CN114672456A - Method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by utilizing ultrasonic stimulation and application - Google Patents

Abstract

The invention discloses a method and application of using ultrasonic stimulation to improve the efficiency of adipose stem cells to secrete extracellular vesicles. Serum-containing low-sugar DMEM, spread a certain thickness of couplant evenly under the cell culture dish, and then tightly fit the transducer of the ultrasonic generator to the couplant to remove air, and the ultrasonic dose is set to 0.5 ~ 1.5W/ cm ² , the exposure time was 30-120 s; C. Extraction of extracellular vesicles from adipose stem cells. It has been verified that the number of vesicles secreted by adipose stem cells stimulated by ultrasound is more than 60 times higher than that of adipose stem cells without ultrasound stimulation; the protein content of vesicles is increased by more than 3 times, and vesicle-specific markers are highly expressed, and It is effective in promoting skin wound healing.

Description

Method and application of enhancing the efficiency of extracellular vesicle secretion from adipose stem cells by ultrasonic stimulation

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a method for improving the efficiency of adipose stem cells to secrete extracellular vesicles by using ultrasonic stimulation and a specific application of the extracellular vesicles.

Background technique

Stem cells have shown great application prospects in regenerative repair medicine because of their multi-directional differentiation potential. Among them, Adipose derived-stem cells (ADSCs) are rich in sources, easy to obtain, and the liposuction process is minimally invasive and easily accepted by patients, making them more suitable for clinical translational applications.

Previous research believed that stem cells migrate to damaged sites in the body and differentiate into damaged tissues and cells. However, numerous studies tracking stem cells in vivo found that fewer stem cells survived and turned into damaged cells. Recent studies have shown that the role of stem cells is mainly through the secretion of extracellular vesicles and growth factors, rather than direct transformation into damaged tissue cells.

Extracellular vesicles (EVs) are mixed particles secreted by cells, including microvesicles (MVs) and exosomes (Exosomes), which are released by budding and forming multivesicular bodies, respectively. Extracellular, carrying its encapsulated contents to act on recipient cells. Extracellular vesicles are signaling mediators secreted by cells, which are fundamentally different from cells. The use of extracellular vesicles avoids many of the safety issues involved in cell therapy: stem cell therapy, for example, has strong immunogenicity, the risk of tumor formation, and the possible blockage of tiny blood sinuses. In contrast, extracellular vesicles have low immunogenicity and can be used for allogeneic applications. Compared with the instability of growth factors, proteins and nucleic acids in extracellular vesicles are protected by membrane structures and are not easily degraded. They can be used as ready-made preparations and have broad application and transformation prospects.

Currently, for the purpose of regenerative therapy, the production of extracellular vesicles requires culturing a large number of cells and extracting and enriching them from the cell supernatant. However, the production of extracellular vesicles secreted by cells is low, and it is difficult to meet the needs of clinical treatment. Previous studies have reported that hypoxia stimulation and the addition of chemical drugs can promote cells to secrete extracellular vesicles to a certain extent, but these methods still cannot meet the needs of clinical translation. At the same time, exogenous addition of chemical drugs may also affect the composition and purity of extracellular vesicles.

SUMMARY OF THE INVENTION

Based on the above research, the present invention provides a method for improving the efficiency of adipose stem cells to secrete extracellular vesicles by using ultrasonic stimulation, and the application of the extracellular vesicles in skin wound healing.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A first aspect of the present invention provides a method for improving the efficiency of adipose stem cells to secrete extracellular vesicles by using ultrasonic stimulation, comprising the following steps:

A. Adipose stem cell extraction

The granular fat discarded after liposuction was digested by enzymatic digestion, and adipose stem cells were extracted, and then routinely passaged to the 3rd generation after full growth;

B. Ultrasound stimulates adipose stem cells

After the third-generation adipose stem cells are full, wash the cells twice with PBS, then replace the serum-free low-sugar DMEM, spread a certain thickness of coupling agent evenly under the cell culture dish, and then put the transducer of the ultrasonic generating device It is closely attached to the coupling agent to remove the air. Since the ultrasonic dose acting on the cells is too large, the cells will undergo obvious apoptosis. In order to reduce the apoptosis, the ultrasonic dose is preferably set to 0.5~1.5W/cm ² and the exposure time is 30~ 120s, most preferably 1.5W/cm ² , 30S.

C. Extraction of adipose stem cell extracellular vesicles

The adipose stem cells were stimulated by ultrasound and cultured for 48 hours, and then the cell supernatant was collected; the extracellular vesicles in the supernatant were extracted by ultrafiltration and dissolved in PBS.

Preferably, in step A, the digestive enzyme adopted is type IV collagenase;

The digestion treatment conditions are as follows: 0.2% (m/v) type IV collagenase and granular adipose tissue are mixed at a volume ratio of 1:1, shaken well on a shaker at 37°C, and digested for 2 hours.

Preferably, in step A, when the adipose stem cells are routinely passaged to the third passage, flow cytometry and three-way induction of osteogenesis, adipogenic and chondrogenesis are used to identify the third passage of adipose stem cells.

Preferably, in step B, ultrasonic stimulation is performed on the cells using an ultrasonic generating device with a pulse repetition frequency of 1 MHz, a pulse repetition frequency of 10 Hz, a duty cycle of 60%, and a cross-sectional area of 9 mm ² .

When performing ultrasonic stimulation, 0.5-1cm thick couplant is evenly spread under the 10cm cell culture dish, and then the transducer of the ultrasonic generating device is tightly attached to the couplant to exclude air.

Preferably, in step C, the supernatant is filtered with a 0.22 μm filter and then added to an ultrafiltration centrifuge tube with a 100KD membrane, centrifuged at 3000 g at 4°C to a volume of 500 μL in the inner tube, transferred to a sterile cryopreservation tube, and transferred to a -80°C refrigerator Freeze.

The extracted extracellular vesicles were detected: NTA test showed that the number of vesicles secreted by adipose stem cells stimulated by the above ultrasonic stimulation conditions increased by more than 60 times; the protein quantitative results showed that after the above ultrasonic stimulation of adipose stem cells, the secreted vesicles The protein content of vesicles increased by more than 3 times; Western Blot results showed that vesicles highly expressed vesicle-specific markers.

The second aspect of the present invention provides adipose stem cell extracellular vesicles extracted according to the above method, the extracellular vesicles show an average diameter of 131 nm, and highly express CD63, CD81 and TSG101 but not GM130.

The third aspect of the present invention provides the application of the adipose stem cell extracellular vesicles prepared according to the above method in the preparation of a drug for promoting skin wound healing.

Preferably, the bioactive preparation for promoting skin wound healing is a bioactive preparation for promoting the viability and proliferation of epidermal cell lines and skin fibroblasts, a bioactive preparation for improving the migration ability of skin fibroblasts, or a bioactive preparation for promoting the formation of umbilical vein endothelial cell lines Bioactive agents for vascular capacity.

The fourth aspect of the present invention provides a bioactive preparation for promoting skin wound healing, which contains the above-mentioned adipose stem cell extracellular vesicles, and the added mass of the adipose stem cell extracellular vesicles is 200 μg.

Owing to adopting the above-mentioned technical scheme, the present invention has the following advantages and beneficial effects:

In terms of effects, it has been verified that the number of vesicles secreted by adipose stem cells stimulated by ultrasound is more than 60 times higher than that of adipose stem cells without ultrasound stimulation; the protein content of the vesicles is increased by more than 3 times, and the specific markers of vesicles are highly expressed. thing. In vitro cell experiments showed that the extracellular vesicles can effectively promote the viability and proliferation of epidermal cell lines and skin fibroblasts, improve the migration ability of skin fibroblasts, or promote the angiogenesis ability of umbilical vein endothelial cells, and can promote skin wound healing. The effect is remarkable.

In terms of specific implementation, ultrasonic stimulation is simple to operate and easy to popularize on a large scale.

Description of drawings

Figure 1 shows that ultrasonic stimulation significantly promotes the secretion of extracellular vesicles from adipose stem cells: A. The appearance of extracellular vesicles extracted from adipose stem cells after ultrasonic stimulation under transmission electron microscope; B. NTA detection of extracellular vesicles shows that their average diameter is 131nm; C. Western Blot results showed that extracellular vesicles highly expressed CD63, CD81 and TSG101 but not GM130; D. Endocytosis experiments showed that vesicles could be taken up by skin fibroblasts; E. NTA detection showed that: 1.5W/cm ² for 30s stimulated adipose stem cells, and the number of secreted vesicles increased by more than 60 times; F. The quantitative results of protein showed that: 1.5W/cm ² for 30s stimulated adipose stem cells, the secreted vesicles increased by more than 60 times. The protein content increased by more than 3 times; G.Western Blot results showed that after adipose stem cells were stimulated at 1.5W/cm ² for 30s, the secreted vesicles highly expressed vesicle-specific markers.

Figure 2 shows that ultrasound-stimulated adipose stem cells-secreted vesicles (US-EVs) significantly promoted the viability and proliferation of epidermal cell lines (HACATs) and skin fibroblasts (FBs): A. Effects of different concentrations of US-EVs and EVs on FBs The effect of different concentrations of US-EVs and EVs on the cell viability and effect comparison of HACATs; C, D. The comparison of the effect of different concentrations of US-EVs and EVs on the cell proliferation ability of FBs; E, F. Comparison of the effect of different concentrations of US-EVs and EVs on the cell proliferation of HACATs.

Figure 3 shows that ultrasound-stimulated adipose stem cells secreted vesicles (US-EVs) significantly promoted the migration ability of skin fibroblasts (FBs): A. The results of different concentrations of US-EVs and EVs on the migration of FBs; B. Different concentrations EVs and EVs had no significant effect on the migration of HACATs; C, US-EVs and EVs compared statistics on the promotion effect of FBs migration; D, statistics of the effect of US-EVs and EVs on the migration ability of HACATs.

Figure 4 shows that ultrasound-stimulated vesicles (US-EVs) secreted from adipose stem cells significantly promoted the angiogenesis of umbilical vein endothelial cell lines (HUVECs): A. The effects and effects of different concentrations of US-EVs and EVs on the cell viability of HUVECs Comparison; B. The effect of different concentrations of US-EVs and EVs on HUVECs migration; C. Microscopic images of the effect of different concentrations of US-EVs and EVs on HUVECs angiogenesis in vitro; D. Different concentrations of US-EVs and EVs on HUVECs cell migration E, the effect of different concentrations of US-EVs and EVs on the number of blood vessels; F, the effect of different concentrations of US-EVs and EVs on the formation of blood vessel branch points.

Figure 5 shows that ultrasound-stimulated vesicles (US-EVs) secreted by adipose stem cells significantly promoted the healing of skin wounds in diabetic mice: A pictures of changes in wound size in different experimental groups from 0 to 14 days; B: different experimental groups in different periods Comparison of statistical results of wound area.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to the preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

Example 1: Using ultrasonic stimulation to improve the efficiency of extracellular vesicle secretion by adipose stem cells

1. Cell extraction and identification

Extraction and identification of adipose stem cells: Enzymatic digestion was used to digest the granular fat discarded after liposuction to extract adipose stem cells, which were routinely passaged after confluency, and were subjected to flow cytometry and three-way induction (osteogenesis, adipogenic and chondrogenesis) to identify the third generation of adipose stem cells (P3).

When performing the digestion treatment, the digestive enzyme used is type IV collagenase; the conditions of the digestion treatment are as follows: 0.2% (m/v) type IV collagenase and granular adipose tissue are mixed at a volume ratio of 1:1, at 37° C. Shake well on a shaker and digest for 2 hours.

2. Ultrasound stimulates adipose stem cells

The cells were ultrasonically stimulated using a 1MHz, 10Hz pulse repetition frequency, 60% duty cycle, and 9mm cross ^- sectional area ultrasonic generator. The specific process was as follows: 0.5-1cm thick couplant was evenly spread on a 10cm cell culture dish Then, the transducer of the ultrasonic generating device is closely attached to the coupling agent to remove air, and the ultrasonic stimulation intensity used is: 0.5-1.5W/cm ^{2 , 30-120s, preferably 1.5W/cm 2 ,} 30s.

When ultrasonic stimulation is performed ^on adipose stem cells, the stimulation intensity and time should not be too large or too strong, so as to avoid cell apoptosis or rupture.

3. Extraction of extracellular vesicles from adipose stem cells

After the third passage of adipose-derived stem cells were confluent, the cells were washed twice with PBS, and then replaced with serum-free low-glucose DMEM. (1) For cells without ultrasonic stimulation: After 48 hours of continuous culture, the cell supernatant was collected. (2) For ultrasonically stimulated cells: perform ultrasonic stimulation according to step 2, and then collect the cell supernatant after culturing for 48 hours. The extracellular vesicles in the supernatant of the cells treated differently were extracted by ultrafiltration, and the extracted vesicles were dissolved in PBS and stored in a -80°C refrigerator.

Example 2: Identification and analysis of adipose stem cell extracellular vesicles

1. Identification of adipose stem cell extracellular vesicles

The shape and size of vesicles were observed by transmission electron microscopy, the average diameter of vesicles was calculated by nanoparticle tracking analysis (NTA), the specific markers related to vesicles were detected by Western Blot, and endocytosis experiments were used to verify whether extracellular vesicles could be absorbed by recipient cells. ingested.

The endocytosis experiment was carried out with skin fibroblasts, and the extraction method was as follows: after soaking and digesting the foreskin with Dispase for 24 hours, the epidermis was removed, the remaining dermis was digested with collagenase, and the skin fibroblasts were extracted. To study the effect of extracellular vesicles on the proliferation and migration of skin fibroblasts in vitro.

2. Quantitative analysis of extracellular vesicles from adipose stem cells

Quantitative analysis of extracted vesicles by NTA and protein quantification, respectively

(1) NTA quantification: Take a small amount of vesicle solution, dilute it with PBS buffer to a volume of about 300 μL, analyze the distribution of particle number and particle diameter, etc., and record the dilution ratio on the machine to facilitate conversion with protein quantification.

(2) Protein quantification (BCA method): The BCA protein quantification kit of Biyuntian Company was used for quantitative detection, protein standards of different concentrations were prepared, and the vesicles were diluted by a certain number of times, and the appropriate volume of protein standards and The vesicles were added to a 96-well plate, and the working solution was added for the reaction, and the protein concentration of the vesicles was calculated by the absorbance value of each well.

The results are shown in Figure 1. The transmission electron microscope showed that the extracellular vesicles extracted after ultrasonic stimulation of adipose stem cells were irregular in shape (Figure 1A); NTA detection of extracellular vesicles showed that their average diameter was 131 nm (Figure 1B); Western Blot results showed that extracellular vesicles highly expressed CD63, CD81 and TSG101 but not GM130 (Fig. 1C). After adipose stem cells were stimulated at 1.5W/cm ² for 30s, the secreted vesicles highly expressed vesicle-specific markers (Fig. 1G); Endocytosis experiments showed that vesicles could be taken up by skin fibroblasts (Fig. 1D); NTA assay showed that: adipose stem cells were stimulated at 1.5W/cm ² for 30s, and the number of vesicles secreted The protein content was increased by more than 60 times (Fig. 1E); the protein quantification results showed that the amount of protein contained in the secreted vesicles of adipose stem cells was stimulated by 1.5W/cm ² for 30s, and the amount of protein was increased by more than 3 times (Fig. 1F).

Example 3: Effect Verification

1. Cell viability detection

Epidermal cell lines (HACATs) and skin fibroblasts (FBs) were seeded in 96-well plates, respectively, and different concentrations (20, 50, 100, 150, 200 μg/mL) of untreated adipose stem cell extracellular vesicles were added. (EVs) and ultrasound-stimulated adipose stem cell extracellular vesicles (US-EVs), cultured for 72 hours, and measured the cell OD value at 450 nm with the CCK-8 kit according to the instructions. The cell viability was calculated by the following formula:

Cell viability=(OD value of cells in treatment group/OD value of control group)×100%

The dose of vesicles used in subsequent in vitro experiments was determined by the results of cell viability.

2. Cell proliferation assay

Epidermal cell lines (HACATs) and skin fibroblasts (FBs) were seeded in 96-well plates, respectively, with different concentrations (determined by the above cell viability assay) of untreated adipose stem cell extracellular vesicles (EVs) and sonication. The stimulated adipose stem cell extracellular vesicles (US-EVs) were incubated for 48 hours, and the cells were labeled and fixed with fluorescent dyes according to the instructions of the EdU detection kit, and the cell proliferation rate was observed and calculated by fluorescence microscope. The cell proliferation rate was calculated according to the following formula:

Cell proliferation rate = (number of EdU positive cells/total number of inoculated cells) × 100%

The above experimental results are shown in Figure 2. Compared with EVs, US-EVs significantly promoted the cell viability (Figure 2A) and cell proliferation (Figure 2C, D) of FBs in a dose-dependent manner; Promote the viability of HACATs cells, but there is no significant difference compared with EVs, and the effect of different doses of US-EVs on cell viability is not significantly different. US-EVs (Fig. 2B), but could significantly promote the cell proliferation ability of HACATs (Fig. 2E, F).

3. Cell migration assay

Cell migration was detected by scratch assay. Epidermal cell lines (HACATs), skin fibroblasts (FBs), and umbilical vein endothelial cell lines (HUVECs) were seeded into 6-well plates. Scratch the cells, add serum-free/1% serum-containing DMEM, and take pictures after scratching as 0 h. The experimental group was added with different concentrations of EVs and US-EVs, and the control group was added with an equal volume of PBS. After 24 hours, the photos were taken as 24h, the scratch areas at 0h and 24h were measured, and the cell migration rate was calculated by the following formula:

Cell mobility=(0h scratch area-24h scratch area)/0h scratch area×100%.

The results are shown in Figure 3. Compared with EVs, US-EVs significantly promoted the migration ability of FBs, and high concentration of US-EVs promoted migration better than low concentrations (Figure 3A, C). Compared with EVs, US-EVs had no significant significantly altered the migratory capacity of HACATs (Fig. 3B,D).

4. Detection of tube-forming ability of umbilical vein endothelial cells

Matrigel was spread on the bottom of a 96-well plate and placed in an incubator for 20 minutes. After it solidified, the umbilical vein endothelial cell line (HUVECs) was inoculated on Matrigel, and low-glucose DMEM was added. The experimental group was added with different concentrations of EVs and US- EVs and control group were added with equal volume of PBS. After 6 h, the tube formation was observed under the microscope and photographed, and the number of tubes was counted by software.

The results are shown in Figure 4. Compared with EVs, US-EVs significantly promoted the cell viability of HUVECs (Figure 4A); US-EVs significantly promoted the migration ability of HUVECs compared with EVs (Figures B, D). Compared with EVs, EVs significantly promoted the angiogenesis of HUVECs (Fig. 4C), and the number of blood vessels and branch points were significantly better than those of EVs (Fig. 4E, F).

5. Construction and treatment steps of diabetic mouse skin wound model

32 female c57 mice (diabetic mice) with a 7-week-old knockout of the db gene were purchased, and were routinely raised in an SPF environment for 1 week to adapt to the environment. From week 8, mice were randomly divided into 4 groups:

(1) PBS group: wounds were made on the back of mice and treated with PBS;

(2) HA+PBS group: wounds were made on the back of mice and treated with 100 μL hyaluronic acid gel (HA)+100 μL PBS;

(3) HA+EV group: wounds were made on the back of mice and treated with 100 μL HA+200 μL EVs (the solution volume was 100 μL, and the final concentration of EVs was 1 μg/μL);

(4) HA+US-EV group: wounds were made on the back of mice, and 100 μL HA+200 μg US-EVs (the solution volume was 100 μL, the final concentration of EVs was 1 μg/μL) was treated;

The skin wound model was established as follows: the mice were anesthetized with a small animal anesthetic, a circular scratch was made on the back of the mouse with a corneal trephine with a diameter of 6 mm, and the skin was cut along the scratch with ophthalmic scissors, reaching deep tendons. The film layer was cut off, and the skin wounds were treated according to the above groups. Parts of different preparations were applied on the surface of the wounds, and some were injected subcutaneously around the wound edges.

6. Evaluation of skin wound treatment effect:

General view: The skin wounds on the 0th, 3rd, 7th, 10th and 14th days were photographed, and the healing, exudation and infection of the skin wounds were observed with the naked eye. Pro Plus 6.0 calculates the size of the wound area and evaluates the changes in the size of the wound in each group.

The results are shown in Figure 5. On the 3rd day, the skin healing of the four experimental groups was similar. Over time, the skin healing of the US-EVs group was significantly better than that of the other groups. On the 14th day, the skin of this group was almost completely healed, and the other three There were still skin lesions in the group (Fig. 5A, B).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Within the scope of the technical solution of the present invention, personnel can make some changes or modifications to equivalent examples of equivalent changes by using the above-mentioned technical content, but any content that does not depart from the technical solution of the present invention is based on the technical solution of the present invention. Substantially any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solutions of the present invention.

Claims (10)

1. a method utilizing ultrasonic stimulation to improve the efficiency of adipose stem cells to secrete extracellular vesicles, is characterized in that, comprises the steps: A. Adipose stem cell extraction The granular fat discarded after liposuction was digested by enzymatic digestion, and adipose stem cells were extracted, and then routinely passaged to the 3rd generation after full growth; B. Ultrasound stimulates adipose stem cells After the third-generation adipose stem cells are full, wash the cells twice with PBS, then replace the serum-free low-sugar DMEM, spread a certain thickness of coupling agent evenly under the cell culture dish, and then put the transducer of the ultrasonic generating device It is closely attached to the couplant to exclude air, the dose of ultrasound is set to 0.5-1.5W/cm ² , and the exposure time is 30-120s; C. Extraction of adipose stem cell extracellular vesicles The adipose stem cells were stimulated by ultrasound and cultured for 48 hours, and then the cell supernatant was collected; the extracellular vesicles in the supernatant were extracted by ultrafiltration and dissolved in PBS. 2. The method according to claim 1, characterized in that: Wherein, in step A, the digestive enzyme adopted is type IV collagenase; The digestion treatment conditions are as follows: 0.2% mass-volume ratio of type IV collagenase and granular adipose tissue are mixed at a volume ratio of 1:1, fully shaken on a shaker at 37°C, and digested for 2 hours. 3. The method according to claim 1, characterized in that: Wherein, in step A, when the adipose stem cells are routinely passaged to the third generation, flow cytometry and three-way induction of osteogenesis, adipogenic and chondrogenesis are used to identify the third generation of adipose stem cells. 4. The method according to claim 1, characterized in that: Wherein, in step B, ultrasonic stimulation is performed on the cells by using an ultrasonic generating device with a pulse repetition frequency of 1 MHz, a pulse repetition frequency of 10 Hz, a duty cycle of 60%, and a cross-sectional area of 9 mm ² . 5. The method according to claim 4, characterized in that: Among them, when performing ultrasonic stimulation, 0.5-1cm thick couplant is evenly spread under the 10cm cell culture dish, and then the transducer of the ultrasonic generating device is closely attached to the couplant to remove air, and the ultrasonic dose is set to 1.5W/cm ² , the exposure time is 30s. 6. The method according to claim 1, characterized in that: Wherein, in step C, the supernatant is filtered with a 0.22 μm filter and then added to an ultrafiltration centrifuge tube with a 100KD membrane, centrifuged at 3000 g at 4°C to a volume of 500 μL in the inner tube, transferred to a sterile cryopreservation tube, and transferred to a -80°C refrigerator for freezing live. 7. Adipose stem cell extracellular vesicles extracted by the method according to any one of claims 1 to 6. 8. The application of the adipose stem cell extracellular vesicle according to claim 7 in the preparation of a bioactive preparation for promoting skin wound healing. 9. Use according to claim 7, characterized in that: Wherein, the bioactive preparation for promoting skin wound healing is a preparation for promoting the viability and proliferation of epidermal cell lines and skin fibroblasts, a preparation for improving the migration ability of skin fibroblasts, or a preparation for promoting the angiogenesis ability of umbilical vein endothelial cells . 10 . A bioactive preparation for promoting skin wound healing, characterized in that it contains the adipose stem cell extracellular vesicles according to claim 7 , and the added mass of the adipose stem cell extracellular vesicles is 200 μg.

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