentioned in vitro calcification inducing agent, and / or the use of the above-mentioned in vitro calcification inducing culture medium for constructing an in vitro calcification induction model of aortic valve, wherein the cells are selected from mesenchymal cells, osteoblast-associated cells or calcification-susceptible cells.

[0011] Preferably, the cells are aortic valve interstitial cells.

[0012] The present invention also provides a modeling method for an in vitro aortic valve calcification induction model, comprising: culturing aortic valve interstitial cells using the above-mentioned cell in vitro calcification induction culture medium.

[0013] Preferably, the steps include: Step 1: Culture aortic valve interstitial cells to a density of 70%-80% in basal culture medium; Step 2: Replace the culture medium with the cell in vitro calcification induction medium and continue culturing to obtain the final product.

[0014] Preferably, in step 2, the culture time is 1.5-3 days.

[0015] The present invention also provides an in vitro aortic valve calcification induction model established according to the modeling method of the above-described in vitro aortic valve calcification induction model.

[0016] By adopting the technical solution of the present invention, the following beneficial technical effects can be achieved: 1. The synergistic effect of two factors significantly accelerates the osteogenic / calcification induction process. In traditional osteogenic induction systems, aortic valve interstitial cells (HVICs) typically require continuous culture for 7–21 days before an alkaline phosphatase (ALP) positive reaction and mineralized nodule formation can be observed. This invention is the first to demonstrate that the simultaneous addition of VC (vitamin C) and VD3 (vitamin D3) to the β-GP + DEX system can induce significant ALP positive signals and upregulate RUNX2 and ALPL protein expression within 1.5–3 days. This indicates that the synergistic effect of the two factors significantly shortens the osteogenic induction cycle, improves cellular response speed and model sensitivity, and represents a technological breakthrough from a "weekly response" to a "daily response."

[0017] 2. Significantly improves induction efficiency and signal strength Western blot results showed that the inducer of this invention significantly increased the levels of RUNX2 and ALPL proteins after 1.5 days of induction, which were significantly higher than those in the normal cell group; after 3 days, the expression was further enhanced and tended to stabilize, showing obvious early activation characteristics. These results indicate that the dual-factor synergy can achieve stable and significant upregulation of osteogenic signals in a very short time, greatly improving induction sensitivity.

[0018] 3. Excellent system stability and repeatability The inducer of this invention showed consistent ALP staining results and protein expression trends in multiple batches of experiments, with good cell morphology, low toxicity, and small batch-to-batch differences. Compared with multi-factor systems containing TGF-β or CaCl2, this system is more stable, easier to operate, and has more controllable components, making it suitable for standardized establishment and long-term use.

[0019] 4. Economic viability and scalability The reagents used in the inducing agent of this invention are all conventional experimental grade components, which are low in cost, stable in storage, and have a simple system formulation. They are suitable for operation under normal conditions in various laboratories and have good universality and promotion potential.

[0020] In summary, the inducer and the established in vitro aortic valve calcification induction model of this invention have wide applications, such as: in vitro screening of anti-calcification drugs and signaling pathway inhibitors; research on osteogenic differentiation mechanisms, calcification regulatory factors, and transcriptional networks; and validation of early intervention strategies for cardiovascular valve calcification. This system provides fundamental technical support for establishing a rapid, stable, and quantifiable aortic valve calcification drug screening platform.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 Images showing ALP staining results after 3 days of induction culture with different inducing agents in Experiment Example 1; Figure 2 Microscopic images of ALP staining results after 3 days of induction culture with different inducing agents in Experiment Example 1; Figure 3 The results of ALP staining and quantitative analysis in the three experimental groups (β-GP + DEX + VC + VD3, β-GP + DEX + VC, and β-GP + DEX + VD3) in Experimental Example 1 are shown below; This means p < 0.0001.

[0024] Figure 4 The results of WB detection of RUNX2 and ALPL expression in the three experimental groups β-GP + DEX + VC + VD3, β-GP + DEX + VC + TGF-β, and β-GP + DEX + CaCl2 + VD3 in Experimental Example 1 are shown. Detailed Implementation

[0025] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0026] Example 1: In vitro calcification inducing agent and induction culture medium for aortic valve interstitial cells The in vitro calcification inducing agent for aortic valve interstitial cells provided in this embodiment has the following composition: Sodium β-glycerophosphate (β-glycerophosphate) 2160.4 parts Dexamethasone 0.039 parts Vitamin C (Ascorbic acid) 50 servings Vitamin D3 (Cholecalciferol) 4 parts.

[0027] The induction medium is prepared by adding an inducer to the basal medium. The basal medium is DMEM / F12 + 5% FBS + 1% P / S. The concentration of the inducer added is as follows: Sodium β-glycerophosphate: 10.0 mmol / L Dexamethasone: 100.0 nmol / L Vitamin C: 50 mg / L Vitamin D3: 4.0 μg / mL.

[0028] The in vitro induction model of aortic valve calcification using the induction culture medium of this embodiment includes the following steps: 1. Cell preparation Take HVIC in the logarithmic growth phase, digest with trypsin, and count.

[0029] With 2 × 10 4 Cells / well were seeded at a density of 200 μL of basal medium per well in 48-well plates.

[0030] Incubate at 37 ℃ in a 5% CO2 incubator and let stand overnight.

[0031] 2. Inducing calcification Once the cells reach a density of 70%-80%, discard the culture medium and replace it with induction medium to continue culturing. The induction time is 1.5-3 days.

[0032] 3. Conduct follow-up testing.

[0033] The technical solution of the present invention will be further illustrated by the following experiments.

[0034] Experiment Example 1: Screening Experiment of Inducing Agent Formulation I. Experimental Methods 1. Experimental group setup The basal culture medium for each experimental group was DMEM / F12 + 5% FBS + 1% P / S, and the composition of the inducer is shown in Table 1.

[0035] Table 1 Experimental group setup Note: "+" indicates that the substance is added, and "-" indicates that the substance is not added.

[0036] 2. Induction methods HVIC induced calcification was performed according to the method provided in Example 1.

[0037] 3. Detection Method The induction results were verified by Beyotime ALP staining and Western blotting.

[0038] II. Experimental Results 1. ALP staining results Based on sodium β-glycerophosphate (β-GP) and dexamethasone (DEX), 16 inducers were designed and systematically screened and validated by adding different cofactors, including vitamin C (VC), vitamin D3 (VD3), transforming growth factor β (TGF-β) and calcium chloride (CaCl2).

[0039] ALP staining results are as follows Figures 1-3 As shown, the osteogenic activity of each induction combination was compared and evaluated by ALP staining and cell morphology observation. The results showed that different additives had significantly different effects on the osteogenic / calcification differentiation of aortic valve interstitial cells, as detailed below: The normal group (CM) could only induce mild ALP activity and had a low level of osteogenic differentiation, suggesting that the system could provide the basic conditions for osteogenic differentiation, but was not sufficient to significantly induce calcification.

[0040] After the addition of VC (β-cell osteogenic differentiation level is low, it is added), ALP staining was significantly deepened and the positive signal was enhanced, suggesting that VC can promote intracellular collagen synthesis and matrix formation, thereby significantly improving early osteogenic activity.

[0041] After the addition of VD3 (β-vitamin 3, which promotes intracellular collagen synthesis and matrix formation), ALP staining was significantly enhanced, indicating that VD3 can upregulate the expression of osteogenic-related genes (such as RUNX2 and ALP) and promote the transformation of cells to an osteogenic phenotype.

[0042] Comparing the results of the three experimental groups—β-GP + DEX + VC + VD3, β-GP + DEX + VC, and β-GP + DEX + VD3—as follows: Figure 3As shown, when VC and VD3 were added together (β-GP + DEX + VC + VD3), the ALP positive signal was most pronounced, with a signal intensity higher than the sum of the ALP positive signals in the two experimental groups where VC or VD3 was used alone (β-GP + DEX + VC or β-GP + DEX + VD3). This phenomenon indicates a synergistic effect between VC and VD3: VC promotes matrix formation, and VD3 promotes calcium salt deposition, synergistically enhancing the osteogenic differentiation capacity of cells. This combination showed a stable and reproducible strong positive reaction in multiple repeated experiments.

[0043] In systems incorporating TGF-β (such as β-GP + DEX + TGF-β), ALP staining was significantly reduced, indicating that TGF-β has a certain inhibitory effect on osteogenic induction in the early stages, possibly by intervening in osteogenic signaling pathways to inhibit ALP expression. However, when used in combination with VC or VD3, the inhibitory effect can be partially offset, but the overall effect is still not as good as the VC or VD3 groups alone.

[0044] In systems with added CaCl2 (such as β-GP + DEX + CaCl2), ALP activity was slightly enhanced compared to the baseline group, indicating that calcium ions can promote mineralization reactions to a certain extent, but the effect is limited. Furthermore, excessively high CaCl2 concentrations can easily cause cell stress or death, resulting in poor experimental reproducibility.

[0045] Although multi-factor combination systems (such as β-GP + DEX + VC + TGF-β + CaCl2 + VD3) show strong ALP staining in some replicate experiments, they are complex, involve complicated procedures, have low reagent stability, and require high control over culture conditions and concentrations. This results in poor experimental reproducibility, high costs, and is not conducive to standardized operation and large-scale drug screening applications.

[0046] After comprehensively comparing the osteogenic induction effects, system stability, ease of operation, and economy of different induction combinations, the β-GP + DEX + VC + VD3 system was determined to have significant advantages in the following aspects: 1) Strongest induction efficacy: ALP staining was most obvious, with a wide range of positive areas, and osteogenic activity was significantly higher than other groups; 2) High system stability: The results were consistent in multiple repeated experiments, the cells were in good condition, and there were no obvious toxic reactions; 3) Simple operation and low cost: Only conventional reagents are required, storage conditions are stable, and the experimental procedure is simple; 4) Wide applicability: It can be used as a standard system for in vitro induction model of aortic valve calcification, and can be used for drug screening, signaling pathway research and verification of anti-calcification mechanism.

[0047] 2. Western blotting experiments were used to verify the effects of RUNX2 and ALPL expression on β-GP + DEX + VC + VD3, β-GP + DEX + VC + TGF-β, and β-GP + DEX + CaCl2 + VD3. To further verify the differences in the expression of osteogenic-related genes under different induction systems, Western blot analysis was performed on cells induced for 1.5 days and 3 days, mainly detecting key early osteogenic proteins RUNX2 and ALPL (alkaline phosphatase).

[0048] Test results as follows Figure 4 As shown: At 1.5 days of induction, β-GP + DEX + VC + VD3 ( Figure 4 In the VC+D group, RUNX2 and ALPL proteins were significantly upregulated and their expression was relatively stable, showing a significant increase in expression levels compared to the normal CM group, suggesting that this system can rapidly activate the osteogenic differentiation pathway. Three days after induction, the expression of RUNX2 and ALPL proteins in the β-GP + DEX + VC + VD3 group was further enhanced, with clear bands and strong signals. The variation between different experimental batches was small, showing good system stability. Other combinations, such as β-GP + DEX + CaCl2 + VD3 ( Figure 4 The system represented as VD3+CaCl2 showed some upregulation of RUNX2, but the magnitude and reproducibility were lower than those of the β-GP + DEX + VC + VD3 group; and its ALP expression was significantly lower than that of the β-GP + DEX + VC + VD3 group.

[0049] In systems containing TGF-β, such as β-GP + DEX + VC + TGF-β ( Figure 4 The expression of RUNX2 was significantly suppressed, but ALPL expression was significantly suppressed, which was consistent with the trend of ALP staining.

[0050] These results indicate that VC and VD3 have a significant synergistic effect under β-GP + DEX induction, and can jointly promote the continuous and stable upregulation of osteogenic signaling molecules (RUNX2, ALPL) in aortic valve interstitial cells, thereby achieving more efficient early calcification induction.

[0051] Based on the combined results of ALP staining, Western blotting (WB) protein detection, system stability, and cost-effectiveness, the optimal induction system for aortic valve interstitial cell osteogenic / calcification was determined to be β-GP + DEX + VC + VD3. This system can stably induce osteogenic differentiation of aortic valve interstitial cells within a short period, exhibiting excellent ALP activity and a RUNX2 / ALPL upregulation trend. It provides an efficient, reproducible, and standardized induction protocol for in vitro aortic valve calcification models and drug screening, demonstrating promising application prospects.

Claims

1. An in vitro calcification inducer, characterized in that, The components include the following parts by weight: Sodium β-glycerophosphate 1080.2-2160.4 parts Dexamethasone 0.0039-0.039 parts Vitamin C 25-100 servings Vitamin D3 1-10 servings.

2. The in vitro calcification inducer according to claim 1, characterized in that, The components include the following parts by weight: 2160.4 parts of sodium β-glycerophosphate Dexamethasone 0.039 parts, Vitamin C 50 servings Vitamin D3 (4 servings).

3. The in vitro calcification inducer according to claim 1 or 2, characterized in that, It also includes at least one of the following components in parts by weight: Transforming growth factor β 0.001-0.01 parts, Alternatively, 444-1110 parts of calcium chloride.

4. A cell in vitro calcification induction culture medium, characterized in that, The medium includes a basal culture medium and the in vitro calcification inducer according to any one of claims 1-3, wherein the final concentration of the in vitro calcification inducer in the culture medium is: β-glycerophosphate sodium 5.0-10.0 mmol / L Dexamethasone 10.0-100.0 nmol / L Vitamin C 25.0-100.0 mg / L Vitamin D3 1.0-10.0 μg / mL.

5. The use of the in vitro calcification inducing agent according to any one of claims 1-3, and / or the in vitro calcification inducing culture medium according to claim 4 or 5, in constructing an in vitro aortic valve calcification induction model, characterized in that, The cells are selected from mesenchymal cells, osteoblast-associated cells, or calcification-susceptible cells.

6. The use according to claim 5, characterized in that: The cells in question are aortic valve interstitial cells.

7. A modeling method for an in vitro induction model of aortic valve calcification, characterized in that, include: Aortic valve interstitial cells were cultured using the in vitro calcification induction medium as described in claim 4 or 5.

8. The modeling method for the in vitro induction model of aortic valve calcification according to claim 7, characterized in that, Includes the following steps: Step 1: Culture aortic valve interstitial cells to a density of 70%-80% in basal culture medium; Step 2: Replace the culture medium with the cell in vitro calcification induction medium and continue culturing to obtain the final product.

9. The modeling method for the in vitro induction model of aortic valve calcification according to claim 8, characterized in that, In step 2, the incubation period is 1.5-3 days.

10. An in vitro aortic valve calcification induction model established according to the modeling method of any one of claims 7-9.

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