Methods and formulations and uses to promote exosome endocytosis and stem cell osteogenic differentiation

Abstract

The application provides a method, preparation and use for promoting endocytosis of exosomes and osteogenic differentiation of stem cells. Experiments prove that Moesin protein plays a role in regulating phagocytosis of exosomes, promotes the number of exosomes phagocytosed by cells, and plays a key role in regulating osteogenic differentiation of stem cells, and promotes osteogenic differentiation of stem cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a method for promoting endocytosis of exosomes and osteogenic differentiation of stem cells, a preparation and use thereof. BACKGROUND

[0002] Moesin protein is a member of the ERM family, which is distributed on the surface of membranes such as cell membranes, and links the cytoskeleton and the plasma membrane. At present, the research of Moesin protein mainly focuses on tumors. It is involved in regulating the movement of oral cancer cells (Li, Yao-yin et al. "Moesin regulates the motility of oral cancer cells via MT1-MMP and E-cadherin / p120-catenin adhesion complex." Oral oncology vol. 51, 10 (2015): 935-43.), metastasis of breast cancer (Luo, Shuya et al. "Activation of TMEM16A Ca2+-activated Cl-channels by ROCK1 / moesin promotes breast cancer metastasis." Journal of advanced research vol. 33 253-264. 17 Mar. 2021), fibrosis of liver (Karvar, Serhan et al. "Moesin, an Ezrin / Radixin / Moesin Family Member, Regulates Hepatic Fibrosis." Hepatology (Baltimore, Md.) vol. 72, 3 (2020)), etc., and is mainly used as a potential target for tumor progression and treatment. However, there is no specific application in other aspects at present.

[0003] Exosomes are nanovesicles of 30-150 nm in diameter secreted by various types of cells (Johnstone, 1992). They play a key role in intercellular communication by transferring bioactive molecules (such as lipids, proteins, and genetic information) between cells (Tkach and Thery, 2016). The regulation of exosome biogenesis is crucial for intercellular communication (Zhang et al., 2015). Various microenvironmental factors, including hypoxia, inflammatory cytokines, and infectious agents, can promote exosome biogenesis (Colombo et al., 2014). However, whether Moesin protein is associated with exosome endocytosis needs further exploration. SUMMARY

[0004] The present application aims to provide a method, a preparation and a use for promoting exosome endocytosis and osteogenic differentiation of stem cells.

[0005] To achieve the above-mentioned purpose, the present application provides a method for promoting exosome endocytosis, which comprises the step of using Moesin protein.

[0006] According to some embodiments of the present application, the concentration of the Moesin protein is 4-6 μg / mL.

[0007] The present application also provides a method for promoting osteogenic differentiation of stem cells, which comprises the step of using Moesin protein as defined above.

[0008] The present application also provides a preparation for promoting exosome endocytosis, which comprises Moesin protein.

[0009] The present application also provides a preparation for promoting osteogenic differentiation of stem cells, which comprises Moesin protein.

[0010] According to some embodiments of the present application, the dosage form of the above-mentioned preparation is selected from any one of oral liquid, pill, powder, tablet, granule, capsule, decoction and injection.

[0011] According to some embodiments of the present application, the above-mentioned Moesin protein is a natural protein without modification.

[0012] The present application also provides a use of Moesin protein in the preparation of a preparation for promoting exosome endocytosis.

[0013] The present application also provides a use of Moesin protein in the preparation of a preparation for promoting osteogenic differentiation of stem cells.

[0014] The present application plays a role in regulating the phagocytosis of exosomes, promotes the number of exosomes phagocytosed by cells, and also plays a key role in regulating the osteogenic differentiation of stem cells, and promotes the osteogenic differentiation of stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Fluorescent staining diagram for Moesin protein of the present application regulating exosome endocytosis;

[0016] Figure 2 Comparison diagram for the number of exosomes endocytosed after H2S treatment increases the expression of Moesin protein;

[0017] Figure 3 Comparison diagram for the number of exosomes endocytosed after knocking down Moesin protein;

[0018] Figure 4 Fluorescent staining image of exosomes being endocytosed after Moesin protein knockdown;

[0019] Figure 5 This is the result of the Moesin protein toxicity test in this invention;

[0020] Figure 6 Alizarin Red staining and bar graph showing how Moesin protein promotes osteogenic differentiation of stem cells according to the present invention.

[0021] Figure 7 Histological staining and bar graphs showing the effect of Moesin protein in promoting osteogenic differentiation of stem cells according to the present invention. Detailed Implementation

[0022] To more clearly understand the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in further detail. It should be understood that the following specific embodiments are merely exemplary, and the technical solution of the present invention is not limited to the specific embodiments listed below.

[0023] Unless otherwise specified, the experimental methods and apparatus used in the following examples and comparative examples are conventional methods and apparatus.

[0024] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available.

[0025] Example 1

[0026] The regulatory role of Moesin protein in exocytosis

[0027] The amount of exosomes phagocytosed in different samples was observed using a laser confocal microscope (200x) (co-culture time was 12 hours). The results are as follows: Figure 1 As shown:

[0028] M0 macrophage exosomes, M2 macrophage exosomes, and macrophage exosomes treated with H2S (GYY4137, Abcam, used to enhance the expression of Moesin protein in exosomes) were stained red. Bone marrow stem cells were treated for 12 hours, and the amount of exosomes phagocytosed was observed using a laser confocal microscope (200x).

[0029] Figure 1 The image shows fluorescence staining images after staining with different dyes (PKH-26, Phalloidin, DAPI, Merge (overlay)), where red fluorescence indicates the location and expression of exosomes. Figure 1It can be seen that, compared with the untreated mesenchymal stem cell control group (M0-exo) and the mesenchymal stem cells with added macrophage exosomes (M2-exo), the amount of exosomes phagocytosed in the mesenchymal stem cell sample with macrophage exosomes treated with H2S (GYY-M2-exo) was significantly increased. Figure 1 The ratio of positive reaction area to total area shown on the right side of the image (PKH-26 positive area / total area) also shows that the amount of exosomes phagocytosed in the GYY-treated samples is significantly increased.

[0030] The effect of GYY on promoting Moesin protein expression was verified. IL-4 was used as a stimulant guiding the differentiation of macrophage M2 cells, and β-actin was used as an internal standard. (Reference) Figure 2 The protein gel electrophoresis diagram shown indicates that the expression of Moesin protein was enhanced after GYY treatment.

[0031] The effect of siRNA knockdown of Moeisn protein was verified. (Reference) Figure 3 β-actin was used as an internal standard. Proteins from different samples (ectoderm exomoes and cells) were subjected to gel electrophoresis. Figure 3 It can be seen that the expression of Moesin protein was reduced after siRNA knockdown treatment.

[0032] refer to Figure 4 Compared to the control group (untreated exosomes, Control-exo), the experimental group, after knocking down Moeisin protein with siRNA (si-Moesin-exo), showed a reduction in the amount of exosomes phagocytosed. Figure 4 The ratio of positive reaction area to total area shown on the right (PKH-26 positive area / total area) also indicates that the amount of exosomes phagocytosed in the siRNA knockdown treated samples was significantly reduced.

[0033] pass Figures 1-4 It was found that, compared with the control group, the amount of exosomes endocytosed increased with each increase in Moesin protein level; after knocking down Moesin protein with Sirna, the amount of exosomes phagocytosed decreased compared with the control group, and the results were statistically significant (*P<0.05, **P<0.01, ***P<0.001).

[0034] Example 2

[0035] Moesin protein promotes osteogenic differentiation of stem cells

[0036] Firstly, the appropriate concentration of Moesin protein (aa 1-346, His Tag, Sino Biological, China) was explored by CCK8 experiment, which was divided into control group (con), 5 μg / ml, 10 μg / ml, 15 μg / ml and 20 μg / ml. The results showed that the concentration of 5 μg / ml was more appropriate. Through Figure 5 It can be seen that the absorbance curve of 5 μg / ml concentration is almost consistent with the control group.

[0037] CCK8 experiment steps:

[0038] 1. The cell suspension (100 μL / well) was inoculated in 96-hole plates, and different concentrations of Moesin protein were added. The plate was pre-incubated in a humidified incubator (at 37°C, 5% CO2).

[0039] 2. 10 μL CCK8 solution was added to each well of the plate.

[0040] 3. Incubate the culture plate in the incubator for 1-4 hours.

[0041] 4. Use the enzyme marker to measure the absorbance at 450 nm.

[0042] Alizarin red staining:

[0043] Osteogenic induction medium: α-MEM medium containing 15% fetal bovine serum, 100 U / ml penicillin, 100 U / ml streptomycin, 2 mM glutamine, 5 mM β-glycerol, 500 μM vitamin C, 10 nM dexamethasone sodium phosphate. The osteogenic induction medium was changed every two days, and the cell alizarin red staining experiment was performed at 14 days: the culture medium in the well plate was aspirated, washed with PBS for 3 times, 4% paraformaldehyde was added for fixation for 20 minutes;

[0044] Discard the fixing solution, wash with PBS for 3 times, add 1% alizarin red staining, incubate on the shaking table at room temperature for 5-10 minutes; after discarding the staining solution, wash with double distilled water for 3 times, observe and take pictures under the optical microscope.

[0045] Figure 6 Further in vitro experiment of Moesin protein to promote the osteogenic differentiation of stem cells. Figure 6 From left to right, the control group, the Moesin protein group (5 μg / ml), the osteogenic induction medium group, and the osteogenic induction medium + Moesin protein group were divided. After 7 days, the alizarin red staining was used to observe the osteogenic differentiation effect, and the Figure 6 The results showed that Moesin protein has the effect of promoting the osteogenesis of stem cells. Figure 6It can be seen that compared with the control group, a small amount of calcified nodules were formed in the osteogenic medium group, no obvious calcified nodules were formed in the Moesin protein alone treatment group, and a large amount of calcified nodules were formed in the osteogenic medium group with Moesin protein, and the results had statistical significance by ANOVA test (*P<0.05, **P<0.01, ***P<0.001).

[0046] The untreated macrophage exosomes (M0-exo), the macrophage exosomes added to the bone marrow mesenchymal stem cells (M2-exo), and the stem cells implanted in the back of the nude mice subcutaneously treated by the exosomes with high expression of Moesin protein were implanted into the back of the nude mice subcutaneously, and the osteogenic tissue samples were collected after two months, and the bone formation of each group was observed by HE staining (Arg-1 staining was the control group). Figure 7 It can be seen from the HE staining and the column contrast chart (CT is connective tissue, B is bone tissue, and HA is a biological scaffold material) that the bone formation of the exosome treatment group with Moesin protein was more, and the results had statistical significance (*P<0.05, **P<0.01, ***P<0.001).

[0047] The above only describes the preferred embodiments of the present application. It should be noted that those skilled in the art can make various modifications, combinations, changes or replacements to the details and features of the present application without departing from the spirit and essence of the present application. These modifications, combinations, changes or replacements should also be understood as included in the scope of protection of the present application.

Claims

1. Use of Moesin protein in the preparation of formulations that promote osteogenic differentiation of stem cells, wherein the stem cells are bone marrow mesenchymal stem cells.

2. The use according to claim 1, characterized in that, The concentration of the Moesin protein was 4-6 μg / mL.

3. The use according to claim 1, characterized in that, The dosage form of the preparation is an injection.

4. The use according to any one of claims 1-3, characterized in that, The Moesin protein is an unmodified natural protein.

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