CN114540296A - Preparation method of composite exosome and application of composite exosome in directionally enhancing angiogenesis capacity - Google Patents
Preparation method of composite exosome and application of composite exosome in directionally enhancing angiogenesis capacity Download PDFInfo
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- CN114540296A CN114540296A CN202210281021.XA CN202210281021A CN114540296A CN 114540296 A CN114540296 A CN 114540296A CN 202210281021 A CN202210281021 A CN 202210281021A CN 114540296 A CN114540296 A CN 114540296A
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Abstract
The application relates to the technical field of stem cell exosomes, and particularly discloses a preparation method of a composite exosome and application of the composite exosome in directionally enhancing angiogenesis capacity. The preparation method of the composite exosome comprises the following steps: culturing cells and preparing a composite exosome; the culture medium adopted in the cell culture step is a mesenchymal stem cell serum-free culture medium; the mesenchymal stem cell serum-free culture medium further comprises nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium; the composite exosome prepared by the preparation method of the composite exosome and the application of the composite exosome in directionally enhancing the angiogenesis capacity. The high-content human umbilical cord mesenchymal stem cell exosome can be obtained, and the human umbilical cord mesenchymal stem cell exosome can promote the generation and differentiation of blood vessels and enhance the generation capacity of the blood vessels.
Description
Technical Field
The application relates to the technical field of stem cell exosomes, in particular to a preparation method of a composite exosome and application of the composite exosome in directionally enhancing angiogenesis capacity.
Background
Mesenchymal stem cells are adult stem cells having excellent properties, and are widely present in tissues such as umbilical cords, bone marrow, fat, and blood, and in recent years, research on mesenchymal stem cells and their plasticity has become one of the hot spots in the biomedical field. The mesenchymal stem cells not only can continuously carry out self-renewal, but also can be cultured, amplified and directionally induced and differentiated into various cells in vivo. In clinical operation, mesenchymal stem cells are often combined with hematopoietic stem cells to improve the success rate of surgical transplantation and accelerate hematopoietic reconstruction; in addition, the mesenchymal stem cells can be differentiated and induced into bone in vitro, and further have wide application.
Exosomes are microvesicles secreted by a variety of cells under normal or pathological conditions. Research shows that the human umbilical cord mesenchymal stem cells can secrete an exosome, and the exosome can repair tissues and promote angiogenesis like the human umbilical cord mesenchymal stem cells, and is more stable, safer and easier to store than the human umbilical cord mesenchymal stem cells. However, at present, the extraction of high-content human umbilical cord mesenchymal stem cell exosomes is difficult, firstly, the operation of the extraction process is complex, and secondly, the time consumption of the extraction process is long, so that the large-scale production is not facilitated.
Disclosure of Invention
In order to rapidly obtain high-content human umbilical cord mesenchymal stem cell exosomes, the application provides a preparation method of a composite exosome and application of the composite exosome in directionally enhancing angiogenesis capacity.
In a first aspect, the present application provides a composite exosome, employing the following technical scheme:
a preparation method of a composite exosome comprises the following steps: culturing cells and preparing a composite exosome;
the culture medium adopted in the cell culture step is a mesenchymal stem cell serum-free culture medium; the serum-free culture medium for the mesenchymal stem cells also comprises nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium.
The preparation method of the composite exosome can obviously improve the content of the exosome obtained by extraction, and further obtains the composite exosome with high exosome content, and the composite exosome has the capacity of directionally enhancing human vascular skin proliferation, migration and angiogenesis. The culture medium adopted in the cell culture step in the preparation method is a serum-free culture medium of the mesenchymal stem cells, and the culture medium does not contain serum and has few chemical components, so the preparation method has high safety; meanwhile, the medium is also added with nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium, and the components can effectively promote the growth and differentiation of the mesenchymal stem cells, so that the propagation of the mesenchymal stem cells is promoted.
Nicotinamide is a vitamin derivative, the main component of which is coenzyme, is an active substance for maintaining the growth of the mesenchymal stem cells, and is beneficial to the rapid growth and propagation of the mesenchymal stem cells. The N-N-octyl-D-glucosamine can provide nutrition required by growth for the mesenchymal stem cells and promote the growth and propagation of the mesenchymal stem cells; the medical heparin sodium can block the generation of thromboplastin, inhibit prothrombin from forming thrombin, and further play a role in anticoagulation and inhibition of platelet aggregation, so that the mesenchymal stem cells are always in a dispersed state, the growth and the propagation of the mesenchymal stem cells are facilitated, and the protective effect is good.
Preferably, the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 1-4 parts of nicotinamide, 2-6 parts of N-N-octyl-D-glucosamine and 1-3 parts of medical heparin sodium.
Further preferably, the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 2.5-3.5 parts of nicotinamide, 3-5 parts of N-N-octyl-D-glucosamine and 1.5-2.5 parts of medical heparin sodium.
Still further preferably, the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 3 parts of nicotinamide, 3.5 parts of N-N-octyl-D-glucosamine and 2 parts of medical heparin sodium.
In a specific embodiment, in the mesenchymal stem cell serum-free medium, the nicotinamide may be 1 part, 2.5 parts, 3 parts, 3.5 parts or 4 parts.
In some specific embodiments, the nicotinamide may also be 1-2.5 parts, 1-3 parts, 1-3.5 parts, 2.5-3 parts, 2.5-4 parts, 3-3.5 parts, 3-4 parts, or 3.5-4 parts in the mesenchymal stem cell serum-free medium.
In a specific embodiment, the N-octyl-D-glucosamine may be 2 parts, 3 parts, 4 parts, 5 parts, or 6 parts in the mesenchymal stem cell serum-free medium.
In some specific embodiments, the N-octyl-D-glucosamine may also be 2-3 parts, 2-4 parts, 2-5 parts, 3-4 parts, 3-5 parts, 3-6 parts, 4-5 parts, 4-6 parts, or 5-6 parts in the mesenchymal stem cell serum-free medium.
In a specific embodiment, in the mesenchymal stem cell serum-free medium, the medical heparin sodium may be 1 part, 1.5 parts, 2 parts, 2.5 parts or 3 parts.
In some specific embodiments, the N-octyl-D-glucosamine may also be 1-1.5 parts, 1-2 parts, 1-2.5 parts, 1.5-2 parts, 1.5-2.5 parts, 1.5-3 parts, 2-2.5 parts, 2-3 parts, or 2.5-3 parts in the mesenchymal stem cell serum-free medium.
In the application, the weight parts of nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium in the serum-free culture medium of the mesenchymal stem cells are controlled within the range, so that the cell proliferation and differentiation can be effectively promoted, and a large amount of mesenchymal stem cells can be obtained.
Preferably, the step of extracting exosomes is: and (3) carrying out low-speed centrifugation on the supernatant in the culture medium for 3 times, then carrying out ultracentrifugation on the supernatant after the low-speed centrifugation for 1 time, and harvesting the centrifugal precipitate to obtain the exosome.
By adopting the separation method of gradient centrifugation, the exosome of the human umbilical cord mesenchymal stem cell can be effectively extracted from the human umbilical cord mesenchymal stem cell, the method is simple to operate, and the content of the obtained human umbilical cord mesenchymal stem cell exosome is high.
Preferably, the preparation of the complex exosome requires preparation of a somatomedin complex; the somatomedin complex comprises vascular endothelial growth factor and placental growth factor.
Preferably, the somatomedin complex comprises the following components in parts by weight: 3-5 parts of vascular endothelial growth factor
And 1-2 parts of placenta growth factor.
In a specific embodiment, the vascular endothelial growth factor may be present in 3 parts, 4 parts or 5 parts by weight.
In some specific embodiments, the weight part of the vascular endothelial growth factor can be 3 to 4 parts or 4 to 5 parts.
In a particular embodiment, the placenta growth factor may be present in 1 part, 1.5 parts, or 2 parts by weight.
In some specific embodiments, the placenta growth factor may also be present in 1 to 1.5 parts or 1.5 to 2 parts by weight.
The application provides a growth-promoting factor compound prepared from vascular endothelial growth factor and placenta growth factor, and the growth-promoting factor compound has the functions of promoting migration, proliferation and angiogenesis of vascular endothelial cells.
Preferably, the specific steps for preparing the composite exosome are as follows: the somatomedin complexes are entrapped on the exosomes using electroporation techniques.
Further, the exosome is a human umbilical cord mesenchymal stem cell exosome.
According to the method, the human umbilical cord mesenchymal stem cells are cultured, then the human umbilical cord mesenchymal stem cell exosomes are extracted, and the composite exosomes are prepared by utilizing the exosomes. The selected human umbilical cord belongs to the reutilization of waste resources, and has the advantages of convenient human umbilical cord source, excellent performance and no ethical problem, thereby having wide application prospect in the field of cell therapy.
Further, the electroporation conditions are as follows: voltage 120V, capacitance 115 μ F, discharge time 0.8ms, number of discharges 1 time.
Preferably, in the composite exosome, 50-200 parts of human umbilical cord mesenchymal stem cell exosome and 10-15 parts of somatomedin composite are used.
In a specific embodiment, the weight part of the human umbilical cord mesenchymal stem cell exosome may be 100 parts.
In a particular embodiment, the somatomedin complex may be present in 13 parts by weight.
According to the application, the growth-promoting factor compound is coated on the human umbilical cord mesenchymal stem cell exosome by adopting an electroporation technology, the repairing tissues of the human umbilical cord mesenchymal stem cell exosome and the functions of promoting angiogenesis and the functions of promoting vascular endothelial cell migration, proliferation and angiogenesis of the growth-promoting factor compound are fully combined, and the compound exosome is prepared.
In a second aspect, the present application provides a composite exosome prepared by the preparation method of the composite exosome.
In a third aspect, the application provides a preparation method of the composite exosome and an application of the composite exosome in directionally enhancing angiogenesis capacity.
In summary, the present application has the following beneficial effects:
1. the culture medium is used for culturing the human umbilical cord mesenchymal stem cells by adopting the serum-free culture medium containing the nicotinamide, the N-N-octyl-D-glucosamine and the medical heparin sodium, and can effectively promote the growth and differentiation of the human umbilical cord mesenchymal stem cells, so that the human umbilical cord mesenchymal stem cells have stronger proliferation capacity; meanwhile, the culture medium does not contain animal serum, so that the safety of the human umbilical cord mesenchymal stem cells obtained by culture is high.
2. The method is simple to operate, and the content of the extracted human umbilical cord mesenchymal stem cell exosomes is high.
3. The composite growth factor is prepared, and the composite growth factor is wrapped on the human umbilical cord mesenchymal stem cell exosome by adopting the electroporation technology, so that the advantages of the composite growth factor and the human umbilical cord mesenchymal stem cell exosome are effectively combined, and the composite exosome which is safe and can effectively promote angiogenesis is prepared.
Drawings
Fig. 1 is a flow chart of a method for preparing a complex exosome provided herein.
Detailed Description
The application provides a preparation method of a composite exosome, which specifically comprises the following steps:
(1) signing an informed consent with the family members of the newborn, and registering and collecting the umbilical cord of the newborn in a standard manner; sterilizing newborn umbilical cords with iodophor, repeatedly washing with PBS solution, ligating two ends of umbilical cords with sterile thread, placing in a sampling bottle containing collecting device, and sending to laboratory for experiment at 5-15 deg.C within 24 hr; meanwhile, collecting the peripheral blood of the mother for virus detection.
(2) Taking out the umbilical cord from the collected variety, and soaking in a culture dish of 65-70% alcohol for 1-2 min; then taking out the umbilical cord, and cleaning the umbilical cord with physiological saline; cutting the umbilical cord into 2-4cm small-section tissue blocks, removing blood vessels in the tissue blocks by using forceps, and then placing the tissue blocks in a culture dish containing physiological saline to remove residual extravasated blood; a pre-treated human umbilical cord tissue mass is obtained.
(3) Placing the pretreated human umbilical cord tissue block in a mixed solution of 1% collagenase II and 0.3% trypsin for digesting for 20min, then attaching the human umbilical cord tissue block to the bottom wall of a culture bottle containing a mesenchymal stem cell serum-free culture medium, then placing the culture bottle in an incubator at 37 ℃, and turning over once after 12 h; changing the culture medium to culture flask every 48h, continuously observing cell climbing-out condition around the patch, and removing the patch after 2 weeks; the cells were then continued to the log phase and cell culture supernatant was collected.
Wherein the serum-free culture medium of the mesenchymal stem cells also comprises nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium; the mesenchymal stem cell serum-free culture medium further comprises 1-4 parts of nicotinamide, 2-6 parts of N-N-octyl-D-glucosamine and 1-3 parts of medical heparin sodium; furthermore, the mesenchymal stem cell serum-free culture medium can also comprise 2.5-3.5 parts of nicotinamide, 3-5 parts of N-N-octyl-D-glucosamine and 1.5-2.5 parts of medical heparin sodium.
(4) Centrifuging the cell culture supernatant at low speed of 400 Xg, 1000 Xg, 2000 Xg for 5min, removing precipitate, and collecting supernatant; and then carrying out ultracentrifugation on the supernatant for 1 time at 100000 Xg for 30min, discarding the supernatant after the centrifugation is finished, and harvesting the precipitate to obtain the human umbilical cord mesenchymal stem cell exosome.
(5) Mixing 100 parts of human umbilical cord mesenchymal stem cell exosome and 13 parts of growth-promoting factor compound, dissolving in 1-3mL of PBS solution to obtain mixed reaction liquid, adding the mixed reaction liquid into a perforating dish for electroporation, and completing electroporation entrapment to obtain pre-composite exosome; wherein, the conditions of electroporation are as follows: voltage 120V, capacitance 115 μ F, discharge time 0.8ms, number of discharges 1 time.
The somatomedin compound comprises the following components in parts by weight: 3-5 parts of vascular endothelial growth factor and 1-2 parts of placenta growth factor.
(6) And (3) placing the composite exosome in a cell culture box to incubate for 1h, then carrying out ultracentrifugation for 2 times under the centrifugal force of 10-12 ten thousand Xg, centrifuging for 30min each time, and removing supernatant to obtain precipitate, namely the composite exosome.
The composite exosome is prepared by the preparation method of the composite exosome.
The preparation method of the composite exosome and the application of the composite exosome in promoting angiogenesis.
In the application, the serum-free culture medium of the mesenchymal stem cells, the nicotinamide, the N-N-octyl-D-glucosamine, the medical heparin sodium, the vascular endothelial growth factor and the placenta growth factor are purchased from Beijing YinuoKai science and technology Limited; the electroporator is a Gemini X2 electroporator by BTX, USA; the remaining reagents, solvents, etc. are commercially available.
The present application will be described in further detail below with reference to examples 1 to 21, comparative examples 1 to 5, drawings and test results.
Examples
Examples 1 to 13
Examples 1-13 provide a method for preparing a complex exosome, respectively.
The above embodiments differ in that: the amounts of the components added to the medium are shown in Table 1.
Examples 1-13 provide a method for the preparation of complex exosomes as follows:
(1) sampling: signing an informed consent with the family members of the newborn, and registering and collecting the umbilical cord of the newborn in a standard manner; sterilizing newborn umbilical cords by iodophor, repeatedly washing by PBS solution, ligating two ends of umbilical cords by sterile threads, placing in a sampling bottle containing a collecting device, and sending to a laboratory for experimental operation at 10 ℃ within 24 h; meanwhile, collecting the peripheral blood of the mother for virus detection.
(2) Sample pretreatment: taking out the umbilical cord from the collected variety, and soaking in a culture dish of 70% alcohol for 1 min; then taking out the umbilical cord, and cleaning the umbilical cord with physiological saline; then, cutting the umbilical cord into a small-section tissue block of 3cm, removing blood vessels in the tissue block by using forceps, and then placing the tissue block in a culture dish containing physiological saline to remove residual extravasated blood; a pre-treated human umbilical cord tissue mass is obtained.
(3) Cell culture: placing the pretreated human umbilical cord tissue block in a mixed solution of 1% collagenase II and 0.3% trypsin for digesting for 20min, then attaching the tissue block to the bottom wall of a culture bottle containing 100mL of mesenchymal stem cell serum-free culture medium, then placing the culture bottle in an incubator at 37 ℃, and turning over once after 12 h; changing the culture medium to culture flask every 48h, continuously observing cell climbing-out condition around the patch, and removing the patch after 2 weeks; the cells were then continued to the log phase and cell culture supernatant was collected.
Wherein the serum-free culture medium of the mesenchymal stem cells comprises 300 mug of nicotinamide, 400 mug of N-N-octyl-D-glucosamine and 200 mug of medical heparin sodium.
(4) And (3) extracting exosomes: centrifuging the cell culture supernatant at 400 Xg for 5min, removing precipitate such as cell debris, and collecting supernatant; centrifuging the supernatant at 1000 Xg for 5min, removing precipitate, and collecting supernatant; continuing to centrifugate at low speed of 2000 Xg for 5min, removing precipitate, and collecting supernatant; finally, the supernatant is ultracentrifuged for 1 time under 100000 Xg, the supernatant is discarded, and the precipitate is harvested to obtain the exosome.
(5) Preparing a composite exosome: dissolving 100 mu g of human umbilical cord mesenchymal stem cell exosome and 13 mu g of growth-promoting factor compound in 1-3mL of PBS solution to obtain mixed reaction liquid, adding the mixed reaction liquid into a perforating dish for electroporation, and completing electroporation entrapment to obtain pre-composite exosome; wherein, the conditions of electroporation are as follows: voltage 120V, capacitance 115 μ F, discharge time 0.8ms, number of discharges 1 time.
The growth-promoting factor compound comprises the following components in parts by weight: 40 ug of vascular endothelial growth factor and 15 ug of placenta growth factor.
(6) Culturing and purifying: and (3) placing the composite exosome in a cell culture box to incubate for 1h, then centrifuging at an ultra-high speed for 2 times under the centrifugal force of 12 ten thousand Xg, centrifuging for 30min each time, and removing supernatant to obtain a precipitate, namely the composite exosome.
TABLE 1 preparation methods provided in examples 1-13 the amounts of the components added to the media
Examples 14 to 21
Examples 14-21 provide a method for preparing a complex exosome, respectively.
The above embodiment is different from embodiment 3 in that: the addition amounts of the components of the somatomedin complex are shown in table 2.
TABLE 2 EXAMPLE 3, EXAMPLES 14-21 methods of preparation provided by the addition of growth-promoting factor Complex Components
Comparative example
Comparative example 1
Comparative example 1 provides a method for the preparation of a complex exosome.
The above comparative example differs from example 3 in that: the culture medium adopted in the culture step is a serum-free culture medium of mesenchymal stem cells, and does not comprise nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium.
Comparative example 2
Comparative example 2 provides a method for the preparation of a complex exosome.
The above comparative example differs from example 3 in that: the culture medium adopted in the culture step is a serum-free culture medium of mesenchymal stem cells, and nicotinamide and N-N-octyl-D-glucosamine are also added.
Comparative example 3
Comparative example 3 provides a method for the preparation of a complex exosome.
The above comparative example differs from example 3 in that: the culture medium adopted in the culture step is a mesenchymal stem cell serum-free culture medium and is also added with N-N-octyl-D-glucosamine and medical heparin sodium.
Comparative example 4
Comparative example 4 provides a method for the preparation of a complex exosome.
The above comparative example differs from example 3 in that: the culture medium adopted in the culture step is a serum-free culture medium of mesenchymal stem cells, and nicotinamide and medical heparin sodium are also added.
Comparative example 5
Comparative example 5 provides a method for the preparation of a complex exosome.
The preparation method of the compound exosome comprises the following steps:
(1) firstly, preparing DMEM/F12 culture solution; the DMEM/F12 culture solution contains 20% FBS by mass concentration, 100U/ml penicillin and 100 mu g/ml streptomycin.
(2) Carrying out collagenase II digestion, pancreatin digestion, filtration and centrifugation treatment on human umbilical cord mesenchymal stem cells in sequence, and taking centrifugal precipitates.
(3) And (3) carrying out primary culture on the centrifugal precipitate in the step (2) by using the DMEM/F12 culture solution in the step (1) to obtain the human umbilical cord mesenchymal stem cells.
(4) And (4) sequentially culturing the human umbilical cord mesenchymal stem cells obtained in the step (3) in a serum-free starvation culture solution and a DMEM/F12 culture solution containing 10ng/mL of epidermal cell growth factors to a logarithmic phase, collecting supernatant, centrifuging for 100min under the centrifugal force of 5000 Xg, and harvesting precipitates to obtain the umbilical cord mesenchymal stem cell exosomes.
(5) Dissolving umbilical cord mesenchymal stem cell exosome and mir-181b in 1ml of phosphate buffer salt solution containing 50mM trehalose to obtain a mixed solution; adding the mixed solution into a perforating dish of an electroporator to complete entrapment, and obtaining a composite exosome; wherein the conditions of the electroporation are as follows: 100V, a capacitor 125 muF and a discharge time of 1 ms.
(6) And (3) performing membrane repair, extraction and purification on the composite exosome to obtain the mir-181 b-entrapped composite exosome.
Detection test
Exosome content detection
The composite exosomes prepared by the preparation methods of the composite exosomes provided in examples 1-21 and comparative examples 1-5 were subjected to content detection (in terms of protein), and the results are shown in table 3.
The assay for exosome content is referenced to the BCA assay. The BAC working solution comprises a reagent A + a reagent B. Wherein the reagent A is a mixed solution of 1% BCA disodium salt, 2% anhydrous sodium carbonate 0.16% sodium tartrate, 0.4% sodium hydroxide and 0.95% sodium bicarbonate, and the pH value is 11.25; reagent B was 4% copper sulfate.
TABLE 3 detection results of exosome content of the composite exosomes prepared in examples 1-21 and comparative examples 1-5
Angiogenesis test the complex exosomes prepared in examples 1-21 and comparative examples 1-5 were prepared into suspensions with a concentration of 0.3mg/mL using PBS solutions, and then angiogenesis test was performed on the suspensions, and the test results are shown in table 4.
Adding 10 μ L matrigel without growth factor into micropores of angiogenesis glass slide, gelatinizing at 37 deg.C, inoculating one of the above suspensions in each well, culturing for 2.5 hr, taking picture, inverting microscope, and observing angiogenesis.
TABLE 4 detection of angiogenesis by Complex exosomes obtained in examples 1-21 and comparative examples 1-2
By combining tables 3 and 4 and comparing detection results of examples 1 to 21 and comparative examples 1 to 5, it can be known that the preparation method of the composite exosome provided by the application can obtain a high-content human umbilical cord mesenchymal stem cell exosome, and further can prepare the composite exosome with a high-content exosome content, and the composite exosome can remarkably promote angiogenesis and differentiation and enhance angiogenesis capacity.
According to the detection results of the examples 1-5 and the comparative example 3, the content of the exosome in the composite exosome is gradually increased and then kept unchanged along with the increase of the content of the nicotinamide in the culture medium, which indicates that the nicotinamide can effectively promote the mesenchymal stem cells to secrete the exosome; further comparison shows that the content of the exosome in the prepared composite exosome is higher by controlling nicotinamide in the culture medium to be 2.5-3.5 parts. Meanwhile, angiogenesis tests show that when the addition amount of nicotinamide in the culture medium is 2.5-3.5 parts, the compound exosome can remarkably promote the generation and differentiation of blood vessels and has a remarkable effect on the culture of angiogenesis capacity.
According to the detection results of example 3, examples 6 to 9 and comparative example 4, the content of the exosomes in the composite exosomes shows a trend of increasing and then decreasing with the increase of the content of the N-N-octyl-D-glucosamine in the culture medium, which indicates that the N-N-octyl-D-glucosamine can effectively promote the secretion of the exosomes by the mesenchymal stem cells; by further comparison, the content of the exosome in the prepared composite exosome is higher by controlling the content of the N-N-octyl-D-glucosamine in the culture medium to be between 3 and 4 parts. Meanwhile, angiogenesis tests show that when the addition amount of the N-N-octyl-D-glucosamine in the culture medium is between 3 and 4 parts, the composite exosome has an obvious effect of promoting angiogenesis. Therefore, the application shows that when the adding amount of the N-N-octyl-D-glucosamine is controlled to be between 3 and 4 parts, the obtained composite exosome contains a large amount of human umbilical cord mesenchymal stem cell exosomes, and the composite exosome can effectively promote the generation and differentiation of blood vessels and has a remarkable action effect on the culture of angiogenesis capacity.
By combining the detection results of the embodiment 3, the embodiments 10-13 and the comparative example 2, it can be known that the content of exosomes in the composite exosomes shows a trend of increasing first and then keeping unchanged along with the increase of the content of heparin sodium for traditional Chinese medicine in the culture medium, which indicates that the medical heparin sodium can promote mesenchymal stem cells to secrete exosomes; further comparison shows that the exosome content in the prepared composite exosome is higher by controlling the addition amount of the heparin sodium for the traditional Chinese medicine of the culture medium to be 1.5-2.5 parts. Meanwhile, an angiogenesis test finds that when the addition amount of the heparin sodium for the traditional Chinese medicine culture medium is 1.5-2.5 parts, the effect of the compound exosome on promoting angiogenesis is more obvious. Therefore, the application shows that when the adding amount of the medical heparin sodium is controlled to be 1.5-3.5 parts, the obtained composite exosome contains a large amount of human umbilical cord mesenchymal stem cell exosomes, and the composite exosome can effectively promote the generation and differentiation of blood vessels and has a remarkable action effect on the culture of angiogenesis capacity.
As can be seen from the results of comparing example 3 and examples 14 to 17, the composite exosomes prepared by the preparation methods of the composite exosomes provided in examples 14 to 17 have the same exosome content; the angiogenesis test result shows that the content of the vascular endothelial growth factor in the growth-promoting factor compound of the compound exosome is increased, the effect of the compound exosome on promoting angiogenesis is gradually increased, a large number of branches are generated in matrigel, and the capacity of the compound exosome on promoting blood vessels is gradually reduced when the addition amount of the vascular endothelial growth factor exceeds 50 ng/mL. Therefore, the effect of the obtained composite exosome on promoting angiogenesis and differentiation is better when the addition amount of the vascular endothelial growth factor in the growth-promoting factor composite of the composite exosome is controlled to be 3-5 parts.
According to the detection results of example 3 and examples 18 to 21, the content of exosomes in the composite exosomes prepared by the preparation method of the composite exosomes provided in examples 18 to 21 is the same; the angiogenesis test result shows that the content of the vascular endothelial growth factor in the growth-promoting factor compound of the compound exosome is increased, the effect of the compound exosome on promoting angiogenesis is gradually increased, a large number of branches are generated in matrigel, and the capacity of the compound exosome on promoting blood vessels is gradually reduced when the addition amount of the placenta growth factor exceeds 20 ng/mL. Therefore, the application shows that when the adding amount of the placenta growth factor in the growth-promoting factor compound of the compound exosome is controlled to be 1-2 parts, the obtained compound exosome has better effect on promoting angiogenesis and differentiation.
Comparing the test results of examples 1-21 and comparative example 1, it can be seen that the exosome content in the composite exosomes prepared by the preparation methods provided in examples 1-21 is significantly higher than that in the composite exosomes prepared by the preparation method provided in comparative example 1, which indicates that the addition of nicotinamide, N-octyl-D-glucosamine and medical heparin sodium to the mesenchymal stem cell serum-free preparation medium can effectively promote the secretion of exosomes by cells, further promote the generation and differentiation of blood vessels, and enhance the generating capacity of blood vessels.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.
Claims (10)
1. A preparation method of a composite exosome is characterized by comprising the following steps: culturing cells and preparing a composite exosome;
the culture medium adopted in the cell culture step is a mesenchymal stem cell serum-free culture medium; the serum-free culture medium for the mesenchymal stem cells also comprises nicotinamide, N-N-octyl-D-glucosamine and medical heparin sodium.
2. The preparation method of the composite exosome according to claim 1, wherein the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 1-4 parts of nicotinamide, 2-6 parts of N-N-octyl-D-glucosamine and 1-3 parts of medical heparin sodium.
3. The preparation method of the composite exosome according to claim 1, wherein the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 2.5-3.5 parts of nicotinamide, 3-5 parts of N-N-octyl-D-glucosamine and 1.5-2.5 parts of medical heparin sodium.
4. The preparation method of the composite exosome according to claim 1, wherein the mesenchymal stem cell serum-free medium comprises the following components in parts by weight: 3 parts of nicotinamide, 3.5 parts of N-N-octyl-D-glucosamine and 2 parts of medical heparin sodium.
5. The method for preparing a composite exosome according to claim 1, wherein the step of extracting an exosome is: and (3) carrying out low-speed centrifugation on the supernatant in the culture medium for 3 times, then carrying out ultracentrifugation on the supernatant after the low-speed centrifugation for 1 time, and harvesting the centrifugal precipitate to obtain the exosome.
6. The method for preparing a complex exosome according to claim 1, wherein a somatomedin complex is prepared before preparing the complex exosome; the somatomedin complex comprises vascular endothelial growth factor and placental growth factor.
7. The method for preparing a composite exosome according to claim 6, wherein the somatomedin composite comprises the following components in parts by weight: 3-5 parts of vascular endothelial growth factor and 1-2 parts of placenta growth factor.
8. The method for preparing a composite exosome according to claim 1, wherein the specific steps of preparing the composite exosome are as follows: the somatomedin complexes are entrapped on the exosomes using electroporation techniques.
9. A complex exosome, characterized in that it is produced by a method for producing a complex exosome according to any one of claims 1 to 8.
10. A method for preparing a composite exosome according to any one of claims 1-8 and the use of a composite exosome according to claim 9 for directionally enhancing angiogenic capacity.
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