Preparation and application of epimedium vesicles

Abstract

The present invention belongs to the field of biomedicine and provides an application of epimedium vesicles in promoting the proliferation or osteogenic ability of mesenchymal stem cells. The present invention also provides a method for preparing mesenchymal stem cells loaded with epimedium vesicles: culturing mesenchymal stem cells until the confluence reaches 80%-95%; adding epimedium vesicles and co-culturing at 4°C and pH 6.8-7.4. The above-mentioned mesenchymal stem cells loaded with epimedium vesicles can be used to treat bone defects. The present invention solves the problem of maintaining the proliferation characteristics and osteogenic differentiation ability of UC-MSCs during long-term culture and passage.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the preparation of epimedium vesicles and the application of epimedium vesicles in enhancing the osteogenic differentiation ability of mesenchymal stem cells. Background Art

[0002] Trauma, infection, tumor and many congenital diseases can lead to bone defects. Bone defects seriously affect the quality of life of patients and increase their economic burden. The effective treatment of bone defects has always been a difficult problem in orthopedic clinics. Mesenchymal stem cells (MSCs) are a type of stem cells with strong proliferation ability and multidirectional differentiation potential. They are widely used in clinical and basic research. In recent years, the use of MSCs to treat a variety of clinically refractory diseases has become a research hotspot. Due to their strong osteogenic activity, bone marrow mesenchymal stem cells were the first stem cells used in orthopedic diseases. However, the collection of bone marrow involves an invasive process, and the proliferation and differentiation potential of bone marrow mesenchymal stem cells significantly decreases with age. Due to the low content of bone marrow stem cells per unit volume of bone marrow blood, the separation, purification and expansion of seed cells are more difficult, and the clinical application of bone marrow mesenchymal stem cells is relatively limited. Studies have shown that a large number of mesenchymal stem cells (umbilical cord mesenchymal stem cells, UC-MSCs) can be easily isolated from umbilical cords, which can replace bone marrow mesenchymal stem cells for cell technology treatment of bone defects. However, during the culture process of UC-MSCs, with the increase of cell generations, the difference in osteogenic and chondrogenic differentiation abilities of P10 UC-MSCs compared with P5 UC-MSCs was significantly weakened. How to maintain their proliferation characteristics and osteogenic differentiation ability during culture and passaging is an urgent problem that needs to be solved in the clinical treatment of orthopedic diseases using UC-MSCs.

[0003] The natural juice of fresh plant-based Chinese herbal medicines and fresh animal or insect medicines are called fresh Chinese medicines. In particular, fresh plant-based Chinese herbal medicines have a certain growth cycle, and the medicinal materials are picked according to the season. Due to the backward preservation technology of fresh medicines, the storage and transportation costs are high, which greatly limits the application of fresh Chinese medicines. Therefore, Chinese medicines need to be processed when applied, and they need to be processed and processed in depth according to the properties of Chinese medicinal materials and clinical needs to make them easier to use and store. The processing of Chinese medicines involves many processes such as cleaning, cutting, and processing. However, the current processing standards for Chinese medicines are mainly formulated by Chinese medicine personnel based on their own practical experience. The unclear processing standards directly lead to the gap in the quality of Chinese medicines, which limits the promotion and application of Chinese medicines. Therefore, finding a way to more effectively exert the effects of fresh Chinese medicines and reduce the storage and transportation costs of fresh Chinese medicines is conducive to the establishment of a modern system of Chinese medicines and the realization of the value of Chinese medicines.

[0004] Extracellular vesicles refer to a general term for tiny vesicles with membrane structures that are actively secreted by cells. According to their size, biological characteristics and formation process, they are mainly divided into three categories: exosomes, microvesicles and apoptotic bodies. Extracellular vesicles have a typical lipid bilayer structure, carry a variety of substances such as proteins, lipids, RNA, etc., and play an important role in the transfer of substances and information between cells. In recent years, with the development of extracellular vesicle separation and detection technology, extracellular vesicles have been separated and identified from cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid and other biological fluids. Researchers have also been separating and identifying plant extracellular vesicles. For example, ginseng-derived extracellular vesicles can target the liver and spleen, and promote the polarization of tumor-associated macrophage phenotype M2 to M1, thereby promoting the apoptosis of melanoma cells. Extracellular vesicles of Drynaria fortunei are bone-targeted, can regulate the ER pathway, and have a certain therapeutic effect on osteoporosis.

[0005] Epimedium is a traditional Chinese herbal medicine for strengthening the kidney and bones. It is the leaves of the plant Epimedium of the Berberidaceae family. Icariin is the main component of Epimedium, which is mainly composed of flavonoids, lignans and alkaloids. Traditional Chinese medicine believes that Epimedium has the effects of strengthening tendons and bones, strengthening the kidney and promoting yang. In recent years, studies have shown that icariin can be used as a bone trophic factor. In vivo and in vitro experiments have confirmed that it can promote the osteogenic differentiation of mesenchymal stem cells. This therapeutic effect provides new hope for the treatment of bone defects. However, there is no literature reporting on extracellular vesicles from Epimedium. Therefore, it is still unknown whether it is feasible to extract extracellular vesicles from fresh Epimedium, whether the extracted Epimedium extracellular vesicles (EPEVs) have biological activity and whether they can maintain the osteogenic differentiation ability of UC-MSCs. Therefore, how to provide a method for extracting extracellular vesicles from fresh Chinese herbal medicine Epimedium, and conduct in-depth research on the effect of the extracted extracellular vesicles of Epimedium on the osteogenic differentiation ability of UC-MSCs, so as to explore the application potential of Epimedium and UC-MSCs in the treatment of orthopedic diseases, has become an urgent problem to be solved in this field. Summary of the invention

[0006] In view of the problem that mesenchymal stem cells have poor osteogenic ability after long-term culture, the present invention provides an application of epimedium vesicles in promoting the osteogenic ability of mesenchymal stem cells. The epimedium vesicles have the ability to promote the proliferation and osteogenic differentiation of mesenchymal stem cells. The mesenchymal stem cells loaded with epimedium vesicles have good bone targeting and can be used to treat bone defects.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A use of epimedium vesicles in promoting the proliferation or osteogenic ability of mesenchymal stem cells.

[0009] The above-mentioned epimedium vesicles can be obtained by conventional methods commonly used in the art, such as differential centrifugation.

[0010] Preferably, the method for preparing the epimedium vesicles comprises the following steps:

[0011] (1) Add water to fresh Epimedium leaves to squeeze out juice, and filter to obtain filtrate;

[0012] (2) The filtrate is subjected to low-temperature differential centrifugation to obtain the supernatant;

[0013] (3) The supernatant is subjected to low-temperature ultracentrifugation to obtain a precipitate, namely, the epimedium vesicles.

[0014] In step (2), the differential centrifugation is performed at 2000 g for 15 min and at 10000 g for 30 min.

[0015] In step (3), ultracentrifugation is performed at 100,000-120,000 g for 70 min.

[0016] A method for preparing mesenchymal stem cells loaded with epimedium vesicles comprises the following steps:

[0017] (i) Cultivating mesenchymal stem cells until confluence reaches 80%-95%;

[0018] (ii) Add Epimedium vesicles and co-culture at 37°C and pH 6.8-7.4 for 24-48 h.

[0019] Preferably, in step (ii), the pH is 6.8.

[0020] The mesenchymal stem cells loaded with epimedium vesicles obtained by the above preparation method.

[0021] The mesenchymal stem cells loaded with epimedium vesicles can be used to treat bone defects.

[0022] The above-mentioned mesenchymal stem cells are derived from placenta, umbilical cord, umbilical cord blood, bone marrow and fat.

[0023] The mesenchymal stem cells are passaged 4-10 times.

[0024] The present invention has the following advantages:

[0025] The present invention provides a method for extracting extracellular vesicles from fresh Chinese medicinal herb Epimedium; further provides application of the extracellular vesicles of Epimedium in promoting the osteogenic differentiation ability of UC-MSCs; the present invention promotes the uptake of EPEVs by UC-MSCs by combining starvation induction with acidic pH acclimation to prepare EPEVs-loaded UC-MSCs; the present invention solves the problem of maintaining the proliferation characteristics and osteogenic differentiation ability of UC-MSCs during long-term culture and passage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an electron micrograph of extracellular vesicles of Epimedium;

[0027] Figure 2 It is the NanoFCM particle size distribution of epimedium extracellular vesicles;

[0028] Figure 3 CCK8 was used to detect the effect of EPEVs on the proliferation of UC-MSCs;

[0029] Figure 4 is the effect of EPEVs on the mRNA of osteogenic differentiation genes OCN, OPN, and Runx2 in UC-MSCs;

[0030] Figure 5 is the uptake of EPEVs by UC-MSCs;

[0031] Figure 6 These are the in vivo fluorescence images of nude mice after injection of different UC-MSCs. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0033] Example 1 Preparation and identification of epimedium extracellular vesicles (EPEVs)

[0034] 1. Preparation of Epimedium Extracellular Vesicles

[0035] (1) Take 500 g of fresh Epimedium leaves, wash them thoroughly with sterile water, chop them, add 500 mL of sterile water (precooled at 4°C), and use a spiral juicer to extract the juice (voltage / frequency / power: 220V / 50HZ / 1.2KW). Collect the juice and filter it with a 0.45μm filter membrane to collect the filtrate;

[0036] (2) Place the filtrate in a 50 mL centrifuge tube and centrifuge at 2000 g for 15 min at 4 °C to collect the supernatant I.

[0037] (3) Place supernatant I in a 50 mL centrifuge tube and centrifuge at 10,000 g for 30 min at 4°C to collect supernatant II.

[0038] (4) Place supernatant II in an ultracentrifuge tube, centrifuge at 120,000 g for 70 min at 4°C, and collect the precipitate;

[0039] (5) Resuspend the precipitate in 10 mL of pre-cooled PBS and filter through a 0.22 μm filter membrane. The filtrate is the solution containing the extracellular vesicles of Epimedium, which should be stored at -80°C until use.

[0040] 2. Identification of Epimedium Extracellular Vesicles

[0041] The morphology, particle size and particle concentration of the prepared epimedium extracellular vesicles were characterized. Figure 1 As shown: It can be observed that EPEVs have a typical extracellular vesicle structure, which is round or oval, and the particle size is between 0-200nm, which is consistent with the extracellular vesicle particle size range reported in the literature;

[0042] The particle size distribution and particle concentration of EPEVs were analyzed using Nanoflow cytometry (NanoFCM). Figure 2 As shown: the particle size distribution is between 40-200nm, the average particle size is 90.31nm, which is consistent with the electron microscopy results, and the concentration is 7.59×10 10 particles / mL;

[0043] The surfactant Triton X-100 was used to further determine the number of particles with intact membrane structures. This test can directly reflect the purity of the extracted extracellular vesicles. 5µL of 10% Triton X-100 was added to 45µL of EPEVs solution for 60 min, and 5µL PBS was added to the control group (Control). The NanoFCM detection method was used to evaluate the changes in the number of particles before and after Triton X-100 treatment. Purity was calculated according to the following formula: Purity = (number of vesicles before treatment - number of vesicles after treatment) / number of vesicles before treatment * 100%.

[0044] Table 1 Particle number and EPEVs purity before and after Triton X-100 treatment (n=3)

[0045]

[0046] The results are shown in Table 1: Compared with the control group, the number of EPEVs particles in the 1% Triton X-100 group decreased significantly (p<0.001). This result proves that the extracted EPEVs have a complete membrane structure with a purity of (60.70±1.91)%.

[0047] Example 2 Effect of EPEVs on the proliferation of human umbilical cord mesenchymal stem cells (UC-MSCs)

[0048] The CCK-8 assay was used to evaluate the effect of EPEVs on MSC proliferation. P9-UC-MSCs cells were cultured at 37°C and 5% CO2. When the cell confluence reached 90%, they were digested with TrypLE, collected by centrifugation, and counted. Subsequently, P10 MSCs (3×10 4 cells / well) in a 96-well plate in basal medium, and different doses of EPEVs prepared in Example 1 were added (low-dose group: 5×10 7 particles / mL; medium dose group: 5×10 8 particles / mL; high dose group: 5×10 9 particles / mL) for 12, 24, and 48 hours. At the corresponding time point, each well was replaced with normal culture medium containing 10% CCK-8 solution and incubated at 37°C for 3 hours. The proliferation rate was measured by absorbance at 450 nm using a microplate reader (Promega). The results are shown in Figure 3 As shown in the figure, compared with the control group, EPEVs in the low, medium and high dose groups could promote the proliferation of UC-MSCs, and the difference was statistically significant (p<0.05). 8 particles / mL EPEVs has the best effect in promoting proliferation and is the optimal concentration.

[0049] Example 3 Effect of EPEVs on osteogenic differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs)

[0050] We conducted an osteogenic differentiation induction experiment to evaluate the beneficial effects of EPEVs on the osteogenic differentiation of P10 UC-MSCs cells. 1 mL of gelatin was evenly added to a 6-well plate to avoid bubbles. After adding gelatin, the plate was left to stand for 30 minutes on a clean bench. The gelatin was aspirated and dried for later use. P9-UC-MSCs cells were cultured at 37°C and 5% CO2. When the cell confluence reached 90%, they were digested with TrypLE, and the cells were collected by centrifugation and counted. Subsequently, the cell density was 8000 cells / cm 2 P10 MSC cells were seeded in the above 6-well plates.

[0051] When the cell density reached 90%, the medium was replaced with osteogenic differentiation induction medium (Sciencell, 7531 mesenchymal stem cell-osteoblast differentiation medium). The control group only added the above induction medium, while the experimental group added 5×10 8particles / mL EPEVs for induction of differentiation; thereafter, the culture medium was changed every 3 days. At 0, 7, and 14 days of osteogenic differentiation, cells were collected to extract RNA for reverse transcription and then qRT-PCR was performed. The sequences in Table 2 were used as primers and GAPDH was used as the internal reference gene to detect the expression of osteogenic differentiation-related genes OCN, OPN, and Runx2.

[0052] Table 2 qRT-PCR primer sequences

[0053]

[0054] The results are as follows Figure 4 As shown: On the 7th and 14th days, the mRNA levels of OCN, OPN and Runx2 in the experimental group cells gradually increased, which were significantly higher than those in the control group cells (p<0.05), indicating that EPEVs can upregulate the expression of osteogenic genes in UC-MSCs.

[0055] Example 4 Preparation of EPEVs-loaded UC-MSCs

[0056] The EPEVs prepared in Example 1 were labeled with PKH26 fluorescent dye, which can stably bind to the lipid region of the exosome membrane and emit red fluorescence, to obtain PKH26-labeled EPEVs (PKH26-EPEVs). The P10 UC-MSCs cell suspension was plated in a T175 flask and cultured at 37°C, 5% CO2 until the confluence was above 90%. The serum-free culture medium was replaced to starve the cells. The cells were cultured in DPBS (2.67 mM KCl, 1.47 mM KH2PO4) at pH 6.8, 7.4, and 7.8, respectively. 4， UC-MSCs loaded with PKH26-EPEVs were prepared under the medium of 137.93 mM NaCl, 8.06 mM Na2HPO4-7H2O at 37°C. Under confocal microscopy, the green fluorescence was FITC-labeled phalloidin showing the cytoskeleton, the blue fluorescence was DAPI-stained nuclei, and the red fluorescence was PKH26-labeled exosomes. The results are shown in Figure 5 The cells shown can take up PKH26-labeled exosomes. Compared with pH 7.4 and 7.8, the red particles accumulated in the cells are the largest at pH 6.8, indicating that the uptake of PKH26-EPEVs by UC-MSCs is affected by pH, and pH 6.8 is the optimal condition for UC-MSCs to take up EPEVs.

[0057] Example 5 Bone targeting of EPEVs-loaded UC-MSCs

[0058] The EPEVs prepared in Example 1 were labeled with DiD fluorescent dye to obtain DiD-labeled EPEVs (DiR-EPEVs). The P10 UC-MSCs cell suspension was plated in a T175 flask and cultured at 37°C, 5% CO2 until the confluence was above 90%. The serum-free medium was replaced to starve the cells, and DiD-EPEVs-loaded UC-MSCs cells (DiD-EPEVs-MSCs) were prepared at 4°C pH 6.8. At the same time, the same batch of UC-MSCs cells (DiD-MSCs) were labeled with DiD fluorescent dye. According to 5.0×10 7 The above cells DiD-EPEVs-MSCs (experimental group) and DiD-MSCs (control group) were injected into the tail vein of nude mice at a dose of cells / kg, and fluorescence imaging was performed 12 hours after administration. The group injected with PBS was used as a blank control. DiD is a lipophilic carbocyanine dye with a very long lipophilic hydrocarbon chain. After entering the cell membrane, it can diffuse laterally and gradually stain the cell membrane of the entire cell. The staining is very stable. After being excited, DiD can emit far-infrared fluorescence, which can penetrate the skin of nude mice and be detected by fluorescence imaging instruments. Different intensities of fluorescence signals are displayed by different colors, and from weak to strong, it is displayed as a change from blue to green and then to red. The results are shown in Figure 6 As shown, the blank group basically had no fluorescence detected. In the control group and the experimental group, obvious fluorescence signals could be detected in the abdominal cavity where the tissues and organs of the mice were concentrated at 12 hours. In the experimental group, the nude mice were injected with DiD-EPEVs-MSCs through the tail vein, and obvious accumulation of fluorescence signals could be observed in the femur, indicating that EPEVs enhanced the bone targeting of UC-MSCs.

Claims

1. An application of epimedium vesicles in promoting the proliferation of mesenchymal stem cells in vitro, characterized in that: The preparation method of the epimedium vesicle comprises the following steps: (1) Add water to fresh Epimedium leaves to squeeze out juice, and filter to obtain filtrate; (2) The filtrate is subjected to low-temperature differential centrifugation to obtain the supernatant; (3) The supernatant is subjected to low-temperature ultracentrifugation to obtain a precipitate, namely, the epimedium vesicles; In step (2), the differential centrifugation is 2000g for 15 min and 10000g for 30 min. In step (3), ultracentrifugation is performed at 100,000-120,000 g for 70 min; The sources of the mesenchymal stem cells are placenta, umbilical cord and umbilical cord blood.

2. A method for preparing mesenchymal stem cells loaded with epimedium vesicles, characterized in that: The following steps are involved: (i) Cultivating mesenchymal stem cells until confluence reaches 80%-95%; (ii) adding epimedium vesicles and co-culturing at 37°C and pH 6.8 for 24-48 h; The preparation method of the epimedium vesicle comprises the following steps: (1) Add water to fresh Epimedium leaves to squeeze out juice, and filter to obtain filtrate; (2) The filtrate is subjected to low-temperature differential centrifugation to obtain the supernatant; (3) The supernatant is subjected to low-temperature ultracentrifugation to obtain a precipitate, namely, the epimedium vesicles; In step (2), the differential centrifugation is 2000g for 15 min and 10000g for 30 min. In step (3), ultracentrifugation is performed at 100,000-120,000 g for 70 min; The sources of the mesenchymal stem cells are placenta, umbilical cord and umbilical cord blood.

3. A mesenchymal stem cell loaded with epimedium vesicles obtained by the preparation method as claimed in claim 2.

4. The mesenchymal stem cell according to claim 3, characterized in that The number of passages of mesenchymal stem cells is 4-10 times.

5. Use of the mesenchymal stem cells loaded with epimedium vesicles as claimed in claim 3 in the preparation of a medicament for treating bone defects.

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