Application of CRISP3 gene as molecular marker for osteoporosis
By using the CRISP3 gene as a molecular marker of osteoporosis, the shortcomings in the diagnosis and treatment of osteoporosis in the prior art have been solved, and more accurate and effective diagnosis and treatment have been achieved.
Patent Information
- Application Number
- CN202510274534.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art is difficult to effectively prevent and treat osteoporosis, and there is a lack of rapid and accurate diagnosis methods.
Using the CRISP3 gene as a molecular marker of osteoporosis, products and drugs for the diagnosis and treatment of osteoporosis were developed by detecting the expression level of the CRISP3 gene.
The CRISP3 gene is significantly highly expressed in patients with osteoporosis and can be used as a marker for diagnosing osteoporosis. Its inhibitor can be used to treat osteoporosis and improve the accuracy and effectiveness of diagnosis and treatment.
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Abstract
Description
Technical Field
[0001] The invention relates to application of CRISP3 gene as a molecular marker for osteoporosis, and belongs to the technical field of biomedicine. Background Art
[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass, destruction of bone tissue microstructure, and increased bone brittleness, and the risk of fractures in patients is significantly increased. With the increasing aging of the world, the incidence of osteoporosis has increased year by year. The results of an epidemiological survey in 2021 showed that the global incidence of osteoporosis in the past 20 years was about 18.3%, of which about 23.1% were women and about 11.7% were men. At first, the bone loss of osteoporosis patients was very slow, and the patients had no obvious clinical symptoms. As the disease progresses, when osteoporosis causes fractures in patients, clinical symptoms are gradually detected. The fractures of patients with osteoporosis heal more slowly and may cause deformities such as spinal curvature. At present, the treatment of osteoporosis is mainly based on drugs such as alendronate sodium tablets and calcium carbonate D3 tablets. These drugs have limitations in the treatment of osteoporosis. At the same time, long-term use of Western medicine has greater side effects on patients with osteoporosis and a higher risk of inducing complications. Therefore, how to effectively prevent and treat osteoporosis is a top priority, and the clinic urgently needs to find a new molecular marker for rapid, effective and accurate diagnosis of osteoporosis. The combination of new biomarkers and traditional screening programs will effectively increase the early diagnosis rate of osteoporosis, thereby improving the cure rate of osteoporosis.
[0003] Cysteine rich secretory protein 3 (CRISP3) is the third member of the cysteine rich secretory proteins (CRISPs) family and is located on human chromosome 6. CRISP3 is widely distributed in human tissues, mainly in secretions such as salivary glands, pancreas, prostate, blood and sweat. CRISP3 encodes a member of the cysteine rich secretory protein (CRISP) family in the CRISP, antigen 5 and pathogenesis-related protein 1 superfamily. The encoded protein has an N-terminal CRISP, antigen 5 and pathogenesis-related protein 1 protein domain, a hinge region and a C-terminal ion channel regulatory domain. The protein contains cysteine residues located in the N-terminal and C-terminal domains, forming eight disulfide bonds, which is a significant feature of this family. At present, there are few reports on CRISP3, and its role in the pathogenesis, disease diagnosis and prevention of osteoporosis is still unknown. Therefore, it is of great significance and application value to explore the role of CRISP3 in the pathogenesis, disease diagnosis and prevention of osteoporosis. Summary of the invention
[0004] In view of the deficiencies of the prior art, the present invention provides the use of CRISP3 gene as a molecular marker for osteoporosis.
[0005] The technical solution of the present invention is as follows:
[0006] The application of CRISP3 gene as a molecular marker for osteoporosis includes the application of CRISP3 gene detection reagent in the preparation of osteoporosis diagnosis products, bone metabolism disease diagnosis products or products for detecting osteogenic differentiation, bone formation and bone regeneration levels, or the application of CRISP3 gene inhibitors in the preparation of drugs for treating osteoporosis.
[0007] Use of a reagent for detecting the expression level of CRISP3 gene in preparing a product for diagnosing osteoporosis, wherein the nucleotide sequence of the CRISP3 gene is shown in SEQ NO.1.
[0008] The invention relates to the use of a reagent for detecting the expression level of the CRISP3 gene in the preparation of a diagnostic product for bone metabolic diseases, wherein the nucleotide sequence of the CRISP3 gene is shown in SEQ NO.1.
[0009] Preferably according to the present invention, the bone metabolic disease is osteoporosis, osteoarthritis, osteopenia or bone defect.
[0010] The invention relates to the use of a reagent for detecting the expression level of the CRISP3 gene in the preparation of a product for detecting osteogenic differentiation, bone formation and bone regeneration levels. The nucleotide sequence of the CRISP3 gene is shown in SEQ NO.1.
[0011] Preferably according to the present invention, the reagent for detecting the expression level of CRISP3 gene comprises a real-time fluorescence quantitative PCR detection reagent and a primer pair for specifically amplifying CRISP3 gene, which can detect the level of osteogenic differentiation, bone formation and bone regeneration.
[0012] According to the preferred embodiment of the present invention, the fluorescent quantitative PCR detection reagent includes a fluorescent dye and a real-time fluorescent quantitative PCR reaction solution; the real-time fluorescent quantitative PCR reaction solution includes deoxynucleoside triphosphates (dNTPs), MgCl 2 , TaqDNA polymerase and buffer, and the fluorescent dye is green fluorescent nucleic acid staining solution (SYBR).
[0013] Preferably, according to the present invention, the primer pair for specific amplification of CRISP3 is the primer pair shown in SEQ ID NO.2-3 or the primer pair shown in SEQ ID NO.4-5;
[0014] The specific primer sequences are as follows:
[0015] Upstream primer 1: 5′-TACAGACACAGTAACCCAAAGGA-3′ (SEQ ID NO. 2),
[0016] Downstream primer 1: 5′-TGGATTGCTTGTGACCATGAG-3′ (SEQ ID NO. 3).
[0017] Upstream primer 2: 5′-CCAAGCAGGCCATACCTAGC-3′ (SEQ ID NO. 4),
[0018] Downstream primer 2: 5′-CGTATTCCAGACGTTTACACCAA-3′ (SEQ ID NO. 5).
[0019] Preferably according to the present invention, the test sample of the osteoporosis diagnostic product is selected from serum, plasma, whole blood or bone tissue.
[0020] An osteoporosis diagnostic kit comprises a reagent for detecting the expression level of CRISP3 gene.
[0021] The invention relates to the use of a CRISP3 gene inhibitor in the preparation of a drug for treating osteoporosis, wherein the nucleotide sequence of the CRISP3 gene is shown in SEQ NO.1.
[0022] Preferably according to the present invention, the CRISP3 gene inhibitor is a gene inhibitor and / or a chemical inhibitor that inhibits CRISP3 gene expression.
[0023] Further preferably, the gene inhibitor is CRISP3 gene knockout virus sh-CRISP3, and CRISP3 gene knockout virus sh-CRISP3#1 and sh-CRISP3#2 are both double-stranded, and their nucleotide sequences are as shown in SEQ ID NO.8-9 and SEQ ID NO.10-11.
[0024] A drug for treating osteoporosis, comprising a CRISP3 gene inhibitor or a chemical inhibitor that reduces the expression level of the CRISP3 gene.
[0025] Beneficial effects:
[0026] 1. The present invention first discovered that the CRISP3 gene is significantly highly expressed in the serum of osteoporosis patients, the serum of osteoporosis mice and bone tissue, which indicates that the CRISP3 gene can be used as a molecular marker for diagnosing osteoporosis. The present invention also provides the use of a detection reagent for the CRISP3 gene in the preparation of an osteoporosis diagnosis product, mainly using the CRISP3 gene as a detection marker to prepare a corresponding fluorescent quantitative PCR detection kit for diagnosing osteoporosis. In addition, overexpression of the CRISP3 gene can inhibit the proliferation and osteogenesis of human osteoblasts, while knocking down CRISP3 can significantly improve the proliferation and osteogenesis of human osteoblasts, indicating that the CRISP3 gene inhibitor can be used to prepare drugs for the treatment of osteoporosis, which is of great significance in the research and development of drugs for the treatment of osteoporosis.
[0027] 2. The present invention discovered for the first time that the CRISP3 gene was significantly lowly expressed during osteogenic differentiation, which indicates that the CRISP3 gene can be used as a molecular marker for detecting osteogenic differentiation, bone formation or bone regeneration levels, and can be used to prepare diagnostic products for bone metabolic diseases such as osteoarthritis, osteopenia or bone defects, and can also be used to prepare products for detecting osteogenic differentiation, bone formation and bone regeneration levels.
[0028] 3. The present invention designs a primer pair for specific amplification of the CRISP3 gene, which can specifically and effectively detect the expression of CRISP3. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is a statistical chart showing the difference in CRISP3 gene expression levels in the serum of normal subjects and osteoporosis patients.
[0030] Figure 2 This is a statistical diagram of the difference in CRISP3 gene expression levels in serum and bone tissue between mice in the sham operation group and mice in the osteoporosis group;
[0031] In the figure, A is a statistical diagram showing the difference in CRISP3 gene expression levels in serum between sham-operated mice and osteoporosis mice, and B is a statistical diagram showing the difference in CRISP3 gene expression levels in bone tissue between sham-operated mice and osteoporosis mice.
[0032] Figure 3 This is a graph showing the specificity and sensitivity analysis results of CRISP3 gene expression levels in the diagnosis of osteoporosis.
[0033] Figure 4 This is a result diagram showing that overexpression of the CRISP3 gene inhibits the proliferation and osteogenesis of human osteoblasts;
[0034] In the figure, A is the transfection efficiency of overexpressing CRISP3 gene, B is the statistical result of CCK8 experiment, C is the result of Alizarin red staining, and D is the result of alkaline phosphatase staining.
[0035] Figure 5 This is the result of knocking down the CRISP3 gene to improve the proliferation and osteogenesis of human osteoblasts;
[0036] In the figure, A is the transfection efficiency of knocking down the CRISP3 gene, B is the statistical result of the CCK8 experiment, C is the result of Alizarin red staining, and D is the result of alkaline phosphatase staining.
[0037] Figure 6 This is a graph showing changes in CRISP3 gene expression during osteogenic differentiation of human bone marrow mesenchymal stem cells.
[0038] Figure 7 This is a graph showing changes in CRISP3 gene expression during osteogenic differentiation of mouse bone marrow mesenchymal stem cells. DETAILED DESCRIPTION
[0039] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.
[0040] The human osteoblasts used in the examples were purchased from Wuhan Pronocell Life Science Co., Ltd., China.
[0041] The human bone marrow mesenchymal stem cells used in the examples were purchased from China Saiye Biotechnology Co., Ltd.
[0042] The CRISP3 gene overexpression lentivirus (OE-CRISP3) and negative control (OE-NC) used in the examples were purchased from Shanghai GenePharma, China, and artificially synthesized by Shanghai GenePharma, China according to the sequence information.
[0043] The CRISP3 gene knockdown lentivirus (sh-CRISP3) and negative control (si-NC) used in the examples were purchased from Shanghai GenePharma, China, and artificially synthesized by Shanghai GenePharma, China according to the sequence information.
[0044] Example 1
[0045] After extensive research, the present invention found that the CRISP3 gene shown in SEQ NO.1 is abnormally highly expressed in the osteoporosis environment, and is related to the proliferation and osteogenesis of human osteoblasts. It is speculated that it may be used as a marker for the diagnosis and prognosis of osteoporosis, and its inhibitor can be used as a drug for the treatment of osteoporosis. In addition, the CRISP3 gene is abnormally low expressed during osteogenic differentiation, and it is speculated that it can be used as a molecular marker for diagnosing osteogenic differentiation, bone formation or bone regeneration levels.
[0046] Next, the expression of CRISP3 gene in the serum of osteoporosis patients was verified by real-time fluorescence quantitative PCR. This experiment was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University, and the collection of all clinical samples obtained the informed consent of the patients.
[0047] The specific implementation process is as follows:
[0048] (1) Whole blood from clinical osteoporosis patients (OP, n = 21) and normal volunteers (CTL, n = 21) was collected and centrifuged to obtain two groups of serum samples;
[0049] (2) Extraction of total RNA: Total RNA was extracted from the serum of the two groups of patients using the TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps described in the kit instructions, and the concentration and purity of RNA were detected using NanoDrop2000.
[0050] (3) Real-time quantitative PCR: A reverse transcription kit (ABI, USA) was used to synthesize cDNA according to the instructions of the kit. Power SYBR TM Green PCR premix (Thermo Fisher Scientific) and specific primers such as SEQ ID NO.2 and SEQ ID NO.3 were used for fluorescence quantitative PCR, with 18S as the internal reference and 2 -ΔΔCT Methods The relative expression levels of CRISP3 gene in the serum of osteoporosis group (OP) and control group (CTL) were calculated. Figure 1 shown.
[0051] The nucleotide sequences of the primer pairs are as follows:
[0052] Upstream primer 1: 5′-TACAGACACAGTAACCCAAAGGA-3′ (SEQ ID NO. 2),
[0053] Downstream primer 1: 5′-TGGATTGCTTGTGACCATGAG-3′ (SEQ ID NO. 3).
[0054] The reverse transcription PCR system and the real-time fluorescence quantitative PCR system are established according to the kits.
[0055] Reverse transcription PCR program: 95°C for 10 minutes; 37°C for 1 hour, 85°C for 5 minutes, 2 cycles; cool to 4°C.
[0056] Real-time fluorescence quantitative PCR program: 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds, for a total of 40 cycles; 72°C for 5 minutes.
[0057] The present invention firstly found that compared with the control group (CTL), the expression level of CRISP3 gene in the serum of patients with osteoporosis (OP group) was significantly increased, and the difference was statistically significant (such as Figure 1 ). The results indicate that the CRISP3 gene is significantly overexpressed in the serum of osteoporosis patients.
[0058] Example 2
[0059] The expression of CRISP3 gene in serum and bone tissue of osteoporotic mice was verified by real-time fluorescence quantitative PCR.
[0060] The specific implementation process is as follows:
[0061] (1) Construction of postmenopausal osteoporosis mouse model: 8-week-old female C57BL / 6J mice were selected, and after 1 week of adaptive feeding, intraperitoneal injection of anesthesia was performed; the anesthetized mice were fixed in a prone position on the operating table, and the hair and skin were shaved; after sufficient disinfection, a longitudinal incision was made on both sides of the spine, the muscles were separated, and the abdominal cavity was opened. The bilateral ovaries were found, the uterus and arteries were ligated, the bilateral ovaries were removed, and the abdomen was closed layer by layer. The mice with bilateral ovarian removal were the osteoporosis model group (OVX); the mice with the peri-ovarian fat tissue removed but the ovaries retained were the sham operation group (SHAM); 8 weeks later, the postmenopausal osteoporosis model group (OVX, n = 12) and sham operation group (SHAM, n = 12) mouse models were obtained;
[0062] (2) Extraction of total RNA: Total RNA was extracted from the serum and bone tissue of the two groups of mice using the TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps described in the kit instructions. The concentration and purity of RNA were detected using NanoDrop2000.
[0063] (3) Real-time quantitative PCR: A reverse transcription kit (ABI, USA) was used to synthesize cDNA according to the instructions of the kit. Power SYBR TM Green PCR premix (Thermo Fisher Scientific) and specific primers such as SEQ ID NO.4 and SEQ ID NO.5 were used for fluorescence quantitative PCR, with 18S as the internal reference and 2 -ΔΔCT Methods The relative expression levels of CRISP3 gene in serum of osteoporosis group (OVX) and sham operation group (SHAM) were calculated. Figure 2 shown.
[0064] The nucleotide sequences of the primer pairs are as follows:
[0065] Upstream primer 2: 5′-CCAAGCAGGCCATACCTAGC-3′ (SEQ ID NO. 4),
[0066] Downstream primer 2: 5′-CGTATTCCAGACGTTTACACCAA-3′ (SEQ ID NO. 5).
[0067] The reverse transcription PCR system and the real-time fluorescence quantitative PCR system are established according to the kits.
[0068] Reverse transcription PCR program: 95°C for 10 minutes; 37°C for 1 hour, 85°C for 5 minutes, 2 cycles; cool to 4°C.
[0069] Real-time fluorescence quantitative PCR program: 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds, for a total of 40 cycles; 72°C for 5 minutes.
[0070] The present invention first found that compared with the sham operation group (SHAM), the expression level of CRISP3 gene in the serum and bone tissue of osteoporosis group (OVX) mice was significantly increased, and the difference was statistically significant (such as Figure 2 ). This result indicates that the CRISP3 gene is significantly overexpressed in the serum of osteoporosis mice.
[0071] Example 3
[0072] High-throughput sequencing data of CRISP3 gene expression level in osteoporosis were obtained from the GEO database (GSE56815). The receiver operating characteristic curve (ROC) was used to evaluate the specificity and sensitivity of CRISP3 gene expression level in the diagnosis of osteoporosis. The test results are shown in Figure 2. Figure 3 shown.
[0073] Depend on Figure 3 It can be seen that the area under the ROC curve (AUC) is 0.686 (CI: 0.566-0.805). When the Youden index is the largest, its specificity is 75% and its sensitivity is 65%, which indicates that the expression level of the CRISP3 gene has a good efficacy in the clinical diagnosis of osteoporosis.
[0074] Example 4
[0075] Verification of the inhibition of human osteoblast proliferation and osteogenic capacity by overexpression of CRISP3 gene. The CRISP3 gene overexpression lentivirus (OE-CRISP3) and negative control (OE-NC) are both double-stranded, the OE-CRISP3 sequence is shown in SEQ ID NO.6-7, and OE-NC is an existing publicly available sequence.
[0076] The specific implementation process is as follows:
[0077] (1) Using Lipofectamine 2000 (Invitrogen, USA) reagent, CRISP3 gene overexpression lentivirus (OE-CRISP3) and negative control (OE-NC) were transfected into human osteoblasts, respectively. After 48 hours of transfection, human osteoblasts overexpressing CRISP3 gene and negative control human osteoblasts were obtained for subsequent experiments; the specific transfection process was carried out according to the instructions of PEI transfection reagent;
[0078] (2) Determination of transfection efficiency: Total RNA was extracted from human osteoblasts overexpressing CRISP3 and negative control human osteoblasts using the TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps in the kit instructions. The concentration and purity of RNA were detected using NanoDrop2000. Real-time quantitative PCR was used to detect the expression of CRISP3 gene in human osteoblasts overexpressing CRISP3 and negative control human osteoblasts (i.e., to detect the transfection efficiency of overexpressing CRISP3 gene). The specific implementation method was the same as step (3) of Example 1. The results are shown in FIG. Figure 4 As shown in A;
[0079] (3) Proliferation capacity assay: Human osteoblasts overexpressing the CRISP3 gene and negative control human osteoblasts were cultured at 5×10 3 The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured with DMEM medium (Hyclone, USA). 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance (OD) value at a wavelength of 450 nm was measured using an ELISA reader (TECAN, Switzerland). The results are shown in Table 1. Figure 4 As shown in B;
[0080] (4) Alizarin red staining: The human osteoblasts overexpressing the CRISP3 gene and the negative control human osteoblasts were rinsed with PBS buffer for 3 times, fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS buffer for 3 times, and stained with 2% alizarin red staining solution at room temperature for 20 min. Finally, the osteoblasts were rinsed with PBS buffer for 3 times, and the cells were observed and photographed under an optical microscope. The results are shown in the figure. Figure 4 As shown in C;
[0081] (5) Alkaline phosphatase staining: The human osteoblasts overexpressing CRISP3 and the negative control human osteoblasts were rinsed with PBS buffer three times, fixed with 4% paraformaldehyde at room temperature for 10 min, rinsed with PBS buffer three times, and stained with alkaline phosphatase staining solution at 37°C for 30 min. Finally, the osteoblasts were rinsed with PBS buffer three times, and the cells were observed and photographed under an optical microscope. The results are shown in Figure 2. Figure 4 As shown in D;
[0082] Depend on Figure 4 The real-time quantitative PCR experimental results in Figure A show that compared with the negative control, the expression of CRISP3 in human osteoblasts was significantly increased after overexpression of the CRISP3 gene.
[0083] Depend on Figure 4 The CCK8 experimental results in Figure B show that compared with the negative control, overexpression of the CRISP3 gene can significantly reduce the proliferation ability of human osteoblasts.
[0084] Depend on Figure 4 The results of Alizarin red staining in C and alkaline phosphatase staining in D show that compared with the negative control, overexpression of the CRISP3 gene can significantly inhibit the osteogenic ability of human osteoblasts.
[0085] Example 5
[0086] Knockdown of CRISP3 gene expression level improves the proliferation and osteogenesis of human osteoblasts. The CRISP3 gene knockdown virus (sh-CRISP3) and negative control (si-NC) are both double-stranded, the sh-CRISP3#1 sequence is shown in SEQ ID NO.8-9, the sh-CRISP3#2 sequence is shown in SEQ ID NO.10-11, and si-NC is an existing publicly available sequence.
[0087] The specific implementation process is as follows:
[0088] (1) Using Lipofectamine 2000 (Invitrogen, USA) reagent, CRISP3 gene knockdown lentivirus (sh-CRISP3) and negative control (si-NC) were transfected into human osteoblasts, respectively. After 48 hours of transfection, human osteoblasts with knockdown of CRISP3 gene expression and negative control human osteoblasts were obtained for subsequent experiments. The specific transfection process was carried out according to the instructions of PEI transfection reagent;
[0089] (2) Determination of transfection efficiency: Total RNA was extracted from human osteoblasts with knockdown of CRISP3 gene expression and negative control human osteoblasts using TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps in the kit instructions. The concentration and purity of RNA were detected using NanoDrop2000. Real-time quantitative PCR was used to detect the expression of CRISP3 gene in human osteoblasts with knockdown of CRISP3 gene expression and negative control human osteoblasts (i.e., to detect the transfection efficiency of knockdown of CRISP3 gene). The specific implementation method was the same as step (3) of Example 1. The results are shown in FIG. Figure 5 As shown in A;
[0090] (3) Proliferation capacity assay: Human osteoblasts with knockdown of CRISP3 gene expression and negative control human osteoblasts were cultured at 5×10 3 The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured with DMEM medium (Hyclone, USA). 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance (OD) value at a wavelength of 450 nm was measured using an ELISA reader (TECAN, Switzerland). The results are shown in Table 1. Figure 5 As shown in B;
[0091] (4) Alizarin red staining: The human osteoblasts with knockdown of CRISP3 gene expression and the negative control human osteoblasts were rinsed with PBS buffer for 3 times, fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS buffer for 3 times, and stained with 2% Alizarin red staining solution at room temperature for 20 min. Finally, the osteoblasts were rinsed with PBS buffer for 3 times, and the cells were observed and photographed under an optical microscope. The results are shown in the figure. Figure 5 As shown in C;
[0092] (5) Alkaline phosphatase staining: The human osteoblasts with knockdown of CRISP3 gene expression and the negative control human osteoblasts were rinsed with PBS buffer for 3 times, fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS buffer for 3 times, and stained with alkaline phosphatase staining solution at 37°C for 30 min. Finally, the osteoblasts were rinsed with PBS buffer for 3 times, and the cells were observed under an optical microscope and photographed. The results are shown in Figure 2. Figure 5 As shown in D;
[0093] Depend on Figure 5 The real-time quantitative PCR experimental results in Figure A show that compared with the negative control, the expression of CRISP3 in human osteoblasts was significantly reduced after knocking down the CRISP3 gene.
[0094] Depend on Figure 5 The CCK8 experimental results in Figure B show that, compared with the negative control, knocking down the expression level of the CRISP3 gene can significantly increase the proliferation ability of human osteoblasts.
[0095] From the results of Alizarin red staining in C and alkaline phosphatase staining in D in 5, it can be seen that compared with the negative control, knocking down the expression level of the CRISP3 gene can significantly improve the osteogenic ability of human osteoblasts.
[0096] Example 6
[0097] The expression changes of CRISP3 gene during the osteogenic differentiation of human bone marrow mesenchymal stem cells were verified by real-time fluorescence quantitative PCR.
[0098] The specific implementation process is as follows:
[0099] (1) Cultivation of human bone marrow mesenchymal stem cells: F12 culture medium (Hyclone, USA) containing 10% fetal bovine serum (BI, USA) and 1% penicillin-streptomycin (Biyuntian, China) was used for culturing at 37°C and 5% CO 2 Cultured in a cell culture incubator (Thermo, USA);
[0100] (2) Osteogenic differentiation: When the confluence of human bone marrow mesenchymal stem cells reached 80%, they were induced with osteogenic induction solution (10% fetal bovine serum, α-MEM culture medium, 100 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg / mL vitamin C, 100 U / mL penicillin, and 100 μg / mL streptomycin). The culture medium was replaced every 3 days. The human bone marrow mesenchymal stem cells were collected at different days of osteogenic differentiation after induction at 0, 3, 7, and 14 days.
[0101] (3) Extraction of total RNA: Total RNA was extracted from human bone marrow mesenchymal stem cells using the TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps described in the kit instructions. The concentration and purity of RNA were detected using NanoDrop2000.
[0102] (4) Real-time quantitative PCR: A reverse transcription kit (ABI, USA) was used to synthesize cDNA according to the instructions of the kit. Power SYBR TM Green PCR premix (Thermo Fisher Scientific) and specific primers such as SEQ ID NO.2 and SEQ ID NO.3 were used for fluorescence quantitative PCR, with 18S as the internal reference and 2 -ΔΔCT Methods The expression level of CRISP3 gene in human bone marrow mesenchymal stem cells at different days of osteogenic differentiation was calculated. Figure 6 shown.
[0103] The nucleotide sequences of the primer pairs are as follows:
[0104] Upstream primer 1: 5′-TACAGACACAGTAACCCAAAGGA-3′ (SEQ ID NO. 2),
[0105] Downstream primer 1: 5′-TGGATTGCTTGTGACCATGAG-3′ (SEQ ID NO. 3).
[0106] The reverse transcription PCR system and the real-time fluorescence quantitative PCR system are established according to the kits.
[0107] Reverse transcription PCR program: 95°C for 10 minutes; 37°C for 1 hour, 85°C for 5 minutes, 2 cycles; cool to 4°C.
[0108] Real-time fluorescence quantitative PCR program: 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds, for a total of 40 cycles; 72°C for 5 minutes.
[0109] The present invention firstly found that compared with the 0th day of osteogenic induction, the expression level of CRISP3 gene decreased significantly with the increase of osteogenic induction days of human bone marrow mesenchymal stem cells, and the difference was statistically significant (such as Figure 6 ). The results indicate that the CRISP3 gene is significantly decreased during the osteogenic differentiation of human bone marrow mesenchymal stem cells.
[0110] Example 7
[0111] The expression changes of CRISP3 gene during the osteogenic differentiation of mouse bone marrow mesenchymal stem cells were detected by real-time fluorescence quantitative PCR for verification.
[0112] The specific implementation process is as follows:
[0113] (1) Isolation and extraction of mouse bone marrow mesenchymal stem cells: 8-week-old female C57BL / 6J mice were obtained, and the bilateral tibia and femur of the mice were separated. The surrounding tissues were removed to expose the bone marrow cavity. The whole bone marrow was collected and placed in a centrifuge at 1000 r / min for 5 min. The cells were inoculated into a cell culture bottle and cultured in F12 culture medium (Hyclone, USA) containing 10% fetal bovine serum (BI, USA) and 1% penicillin-streptomycin (Biyuntian, China). The cells were placed in a 37°C, 5% CO 2 Cultured in a cell culture incubator (Thermo, USA);
[0114] (2) Osteogenic differentiation: When the confluence of mouse bone marrow mesenchymal stem cells reached 80%, they were induced with osteogenic induction solution (10% fetal bovine serum, α-MEM culture medium, 100 nM dexamethasone, 10 mM β-glycerophosphate, 50 μg / mL vitamin C, 100 U / mL penicillin, and 100 μg / mL streptomycin). The culture medium was replaced every 3 days. The bone marrow mesenchymal stem cells of mice were collected at different days of osteogenic differentiation after induction for 0, 3, 7, and 14 days.
[0115] (3) Extraction of total RNA: Total RNA was extracted from mouse bone marrow mesenchymal stem cells using the TRIzol kit (Invitrogen, USA). The extraction method was strictly carried out according to the steps described in the kit instructions. The concentration and purity of RNA were detected using NanoDrop2000.
[0116] (4) Real-time quantitative PCR: A reverse transcription kit (ABI, USA) was used to synthesize cDNA according to the instructions of the kit. Power SYBR TMGreen PCR premix (Thermo Fisher Scientific) and specific primers such as SEQ ID NO.4 and SEQ ID NO.5 were used for fluorescence quantitative PCR, with 18S as the internal reference and 2 -ΔΔCT Methods The expression level of CRISP3 gene in human bone marrow mesenchymal stem cells at different days of osteogenic differentiation was calculated. Figure 7 shown.
[0117] The nucleotide sequences of the primer pairs are as follows:
[0118] Upstream primer 2: 5′-CCAAGCAGGCCATACCTAGC-3′ (SEQ ID NO. 4),
[0119] Downstream primer 2: 5′-CGTATTCCAGACGTTTACACCAA-3′ (SEQ ID NO. 5).
[0120] The reverse transcription PCR system and the real-time fluorescence quantitative PCR system are established according to the kits.
[0121] Reverse transcription PCR program: 95°C for 10 minutes; 37°C for 1 hour, 85°C for 5 minutes, 2 cycles; cool to 4°C.
[0122] Real-time fluorescence quantitative PCR program: 95°C for 10 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds, for a total of 40 cycles; 72°C for 5 minutes.
[0123] The present invention firstly found that compared with the 0th day of osteogenic induction, the expression level of CRISP3 gene decreased significantly with the increase of the number of days of osteogenic induction of mouse bone marrow mesenchymal stem cells, and the difference was statistically significant (such as Figure 7 ). This result indicates that the CRISP3 gene is significantly reduced during the osteogenic differentiation of mouse bone marrow mesenchymal stem cells.
[0124] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. Use of a reagent for detecting CRISP3 gene expression level in the preparation of an osteoporosis diagnostic product, characterized in that: The nucleotide sequence of the CRISP3 gene is shown in SEQ NO.
1.
2. Use of a reagent for detecting CRISP3 gene expression level in the preparation of a bone metabolic disease diagnosis product, characterized in that: The nucleotide sequence of the CRISP3 gene is shown in SEQ NO.
1.
3. Use of a reagent for detecting CRISP3 gene expression level in the preparation of a product for detecting osteogenic differentiation, bone formation and bone regeneration level, characterized in that: The nucleotide sequence of the CRISP3 gene is shown in SEQ NO.
1.
4. Use of the reagent for detecting CRISP3 gene expression level according to claim 2 in preparing a diagnostic product for bone metabolic diseases, characterized in that: The bone metabolic disease is osteoporosis, osteoarthritis, osteopenia or bone defect.
5. The use according to any one of claims 1 to 3, characterized in that: The reagent for detecting the expression level of the CRISP3 gene comprises a real-time fluorescence quantitative PCR detection reagent and a primer pair for specifically amplifying the CRISP3 gene, and can detect the levels of osteogenic differentiation, bone formation and bone regeneration.
6. The use according to claim 5, characterized in that The method comprises a fluorescent quantitative PCR detection reagent, a fluorescent dye, and a real-time fluorescent quantitative PCR reaction solution; the real-time fluorescent quantitative PCR reaction solution comprises deoxynucleoside triphosphates, MgCl2, TaqDNA polymerase and a buffer solution, and the fluorescent dye is a green fluorescent nucleic acid staining solution; The primer pair for specifically amplifying CRISP3 is the primer pair shown in SEQ ID NOs. 2-3 or the primer pair shown in SEQ ID NOs. 4-5.
7. An osteoporosis diagnostic kit, characterized in that: The kit comprises a reagent for detecting the expression level of CRISP3 gene.
8. Use of a CRISP3 gene inhibitor in the preparation of a drug for treating osteoporosis, characterized in that: The nucleotide sequence of the CRISP3 gene is shown in SEQ NO.
1.
9. Use of the CRISP3 gene inhibitor according to claim 8 in the preparation of a drug for treating osteoporosis, characterized in that: The CRISP3 gene inhibitor is a gene inhibitor and / or a chemical inhibitor that inhibits CRISP3 gene expression; The gene inhibitor is a CRISP3 gene knockout virus sh-CRISP3, which is double-stranded and has a nucleotide sequence such as SEQ ID NOs. 8-9 or SEQ ID NOs. 10-11.
10. A drug for treating osteoporosis, characterized in that: The drugs include inhibitors of CRISP3 gene or chemical inhibitors that reduce the expression level of CRISP3 gene.