CN115125198A - Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof - Google Patents
Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human umbilical cord mesenchymal stem cell exosome and a preparation method and application thereof. The preparation method comprises the following steps: the human umbilical cord mesenchymal stem cells are separated under the aseptic condition, and then primary culture and subculture are carried out under the continuous hypoxia condition. The method can prepare sufficient mesenchymal stem cells with good immunoregulation function, and the mesenchymal stem cell exosome prepared by the method has good treatment effect on diabetic skin injury and acute lung injury.
Description
Technical Field
The invention belongs to the technical field of biological application, and particularly relates to a preparation method and application of a human umbilical cord mesenchymal stem cell exosome.
Background
Acute lung injury is a clinical syndrome mainly characterized by hypoxemia, is usually secondary to various pathological states such as systemic infection, severe trauma, severe shock and the like, is characterized by diffuse alveolar injury and pulmonary interstitial edema, and is accompanied by injury of capillary endothelial cells and alveolar epithelial cells. Clinical trials of acute lung injury have been greatly advanced in the past decades, but treatment methods are still mainly symptomatic treatment and respiratory support, and effective drug therapy is still lacking. Preclinical and clinical tests of acute lung injury show that the umbilical cord mesenchymal stem cells can control and relieve inflammatory reaction of organisms, and adverse reactions are rare.
The mesenchymal stem cells can be separated from tissues such as bone marrow, adipose tissue, umbilical cord, placenta, dental pulp and the like, have the functions of multidirectional differentiation, immunoregulation, anti-inflammation, anti-apoptosis, promotion of tissue repair and the like, and have important application values in the fields of tissue engineering and cell therapy. Mesenchymal stem cells exert the above-mentioned effects through intercellular contact and paracrine effects. Paracrine substances of mesenchymal stem cells can increase angiogenesis in damaged tissues, promote granulation tissue formation, regulate extracellular matrix remodeling, and promote skin wound healing and skin regeneration.
The survival rate of the exogenous mesenchymal stem cells after entering the body is low, and the maximum exertion of the treatment effect of the mesenchymal stem cells is limited to a certain extent. A large number of researches show that the mesenchymal stem cell paracrine product can play a protection role equivalent to that of the mesenchymal stem cell, so that the mesenchymal stem cell paracrine product with excellent biological treatment function has important clinical significance.
Oxygen is a necessary condition for cell growth, and the oxygen concentration in the growth environment is an important factor affecting the viability and function of cells. Research shows that the oxygen concentration in the mesenchymal stem cell nest is 1-9%, and the hypoxia culture promotes the proliferation of the umbilical cord mesenchymal stem cells and delays the cell aging. However, the oxygen concentration used for conventional in vitro cell culture was 21%. Accordingly, we speculate that the persistent hypoxic environment is more favorable for maintaining the immunomodulatory function of the mesenchymal stem cells.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a preparation method and application of a human umbilical cord mesenchymal stem cell exosome. The mesenchymal stem cell exosome has an excellent immunoregulation function, and can be used for treating diabetic skin injury and acute lung injury.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of human umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) separating human umbilical cord mesenchymal stem cells from human umbilical cord tissues, and carrying out primary culture and subculture in a hypoxia environment to obtain human umbilical cord mesenchymal stem cells with generation of P3-P9;
(2) inoculating the human umbilical cord mesenchymal stem cells which are obtained in the step (1) and substituted by P3-P9 into a culture bottle, and then culturing in serum-free MEM-alpha culture medium; collecting cell culture solution after the culture is finished, and centrifuging to obtain supernatant, namely umbilical cord mesenchymal stem cell culture supernatant;
(3) and (3) carrying out gradient centrifugation treatment on the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2), wherein the obtained precipitate is an exosome secreted by the human umbilical cord mesenchymal stem cells.
Preferably, in step (1), the method for isolating human umbilical cord mesenchymal stem cells comprises: washing umbilical cord with PBS buffer solution, removing blood vessel in umbilical cord, and cutting umbilical cord into 3-7mm pieces 2 The tissue block is placed in an MEM-alpha culture medium and then is placed in a hypoxic environment for culture, and the liquid is changed every 3 to 4 days; when the cells fused to 70-90%, the supernatant and tissue mass were removed and subcultured with 0.25% trypsin-EDTA, at which time the subcultured cells were designated P1.
Preferably, in the low-oxygen environment in the steps (1) and (2), the temperature is 36-38 ℃, the volume fraction of carbon dioxide is 3-7%, the volume fraction of oxygen is not more than 1%, and the rest gas is nitrogen.
Preferably, in the step (2), the density of the inoculation of the human umbilical cord mesenchymal stem cells is 5 x 10 4 -2×10 5 Per mL; the bottom area of the culture vessel is 50-175cm 2 (ii) a The culture time of the human umbilical cord mesenchymal stem cells in the MEM-alpha culture medium is 24-72 h; the centrifugal force used for centrifugation is 100-300g, and the centrifugation time is 1-10 min.
Preferably, in step (3), the step of gradient centrifugation is: centrifuging the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2) for 5-15min by 400g of 200-.
Preferably, in step (3), the temperature of the gradient centrifugation is 2-6 ℃.
Meanwhile, the invention claims a human umbilical cord mesenchymal stem cell exosome prepared by any one of the methods.
Meanwhile, the invention claims the application of the preparation method in the preparation of the mesenchymal stem cell exosome.
Compared with the prior art, the invention has the following beneficial effects:
1. the human umbilical cord mesenchymal stem cell exosome prepared by the method has a repairing effect on diabetic skin defects, has an improvement effect on lipopolysaccharide-induced acute lung injury, greatly exerts the treatment potential of umbilical cord mesenchymal stem cells, provides a new thought and method for treating the diabetic skin defects and the acute lung injury by the mesenchymal stem cell exosome, and has a wide development prospect.
2. The method is simple, strong in operability and low in manufacturing cost, and can prepare a large amount of mesenchymal stem cells and exosomes thereof in a short time, so that the method has a good market prospect.
Drawings
FIG. 1 is a schematic diagram of morphological observation of umbilical cord mesenchymal stem cell exosomes under a transmission electron microscope;
FIG. 2 is a schematic diagram of the general observation of the repair effect of umbilical cord mesenchymal stem cell exosomes on skin defects of diabetic mice;
FIG. 3 is a schematic diagram of the effect of umbilical cord mesenchymal stem cell exosomes on pathological changes in the skin defect wound surface of diabetic mice (HE staining);
FIG. 4 is a schematic representation of the effect of umbilical cord mesenchymal stem cell exosomes on pathological changes in lung tissue (HE staining and Masson staining) in acute lung injury mice;
FIG. 5 is a schematic illustration of the effect of umbilical cord mesenchymal stem cell exosomes on the level of inflammation in acute lung injury mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. Of course, the specific embodiments described herein are merely illustrative and not restrictive of the invention.
Although the steps in the present invention are arranged by using reference numbers, the order of the steps is not limited, and the relative order of the steps can be adjusted unless the order of the steps is explicitly stated or other steps are required for the execution of a certain step. It will be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
Unless otherwise specified, the chemicals and materials of the present invention are commercially available.
Example 1
A preparation method of human umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) cleaning the healthy neonatal umbilical cord with sterile PBS for three times; removing blood vessels in umbilical cord, and cutting umbilical cord into pieces of 3mm 2 The left and right tissue blocks were placed in a cell culture flask, MEM-alpha complete medium containing 10% FBS was added, and hypoxia (37 ℃ C., CO) was added 2 Volume fraction of 5% and N 2 Volume fraction of 94% and O 2 Volume fraction is 1%) and changing the culture solution every 3 days; when the fibroblasts climb out and are fused in large pieces around the tissue block, removing the supernatant and the tissue block, carrying out passage by using 0.25% trypsin-EDTA (ethylene diamine tetraacetic acid), wherein the passage cell is marked as P1, and the cells in the following passages are P2 and P3.. Pn in sequence;
(2) carrying out 1 × 10 treatment on the P3-P9 generation human umbilical cord mesenchymal stem cells obtained in the step (1) 5 The density of seeds/mL is 75cm 2 Culturing umbilical cord mesenchymal stem cells in serum-free MEM-alpha medium in the culture flask of (1); collecting cell culture solution after 48 hours, and centrifuging for 5 minutes at 200g to obtain supernatant, namely umbilical cord mesenchymal stem cell culture supernatant;
(3) carrying out gradient centrifugation treatment on the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2), wherein the obtained precipitate is an exosome secreted by the mesenchymal stem cells;
the step of gradient centrifugation is as follows: centrifuging the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2) for 10min at 300g to obtain a supernatant No. 1, centrifuging the supernatant No. 1 at 2000g for 30min to obtain a supernatant No. 2, centrifuging the supernatant No. 2 at 10000g for 30min to obtain a supernatant No. 3, centrifuging the supernatant No. 3 at 120000g for 180min to obtain a precipitate, namely the exosome secreted by the mesenchymal stem cells, wherein the temperature of four times of centrifugation is 4 ℃.
Example 2
A preparation method of human umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) cleaning the healthy neonatal umbilical cord with sterile PBS for three times; removing blood vessels in umbilical cord, and cutting umbilical cord into pieces of 3mm 2 The left and right tissue blocks were placed in a cell culture flask, MEM-alpha complete medium containing 10% FBS was added, and hypoxia (37 ℃ C., CO) was added 2 Volume fraction of 5% and N 2 Volume fraction of 94% and O 2 Volume fraction is 1%) and changing the culture solution every 3 days; when the fibroblasts climb out and are fused in large pieces around the tissue block, removing the supernatant and the tissue block, carrying out passage by using 0.25% trypsin-EDTA (ethylene diamine tetraacetic acid), wherein the passage cell is marked as P1, and the cells in the following passages are P2 and P3.. Pn in sequence;
(2) the generation P3-P9 human umbilical cord mesenchymal stem cells obtained in the step (1) are divided into 5 x 10 4 The density of seeds/mL is 175cm 2 Culturing umbilical cord mesenchymal stem cells in a serum-free MEM-alpha medium in the culture flask of (1); collecting cell culture solution after 48 hours, and centrifuging for 5 minutes at 200g to obtain supernatant, namely umbilical cord mesenchymal stem cell culture supernatant;
(3) carrying out gradient centrifugation treatment on the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2), wherein the obtained precipitate is an exosome secreted by the mesenchymal stem cells;
the gradient centrifugation comprises the following steps: centrifuging the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2) for 10min at 250g to obtain a supernatant No. 1, centrifuging the supernatant No. 1 at 1800g for 20min to obtain a supernatant No. 2, centrifuging the supernatant No. 2 at 8000g for 20min to obtain a supernatant No. 3, centrifuging the supernatant No. 3 at 140000g for 140min to obtain a precipitate, namely an exosome secreted by the mesenchymal stem cells, wherein the temperature of four times of centrifugation is 4 ℃.
Example 3
A preparation method of human umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) cleaning the healthy newborn umbilical cord in term with sterile PBS for three times; removing blood vessels in umbilical cord, and cutting umbilical cord into pieces of 3mm 2 The left and right tissue blocks were placed in a cell culture flask, MEM-alpha complete medium containing 10% FBS was added, and hypoxia (37 ℃ C., CO) was added 2 Volume fraction of 5% and N 2 Volume fraction of 94% and O 2 Volume fraction is 1%) and changing the culture solution every 4 days; when the tissue block is crawled and fused with large sheets of fibrous cells, removing supernatant and the tissue block, and carrying out passage by using 0.25% trypsin-EDTA (ethylene diamine tetraacetic acid), wherein the passage cell is marked as P1, and the cells of the following passages are sequentially marked as P2 and P3.. Pn;
(2) carrying out 2X 10 treatment on the P3-P9 generation human umbilical cord mesenchymal stem cells obtained in the step (1) 5 The density of seeds/mL is 55cm 2 Culturing umbilical cord mesenchymal stem cells in serum-free MEM-alpha medium; collecting cell culture solution after 48 hours, and centrifuging for 5 minutes at 200g to obtain supernatant, namely umbilical cord mesenchymal stem cell culture supernatant;
(3) carrying out gradient centrifugation treatment on the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2), wherein the obtained precipitate is an exosome secreted by the mesenchymal stem cells;
the step of gradient centrifugation is as follows: centrifuging the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2) for 10min at 350g to obtain a supernatant No. 1, centrifuging the supernatant No. 1 at 2200g for 40min to obtain a supernatant No. 2, centrifuging the supernatant No. 2 at 10000g for 40min to obtain a supernatant No. 3, centrifuging the supernatant No. 3 at 160000g for 120min to obtain precipitates, namely exosomes secreted by the mesenchymal stem cells, wherein the temperature of the four times of centrifugation is 4 ℃.
In order to explore the repairing effect of exosomes secreted by the mesenchymal stem cells obtained in example 1 on diabetic skin defects and the improving effect on Lipopolysaccharide (LPS) -induced acute lung injury, the following tests were made:
1. the method for testing the repairing effect of exosomes secreted by mesenchymal stem cells on diabetic skin defects comprises the following steps:
(1) construction of diabetic mouse full-thickness skin defect model and exosome intervention
The C57BL/6J male mice at 7-8 weeks were randomly divided into a control group (PBS), a diabetic group (STZ), a normoxic exosome intervention group (STZ + Nor-Exo) and a hypoxic exosome intervention group (STZ + Hypo-Exo). Constructing a diabetes mouse model by injecting Streptozotocin (STZ) into the abdominal cavity of the diabetes group and the exosome group for 5 consecutive days; the control group was injected with the same volume of PBS. The fasting blood sugar is detected one week after the model is made, and the blood sugar concentration is more than or equal to 250-300mg/dL, namely the success of the construction of the diabetes model is determined.
The above mice were subjected to surgical treatment: after the mouse model is anesthetized, a round full-layer skin defect wound surface with the diameter of 8mm is respectively constructed on two sides of a dorsal midline, and a control group, a normal oxygen exosome intervention group and a low oxygen exosome intervention group are injected with 100uL PBS, Nor-Exo or Hypo-Exo at the edge of the wound surface through intradermal injection respectively. And sewing and fixing the annular silica gel sheet on the edge of the wound surface and fixing the annular silica gel sheet by using the transparent dressing.
(2) Detection of skin wound closure
Photographing and recording mouse wound surfaces on days 0, 3 and 7 after the operation treatment, processing the wound surface pictures by using Image J software, and calculating the wound surface healing rate: the wound healing rate is (original wound-unhealed wound)/original wound × 100%.
(3) Histopathological examination of skin wounds
The wound surface of the mouse and the surrounding normal tissues of the mouse are taken on the 7 th day after the operation treatment, fixed by 4 percent paraformaldehyde, embedded in paraffin for sectioning, and observed under a microscope after HE staining for pathological characteristics of the wound surface.
Results and analysis:
as shown in FIG. 2, in the mice, the Nor-Exo and Hypo-Exo treatments accelerated wound healing compared with PBS, while the Hypo-Exo wound healing rate was higher than that of Nor-Exo. The histopathology of the skin wound of the mouse is shown in figure 3, and Nor-Exo and Hypo-Exo can effectively promote the regeneration of the wound epithelium of the mouse; however, the neoepithelial tissue was more intact and the epidermis was thicker in the Hypo-Exo treated mice than in the Nor-Exo treated mice.
2. Improving effect of exosome secreted by mesenchymal stem cell on acute lung injury induced by LPS (low-cholesterol)
The test method comprises the following steps:
(1) establishment of acute lung injury model and intervention of umbilical cord mesenchymal stem cell exosome
The 7-8 week C57BL/6J male mice were randomly assigned to a normal control group (PBS), a model group (LPS), an normoxic exosome dry group (LPS/Nor-Exo) and a hypoxic exosome dry group (LPS/Hypo-Exo). Mice in the model group, the normoxic exosome intervention group and the hypoxic exosome intervention group are subjected to intratracheal instillation of 50uL LPS (5mg/kg) to induce an acute lung injury model, and a normal control group is instilled with PBS in an equal amount. After 4h of treatment, 50uL of PBS, Nor-Exo and Hypo-Exo are respectively instilled in the trachea in the normal control group, the model group, the normal oxygen exosome intervention group and the hypoxia exosome intervention group. After 48h, the animals were sacrificed by cervical dislocation after anesthesia and lung tissue and serum were taken.
(2) Testing for changes in lung tissue pathology
Mouse lung tissue was isolated and parallel HE and Masson staining.
(3) Testing of in vivo inflammatory factor levels
Mouse serum was isolated and the levels of cytokines IL1 β, TNF α, IL6, IL8, TGF β and IL10 in the serum were tested by ELISA.
Results and analysis:
pathological section and histological score of lung tissue show that the pulmonary alveoli of the PBS group mice have complete structures, inflammatory infiltration of the alveolar cavities and normal pulmonary interstitium (figure 4); the LPS-induced model group mice have a large amount of inflammatory infiltration, alveolar walls are damaged, lung interstitium is thickened, and collagen fiber deposition is obvious; and the two groups of exosomes can improve lung tissue inflammatory reaction induced by LPS (low-temperature plasma) and relieve alveolar inflammatory infiltration of mice and lung alveolar wall damage and fibrosis caused by inflammation, wherein the umbilical cord mesenchymal stem cell exosome from the hypoxia culture system has better treatment effect. ELISA results show that the levels of inflammatory factors IL1 beta, TNF alpha, IL6 and IL8 in the serum of mice in the acute lung injury group are obviously increased; when two umbilical cord mesenchymal stem cell exosomes are predicted, the levels of the proinflammatory factors in serum are obviously reduced, and the levels of anti-inflammatory factors TGF beta and IL10 are increased; the decrease in proinflammatory factors and the increase in anti-inflammatory factors in serum was more pronounced in the Hypo-Exo intervention group compared to the Nor-Exo intervention group (FIG. 5).
It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (8)
1. A preparation method of human umbilical cord mesenchymal stem cell exosome is characterized by comprising the following steps:
(1) separating human umbilical cord mesenchymal stem cells from human umbilical cord tissues, and carrying out primary culture and subculture in a hypoxia environment to obtain human umbilical cord mesenchymal stem cells with generation of P3-P9;
(2) inoculating the human umbilical cord mesenchymal stem cells which are obtained in the step (1) and substituted by P3-P9 into a culture bottle, and then culturing in serum-free MEM-alpha culture medium; collecting cell culture solution after the culture is finished, and centrifuging to obtain supernatant, namely umbilical cord mesenchymal stem cell culture supernatant;
(3) and (3) carrying out gradient centrifugation treatment on the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2), wherein the obtained precipitate is an exosome secreted by the human umbilical cord mesenchymal stem cells.
2. The method for preparing human umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in step (1), the exosomes are prepared from umbilical cord mesenchymal stem cellsThe method for separating the human umbilical cord mesenchymal stem cells comprises the following steps: washing umbilical cord with PBS buffer solution, removing blood vessel in umbilical cord, and cutting umbilical cord into pieces of 3-7mm 2 The tissue block is placed in an MEM-alpha culture medium and then is placed in a low-oxygen environment for culture, and the liquid is changed every 3 to 4 days; when the cells fused to 70-90%, the supernatant and tissue mass were removed and subcultured with 0.25% trypsin-EDTA, at which time the subcultured cells were designated P1.
3. The method for preparing human umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the low-oxygen environment in the steps (1) and (2), the temperature is 36-38 ℃, the volume fraction of carbon dioxide is 3-7%, the volume fraction of oxygen is not more than 1%, and the rest gas is nitrogen.
4. The method for preparing exosome of human umbilical cord mesenchymal stem cells according to claim 1, wherein in the step (2), the density of seeding the human umbilical cord mesenchymal stem cells is 5 x 10 4 -2×10 5 Per mL; the bottom area of the culture vessel is 50-175cm 2 (ii) a The culture time of the human umbilical cord mesenchymal stem cells in the MEM-alpha culture medium is 24-72 h; the centrifugal force used for centrifugation is 100-300g, and the centrifugation time is 1-10 min.
5. The method for preparing human umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the step (3), the step of gradient centrifugation is: centrifuging the culture supernatant of the human umbilical cord mesenchymal stem cells obtained in the step (2) for 5-15min by using 400g of 200-plus materials to obtain a supernatant No. 1, centrifuging the supernatant No. 1 for 20-40min by using 2500g of 1500-plus materials to obtain a supernatant No. 2, centrifuging the supernatant No. 2 for 20-40min by using 12000g of 8000-plus materials to obtain a supernatant No. 3, centrifuging the supernatant No. 3 for 100-plus materials for 180min by using 160000g of 100000-plus materials to obtain a precipitate, namely the exosome secreted by the mesenchymal stem cells.
6. The method for preparing human umbilical cord mesenchymal stem cell exosomes according to claim 1, wherein in the step (3), the temperature of gradient centrifugation is 2-6 ℃.
7. A human umbilical cord mesenchymal stem cell exosome prepared by the method of any one of claims 1 to 6.
8. Use of the preparation method of any one of claims 1-6 in preparation of mesenchymal stem cell exosomes.
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