Application of magnetic exosome to preparation of wound repairing or wound healing product
A technology of wound repair and wound healing, applied in the field of biomedicine, can solve the problems that the safety and curative effect cannot be fully guaranteed, and achieve the effect of great application potential
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Embodiment 1
[0039] Example 1: Preparation of Magnetic Exosomes 1
[0040] In this example, magnetic exosomes utilize magnetic Fe 3 o 4 nanoparticles, and magnetic Fe 3 o 4 Magnetic exosomes produced by MSCs stimulated by nanoparticles combined with a static magnetic field.
[0041] (1) 10 mg of sterilized Fe 3 o 4 Dissolve in 50ml of complete medium α-MEM, then dilute in equal proportions, respectively configure to concentrations of 400, 200, 100, 50, 25μg / ml containing Fe 3 o 4 culture medium. During the passage of cells, the medium was replaced with different gradients. Determine optimal Fe based on cell growth morphology and proliferation experiments 3 o 4 concentration.
[0042] (2) the best Fe 3 o 4 The cells cultured at different concentrations were stimulated by static magnetic fields (SMF) of different strengths (NdFeB permanent magnet materials, Beijing Xinchangli Functional Materials Co., Ltd.), and the magnetic field strengths were 50, 100, and 150 mT in turn. Det...
Embodiment 2
[0043] Example 2: Cell Proliferation and Apoptosis Experiments
[0044] (1) Cell proliferation experiment: take the MSCs of the P3 generation, digest and centrifuge, resuspend the cells, count, and prepare Fe with a corresponding concentration gradient according to Example 1 3 o 4 -MSC. According to 5×10 3 Inoculate the cell sample into a 96-well plate, add 100 μL of α-MEM medium containing 10% FBS to each well, and culture in an incubator. After 24 hours, discard the medium, add 90 μL of fresh medium and 10 μL of CCK-8 to each well, and incubate at 37° C. for 1 h in the dark. Continuous measurement was carried out for 5 days, with 3 replicate wells per day. The OD value of each well was measured at a wavelength of 450nm, and the proliferation curve was drawn.
[0045] (2) Apoptosis experiment: prepare the Fe with the corresponding magnetic field intensity gradient according to the steps in Example 1 3 o 4 - SMF-MSC, collected directly into a 10ml centrifuge tube after ...
Embodiment 3
[0048] Example 3 Transmission Electron Microscopy and Western Blotting
[0049] (1) Observing the morphology of exosomes with the electron microscope uses the optimal concentration of Fe 3 o 4 (50 μg / ml) stimulated exosomes secreted by MSCs were denoted as MSC-Fe 3 o 4 -Exo; optimal concentration Fe 3 o 4 (50 μg / ml) combined with the optimal magnetic field strength (100mT) stimulated the exosomes secreted by MSCs and recorded as MSC-Fe 3 o 4 -SMF-Exo. Take 10 μl of exosomes separated and purified by ultracentrifugation, add an equal volume of balanced salt PBS solution to dilute, and then drop it on a 2 mm sample-loading copper grid. After standing at room temperature for 3 minutes, use filter paper to gently absorb the excess liquid, and use 3% (w / v) 30 μl of sodium phosphotungstate solution (PH6.8) was negatively stained at room temperature for 5 minutes, washed gently with double distilled water, dried at room temperature, observed and photographed under a transmissi...
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