CN109722413B - Osteogenesis inducer and application thereof in osteogenic induced differentiation of adipose mesenchymal stem cells - Google Patents
Osteogenesis inducer and application thereof in osteogenic induced differentiation of adipose mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses an osteogenesis inducer and application thereof in osteogenesis induced differentiation of adipose-derived mesenchymal stem cells. RUNX2 is a key regulatory factor in the osteogenic differentiation process, can regulate the transcription of a plurality of genes, and the deletion of RUNX2 can completely inhibit the differentiation of osteoblasts. The invention discovers that the angelicin can obviously improve the expression level of the adipose mesenchymal stem cell osteogenic differentiation regulating factor RUNX2, and is an effective RUNX2 expression activator; the angelica dahurica is acted on adipose mesenchymal stem cells, and the alizarin red staining result shows that the angelica dahurica can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelica dahurica has obvious induction effect on osteogenic differentiation of ADSCs.
Description
Technical Field
The invention belongs to the field of biology, relates to induction culture of adipose-derived mesenchymal stem cells, and particularly relates to an osteogenic inducer and application thereof in osteogenic induced differentiation of adipose-derived mesenchymal stem cells.
Background
Stem cells are the original source of various tissues and organs of the body, have the ability to proliferate and differentiate, and play an important role in cell transplantation, tissue engineering and regenerative medicine with the rapid development of medical technology. Bone tissue engineering, in particular, is the most rapidly developing discipline in the field of tissue engineering, and stem cells are the main seed cells for tissue engineering.
Adipose-derived stem cells (ADSCs) are derived from the mesoderm during embryonic development and are a class of adult stem cells with self-renewal and multi-differentiation potential. It can induce and differentiate into a plurality of cells such as fat, bone, cartilage, islet beta cells and cardiac muscle under specific conditions, and has low immunogenicity, so that it is widely used in clinic.
The early expression protein BSP, the late expression protein OCN, the nuclear machinery protein SATB2 and the core protein RUNX2 are important regulating factors in the bone differentiation process, and the expression level can reflect the bone forming capacity of cells. BSP is a bone-marker protein, a sulfated phosphoprotein and is highly glycosylated, and can activate osteoblasts and promote bone mineralization. The OCN is generally matrix protein only expressed by osteoblasts, has the function of regulating matrix calcification, and the concentration of the matrix protein can be used as a marker of osteoblast activity. SATB2 is a member of a newly discovered family of specific rich sequence binding proteins that bind to nuclear matrix binding domains and activate gene transcription processes in a dependent manner, positively regulating the expression of a variety of osteoblast specific genes such as BSP and OCN. RUNX2 is a key regulatory factor in the osteogenic differentiation process, can regulate the transcription of a plurality of genes, and in a mouse with RUNX2 deletion, the differentiation of osteoblasts is completely inhibited, and osteogenesis in the periosteum and chondrogenesis do not occur.
Angelicin (angelicin) is a natural compound separated from plants such as radix angelicae pubescentis, angelica dahurica and the like. Researches find that the radix angelicae pubescentis has a certain promotion effect on bone growth. However, the application of the angelicin in inducing the osteogenic differentiation of the adipose mesenchymal stem cells in bone tissue engineering is not found, and the application of the angelicin in regulating and controlling RUNX2 is not reported.
Disclosure of Invention
The invention aims to provide an osteogenesis inducer and application thereof in osteogenesis induced differentiation of adipose tissue-derived mesenchymal stem cells, wherein the inducer is angelica dahurica, and the osteogenesis differentiation of the adipose tissue-derived mesenchymal stem cells is induced by regulating RUNX 2.
The above object of the present invention is achieved by the following technical solutions:
the application of an osteogenic differentiation regulating factor RUNX2 expression activator in osteogenic induced differentiation of adipose tissue-derived mesenchymal stem cells. RUNX2 is a key regulatory factor in the osteogenic differentiation process, can regulate the transcription of a plurality of genes, and the deletion of RUNX2 can completely inhibit the osteoblastic differentiation. Therefore, the osteogenic differentiation regulating factor RUNX2 expression activator can be used for inducing the osteogenic induced differentiation of the adipose mesenchymal stem cells.
Preferably, the osteogenic differentiation regulating factor RUNX2 expression activator is angelicin. In one embodiment, the angelicin can significantly increase the expression level of the osteogenic differentiation regulatory factor RUNX2, and is an effective RUNX2 expression activator.
Use of angelicin as an inducer of osteogenic differentiation of stem cells. In a specific embodiment, the angelicin is acted on the adipose-derived mesenchymal stem cells, and the alizarin red staining result shows that the angelicin can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelicin has an obvious induction effect on the osteogenic differentiation of ADSCs.
Use of angelicin as an inducer of osteogenic differentiation of adipose-derived mesenchymal stem cells. In a specific embodiment, the angelicin is acted on the adipose-derived mesenchymal stem cells, and the alizarin red staining result shows that the angelicin can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelicin has an obvious induction effect on the osteogenic differentiation of ADSCs.
Application of angelicin in inducing osteogenic differentiation of stem cells in vitro is provided. In a specific embodiment, the tigogenin acts on adipose-derived mesenchymal stem cells, and the alizarin red staining result shows that the tigogenin can obviously improve the calcium mineralization nodule degree of cells, which indicates that the tigogenin has an obvious induction effect on the osteogenic differentiation of ADSCs.
Application of angelicin in vitro induction of adipose mesenchymal stem cell osteogenic differentiation is provided. In a specific embodiment, the angelicin is acted on the adipose-derived mesenchymal stem cells, and the alizarin red staining result shows that the angelicin can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelicin has an obvious induction effect on the osteogenic differentiation of ADSCs.
Application of angelicin in preparation of adipose-derived mesenchymal stem cell osteogenic differentiation induction culture medium. Because the angelica dahurica element has obvious induction effect on osteogenic differentiation of ADSCs, the angelica dahurica element can be used for preparing an adipose mesenchymal stem cell osteogenic differentiation induction culture medium.
Has the advantages that:
RUNX2 is a key regulatory factor in the osteogenic differentiation process, can regulate the transcription of a plurality of genes, and the deletion of RUNX2 can completely inhibit the differentiation of osteoblasts. The invention discovers that the angelicin can obviously improve the expression level of the adipose mesenchymal stem cell osteogenic differentiation regulating factor RUNX2, and is an effective RUNX2 expression activator; the angelica dahurica is acted on adipose mesenchymal stem cells, and the alizarin red staining result shows that the angelica dahurica can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelica dahurica has obvious induction effect on osteogenic differentiation of ADSCs.
Drawings
FIG. 1 shows the detection result of the surface markers of ADSCs by immunocytochemistry (flow assay), wherein: a is a picture of CD29-FITC, B is a picture of CD44-FITC, C is a picture of CD14-FITC, and D is a picture of CD 45-FITC; the result shows that the CD29 and the CD44 are strongly expressed, and the CD14 and the CD45 are lowly expressed, thereby conforming to the immunological characteristics of ADSCs.
FIG. 2 shows the results of alizarin red staining. Compared with a blank control group, the positive control group has obvious calcium mineralized nodules; compared with the positive control group, the calcium mineralization nodule degree of the angelica dahurica group is obviously higher. The result shows that the angelica dahurica has obvious induction effect on the osteogenic differentiation of ADSCs.
FIG. 3 shows the Western blot results. Compared with a blank control group, the positive control composition bone differentiation regulating factor RUNX2 has obviously increased expression; compared with the positive control group, the expression of the angelica dahurica constituting the bone differentiation regulating factor RUNX2 is higher. The result shows that the angelica chuanxiong dehurigen induces the osteogenic differentiation of ADSCs by regulating the expression of an osteogenic differentiation regulating factor RUNX 2.
Detailed Description
The following is a detailed description of the essential aspects of the invention, but the scope of the invention is not limited thereto.
First, experimental material
The cleaning-grade rabbits are female and male, the weight is about 1.2 kg, and the rabbits are reserved after 1 week of adaptive feeding.
Fetal bovine serum and DMEM medium are products of Gibo company; CD14 is CALTAG, CD29 and CD44 are CHEMCON, and CD45 is ANTIGENIX. The RUNX2 monoclonal antibody and the secondary antibody are Abcam products. The angelica dahurica (angelin) is a product of Shidande company, and the HPLC purity is more than or equal to 98%.
Second, Experimental methods
1. Separation preparation, culture and phenotype identification of ADSCs
Anesthetizing rabbit, taking out adipose tissue at two side ventral ditches, shearing, homogenizing, digesting with 0.075% collagenase I, sieving with 300 mesh sieve, centrifuging at 4 deg.C for 5min at 1200r/min, discarding upper layer fat and supernatant, washing precipitate with PBS 3 times, washing with DMEM medium (complete medium) containing 10% fetal calf serum, centrifuging, collecting precipitate, inoculating into culture bottle, culturing at 37 deg.C and 5% CO 2 Culturing for 24h under saturated humidity condition, discarding original culture solution, washing with PBS for 2 times to remove non-adherent residue, and addingAnd (4) continuously culturing in the complete culture medium, changing the culture solution for 1 time every 3-4 days, and digesting with 0.25% trypsin for subculture when the bottle wall is basically full of primary culture cells.
Taking 3 rd generation ADSCs, and detecting the surface markers of the ADSCs by an immunocytochemistry method: antibodies CD29, CD44, CD14 and CD45 were added and cell surface specific antigens were detected using flow cytometry as specified.
2. Angelica dahurica extract inducing effect on osteogenic differentiation of ADSCs
Collecting 3 rd generation ADSCs, digesting with 0.25% trypsin to obtain product with concentration of 1 × 10 6 Inoculating the cell suspension/mL into 24-well plate, randomly dividing into positive control group, tigogenin group and blank control group, and changing the positive control group into osteogenic inducing solution (containing 10% fetal calf serum, 10mmol/L beta-sodium glycerophosphate) after 24 hr -7 DMEM culture medium containing dexamethasone and L-ascorbic acid 50mg/L in mol/L), adding 5 μ M of radix Angelicae Dahuricae in the radix Angelicae Dahuricae group on the basis of bone inducing liquid composed of positive control, adding complete culture medium in the blank control group, changing inducing liquid at intervals of 2d, inducing for 14d, staining with alizarin red, observing under inverted microscope, and taking picture. Alizarin red staining method as follows:
preparing 1% alizarin red: 0.1mol/L tris-HCl 100ml is added with 0.1g alizarin red S and stored at 4 ℃;
secondly, sucking out the culture medium, washing the culture medium for 3 times by PBS, fixing the culture medium by 4% paraformaldehyde for 30min, sucking out the fixing solution, washing the culture medium for 2 times by deionized water, dyeing alizarin red for 5min, washing the culture medium for 2 times by deionized water, observing the culture medium under a microscope and taking pictures.
3. Mechanism of action of angelicin for inducing osteogenic differentiation of ADSCs
Collecting 3 rd generation ADSCs, digesting with 0.25% trypsin to obtain product with concentration of 1 × 10 6 Inoculating the cell suspension/mL into 24-well plate, randomly dividing into positive control group, tigogenin group and blank control group, and changing the positive control group into osteogenic inducing solution (containing 10% fetal calf serum, 10mmol/L beta-sodium glycerophosphate) after 24 hr -7 DMEM medium containing dexamethasone and L-ascorbic acid 50 mg/L), and the radix Angelicae Dahuricae group is added with 5 μ M radix Angelicae Dahuricae on the basis of bone inducing liquid composed of positive control, and the blank control group is only addedAnd (3) culturing the whole culture medium for 72 hours, collecting cells, and determining the expression of the osteogenic differentiation regulating factor RUNX2 in each group of cells by using a western blot method.
The Western blot operation method comprises the following steps:
cellular proteins were extracted on ice using RIPA lysis buffer and transferred to polyvinylidene fluoride membranes by electrophoresis, the membranes were then blocked in 50g/L skim milk powder in TBS for 1h, 1: 200 diluted RUNX2 mab and 1: 2000 diluted β -actin mab were added and incubated overnight at 4 ℃. Washing the membrane for 3 times, adding a horseradish peroxidase-labeled secondary antibody diluted by 1: 1000, incubating for 2h at room temperature, developing by using a chemiluminescence ECL reagent, and recording and imaging by using a digital imaging system.
Third, experimental results
1. Phenotypic identification results of ADSCs
A significant feature of ADSCs is that they are not homogeneous populations. Many studies have attempted to analyze the phenotype of ASCs by flow cytometry using cell surface markers, but the only single marker has not been identified. The ADSCs positively express typical mesenchymal markers such as CD13, CD29, CD44, CD63, CD73, CD90 and CD105, and the ADSCs are negative for hematopoietic antigens such as CD14, CD31, CD45 and CD144 expression.
The phenotype identification result of the ADSCs is shown in figure 1, and the flow identification result shows that the ADSCs have strong CD29 and CD44 expression, and low CD14 and CD45 expression, and accords with the immunological characteristics of adipose mesenchymal stem cells.
2. Angelica dahurica extract inducing effect on osteogenic differentiation of ADSCs
Alizarin red staining pattern is shown in figure 2. Compared with the blank control group, the positive control group has obvious calcium mineralized nodules; compared with the positive control group, the calcium mineralization nodule degree of the angelica dahurica group is obviously higher. The result shows that the angelica chuanxiongoides extract has obvious induction effect on the osteogenic differentiation of ADSCs.
3. Mechanism of action of chuanbaizhi element for inducing osteogenic differentiation of ADSCs
Western blot is shown in FIG. 3. Compared with a blank control group, the expression of the bone differentiation regulating factor RUNX2 formed by the positive control group is obviously increased; compared with the positive control group, the expression of the bone differentiation regulating factor RUNX2 composed of the angelica dahurica is higher. The result shows that the angelica chuanxiong dehurigen induces the osteogenic differentiation of ADSCs by regulating the expression of an osteogenic differentiation regulating factor RUNX 2.
RUNX2 is a key regulatory factor in the osteogenic differentiation process, can regulate the transcription of a plurality of genes, and the deletion of RUNX2 can completely inhibit the osteoblastic differentiation. The invention discovers that the angelicin can obviously improve the expression level of the adipose mesenchymal stem cell osteogenic differentiation regulating factor RUNX2, and is an effective RUNX2 expression activator; the angelica dahurica is acted on the adipose mesenchymal stem cells, and the result of alizarin red staining shows that the angelica dahurica can obviously improve the calcium mineralization nodule degree of cells, which indicates that the angelica dahurica has obvious induction effect on osteogenic differentiation of ADSCs.
Because the angelica dahurica element has obvious induction effect on osteogenic differentiation of ADSCs, the angelica dahurica element can be used for preparing an adipose mesenchymal stem cell osteogenic differentiation induction culture medium.
The above embodiments are intended to specifically describe the substance of the present invention, and should not limit the scope of the present invention to the specific embodiments.
Claims (2)
1. Application of angelicin in-vitro induction of adipose mesenchymal stem cell osteogenic differentiation.
2. Application of angelicin in preparation of adipose-derived mesenchymal stem cell osteogenic differentiation induction culture medium.
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| CN104873499A (en) * | 2015-06-11 | 2015-09-02 | 中国医学科学院医药生物技术研究所 | Application of compound for up-regulating transcription activity of Runx2 (Runt-related transcription factor 2) in prevention and treatment of osteoporosis |
| CN105695399A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Adipose-derived mesenchymal stem cell osteogenic induction composition and osteogenic induction method thereof |
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| CN104873499A (en) * | 2015-06-11 | 2015-09-02 | 中国医学科学院医药生物技术研究所 | Application of compound for up-regulating transcription activity of Runx2 (Runt-related transcription factor 2) in prevention and treatment of osteoporosis |
| CN105695399A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Adipose-derived mesenchymal stem cell osteogenic induction composition and osteogenic induction method thereof |
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| 异补骨脂素促进大鼠骨髓间充质干细胞向成骨细胞分化并抑制其向脂肪细胞分化;史春民等;《南京医科大学学报（自然科学版）》;20110531;第31 卷(第5 期);第606-611页 * |
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