Application of stem cell exosome in treating acute lung injury
By using human streptococcal lysates to stimulate umbilical cord mesenchymal stem cells and increase their exosome secretion, the problem of low exosome secretion in the treatment of acute lung injury is solved, and the effect on lung injury repair is significantly enhanced. A highly effective biological agent is provided for the treatment of acute lung injury.
Patent Information
- Application Number
- CN202410953072.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-16
- Publication Date
- 2024-12-31
AI Technical Summary
There is a lack of effective treatment for acute lung injury, and the secretion of stem cell exosomes is low, resulting in low efficacy in treating acute lung injury.
By stimulating the umbilical cord mesenchymal stem cells with human Streptococcus pneumoniae lysates, the secretion of their exosomes is significantly increased, and the preparation method includes isolating, culturing and extracting exosomes to form efficient biological agents.
It significantly increased the secretion of mesenchymal stem cells of umbilical cord, enhanced its effect on repairing human alveolar epithelial cell injury, weakened the inhibitory effect of LPS-induced cell viability, and improved the efficacy of treating acute lung injury.
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Figure CN119215070A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological technology, and in particular relates to an application of stem cell exosomes in treating acute lung injury. Background Art
[0002] Acute lung injury is a common and potentially fatal disease characterized by rupture of the alveolar capillary membrane, pulmonary edema, and impaired gas exchange due to increased apoptosis and lung inflammation. Acute lung injury is an excessive and uncontrolled systemic inflammatory response triggered by direct or indirect lung injury. It is a serious and critical illness. There is no effective and specific treatment for acute lung injury. However, mesenchymal stem cells have been shown to be a potential option.
[0003] Stem cells have become an important breakthrough in the medical field due to their unique self-renewal and multidirectional differentiation capabilities. Multipotent stem cells and mesenchymal stem cells may play a promoting role in the treatment of acute lung injury. Stem cells have paracrine functions, regulating the state of inflammatory cells and immune microenvironment in specific areas, and the ability to differentiate into blood vessel-related cells.
[0004] Studies have reported that among the secretory functions of stem cells, exosomes are one of the main parts that play a major role in paracrine effects. Exosomes have a nanoscale double-layer membrane structure and contain lipids, microRNAs, and proteins that play a key role in intercellular communication. Due to their low immunogenicity and tumorigenicity, as well as their easy storage and management characteristics, exosomes have become an alternative to whole-cell therapy. Umbilical cord mesenchymal stem cell exosomes are expected to play an effective role in the treatment of acute lung injury, but the low secretion of conventional cultured stem cell exosomes and low activity in the treatment of acute lung injury remain to be solved. Therefore, how to increase the secretion of stem cells and induce stem cells to produce exosomes for the treatment of acute lung injury is the key to the application of stem cell exosomes in epidermal injury repair preparations. Summary of the invention
[0005] The purpose of the present invention is to provide an application of stem cell exosomes in the treatment of acute lung injury, so as to better achieve the effect of epidermal wound repair by utilizing umbilical cord mesenchymal stem cell exosomes.
[0006] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides an application of stem cell exosomes in the treatment of acute lung injury, wherein the stem cell exosomes are exosomes produced by umbilical cord mesenchymal stem cells under the stimulation of Streptococcus pneumoniae lysate.
[0007] Furthermore, the method for preparing stem cell exosomes comprises the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) preparing pneumococcal lysate; (3) Umbilical cord mesenchymal stem cells are passaged into cell culture bottles for no more than 5 generations, with a cell density of 1×106 / mL, and cultured in a carbon dioxide incubator for 24 hours until the cell density reaches more than 80%; (4) Add Streptococcus pneumoniae lysate to the cell flask and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. Mix the exosome extraction reagent and cell culture supernatant in a 1:1 ratio. After standing for 16 h, centrifuge at 12,000 rpm for 1 h to collect the precipitate. Dissolve the exosomes in PBS with 1 / 10 volume of the original culture medium.
[0008] Furthermore, the dosage of the Streptococcus pneumoniae lysate added to the umbilical cord mesenchymal stem cell culture medium is 50 μg / mL.
[0009] Furthermore, the method for preparing the pneumococcal lysate comprises the following steps: (1) Take frozen Streptococcus pneumoniae and streak it on a blood agar plate with an inoculation loop. Incubate the plate at 37°C in 5% CO2 for 24 hours.
[0010] (2) Select colonies with typical hemolysis zones and streak them on a new blood agar plate. Incubate the plate at 37°C with 5% CO2 for 24 hours.
[0011] (3) Rinse off the surface bacteria on each 90 mm blood agar plate with 5 ml of PBS.
[0012] (4) The bacterial solution was disrupted by ultrasonic disruptor in an ice bath, centrifuged at 12000 rpm / min for 5 min, and the supernatant was collected.
[0013] (5) The broken bacterial liquid is filtered through a 0.22 μm filter to obtain the pneumococcal lysate. The protein concentration is determined and stored at 4°C for later use.
[0014] Secondly, the present invention provides a biological preparation for increasing the production of exosomes from umbilical cord mesenchymal stem cells, wherein the biological preparation is a non-pathogenic human Streptococcus pneumoniae lysate.
[0015] Furthermore, the added dosage of the biological agent in the umbilical cord mesenchymal stem cell culture medium is 50 μg / mL.
[0016] Furthermore, the method for preparing the biological agent comprises the following steps: (1) Take frozen Streptococcus pneumoniae and streak it on a blood agar plate with an inoculation loop. Incubate the plate at 37°C in 5% CO2 for 24 hours.
[0017] (2) Select colonies with typical hemolysis zones and streak them on a new blood agar plate. Incubate the plate at 37°C with 5% CO2 for 24 hours.
[0018] (3) Rinse off the surface bacteria on each 90 mm blood agar plate with 5 ml of PBS.
[0019] (4) The bacterial solution was disrupted by ultrasonic disruptor in an ice bath, centrifuged at 12000 rpm / min for 5 min, and the supernatant was collected.
[0020] (5) The broken bacterial liquid is filtered through a 0.22 μm filter to obtain the pneumococcal lysate. The protein concentration is determined and stored at 4°C for later use.
[0021] The beneficial effects of the present invention are: The present invention prepares pneumococcal lysate by culturing human pneumococcal bacteria, and uses the human pneumococcal lysate to stimulate umbilical cord mesenchymal stem cells to prepare exosomes. Compared with conventionally cultured umbilical cord mesenchymal stem cell exosomes, the secretion of umbilical cord mesenchymal stem cell exosomes can be significantly increased by stimulating with human pneumococcal lysate. When exosomes are used for LPS-induced human alveolar epithelial cell damage repair, the same dose of conventional exosomes and pneumococcal lysate cultured exosomes can weaken the inhibitory effect of LPS on A549 cell viability, and pneumococcal lysate cultured exosomes have a stronger effect on the repair of human alveolar epithelial cell damage than conventional exosomes. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The results of the cytotoxicity test of Streptococcus pneumoniae lysate on umbilical cord mesenchymal stem cells; Figure 2 The grayscale difference of CD36 protein in exosomes of umbilical cord mesenchymal stem cells after culture with different concentrations of Streptococcus pneumoniae lysate; Figure 3 The effect of umbilical cord mesenchymal stem cell exosomes on the repair of damaged human alveolar epithelial cells. DETAILED DESCRIPTION
[0023] The following is a more detailed description of the present invention, which is illustrated by examples. It should be clear that these examples are only used as examples of the present invention, and their purpose is to illustrate the principles and functions of the present invention, but not to limit the scope of protection of the present invention.
[0024] Example 1 Isolation and culture of umbilical cord mesenchymal stem cells (1) Obtain an appropriate amount of umbilical cord tissue from full-term newborns using sterile instruments.
[0025] (2) The fresh umbilical cord tissue was transferred into the culture medium containing double antibodies, processed with sterile instruments, and then repeatedly rinsed with PBS for 3 times to wash away blood stains and other foreign matter on the surface.
[0026] (3) Use an appropriate amount of trypsin to digest for about 8 hours and then terminate the digestion. Then transfer the digested cells to a centrifuge tube, add 15 times the volume of PBS and centrifuge. After centrifugation, discard the liquid.
[0027] (4) Add an appropriate amount of mesenchymal stem cell culture medium to resuspend the centrifuged cells, inoculate them into a cell culture bottle, and move them to a carbon dioxide incubator with a CO2 concentration of 5% for culture. Check the adhesion and growth of stem cells every 2 hours. Subsequently, check the cell growth status every 48 hours and replace the culture medium.
[0028] (5) When the cells grow to 80%-90% of the bottom of the bottle, they are subcultured and frozen at the second generation. The stem cells used in this study are from the third to fifth generations.
[0029] Example 2 Preparation of Streptococcus pneumoniae lysate (1) Take frozen Streptococcus pneumoniae from the laboratory, streak it on a blood agar plate with an inoculation loop, and incubate it at 37°C with 5% CO2 for 24 hours.
[0030] (2) Select colonies with typical hemolysis zones and streak them on a new blood agar plate. Incubate the plate at 37°C with 5% CO2 for 24 hours.
[0031] (3) Rinse off the surface bacteria on each 90 mm blood agar plate with 5 ml of PBS.
[0032] (4) The bacterial solution was disrupted by ultrasonic disruptor in an ice bath, centrifuged at 12000 rpm / min for 5 min, and the supernatant was collected.
[0033] (5) The broken bacterial solution is filtered through a 0.22 μm filter to obtain the pneumococcal lysate. The protein concentration is determined and stored at 4°C for later use.
[0034] Example 3 Detection of the cytotoxicity of Streptococcus pneumoniae lysate on umbilical cord mesenchymal stem cells (1) After the umbilical cord mesenchymal stem cells preserved in the experiment were revived and cultured to the third generation, the cells were digested to prepare a single cell suspension and the cells were inoculated into a 96-well plate and cultured in a carbon dioxide incubator with a CO2 concentration of 5% until the cell density reached about 80%.
[0035] (2) Use mesenchymal stem cell culture medium to prepare Streptococcus pneumoniae lysate into Streptococcus pneumoniae lysate culture medium with different concentrations: 10 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL and 500 μg / mL.
[0036] (3) Remove the culture medium in the 96-well cell culture plate and replace it with the Streptococcus pneumoniae lysate culture medium in step (2). Replace the control group with the mesenchymal stem cell culture medium.
[0037] (4) After culturing in a cell culture incubator for 24 h, the OD450 value was measured using CCK-8 and the cell survival rate was calculated. Cell survival rate = (OD450 of the experimental group - OD450 of the blank control group) / (OD450nm of the control group - OD450 of the blank control group).
[0038] from Figure 1 It can be seen that with the increase of the concentration of Streptococcus pneumoniae lysate in the range of 10-100 μg / mL, the cell survival rate of umbilical cord mesenchymal stem cells remained basically stable, and there was no statistically significant difference between the groups. However, the survival rate of umbilical cord mesenchymal stem cells in the 250 μg / mL and 500 μg / mL Streptococcus pneumoniae lysate groups decreased to 78.76% and 59.85%, respectively, indicating that the concentration of Streptococcus pneumoniae lysate in the culture medium reaching 250 μg / mL and above will cause toxicity to umbilical cord mesenchymal stem cells.
[0039] Example 4 Preparation of umbilical cord mesenchymal stem cell exosomes (1) Umbilical cord mesenchymal stem cells were passaged into 75T cell culture flasks, with 30 mL of mesenchymal stem cell serum-free medium per flask and a cell density of 1×10 6 / mL, and cultured in a carbon dioxide incubator with a CO2 concentration of 5% for 24 hours until the cell density reached more than 80%. Groups 1 to 4 were added with 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL of pneumococcal lysate, respectively, and group 5 was a cell control without pneumococcal lysate. Each group had 3 replicates. Continue to culture for 24 hours, collect the cell culture supernatant, and according to the instructions of the exosome extraction kit, mix the exosome extraction reagent and ultrafiltered cell culture supernatant in a ratio of 1:1. After standing for 16 hours, centrifuge at 12000 rpm for 1 hour to collect the precipitate, and dissolve the exosomes with 1 / 10 volume of the original culture medium PBS.
[0040] (2) Use RIPA lysis buffer and exosomes in equal volumes, let stand on ice for 1 hour, and shake for 1 to 2 minutes every 10 minutes. Centrifuge at 12000rpm for 15 minutes at 4℃, and collect the supernatant. Mix the sample and 2× Loading buffer at a volume ratio of 1:1, and boil in a water bath for 10 minutes. Perform SDS-PAGE electrophoresis on the prepared sample, transfer to a PVDF membrane, block with 5% skim milk powder solution for 1 hour, use anti-mouse CD63 (ab217345) and GAPDH antibody (ab9485) purchased from Abcam, incubate at 37℃ for 1 hour, rinse 3 times with PBS; use goat anti-rabbit secondary antibody (ab150077) at a dilution of 1:2000, incubate at 37℃ for 1 hour, rinse 3 times with PBS; finally, use ECL luminescent reagent for color development and exposure. Quantity One v462 software was used to analyze the protein gray value and compared with the internal reference protein band to obtain the relative gray ratio. The gray value of the target protein = target protein OD value / corresponding internal reference OD value.
[0041] from Figure 2 It can be seen that the amount of exosome marker protein CD63 produced by umbilical cord mesenchymal stem cells cultured with 12.5 μg / mL, 25 μg / mL, and 50 μg / mL pneumococcal lysate in the culture medium showed a significant increase trend with the increase of pneumococcal lysate concentration, while there was no statistically significant difference in the amount of exosome marker protein CD63 produced by umbilical cord mesenchymal stem cells cultured with 50 μg / mL and 100 μg / mL pneumococcal lysate groups. The gray value of exosome marker protein CD63 produced by umbilical cord mesenchymal stem cells cultured without pneumococcal lysate was 0.7785, and the gray value of exosome marker protein CD63 produced by umbilical cord mesenchymal stem cells cultured with 50 μg / mL pneumococcal lysate was 1.636. The amount of exosome secretion produced by umbilical cord mesenchymal stem cells cultured with 50 μg / mL pneumococcal lysate was 2.1 times that of the group without pneumococcal lysate.
[0042] Example 5 Effect of umbilical cord mesenchymal stem cell exosomes on the repair of damaged human alveolar epithelial cells (1) Adjust the cell density of human alveolar epithelial cell line A549 to 1×10 5 / well, inoculated into a 96-well plate, placed in a 37℃, 5% carbon dioxide incubator for 24h, then the normal cell wells were discarded and LPS 100µg / mL was added to each of the remaining wells.
[0043] (2) The first group was the LPS-induced alveolar epithelial cell A549 injury model group, in which 100 μg / mL LPS was added and then 50 μL of cell culture medium was added; the second group was the conventional exosome group, in which 50 μL of exosomes produced by umbilical cord mesenchymal stem cells without the addition of Streptococcus pneumoniae lysate in Example 4 was added to each well; the third group was the Streptococcus pneumoniae lysate cultured exosome group, in which the exosomes produced by umbilical cord mesenchymal stem cells cultured with 50 μg / mL Streptococcus pneumoniae lysate in Example 4 were diluted with culture medium and diluted to the same gray value of the exosome marker protein CD63 as that of the exosomes in the third group.
[0044] (3) After LPS and exosomes were added to A549 cells, the four groups of cells were cultured in a 37°C, 5% carbon dioxide incubator for 6 hours. The OD450 value was determined using CCK-8, and the cell survival rate was calculated. Cell survival rate = (OD450 of the experimental group - OD450 of the blank control group) / (OD450nm of the control group - OD450 of the blank control group). The data are expressed as the average of three independent experiments.
[0045] from Figure 3 It can be seen that after adding 100µg / mL LPS to A549 cells, the cell survival rate decreased to 56.04%; the cell survival rate of the conventional exosome group was 83.51%, and the cell survival rate of the exosome group cultured with Streptococcus pneumoniae lysate was 100.44%, which was not statistically significant compared with the normal cell control group. It can be seen that the same dose of conventional exosomes and exosomes cultured with Streptococcus pneumoniae lysate can weaken the inhibitory effect of LPS on the viability of A549 cells, and exosomes cultured with Streptococcus pneumoniae lysate have a stronger effect on the repair of human alveolar epithelial cell damage than conventional exosomes.
Claims
1. A use of stem cell exosomes in the treatment of acute lung injury, characterized in that: The stem cell exosomes are exosomes produced by umbilical cord mesenchymal stem cells under the stimulation of Streptococcus pneumoniae lysate.
2. The use according to claim 1, characterized in that: The method for preparing stem cell exosomes comprises the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) preparing pneumococcal lysate; (3) Umbilical cord mesenchymal stem cells were subcultured into cell culture flasks. The number of subcultures did not exceed 5, and the cell subculture density was 1×10 6 / mL, culture in a carbon dioxide incubator for 24 h until the cell density reaches more than 80%; (4) Add Streptococcus pneumoniae lysate to the cell flask and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. Mix the exosome extraction reagent and cell culture supernatant in a 1:1 ratio. After standing for 16 h, centrifuge at 12,000 rpm for 1 h to collect the precipitate. Dissolve the exosomes in PBS with 1 / 10 volume of the original culture medium.
3. The Streptococcus pneumoniae lysate according to claim 2, characterized in that The dosage of the pneumococcal lysate added to the umbilical cord mesenchymal stem cell culture medium is 50 μg / mL.
4. The Streptococcus pneumoniae lysate according to claim 3, characterized in that The method for preparing the pneumococcal lysate comprises the following steps: (1) Take frozen pneumococcus, streak it on a blood agar plate with an inoculation loop, and incubate it at 37°C with 5% CO2 for 24 hours. (2) Select colonies with typical hemolysis zones and streak them on a new blood agar plate. Incubate at 5% CO2 and 37°C for 24 hours. (3) Rinse off the surface bacteria on each 90 mm blood agar plate with 5 ml of PBS; (4) The bacterial solution was disrupted by ultrasonic disruptor in an ice bath, centrifuged at 12000 rpm / min for 5 minutes, and the supernatant was collected; (5) The broken bacterial liquid is filtered through a 0.22 μm filter to obtain the pneumococcal lysate. The protein concentration is determined and stored at 4°C for later use.
5. A biological preparation for increasing the production of exosomes from umbilical cord mesenchymal stem cells, characterized in that: The biological preparation is a lysate of human Streptococcus pneumoniae.
6. The biological agent according to claim 5, characterized in that The dosage of the biological preparation added to the umbilical cord mesenchymal stem cell culture medium is 50 μg / mL.
7. The biological agent according to claim 6, characterized in that The preparation method of the biological preparation The method includes the following steps: (1) Take frozen pneumococcus, streak it on a blood agar plate with an inoculation loop, and incubate it at 37°C with 5% CO2 for 24 hours. (2) Select colonies with typical hemolysis zones and streak them on a new blood agar plate. Incubate at 5% CO2 and 37°C for 24 hours. (3) Rinse off the surface bacteria on each 90 mm blood agar plate with 5 ml of PBS; (4) The bacterial solution was disrupted by ultrasonic disruptor in an ice bath, centrifuged at 12000 rpm / min for 5 minutes, and the supernatant was collected; (5) The broken bacterial liquid is filtered through a 0.22 μm filter to obtain the pneumococcal lysate. The protein concentration is determined and stored at 4°C for later use.