Application of FZD4 gene
By regulating the FZD4 gene in chicken bone marrow mesenchymal stem cells, promoting or inhibiting its osteogenic differentiation, the problem of frequent avian skeletal diseases has been solved, enabling the breeding of chicken breeds with high bone strength and the prevention and treatment of skeletal diseases, thereby improving breeding efficiency and animal welfare.
Patent Information
- Application Number
- CN202610089548.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-22
- Publication Date
- 2026-04-17
AI Technical Summary
In the current technology, skeletal diseases are frequent in poultry, especially osteoporosis in chickens and serious leg bone diseases in broiler chickens, which affect growth performance and animal welfare, and there is a lack of effective gene regulation methods.
By overexpressing or knocking down the FZD4 gene in chicken bone marrow mesenchymal stem cells, osteogenic differentiation and mineralization can be regulated. Using the nucleotide sequence of the FZD4 gene coding region (as shown in SEQ ID NO.1) and small interfering RNA sequence, chicken bone formation can be promoted or inhibited. Overexpression and knockdown vectors can be constructed to prepare biological agents.
The positive regulatory role of the FZD4 gene in chicken skeleton formation was clarified, and gene targets were provided for breeding chicken breeds with high bone strength, preventing bone diseases, and improving the efficiency of intensive farming and animal welfare.
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Figure CN121874266A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering and poultry breeding technology, specifically involving the application of the FZD4 gene. Background Technology
[0002] Chickens are one of the most important poultry breeds globally, and their skeletal health directly impacts growth performance, production efficiency, and animal welfare. In intensive poultry farming systems, problems such as osteoporosis in laying hens and leg bone diseases in broilers are frequent, causing not only economic losses but also sparking animal welfare controversies. Maintaining skeletal homeostasis depends on the directed differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts. Understanding the molecular regulatory mechanisms of BMSC osteogenic differentiation and identifying key regulatory genes are crucial prerequisites for breeding poultry breeds with high bone strength and preventing skeletal diseases.
[0003] Frizzled family receptors are key mediators of the Wnt signaling pathway. Among them, the FZD4 gene has been shown to play an important role in the regulation of mammalian skeletal homeostasis, but there are still many gaps in the research on its function and regulatory mechanism in osteogenic differentiation of avian (especially chicken) BMSCs. Summary of the Invention
[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing an application of the FZD4 gene, clarifying the positive regulatory role of the FZD4 gene in chicken bone formation, and providing a reliable gene target and technical support for the breeding of poultry breeds with high bone strength and the prevention and treatment of bone diseases.
[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an application of the FZD4 gene, wherein the FZD4 gene is used to regulate osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells; Overexpression of the FZD4 gene in chicken bone marrow mesenchymal stem cells was used to promote osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. Or / and by knocking down the FZD4 gene in chicken bone marrow mesenchymal stem cells, osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells can be inhibited. The nucleotide sequence of the coding region of the FZD4 gene is shown in SEQ ID NO.1.
[0006] Preferably, when used to inhibit osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells, the nucleotide sequence of the sense strand of the small interfering RNA sequence of the FZD4 gene is 5'-GCUUUAAAGUGCCACAAUATT-3', and the nucleotide sequence of the antisense strand is 5'-UAUUGUGGCACUUUAAAGCTT-3'.
[0007] Preferably, when the FZD4 gene in chicken bone marrow mesenchymal stem cells is overexpressed, the level of osteogenic marker mRNA in the chicken bone marrow mesenchymal stem cells increases.
[0008] Preferably, the osteogenic markers include early osteogenic markers ALP, Col1A1, Runx2 and late osteogenic marker OCN.
[0009] Preferably, the feature is that when the FZD4 gene in chicken bone marrow mesenchymal stem cells is overexpressed, the expression levels of C-myc, Cyclin D1, and β-catenin mRNA in the Wnt signaling pathway of the chicken bone marrow mesenchymal stem cells are increased. The level of expression has decreased.
[0010] Preferably, an overexpression vector of the FZD4 gene is prepared for use in the preparation of a biological agent that promotes bone formation in poultry.
[0011] Preferably, the biological agent that promotes poultry bone formation further includes a liposome transfection reagent.
[0012] Preferably, the FZD4 gene is used as a molecular marker to breed chickens with high bone strength.
[0013] Preferably, qRT-PCR is used to detect the expression level of the FZD4 gene in the bone tissue of individual chickens to screen breeding parents.
[0014] Compared with the prior art, the present invention has the following advantages: This invention clarifies for the first time the positive regulatory function of the FZD4 gene in osteogenic differentiation of chicken BMSCs, confirming that it functions through the typical Wnt signaling pathway, filling the gap in the skeletal regulatory mechanism of the FZD4 gene in poultry. Overexpression and knockdown vectors constructed based on this gene can respectively promote and inhibit osteogenic differentiation in chickens, providing new biological targets for the prevention and treatment of poultry skeletal diseases. Simultaneously, the FZD4 gene can be used as a molecular marker for the breeding of chicken breeds with high skeletal strength, helping to improve the efficiency of intensive farming and animal welfare.
[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description
[0016] Figure 1 This is a diagram showing the morphological changes of chicken BMSCs cultured at different times / passages in Example 1 of the present invention.
[0017] Figure 2 This is an immunofluorescence staining image of chicken BMSCs CD29 and CD45 in Example 1 of the present invention.
[0018] Figure 3This is a diagram showing the ALP and ARS staining results of chicken BMSCs in Example 1 of the present invention.
[0019] Figure 4 This is a graph showing the relative mRNA expression levels of ALP and FZD4 in chicken BMSCs on day 0 and day 3 in Example 1 of the present invention.
[0020] Figure 5 This is a graph showing the relative protein expression levels of FZD4 in chicken BMSCs on day 0 and day 3 in Example 1 of the present invention.
[0021] Figure 6 This is a graph showing the relative mRNA expression level of the FZD4 gene overexpressed in chicken BMSCs in Example 1 of the present invention.
[0022] Figure 7 This is a graph showing the relative expression levels of osteogenic markers in chicken BMSCs at days 0 and 7 of FZD4 gene overexpression in Example 1 of the present invention.
[0023] Figure 8 This is an ALP staining diagram of chicken BMSCs overexpressing the FZD4 gene in Example 1 of the present invention; wherein, the bar chart is a quantification of the ALP staining results.
[0024] Figure 9 This is an ARS staining diagram of chicken BMSCs overexpressing the FZD4 gene in Example 1 of the present invention; wherein, the bar chart is a quantification of the ARS staining results.
[0025] Figure 10 This is a graph showing the relative mRNA expression level of the knockdown of the FZD4 gene in chicken BMSCs in Example 1 of the present invention.
[0026] Figure 11 This is a graph showing the relative expression levels of osteogenic markers in chicken BMSCs at days 0 and 7 of FZD4 gene knockdown in Example 1 of the present invention.
[0027] Figure 12 This is an ALP staining diagram of chicken BMSCs with knocked-down FZD4 gene in Example 1 of the present invention; wherein, the bar chart is a quantification of the ALP staining results.
[0028] Figure 13 This is an ARS staining diagram of chicken BMSCs with knocked-down FZD4 gene in Example 1 of the present invention; wherein, the bar chart is a quantification of the ARS staining results.
[0029] Figure 14 This is a graph showing the relative mRNA expression levels of key factors in the Wnt signaling pathway in chicken BMSCs overexpressing the FZD4 gene in Example 1 of this invention.
[0030] Figure 15 This is a graph showing the relative protein expression levels of key factors in the Wnt signaling pathway in chicken BMSCs overexpressing the FZD4 gene in Example 1 of this invention.
[0031] Figure 16 This is a graph showing the relative mRNA expression levels of key factors in the Wnt signaling pathway in chicken BMSCs with knocked-down FZD4 gene in Example 1 of this invention.
[0032] Figure 17 This is a graph showing the relative protein expression levels of key factors in the Wnt signaling pathway in chicken BMSCs with knocked-down FZD4 gene in Example 1 of this invention.
[0033] Figure 18 This is a graph showing the relative mRNA expression levels of osteogenic differentiation markers and key factors of the Wnt signaling pathway in chicken BMSCs overexpressing the FZD4 gene at days 0 and 7 after the addition of a Wnt signaling pathway inhibitor in Example 1 of the present invention.
[0034] Figure 19 This is a graph showing the relative protein expression levels of key factors β-catenin and GSK-3β in chicken BMSCs overexpressing the FZD4 gene in Example 1 of the present invention after the addition of a Wnt signaling pathway inhibitor; wherein, the bar graph represents the quantification of the relative protein expression levels of β-catenin and GSK-3β.
[0035] Figure 20 This is an ALP and ARS staining diagram of chicken BMSCs overexpressing the FZD4 gene in Example 1 of the present invention after the addition of a Wnt signaling pathway inhibitor; wherein, the bar charts are the quantitative results of ALP staining and ARS staining, respectively.
[0036] Figure 21 This is a graph showing the relative mRNA expression of osteogenic differentiation markers and key factors of the Wnt signaling pathway in chicken BMSCs with knocked-down FZD4 gene in Example 1 of the present invention at days 0 and 7 after the addition of a Wnt signaling pathway inhibitor.
[0037] Figure 22 This is a graph showing the relative protein expression levels of key factors β-catenin and GSK-3β in chicken BMSCs with knocked-down FZD4 gene in Example 1 of the present invention after the addition of a Wnt signaling pathway inhibitor; wherein, the bar graph represents the quantification of the relative protein expression levels of β-catenin and GSK-3β.
[0038] Figure 23 This is an ALP and ARS staining diagram of chicken BMSCs with knocked-down FZD4 gene in Example 1 of the present invention after the addition of a Wnt signaling pathway inhibitor; wherein, the bar chart represents the quantitative results of ALP staining and ARS staining, respectively. Detailed Implementation
[0039] Example 1 This embodiment describes the application of the FZD4 (Frizzled-4) gene. Overexpression of the FZD4 gene in chicken bone marrow mesenchymal stem cells promotes osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells; knockdown of the FZD4 gene in chicken bone marrow mesenchymal stem cells inhibits osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. The nucleotide sequence of the coding region of the FZD4 gene is shown in SEQ ID NO.1, and the GenBank accession number is NM_204099.2.
[0040] (a) Experimental methods: (1) Laboratory animals and ethical approval: The fertilized chicken embryos used in the experiment were purchased from a chicken farm in Henan Province, with an embryonic age of 18 days (E18). The experimental protocol was approved by the Animal Experiment Ethics Committee of Henan Agricultural University (License No. 18-0120).
[0041] After euthanizing E18 chicken embryos with 0.2% pentobarbital (40 mg / kg), femoral and tibial tissues were collected for subsequent BMSCs (bone marrow mesenchymal stem cell) isolation.
[0042] (2) Isolation, culture and osteogenic induction of chicken BMSCs: Separation: Under aseptic conditions, the femur and tibia of the chicken embryo were removed and the surrounding tissues were removed. The bone marrow was rinsed with a 1 mL syringe until the bone turned white. The bone marrow suspension was filtered through 100-mesh and 200-mesh sieves and centrifuged at 1000 rpm for 5 min to collect the precipitate.
[0043] Culture: After resuspending the precipitate, inoculate it into culture flasks and incubate at 37°C with 5% CO2. After 24 hours, change the medium to remove non-adherent cells. Change the medium again after 2 days. When the cells reach 80% confluence, replace with osteogenic induction medium (MEM-α basal medium containing 10% fetal bovine serum, supplemented with...). Sodium glycerophosphate Induction was performed using a mixture of ascorbic acid and 100 nM dexamethasone.
[0044] Passaging: P3 generation cells were used for subsequent transfection and functional assays.
[0045] (3) Identification of BMSCs: Morphological observation: The morphology of P0 generation cells was observed at 0, 1, 5, 7 and 9 days of culture.
[0046] (4) Immunofluorescence staining: Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 5% BSA (bovine serum albumin) at room temperature for 1 h. Primary antibodies against CD29 (1:200, BD) and CD45 (1:200, Proteintech) were added, and the cells were incubated overnight at 4°C. The next day, the corresponding secondary antibodies (1:200, Absin / Proteintech) were added, and the cells were incubated at room temperature in the dark for 1 h. DAPI (… Stain the nuclei for 5 minutes and take a picture using an inverted fluorescence microscope (Nikon TS).
[0047] (5) Verification of osteogenic differentiation capacity: Alkaline phosphatase (ALP) staining: P3 generation cells were transfected for 6 hours and then replaced with osteogenic induction medium. The NBT / BCIP kit (Beyotime) was used according to the instructions. The cells were observed and photographed under a regular optical microscope (Olympus) to detect early osteogenic differentiation capacity.
[0048] Alizarin Red S (ARS) staining: 14 days after osteogenic induction, cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% Alizarin Red (Beyotime) at room temperature for 30 min, rinsed with distilled water, and photographed to assess the level of cell mineralization (late osteogenic).
[0049] (6) Cell transfection: FZD4 overexpression vector (pcDNA3.1-FZD4), empty vector control (pcDNA3.1), FZD4 small interfering RNA sequence (si-FZD4), and negative control (si-NC) were synthesized by Beijing Qingke Biotechnology. The nucleotide sequence of the positive strand of si-FZD4 is as follows: The nucleotide sequence of the antisense strand is P3 generation cells were seeded into 6-well plates and overexpressed and knocked down using Lip3000 transfection reagent (Invitrogen). After 6 hours of transfection, the medium was replaced with osteogenic induction medium. Cells were collected at 0, 3 and 7 days after transfection for subsequent detection. The expression level of FZD4 mRNA was detected by qRT-PCR.
[0050] The specific steps for Lip3000 transfection are as follows: Cell plating: P3 generation BMSCs were seeded into culture plates, DMEM medium containing fetal bovine serum (without antibiotics) was added, and the plates were incubated at 37°C in a 5% CO2 incubator until the cell confluence reached 55% (50%–60% is acceptable). Transfection complex preparation: using Opti-MEM serum-depleted medium ( Mix Lip3000 transfection reagent with si-FZD4 / si-NC according to the reagent instructions, and incubate at room temperature to form a transfection complex; Transfection procedure: Add the transfection complex to the cell culture wells, mix gently, and then return to the 37°C, 5% CO2 incubator to continue culturing for 48 hours (0 days) to detect overexpression and interference efficiency.
[0051] (7) Classic Wnt pathway inhibition assay: During cell culture, the overexpression plasmid and the Wnt pathway inhibitor XAV939 (Yeasen) were transfected at a certain cell density (60%–70%). After induction and medium change, the control group was transfected with the overexpression plasmid and DMSO (dimethyl sulfoxide) to verify the role of the pathway in FZD4-regulated osteogenic processes.
[0052] (8) qRT-PCR detection of gene expression: Total RNA was extracted from cells using TRIzol reagent (Vazyme) and reverse transcribed into cDNA using HiScript II Q RT SuperMix (Vazyme).
[0053] Using GAPDH as an internal control, qPCR was performed on a LightCycler® 96 system (Roche) using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The method analyzes the relative expression of genes, and the experiment is set up with at least 3 technical replicates; the genes to be detected include osteogenic markers Col1A1 (type I collagen α1 chain), Runx2 (Runt-related transcription factor 2), ALP (alkaline phosphatase), OCN (osteocalcin), and key factors of the classical Wnt pathway C-myc (c-Myc proto-oncogene), Cyclin D1 (cyclin D1), β-catenin (β-catenin), and GSK-3β (glycogen synthase kinase-3β).
[0054] The nucleotide sequence of the upstream primer for the FZD4 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3; the nucleotide sequence of the upstream primer for the Col1A1 gene is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5; the nucleotide sequence of the upstream primer for the ALP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.7; the nucleotide sequence of the upstream primer for the Runx2 gene is shown in SEQ ID NO.8, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.9; the nucleotide sequence of the upstream primer for the OCN gene is shown in SEQ ID NO.10, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.11; the nucleotide sequence of the upstream primer for the C-myc gene is shown in SEQ ID NO.12, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.13; the nucleotide sequence of the upstream primer for the Cyclin D1 gene is shown in SEQ ID NO.14, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.15; the nucleotide sequence of the upstream primer for the β-catenin gene is shown in SEQ ID NO.15. As shown in NO.16, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.17; the nucleotide sequence of the upstream primer of the GSK-3β gene is shown in SEQ ID NO.18, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.19.
[0055] (9) Western blot detection of protein expression: 48 h after transfection, cells were collected and total protein was extracted. After separation by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), the protein was transferred to a PVDF (polyvinylidene fluoride) membrane, blocked with 5% BSA, and incubated overnight at 4°C with primary antibodies (FZD4 1:2000, GSK-3β 1:2000, β-catenin 1:5000, GAPDH 1:10000).
[0056] The following day, HRP-labeled secondary antibody (1:50000, Searcare) was added and incubated at room temperature. Chemiluminescence detection was performed using an ECL kit (ThermoFisher), and the gray values of the bands were analyzed using ImageJ software.
[0057] (10) Statistical analysis: SPSS 26 was used for t-tests (comparison between two groups) and one-way ANOVA (comparison between multiple groups). GraphPadPrism 8.0 was used for plotting. P<0.05 was considered statistically significant, and P<0.01 was considered highly statistically significant.
[0058] (II) Test Results: (1) Morphological observation of BMSCs: The morphological changes of P0 generation cells at different culturing days were observed. (1) to (5) represent 0d, 1d, 5d, 7d and 9d, respectively. The results are as follows: Figure 1 As shown, P3 generation cells (9d) are arranged in a uniform, elongated spindle shape in a whorled pattern (scale bar: ).
[0059] (2) Immunofluorescence staining: Observe and photograph the immunofluorescence staining results under a fluorescence microscope, such as... Figure 2 As shown, the cells highly express the mesenchymal stem cell marker CD29 but do not express the hematopoietic cell marker CD45, consistent with the BMSCs phenotype (scale bar is shown). ).
[0060] (3) Verification of osteogenic differentiation capacity: like Figure 3 As shown, after induction with osteogenic induction medium, ALP (early osteogenic marker) positive staining was detected after 3 days, and ARS positive mineralized nodules were detected after 7 days (scale bar: 100µm); Figure 4 As shown, FZD4 expression levels were synchronously upregulated with ALP expression during the early induction period (0–3 days), confirming that the isolated cells were chicken BMSCs with osteogenic differentiation potential; Figure 5 As shown, FZD4 protein expression increased significantly after 3 days of induction.
[0061] (4) Effect of FZD4 gene overexpression on osteogenic differentiation of chicken BMSCs: like Figure 6 As shown, FZD4 expression in the pcDNA3.1-FZD4 group was significantly increased at 0d and 7d; osteogenic marker expression was detected at 0d and 7d of induction differentiation, as shown in the figure. Figure 7 As shown, Col1A1 and OCN expression were significantly increased at 0 days, and Col1A1 and OCN expression were significantly increased, while Runx2 expression was significantly increased at 7 days. Figure 8 As shown, the ALP staining positivity rate at 7 days was significantly higher than that in the control group (P<0.01) (scale bar: 100µm); Figure 9 As shown, the positivity of ARS staining at 14 days was significantly higher than that in the control group (P<0.05) (scale bar is 100µm), indicating that overexpression of FZD4 promotes osteogenic differentiation and mineralization.
[0062] (5) Effect of FZD4 gene knockdown on osteogenic differentiation of chicken BMSCs: like Figure 10 As shown, FZD4 expression was significantly reduced in the si-FZD4 group at 0d and 7d; Figure 11As shown, the expression of Col1A1, Runx2, and OCN was significantly decreased at 0 days (P<0.05), and the expression of Runx2 and OCN was extremely significantly decreased at 7 days (P<0.01); Figure 12 As shown, the positivity rate of ALP staining at 7 days was significantly lower than that in the control group (P<0.05) (scale bar: 100µm); Figure 13 As shown, there was no significant difference in ARS staining at 14 days (P>0.05) (scale bar is 100µm), indicating that knockdown of the FZD4 gene inhibits osteogenic differentiation.
[0063] (6) The regulatory role of the FZD4 gene on the typical Wnt signaling pathway in chicken BMSCs: like Figure 14 As shown, C-myc (Cmyc) expression was significantly increased at 0d (P<0.01). Extremely significant decrease (P<0.01); at 7 days, C-myc (Cmyc) and β-catenin were extremely significantly increased. Significantly reduced (P<0.01); in the classic Wnt signaling pathway, β-catenin and GSK-3β are core regulatory molecules in the pathway. Signals downstream of the Frizzled receptor directly act on the degradation complex formed by GSK-3β, thereby regulating the phosphorylation, degradation, and nuclear translocation of β-catenin. The expression levels of both (especially post-translational modifications at the protein level, such as β-catenin phosphorylation) directly reflect the activation / inhibition state of the pathway. C-myc and Cyclin D1 are downstream target genes regulated by TCF / LEF transcription factors after β-catenin enters the nucleus; they are effector molecules after pathway activation, and changes in their transcriptional level (mRNA) can indirectly reflect the degree of pathway activation. Figure 15 As shown, FZD4 and β-catenin were significantly elevated at the protein level. Significantly reduced (P<0.01).
[0064] like Figure 16 As shown, β-catenin expression was significantly decreased at 0 days and significantly increased at 7 days (P<0.05); Figure 17 As shown, FZD4 was significantly decreased at the protein level (P<0.01) and β-catenin was significantly decreased (P<0.05). The increase was extremely significant (P<0.01), confirming that FZD4 can activate the classical Wnt pathway.
[0065] Adding the Wnt pathway inhibitor XAV939 to FZD4 gene overexpressing cells yielded the following results: Figure 18As shown, compared with the pcDNA3.1-FZD4+DMSO group, the pcDNA3.1-FZD4+XAV939 group at day 0... Significantly elevated (P<0.05), Runx2, ALP, C-myc (Cmyc), Cyclin D1, and β-catenin were significantly / extremely significantly decreased at 7 days. Significantly increased; such as Figure 19 As shown, the protein level of β-catenin was significantly reduced. Significantly increased; such as Figure 20 As shown, the positivity of ALP staining at 7 days and ARS staining at 14 days was significantly reduced (scale bar is 100 μm), indicating that the pathway inhibitor can reverse the osteogenic promoting effect of FZD4.
[0066] Add the Wnt pathway inhibitor XAV939 to FZD4 gene knockdown cells, such as Figure 21 As shown, compared with the si-FZD4+DMSO group, the si-FZD4+XAV939 group showed a highly significant decrease in β-catenin at day 0 (P<0.01); Figure 22 As shown, the protein levels of β-catenin were significantly decreased, and GSK-3β (GSK3β) were significantly increased (P<0.01); Figure 23 As shown, the positivity of ALP staining at 7 days and ARS staining at 14 days was extremely / significantly reduced (scale bar is 100μm), further confirming that FZD4 regulates osteoogenesis through the classical Wnt pathway.
[0067] In summary, this invention reveals that FZD4 may participate in bone formation in chickens by activating the classical Wnt signaling pathway and regulating the expression of osteogenic markers. This study elucidates the specific regulatory role of FZD4 in avian skeletal development, providing new insights and theoretical support for understanding skeletal development and skeletal health in poultry and livestock.
[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. Use of a FZD4 gene, characterized in that, The FZD4 gene is used to regulate osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. Overexpression of the FZD4 gene in chicken bone marrow mesenchymal stem cells was used to promote osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. Or / and by knocking down the FZD4 gene in chicken bone marrow mesenchymal stem cells, osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells can be inhibited. The nucleotide sequence of the coding region of the FZD4 gene is shown in SEQ ID NO.
1.
2. The application of the FZD4 gene according to claim 1, characterized in that, The nucleotide sequence of the sense strand of the small interfering RNA sequence of the FZD4 gene is The nucleotide sequence of the antisense strand of the small interfering RNA sequence of the FZD4 gene is when used for inhibiting the osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. 3.The application of FZD4 gene according to claim 1, characterized in that, When the FZD4 gene in chicken bone marrow mesenchymal stem cells is overexpressed, the level of osteogenic marker mRNA in the chicken bone marrow mesenchymal stem cells increases. 4.The application of FZD4 gene according to claim 3, characterized in that, The osteogenic markers include early osteogenic markers ALP, Col1A1, Runx2, and late osteogenic marker OCN. 5.The FZD4 gene of claim 1, wherein the FZD4 gene is used for the preparation of a medicament for treating or preventing a disease caused by abnormal Wnt signaling. When the FZD4 gene in chicken bone marrow mesenchymal stem cells is overexpressed, the expression levels of C-myc, Cyclin D1 and β-catenin mRNA in the Wnt signaling pathway of chicken bone marrow mesenchymal stem cells increase, while the expression level of GSK-3β mRNA decreases. 6.The application of FZD4 gene according to claim 1, characterized in that, An overexpression vector for the FZD4 gene was prepared for use in the preparation of a biological agent that promotes bone formation in poultry.
7. Use according to claim 6, characterized in that, The biological agents that promote poultry bone formation also include liposome transfection reagents. 8.The application of FZD4 gene according to claim 1, characterized in that, The FZD4 gene was used as a molecular marker to breed chickens with high bone strength.
9. The application according to claim 8, characterized in that, The expression level of the FZD4 gene in the bone tissue of individual chickens was detected by qRT-PCR to screen breeding parents.