Application of citric acid in promoting chondrogenic induced differentiation of adipose-derived stem cells

Abstract

The invention relates to an application of citric acid in promoting chondrogenesis induced differentiation of adipose-derived stem cells, a culture solution for promoting chondrogenesis induced differentiation of adipose-derived stem cells and a method for promoting chondrogenesis induced differentiation of adipose-derived stem cells, and the method comprises the following steps: culturing adipose-derived stem cells in a culture medium for promoting chondrogenesis induced differentiation of adipose-derived stem cells, bone differentiation cells are obtained, and the bone differentiation cells are chondroblasts; the culture medium takes a chondrogenesis induction culture medium as a basic culture medium, the culture medium further comprises citric acid, and the concentration of the citric acid is 0.5-2mM. The culture solution containing citric acid is used for culturing the adipose-derived stem cells, so that the cells can obtain sufficient nutrition, cell proliferation and differentiation are facilitated, the biocompatibility is good, degradation is achieved, the cell survival time is prolonged, the cell morphology is maintained, the proliferation capacity of the adipose-derived stem cells is improved, the expression quantity of specific genes of cartilage cells is up-regulated, and the differentiation efficiency is improved.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to the application of citric acid in promoting the chondrogenic differentiation of adipose stem cells. Background Art

[0002] Cartilage is a connective tissue that provides cushioning and lubrication in joints. Its damage or degeneration can lead to pain and limited movement. Traditional cartilage repair methods have many limitations, such as donor site damage in autologous chondrocyte transplantation and immune rejection of allogeneic transplantation. Therefore, finding an effective cartilage repair strategy is an important direction of medical research.

[0003] Adipose-derived stem cells (ADSCs) have become a research hotspot in the field of regenerative medicine due to their wide sources, low immunogenicity, multidirectional differentiation potential and self-renewal ability. ADSCs can differentiate into a variety of cell lines under specific conditions, including osteoblasts, chondrocytes and adipocytes. However, the differentiation efficiency and quality of ADSCs into chondrocytes are affected by many factors, among which the selection of differentiation inducers is particularly critical. In existing technologies, commonly used differentiation inducers include dexamethasone, transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs). These inducers can promote the cartilage differentiation of ADSCs to a certain extent, but there are also some problems, such as low differentiation efficiency.

[0004] Citric acid, as a naturally occurring organic acid, is widely found in lemons and other citrus fruits. Citric acid is not only widely used in the food and pharmaceutical industries, but also has the biological function of regulating cell metabolism and promoting cell proliferation and differentiation. Recent studies have found that citric acid can promote the differentiation of various stem cells by affecting intracellular pH, regulating metabolic pathways and activating signal transduction. Citric acid can promote the differentiation of mesenchymal stem cells (MSCs) into osteoblasts by regulating intracellular calcium ion concentration and pH. In neural stem cells (NSCs), citric acid promotes their differentiation into neurons and glial cells by activating the mTOR signaling pathway. Citric acid has antioxidant properties, which can reduce the damage of oxidative stress to stem cells, and exerts anti-inflammatory effects by regulating the expression of inflammatory factors. Citric acid can also accelerate the formation of new blood vessels by promoting the proliferation and migration of endothelial cells. In terms of metabolism, citric acid regulates cell metabolism by participating in the tricarboxylic acid cycle (TCAcycle) and provides energy and precursor molecules for stem cell differentiation.

[0005] Although citric acid has made some progress in promoting stem cell differentiation, there are relatively few studies on its promotion of ADSCs chondrogenic differentiation. Therefore, studying the mechanism of citric acid promoting ADSCs chondrogenic differentiation and optimizing its application methods are of great scientific significance for the development of new cartilage repair strategies. How to increase the expression of chondrocyte-specific genes and further improve differentiation efficiency has also become a problem that needs to be solved in clinical practice. Summary of the invention

[0006] The present application proposes the use of citric acid in promoting the cartilage-inducing differentiation of adipose-derived stem cells, wherein the chemical structural formula of the citric acid is

[0007] The present application also proposes a culture medium for promoting the chondrogenic differentiation of adipose stem cells, wherein the culture medium contains citric acid, and the concentration of the citric acid in the culture medium is 0.5-2 mM.

[0008] The culture medium also contains BMP-6, TGF-β1 and IGF-1, the concentration of BMP-6 is 480-520 μg / L, the concentration of TGF-β1 is 9-11 μg / L, and the concentration of IGF-1 is 95-105 μg / L.

[0009] The culture solution further comprises 6-6.5 μg / L insulin, 48-52 nM / L ascorbic acid, 95-105 nM / L dexamethasone, 98-102 μg / mL penicillin, 98-102 μg / mL streptomycin, and a high-glucose DMEM culture medium containing 9-11% FBS.

[0010] The present application also proposes a method for promoting the chondrogenic differentiation of adipose-derived stem cells, comprising the following steps:

[0011] The adipose-derived stem cells are cultured in a culture medium that promotes the cartilage-induced differentiation of the adipose-derived stem cells to obtain bone-differentiated cells, wherein the bone-differentiated cells are chondroblasts;

[0012] The culture medium is based on chondrogenic induction culture medium, and the culture medium also includes citric acid, and the concentration of the citric acid is 0.5-2mM.

[0013] The basic culture medium is a high-glucose DMEM culture medium, and the culture medium also contains 6-6.5 μg / L insulin, 48-52 nM / L-ascorbic acid, 95-105 nM / L dexamethasone, 98-102 μg / mL penicillin, 98-102 μg / mL streptomycin, and 9-11% FBS.

[0014] The adipose stem cells are obtained by digesting, washing and subculturing adipose tissue obtained aseptically.

[0015] Among them, when the fusion degree of primary cells reaches 80%, subculture is carried out. During subculture, the culture medium needs to be replaced every 3 days. After culture to the third generation, flow cytometry is used for detection and analysis after counting to obtain adipose stem cells.

[0016] The present application utilizes a culture medium containing citric acid to culture adipose stem cells, which can enable the cells to obtain sufficient nutrition, is beneficial to cell proliferation and differentiation, has good biocompatibility, is degradable, prolongs cell survival time, maintains cell morphology, improves the proliferation ability of adipose stem cells, upregulates the expression of specific genes of chondrocytes, and improves differentiation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0018] Figure 1 This is a flow cytometric graph of adipose stem cell markers detected by flow cytometry in an embodiment of the present application;

[0019] Figure 2 is a bar graph of the relative expression of SOX9 during the chondrogenic differentiation process in the examples of the present application;

[0020] Figure 3 is a bar graph of the relative expression of COL2A1 during the chondrogenic differentiation process in the examples of the present application;

[0021] Figure 4 is a bar graph of the relative expression of ACAN during the chondrogenic differentiation process in the examples of the present application;

[0022] Figure 5 It is a western blot diagram of chondrogenic genes during chondrogenic differentiation in the examples of the present application. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0024] The present application example proposes the use of citric acid in promoting the cartilage-inducing differentiation of adipose-derived stem cells, wherein the chemical structure of the citric acid is

[0025] The present application embodiment first proposes a method for obtaining adipose-derived stem cells, which includes culturing and passaging adipose-derived stem cells.

[0026] Specifically, fat tissue samples were collected from the abdomen of volunteers and washed three times thoroughly with phosphate buffered saline (PBS) under sterile conditions to remove red blood cells. Subsequently, the tissue was sheared into small particles using a scalpel. A 0.1% collagenase solution equivalent to twice its volume was added to the shredded tissue, and then shaken at a constant temperature of 37°C for 2 to 3 hours to achieve tissue digestion.

[0027] After digestion, the digestion liquid was collected and centrifuged at 1500 rpm / min for 10 minutes. The supernatant was removed and the precipitate was resuspended in low-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS).

[0028] The resuspended cell solution was filtered through a 200-mesh fine sieve to remove residual tissue impurities, and the filtered cell solution was transferred to a new centrifuge tube, centrifuged again, and the supernatant was removed. The precipitate was resuspended in low-glucose DMEM medium containing 10% FBS, and the cells were placed at 37°C and 5% CO. 2 The cells were cultured in an incubator and the first medium change was performed after 5 days.

[0029] When the fusion degree of primary cells reaches 80%, digestion and passage are performed. Thereafter, the culture medium is replaced every 3 days, and the culture is continued to the third generation (P3), and the growth and morphology of the cells are further observed under an optical microscope. After counting the P3 generation human adipose stem cells, they are placed in a flow cytometer tube, and monoclonal antibodies against human cells are added: CD29, CD34, CD44, CD105, CD73 and HLA-DR, respectively, and then incubated at 4°C in the dark for 30 minutes. Wash twice with PBS, centrifuge at 1000rpm for 3 minutes each time, and finally use a flow cytometer for detection and analysis.

[0030] like Figure 1 As shown, Figure 1 This is a flow cytometric graph of adipose stem cell markers detected by flow cytometry in the embodiment of the present application. The results show that ADSCs are strongly positive for CD73 (98.48%), CD105 (96.68%), and CD90 (98.49%), but do not express CD45 and CD-HLA. The above results indicate that the obtained cells are adipose stem cells.

[0031] In this example, the obtained adipose stem cells were divided into 4 groups, and appropriate amount of culture medium (8×10 5 -1×10 6 Each cell requires about 1 ml of culture medium) and is placed at 37°C and 5% CO 2 The cells were cultured in an incubator with the medium changed every 2 days and cultured continuously for 2 weeks before testing.

[0032] The addition of culture medium in each group is as follows:

[0033] Control group A: incomplete chondrogenic induction culture medium (without induction agent);

[0034] Experimental group B: incomplete chondrogenic induction medium + BMP 6 + TGF β1 + IGF 1;

[0035] Experimental group C: incomplete chondrogenic induction medium + citric acid + BMP6 + TGFβ1 + IGF1

[0036] The components and contents of the incomplete chondrogenic induction culture medium are: 6.25 μg / L insulin, 50 nM / L ascorbic acid, 100 nM / L dexamethasone, 100 μg / mL penicillin, 100 μg / mL streptomycin, and 10% FBS high-glucose DMEM culture medium. 10% FBS means that the volume percentage of FBS is 10%. The final concentrations of the inducers in experimental group B and experimental group C are: BMP-6 (500 μg / L), TGF-β1 (10 μg / L), and IGF-1 (100 μg / L), respectively. Among them, BMP-6 is bone morphogenetic protein 6, encoded by the BMP6 gene, and is a member of the TGF-β superfamily; TGF-β1 is one of the important members of the cytokine transforming growth factor β superfamily and is also a secretory protein; IGF-1 is the abbreviation of insulin growth factor-1.

[0037] The fat stem cells induced by the culture medium of the control group A, experimental group B and experimental group C will be further tested.

[0038] Real-time fluorescence quantitative PCR test

[0039] Total RNA of control group A, experimental group B and experimental group C was extracted, and RNA concentration was tested. The RNA extraction steps are as follows: take out the sample to be tested, place it for a period of time to melt, add chloroform to shake evenly and then let it stand, centrifuge to obtain RNA, use a pipette to gently shake to fully dissolve the RNA, and then immediately measure its concentration and perform reverse transcription reaction. The specific processing time, centrifugation processing parameters, etc. can be selected according to the experimental conditions, and will not be elaborated here.

[0040] RNA concentration determination steps: (1) Select the nucleic acid ID on the main interface, and the computer and instrument will automatically realize online; (2) Dissolve 1μL RNA in the solution (RNase-free water) on the test bench, click test, and then select the sample type in the upper right corner and select RNA-40; (3) Enter the sample name in the sample ID position; (4) Mix the samples, extract 1μL of the spot on the test bench, and measure again. (5) Save the data according to the report.

[0041] Reverse transcription reaction steps:

[0042] (1) Prepare RT reaction solution on ice

[0043]

[0044] (2) Reverse transcription reaction conditions:

[0045]

[0046] (3) RT-PCR Real Time PCR reaction system (prepared on ice), related primers are shown in Table 1.

[0047]

[0048] Reaction conditions

[0049]

[0050] Table 1. Primer sequences for chondrogenic gene fluorescence quantitative PCR

[0051]

[0052] like Figure 2-4 As shown, the experimental results showed that the relative expression levels of SOX9, COL2A1 and ACAN in the control group A, experimental group B and experimental group C increased with time, the expression of SOX9, COL2A1, COL10A1 and ACAN genes induced to differentiate into cartilage were upregulated, and the expression levels of each gene in the experimental group C were significantly higher than those in the control group A and experimental group B.

[0053] Figure 2 is a bar graph of the relative expression of SOX9 during the chondrogenic differentiation process in the present application example. Figure 2 As shown, on the 14th day of adipose stem cell treatment, the relative expression level of the marker gene SOX9 gene of the chondrocytes induced to differentiate by the method of the present application (marked in the figure: experimental group C) was 50.9 times that of the control group A and 2.6 times that of the induced group (marked in the figure: experimental group B).

[0054] Figure 3is a bar graph of the relative expression of COL2A1 during the chondrogenic differentiation process in the examples of the present application, such as Figure 3 As shown, the relative expression level of the marker gene COL2A1 gene of the chondrocytes induced to differentiate by the method of the present application (marked in the figure: experimental group C) is 200.3 times that of the control group A and 3.4 times that of the induced group (marked in the figure: experimental group B).

[0055] Figure 4 is a bar graph of the relative expression of ACAN during the chondrogenic differentiation process in the present application example. Figure 4 As shown, the relative expression level of the marker gene ACAN of chondrocytes induced to differentiate by the method of the present application is 100.3 times that of the control group A and 2.5 times that of the induced group (marked in the figure: experimental group B).

[0056] Experiments have shown that adipose stem cells are successfully induced to differentiate into chondrocytes, and citric acid combined with BMP-6, TGF-β1, and IGF-1 has a stronger ability to induce cartilage formation. The desired chondrocytes can be obtained by culturing for 14 days using the method of the present application.

[0057] Figure 5 is a western blot image of chondrogenic genes during chondrogenic differentiation in the embodiment of the present application, such as Figure 5 As shown, compared with the control group, experimental group B was simply chondrogenic induction. After 14 days of chondrogenic induction, the expression of chondrocyte marker genes SOX9, COL2A1, and ACAN of adipose stem cells was enhanced. Experimental group C added citric acid on the basis of chondrogenic induction, and the expression of chondrocyte marker genes SOX9, COL2A1, and ACAN was further increased. The above results further indicate that citric acid combined with BMP-6, TGF-β1, and IGF-1 has a stronger ability to induce chondrogenic differentiation of adipose stem cells.

[0058] The present application utilizes an incomplete chondrogenesis induction culture medium containing citric acid and transforming growth factor-β superfamily members (TGF-β1, IGF-1, BMP-6). The combined use of citric acid and transforming growth factor-β superfamily members has a synergistic effect, can upregulate Sox-9 gene expression, promote cell proliferation and improve the efficiency of induced differentiation. The use of a culture medium containing citric acid can enable cells to obtain sufficient nutrition, is beneficial to cell proliferation and differentiation, has good biocompatibility, is degradable, prolongs cell survival time, and maintains cell morphology.

[0059] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0060] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0061] Finally, it should be noted that, in this application, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article including the elements.

[0062] Specific examples are used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core idea of ​​this application. At the same time, for those skilled in the art, according to the idea of ​​this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on this application.

Claims

1. Application of citric acid in promoting cartilage differentiation of adipose-derived stem cells, including: The chemical structural formula of the citric acid is 2. A culture medium for promoting cartilage differentiation of adipose-derived stem cells, characterized in that: The culture solution contains citric acid, and the concentration of the citric acid in the culture solution is 0.5-2 mM.

3. The culture medium for promoting chondrogenic differentiation of adipose-derived stem cells according to claim 2, characterized in that: The culture solution also contains BMP-6, TGF-β1 and IGF-1. The concentration of BMP-6 is 480-520 μg / L, the concentration of TGF-β1 is 9-11 μg / L and the concentration of IGF-1 is 95-105 μg / L.

4. The culture medium for promoting chondrogenic differentiation of adipose-derived stem cells according to claim 3, characterized in that: The culture solution also includes 6-6.5 μg / L insulin, 48-52 nM / L ascorbic acid, 95-105 nM / L dexamethasone, 98-102 μg / mL penicillin, 98-102 μg / mL streptomycin, and a high-glucose DMEM culture medium containing 9-11% FBS.

5. A method for promoting cartilage differentiation of adipose-derived stem cells, characterized in that: The following steps are involved: The adipose-derived stem cells are cultured in a culture medium that promotes the cartilage-induced differentiation of the adipose-derived stem cells to obtain bone-differentiated cells, wherein the bone-differentiated cells are chondroblasts; The culture medium is based on chondrogenic induction culture medium, and the culture medium also includes citric acid, and the concentration of the citric acid is 0.5-2mM.

6. The method for promoting chondrogenic differentiation of adipose-derived stem cells according to claim 5, characterized in that: The basic culture medium is a high-glucose DMEM culture medium, and the culture medium also contains 6-6.5 μg / L insulin, 48-52 nM / L-ascorbic acid, 95-105 nM / L dexamethasone, 98-102 μg / mL penicillin, 98-102 μg / mL streptomycin, and 9-11% FBS.

7. The method for promoting cartilage differentiation of adipose-derived stem cells according to claim 5, characterized in that: The adipose stem cells are obtained by digesting, washing and subculturing adipose tissue obtained aseptically.

8. The method for promoting chondrogenic differentiation of adipose-derived stem cells according to claim 7, characterized in that: When the fusion degree of primary cells reaches 80%, subculture is performed. During subculture, the culture medium needs to be replaced every 3 days. After culture to the third generation, the cells are counted and analyzed by flow cytometry to obtain adipose stem cells.

Images