CN104800891B - A kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, preparation method and applications - Google Patents

Abstract

The invention provides an extracellular matrix biological material for enhancing the biological antioxidant function of in vitro cultured mesenchymal stem cells, a preparation method and its application. The preparation steps of the extracellular matrix biological material are as follows: umbilical cord mesenchymal stem cells are cultured in a complete culture medium In medium, when the density is ≥90%, add complete culture medium to culture for 7‑10 days, change the medium every 3 days, and then add decellularized medium to construct extracellular matrix biomaterials. The extracellular matrix biological material of the present invention can enhance the biological antioxidant function of mesenchymal stem cells cultured in vitro.

Description

An extracellular matrix that enhances the biological antioxidant function of mesenchymal stem cells cultured in vitro Quality biological materials, preparation methods and applications

technical field

The invention belongs to the field of biomedicine, and in particular relates to an extracellular matrix biological material for enhancing the biological antioxidant function of in vitro cultured mesenchymal stem cells, a preparation method and an application thereof.

Background technique

Mesenchymal stem cells (MSCs) are adult stem cells responsible for tissue regeneration and repair. They were originally discovered in the bone marrow. Due to their potential for regeneration and immune regulation, they have attracted widespread attention in various fields. Human umbilical cord mesenchymal stem cells, especially those derived from Wharton's jelly (the mucin-like connective tissue surrounding two umbilical arteries and one umbilical vein in the umbilical cord) are considered ideal seed cells for cell therapy and regenerative medicine. The umbilical cord is an extraembryonic tissue, so umbilical cord mesenchymal stem cells have dual characteristics of embryonic stem cells and adult stem cells, and their self-renewal and multidirectional differentiation potentials are significantly better than bone marrow mesenchymal stem cells.

There is increasing evidence that reactive oxygen species (ROS) are essential for cell proliferation, differentiation, death, homeostasis of the matrix environment, redox signaling mechanisms, etc., but abnormal levels of ROS in cells can lead to DNA, protein , Irreversible damage to lipids, and eventually the cells move towards the fate of aging and death.

Although the application of MSCs in regenerative medicine has received extensive attention, they are inevitably exposed to considerable oxidative stress during in vitro expansion and in vivo transplantation, causing ROS (such as hydrogen peroxide, hydroxyl radicals, superoxide anion free radical) exceeded the standard. Increased _H2O2 levels in long _- term cultured MSCs in vitro lead to cellular senescence and inhibition of proliferation. Chondrocytes or neutrophils and macrophages in patients with osteoarthritis and rheumatoid arthritis produce abnormal levels of ROS, affecting the regenerative potential of MSCs. Studies have shown that the accumulation of ROS is caused by exposure to inflammatory cytokines, thereby inhibiting the cell proliferation and multilineage differentiation potential of stem cells.

The balance between ROS generation and elimination is critical for cell survival, proliferation, and differentiation. Superoxide dismutase (SOD), including isozymes containing copper, zinc (SOD1 and SOD2), and manganese (SOD2), protects MSCs from reactive oxygen species generated by H2O2 catalyzed by _{superoxide anion} . Oxidative stress can abolish SOD2-induced decreased chondrogenic differentiation ability and increased expression of matrix metalloproteinases in human synovium-derived mesenchymal stem cells. In addition, the H ₂ O ₂ produced by SOD is neutralized by catalase. A large number of previous studies have shown that increasing the activity of catalase in MSCs is beneficial to alleviate the apoptosis induced by H ₂ O ₂ .

The extracellular matrix (ECM) components derived from MSCs contain type I and type III collagens, fibronectin, and laminin, which can serve as a microenvironment for in vitro culture systems, promote MSC expansion, and guide MSCs to differentiate in specific directions. Decellularized ECM can not only simulate the extracellular microenvironment of stem cells in vivo, but also regulate the biological behavior of growth factors and hormones. This culture method using decellularized ECM can prevent cell replicative senescence and enhance the redifferentiation ability of terminally differentiated cells, such as chondrocytes and nucleus pulposus cells. Recent studies have shown that decellularized ECM can improve the resistance of BMSCs to _{H2O2 -} induced oxidative stress and cell cycle arrest. However, little is known about the effect of ECM on the antioxidant system in umbilical cord mesenchymal stem cells.

Contents of the invention

Technical problem to be solved: Aiming at the disadvantage that existing MSCs are inevitably exposed to considerable oxidative stress during in vitro expansion and in vivo transplantation, resulting in excessive ROS, the present invention provides an enhanced in vitro cultured mesenchymal Extracellular matrix biomaterials with anti-oxidation function of stem cells, preparation method and application, extracellular matrix biomaterials are prepared by secreting matrix of umbilical cord mesenchymal stem cells, which not only have the characteristics of various protein components, but also can be used for mesenchymal stem cells The in vitro expansion has the advantages of good biocompatibility, high proliferation efficiency, and significantly reduced intracellular active oxygen content.

Technical solutions:

The detection content of the present invention includes: (1) the influence of extracellular matrix biological material on the proliferation ability of UC-MSCs; (2) the influence of extracellular matrix biological material on the expression of ROS and H ₂ O ₂ in UC-MSCs; (3) The effect of extracellular matrix biomaterials on the expression of SOD-2 in UC-MSCs; (4) the effect of extracellular matrix biomaterials on Catalase activity in UC-MSCs.

The content described above is achieved through the following steps:

(A) Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were cultured in complete medium (the components of the complete medium were α-MEM and fetal bovine serum, and their volume ratio was (4- 9): 1, and contains antibiotics, the content of antibiotics is 10~200 U/mL), when the density is ≥90%, add complete culture medium containing ascorbic acid (100-200uM) and cultivate for 7-10 days, and change the medium every 3 days Once, add decellularization solution (prepared from phosphate buffer with a pH value of 7.4, containing Triton X-100 0.5-5% volume ratio and ammonia water 10-40mM) to construct extracellular matrix biomaterials;

(B) UC-MSCs were cultured in ordinary well plates (TCPS plates) and well plates with extracellular matrix biomaterials on the bottom (ECM plates), and the DNA content in the well plates was detected at different time points, and diacetic acid fluorescence Cell number and morphological changes at different time points were recorded by FDA staining;

(C) Detection of ROS and H ₂ O ₂ contents in UC-MSCs on TCPS plate and ECM plate;

(D) Detection of SOD-2 content and Catalase activity in UC-MSCs on TCPS plate and ECM plate.

The UC-MSCs are obtained from human, porcine, mouse or rabbit sources.

The extracellular matrix biomaterial can be applied in fields including cell transplantation therapy and regenerative medicine.

The specific analysis steps of the above content are as follows:

(1) FDA staining and DNA quantitative detection were used to analyze the effects of extracellular matrix biomaterials on the proliferation ability of UC-MSCs.

(2) DCF-DA fluorescence quantitative method was used to detect the level of intracellular ROS.

(3) H ₂ O ₂ detection kit detects intracellular H ₂ O ₂ level.

(4) Western bort detection of intracellular SOD-2 content.

(5) Catalase activity detection kit detects the level of Catalase activity in cells.

H ₂ O ₂ is one of the main components of ROS and plays a dual role in cell proliferation. Low levels of H ₂ O ₂ (<10 μM) stimulate fibroblast growth, while cells exposed to high concentrations of H ₂ O ₂ (>400 μM) undergo rapid apoptosis. The protocol of the present invention shows that the decreased intracellular H ₂ O ₂ level of cells cultured with extracellular matrix biomaterial is an important reason for supporting the survival and rapid proliferation of cells in vitro. In addition, superoxide anion affects cell proliferation in a dose-dependent manner, with low levels of superoxide anion stimulating cell proliferation and increasing concentrations inhibiting cell growth.

Enhanced antioxidant capacity causes a decrease in ROS levels in UC-MSCs, which has a wide range of clinical applications. The impaired SOD-2 activity of chondrocytes in osteoarthritis not only leads to their oxidative damage, but also accelerates the pathogenesis of osteoarthritis.

MSCs are widely used in the research and development of cell transplantation therapy and tissue engineering. However, the inflammatory cytokines produced by excessive ROS under pathological conditions change some characteristics of MSCs and affect their survival. Therefore, in the process of in vivo transplantation, it is particularly important to prevent cell oxidative stress damage and improve cell survival rate. Previous studies have demonstrated that cell culture in decellularized ECM derived from synoviocytes or bone marrow cells significantly reduces ROS levels, so it will be interesting to elucidate the underlying molecular mechanism by which ECM regulates intracellular antioxidant defense.

Beneficial effect: the invention provides a kind of extracellular matrix biological material, preparation method and application thereof that enhances the biological antioxidant function of in vitro cultured mesenchymal stem cells. Higher expression levels of enzymes with antioxidant activity in cells, especially SOD- ₂ and Catalase; enhanced SOD- ₂ and Catalase activities lead to decreased ROS and H2O2 levels, so extracellular matrix biomaterials can enhance mesenchymal in vitro Antioxidant function of stem cells.

Description of drawings

Figure 1 is a graph showing the proliferation of UC-MSCs on TCPS plates and ECM plates at different time points recorded by FDA staining.

Figure 2 is a graph showing the proliferation of UC-MSCs on TCPS plates and ECM plates at different time points by DNA quantitative detection and analysis.

Fig. 3 is a graph showing the changes of ROS and H ₂ O ₂ content in UC-MSCs on TCPS plate and ECM plate.

Figure 4 is a graph showing the change of SOD-2 content in UC-MSCs on the TCPS plate and the ECM plate.

Fig. 5 is a graph showing the change of Catalase activity in UC-MSCs on the TCPS plate and the ECM plate.

detailed description

The present invention will be further explained below in conjunction with specific examples, but the specific examples do not limit the present invention in any way. Unless otherwise specified, the reagents and methods involved in the examples are commonly used reagents and methods in the art.

Example 1

The specific experimental steps are as follows:

(1) Human UC-MSCs were cultured in a 175cm ² culture flask in a 37°C, 5% CO ₂ incubator, and the liquid was changed every three days, and the volume ratio of each component of the medium was 89% α-MEM , 10% fetal bovine serum, 1% penicillin, streptomycin;

(2) When the cell density is ≥90%, digest the cells with 0.25% trypsin-EDTA, inoculate them at a density of 5000/cm ² in pretreated well plates of various specifications, and culture them until they reach 90% confluence, then add 100 μM ascorbic acid to continue the culture For 8 days, change the medium once every three days. The inoculation method is to incubate the TCPS plate with 0.2% gelatin at 37°C for 1 hour, then incubate with 1% glutaraldehyde and 1M ethanolamine at room temperature for 30 minutes. Various specifications of the plate Divided into 12-well plates for FDA staining and DNA quantitative detection, 6-well plates for ROS, H ₂ O ₂ , and Catalase content determination, and 75cm ² culture flasks for SOD-2 protein analysis;

(3) After 8 days, in order to obtain free extracellular matrix biomaterials, the cultured cells were added to the decellularized mixture and incubated at 37°C for 5 minutes. The decellularized mixture was prepared from a phosphate buffer with a pH value of 7.4, containing 0.5% Triton X-100 and 20mM ammonia water;

(4) The decellularized extracellular matrix biological material was washed 3 times with PBS, and the last time was washed with PBS containing 1X double antibody and kept in the well plate, and stored in a sterile environment at 4°C for future use.

(5) UC-MSCs were seeded on TCPS plates and ECM plates at a density of 5000/cm ² , and cultured at 37°C and 5% CO ₂ .

The detection items and methods are as follows:

1. Effects of extracellular matrix biomaterials on UC-MSCs proliferation ability, intracellular ROS and H ₂ O ₂ levels.

A. The effect of extracellular matrix biomaterials on the proliferation ability of UC-MSCs.

The cell viability was evaluated by FDA staining. The cells were stained with FDA on the 1st, 3rd, 5th, and 7th day respectively. After the cells were washed once with PBS, they were incubated with 5 μg/ml FDA dye solution at 37°C for 10 minutes in the dark, and then washed once with PBS. Take pictures with an Olympus IX51 inverted microscope, where the FDA uses acetone to make a 1000X storage concentration, and dilute it with culture medium when used;

Cell proliferation was assessed by quantitative DNA assay using the Quant-iT ^TM PicoGreen ^® dsDNA Assay Kit. The cells were digested with trypsin on days 1, 3, 5, and 7 respectively, and the DNA samples were collected in 1.5ml EP tubes. After high-speed centrifugation, the supernatant was removed, and 4 tubes were collected for each group. Add 200 μL of papain lysis buffer (final concentration of 125 μg/ml dissolved in PBS) to each tube, and bathe in water at 60°C for 4 hours to fully lyse the cells. The same amount of lysates and reaction reagents were added to a 96-well plate, incubated at room temperature in the dark for 5 minutes, and the fluorescence intensity of the sample was measured at 485/520 nm (excitation wavelength/emission wavelength) with a SynergyMx multifunctional microplate reader. Set up 6 replicate holes.

As shown in Figure 1 and Figure 2, the test results show that the extracellular matrix biomaterial significantly improves the proliferation ability of UC-MSCs. FDA staining showed that the morphology of stem cells cultured on ECM showed a spindle shape, and the number of cells at each time point was significantly more than that of the TCPS group (Scale bar = 100 μm); the DNA content of each time point on the ECM plate was significantly higher than that on the TCPS plate.

B. Effects of extracellular matrix biomaterials on the changes of ROS and H ₂ O ₂ contents in UC-MSCs.

The intracellular ROS level was detected by DCF-DA fluorescence quantification method. Collect at least 2×10 ⁵ cells per tube, and bathe in 10 μM 2′,7′ dichlorofluorescein diacetic acid (DCF-DA) at 37ºC in the dark for 30 minutes. Fluorescence intensity was measured using a Cytomics FC500 flow cytometer.

Intracellular H ₂ O ₂ levels were detected using the Amplex ^® Red Hydrogen Peroxide Detection Kit. The cells cultured on the TCPS plate and the ECM plate were lysed with the cell lysate and the supernatant was extracted, and the lysate was mixed with the reaction reagent and incubated for 30 minutes. The fluorescence intensity of the samples was measured at 530/590 nm (excitation wavelength/emission wavelength) with a SynergyMx multifunctional microplate reader.

The detection results are shown in Figure 3. The ROS and H ₂ O ₂ contents in UC-MSCs on the ECM plate were significantly lower than those on the TCPS plate.

2. The effect of extracellular matrix biomaterials on the changes of SOD-2 content and Catalase activity in UC-MSCs.

A. Effect of extracellular matrix biomaterials on the change of SOD-2 content in UC-MSCs

The extracellular matrix biomaterial cells cultured in a ^75cm2 culture flask were digested with trypsin and transferred to a 1.5ml EP tube. After high-speed centrifugation, the supernatant was removed, and 100ul of cell lysate containing protease inhibitors was added to each tube to lyse on ice for 30 minutes. , and the supernatant was taken after high-speed centrifugation, and the protein concentration was determined using the BCA protein quantification kit (Beiyuntian). Equal amounts of extracted proteins were denatured and separated in 10% polyacrylamide gel, and then transferred to nitrocellulose membrane by electrophoresis. The membrane was incubated overnight at 4°C in SOD-2 diluted in PBS, α-tubulin antibody, and then The membrane was further incubated in diluted horseradish peroxidase for 1 hour, exposed and developed after adding a chemiluminescent kit, and the target protein was quantified using ImageJ software.

The detection results are shown in Figure 4, the extracellular matrix biomaterial significantly increased the SOD-2 content in UC-MSCs.

B. Effect of extracellular matrix biomaterials on Catalase activity in UC-MSCs

Intracellular Catalase activity level was detected using a commercial Catalase activity detection kit. The cells cultured on the TCPS plate and the ECM plate were lysed with cell lysate and the protein supernatant was extracted. The total protein was determined using the BCA protein quantification kit, and the equal amount of protein samples in each group was reacted with the colorimetric substrate solution in the kit. After mixing, incubate at room temperature for 15 minutes, and measure the absorbance of the sample at 520 nm with a microplate reader as soon as possible.

The detection results are shown in Figure 5, the extracellular matrix biomaterial significantly increased the Catalase activity level in UC-MSCs.

In summary, the above experiments show that extracellular matrix biomaterials can significantly improve the proliferation ability of UC-MSCs, reduce the content of ROS and H ₂ O ₂ in UC-MSCs, increase the content of SOD-2 in UC-MSCs, and increase the activity of Catalase in UC-MSCs level, effectively prevent stem cell oxidative stress damage, and have broad application prospects in the fields of cell transplantation therapy and regenerative medicine.

Claims (4)

1. a kind of extracellular matrix biomaterial for strengthening In vitro culture mesenchymal stem cell biological anti-oxidation function, its feature exists In being prepared from by following steps：Umbilical cord mesenchymal stem cells culture during density >=90%, has been added in complete medium Full nutrient solution culture 7-10 days, changed liquid once per 3 days, adds de-cell liquid afterwards, is built into extracellular matrix biomaterial；

The complete medium component is α-MEM and hyclone, and their volume ratio is（4-9）:1, and contain antibiosis Element, the content of antibiotic is 10 ~ 200 U/mL；

Contain ascorbic acid 100-200uM in the complete culture solution.

2. a kind of enhancing In vitro culture mesenchymal stem cell biological anti-oxidation function according to claim 1 is extracellular Matrix biopolymers material, it is characterised in that：The de-cell liquid is contained by the phosphate buffered saline that pH value is 7.4 0.5 ~ 5% volume ratios of TritonX-100 and ammonia 10-40mM.

3. a kind of enhancing In vitro culture mesenchymal stem cell biological anti-oxidation function according to claim 1 is extracellular Matrix biopolymers material, it is characterised in that：The umbilical cord mesenchymal stem cells take from people source, pig source, Mus source or rabbit source.

4. it is a kind of strengthen In vitro culture mesenchymal stem cell biological anti-oxidation function extracellular matrix biomaterial preparation side Method, it is characterised in that preparation process is as follows：Umbilical cord mesenchymal stem cells culture in complete medium, during density >=90%, plus Enter complete culture solution culture 7-10 days, liquid was changed once per 3 days, add de-cell liquid afterwards, be built into extracellular matrix biological Material；

The complete medium component is α-MEM and hyclone, and their volume ratio is（4-9）:1, and contain antibiosis Element, the content of antibiotic is 10 ~ 200 U/mL；Contain ascorbic acid 100-200uM in the complete culture solution.