CN110982785B - Method for obtaining adipose-derived stem cell secretory protein - Google Patents
Method for obtaining adipose-derived stem cell secretory protein Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, relates to a method for obtaining cell secretory protein, and particularly relates to a method for obtaining adipose-derived stem cell secretory protein. The method separates adipose-derived stem cells from adipose tissues, performs primary culture and subculture on the adipose-derived stem cells, and performs starvation culture on the adipose-derived stem cells, thereby obtaining a large amount of adipose-derived stem cell secretory proteins. The obtained secretory protein has high biological activity, can be applied to the fields of microenvironment establishment for cell damage repair, cosmetics, anti-aging, disease treatment and the like, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to a method for obtaining cell secretory protein, and particularly relates to a method for obtaining adipose-derived stem cell secretory protein.
Background
Adipose-derived stem cells (ADSCs) can secrete a considerable amount of cytokines including Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGF), Placental Growth Factor (PGF), transforming growth factor-beta (TGF-beta), fibroblast growth factor (FGF-2), etc., and these cytokines are favorable for establishing a better damage repair microenvironment. Wherein, the adipose-derived stem cell is a stem cell with the multipotency of multi-way differentiation separated from adipose tissues. The adipose-derived stem cells have convenient material acquisition and the differentiation capacity of the adipose-derived stem cells is equivalent to that of stem cells from other bone marrow. Adipose-derived stem cells play an important role in the field of regenerative medicine, and can be used for tissue and organ reconstruction, skin regeneration, adipose regeneration and the like. However, in the case of ordinary culture, the adipose-derived stem cells can produce only a certain amount of secreted proteins, and a large amount of adipose tissues is collected to collect the adipose-derived stem cells, so that a sufficient amount of secreted proteins is obtained. The collection process of adipose tissues is complex, thus affecting the wide application of secretory proteins of adipose-derived stem cells, and a method for stimulating adipose-derived stem cells to express and secrete a large amount of secretory proteins with biological activity is urgently needed to be developed.
Disclosure of Invention
The invention aims to provide a method for obtaining an adipose-derived stem cell secretory protein, which can improve the expression of the adipose-derived stem cell secretory protein and can obtain the adipose-derived stem cell secretory protein with high biological activity and high yield.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for obtaining an adipose-derived stem cell secretory protein comprises the following steps:
s1: obtaining adipose-derived stem cells; obtaining a matrix blood vessel component from adipose tissue, separating the matrix blood vessel component to obtain primary adipose-derived stem cells, and performing primary subculture on the primary adipose-derived stem cells to obtain adipose-derived stem cells;
s2: pre-culturing the adipose-derived stem cells: inoculating the adipose-derived stem cells obtained in S1 in a phenol red-free complete culture medium, and pre-culturing the adipose-derived stem cells to obtain activated adipose-derived stem cells;
s3: starvation culture of adipose-derived stem cells: removing the phenol red-free complete culture medium of the activated adipose-derived stem cells, washing the activated adipose-derived stem cells with physiological saline, and performing starvation culture on the activated adipose-derived stem cells with compound electrolyte injection; and collecting the supernatant I after the starvation culture is finished.
By adopting the technical scheme, the technical principle is as follows: adipose-derived stem cells are obtained by separating adipose tissues, and the number and the purity of the adipose-derived stem cells are improved through primary culture and one-time subculture. And then the secretory activity of the adipose-derived stem cells is fully activated through pre-culture, and a sufficient amount of adipose-derived stem cells are obtained, so that the adipose-derived stem cells are prepared for subsequent starvation culture in quantity and growth state. And finally removing the culture medium in the activated adipose-derived stem cells, and culturing the adipose-derived stem cells by using a compound electrolyte injection, wherein the compound electrolyte injection only maintains the osmotic pressure balance of the cells and cannot provide nutrients for the cells, the cells generate an emergency response under the condition of nutrient deficiency, so that the activated adipose-derived stem cells are stimulated to express a large amount of secretory proteins, and the collected supernatant I contains the secretory proteins expressed by the adipose-derived stem cells.
The Stromal Vascular Fraction (SVF) is a cell population obtained by extracting an effective component from adipose tissue extracted from a living body and containing a mixture of a plurality of cells having a repairing function and cytokines. The scheme only relates to operations such as cell separation, culture and the like after the adipose tissues are obtained, and does not relate to surgical operation processes for taking the adipose tissues and the like. The primary adipose-derived stem cells in the S1 are P0 generation adipose-derived stem cells, and the adipose-derived stem cells are P1 generation adipose-derived stem cells obtained by one passage of the primary adipose-derived stem cells.
Has the advantages that:
(1) through the culture and stimulation of the compound electrolyte injection, the adipose-derived stem cells can secrete a large amount of secretory proteins with high biological activity, and the secretory proteins can be applied to the repair of organs, tissues, cells and the like and play a good promoting role. In the prior art, the secretory protein of adipose-derived mesenchymal stem cells is used to produce products with anti-inflammatory or tissue repair functions. The inventor finds that the secretory protein of the adipose-derived stem cell has higher biological activity and higher application value than the secretory protein of the adipose-derived mesenchymal stem cell.
(2) Primary adipose-derived stem cells isolated from adipose tissue are of low purity and limited number and cannot be used directly to induce the expression of secreted proteins. However, the adipose-derived stem cells are rapidly differentiated under the condition of in vitro culture and cannot stably exist, and if the adipose-derived stem cells are subcultured for many times, the adipose-derived stem cells are gradually differentiated, the secretion function is reduced, and the activity of secreted proteins is reduced. The inventor finds that the cell activity and the cell purity of the primary adipose-derived stem cells are high through one-time subculture, and the undifferentiated P1-generation adipose-derived stem cells meet the requirements, so that the activity and the quantity of secreted proteins are ensured.
Further, in S1, the method for obtaining stromal vascular fraction from adipose tissue is as follows: cleaning and shearing adipose tissues, adding an enzyme solution into the adipose tissues, and carrying out enzymolysis and digestion treatment on the adipose tissues; stopping enzymolysis digestion by using an adipose-derived stem cell culture medium after enzymolysis digestion treatment, wherein the volume of the adipose-derived stem cell culture medium is 5-10 times of that of the enzyme solution; then filtered through a 100 μm cell strainer to obtain a stromal vascular fraction.
By adopting the technical scheme, the stromal vascular fraction can be fully separated from the adipose tissue.
Further, in S1, the enzyme solution is a collagenase type i solution or a trypsin solution; in the collagenase I solution, the concentration of collagenase I is 2-3 mg/ml; in the trypsin solution, the concentration of trypsin is 2-3 mg/ml; the temperature of enzymolysis and digestion treatment is 35-40 deg.C, and the duration is 40-80 min.
By adopting the technical scheme, the type I collagenase or trypsin with the concentration can sufficiently remove extracellular matrix in adipose tissues, so that stromal vascular components including adipose stem cells are separated. The enzymolysis digestion treatment temperature can ensure the biological activity of type I collagenase or trypsin, and the enzymolysis time can ensure that the extracellular matrix in the adipose tissue is fully degraded.
Further, the method for separating and obtaining the primary adipose-derived stem cells from the stromal vascular fraction comprises the following steps: inoculating the matrix blood vessel component into a fat stem cell culture medium containing double antibodies, and culturing the matrix blood vessel component; after the culture is finished, removing nonadherent cells to obtain adherent primary adipose-derived stem cells; the volume concentration of the double antibody in the adipose-derived stem cell culture medium is 0.5-1.5%.
By adopting the technical scheme, after the culture of the adipose-derived stem cell culture medium containing the double antibody, the primary adipose-derived stem cells grow in an adherent manner, and other hybrid cells are suspended in the culture medium. The use of a double antibody can prevent the contamination of primary adipose-derived stem cells by bacteria. The double-resistant refers to two antibiotics of penicillin and streptomycin.
Further, in S2, the nopal flower extract is added to the phenol red-free complete medium.
By adopting the technical scheme, the adipose-derived stem cells can be fully activated, so that the production amount of secretory proteins of the adipose-derived stem cells in subsequent starvation culture is increased. The nopalina (Mirabilis jalapa L.) is a plant of Mirabilis, contains flavone, terpenoid, organic acid and other components in the nopalina extract, has anti-inflammatory and antioxidant effects, and is widely applied to the production of cosmetics. The inventors add such ingredients to the culture medium to protect the nutrients in the culture medium, because the change of nutrients (such as oxidative deterioration) during the culture process can lead to the differentiation of the adipose-derived stem cells in advance, thereby reducing the activity of the secreted proteins. The inventors have unexpectedly found that the amount of secreted proteins produced by adipose stem cells can be greatly increased after adding the nopalin extract, and no residue of the nopalin extract is found in a later test. The inventors analyzed that this phenomenon occurs because the nopaline extract enhances the signaling pathways for protein synthesis and secretion.
Further, the nopal flower extract is prepared by the following method: leaching a primula maximowiczii flower bud by using an ethanol solution to obtain an extracting solution, and concentrating and drying the extracting solution to obtain a crude extract; dispersing the crude extract in water, filtering to remove solid phase to obtain water solution of the crude extract; and then purifying the crude extract water solution by using macroporous resin to obtain a target eluent, and concentrating and evaporating the target eluent to dryness to obtain the nopal flower extract.
By adopting the technical scheme, the nopalin extract with the function of stimulating the adipose-derived stem cells to produce secretory proteins can be prepared. The primula maximowiczii flower bud is used as a raw material, and the primula maximowiczii flower extract can be obtained through ethanol extraction and macroporous resin purification. Preliminary analysis shows that the nopaline extract contains polysaccharides and flavonoids, and the inventors will make more intensive studies on the active ingredients and action mechanism of the nopaline extract.
Further, the nopal flower extract is dispersed in water to obtain a solution of the nopal flower extract, the concentration of the solution of the nopal flower extract is 300-500 mg/L.
By adopting the technical scheme, the nopaline extract is dissolved in water, so that the functional components of the nopaline extract can be fully dispersed, and the functional components can be fully combined with adipose-derived stem cells; the sterilization treatment ensures that the culture system is not polluted by bacteria.
Further, the volume ratio of the annatto extract solution to the phenol red-free complete medium is 1 (20-30).
By adopting the technical scheme, the functional components in the nopal asiatica extract can be ensured to fully stimulate the adipose-derived stem cells to produce secretory proteins, and meanwhile, cytotoxicity can not be generated on the adipose-derived stem cells.
Further, S3 further includes the step of obtaining supernatant ii: taking the activated adipose-derived stem cells obtained in S2, removing the phenol red-free complete medium, and washing the activated adipose-derived stem cells with physiological saline; adding the supernatant I into the activated adipose-derived stem cells, and carrying out starvation culture on the activated adipose-derived stem cells; and collecting the supernatant II after the starvation culture is finished.
By adopting the technical scheme, the new adipose-derived stem cells are cultured by using the supernatant I, and the secretory protein can be enriched, so that the concentration of the secretory protein is improved to meet the application requirement.
Further, S3 further includes a step of obtaining supernatant iii: taking the activated adipose-derived stem cells obtained in S2, removing the phenol red-free complete medium, and washing the activated adipose-derived stem cells with physiological saline; adding the supernatant II into the activated adipose-derived stem cells, and carrying out starvation culture on the activated adipose-derived stem cells; and collecting the supernatant III after the starvation culture is finished.
By adopting the technical scheme, the new adipose-derived stem cells are cultured by using the supernatant II, and the secretory protein can be enriched, so that the concentration of the secretory protein is improved to meet the application requirement.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1: examples of obtaining secreted proteins from adipose-derived stem cells
S1: obtaining adipose-derived stem cells:
(1) fat tissue treatment: the collected adipose tissues are washed by normal saline, blood stains are washed clean, and double antibodies are added into the normal saline, wherein the volume concentration of the double antibodies in the normal saline is 1%. The double antibody refers to a solution containing penicillin and streptomycin, wherein penicillin is 100UI/ml, and streptomycin is 50 UI/ml. Placing adipose tissue into a centrifuge tube, and cutting with sharp-pointed scissors to 1-3mm3Size.
(2) Fat tissue digestion: adding 2.5mg/ml collagenase I solution into adipose tissue, carrying out water bath oscillation digestion at 37 ℃ for 1h, centrifuging for 10min at 300g, sucking and removing upper fat and liquid layers, adding physiological saline to resuspend cell sediment, filtering by using a 100-micron cell filter, removing undigested tissue masses, centrifuging for 10min at 300g, and removing supernatant to obtain the stromal vascular fraction.
(3) Primary culture: after counting the stromal vascular fraction, the cells were counted at 5X 105The amount of each/ml of the culture medium was inoculated into a cell culture flask, and a double antibody was added at a concentration of 1% by volume in the culture medium. And (3) placing the cell culture bottle in an incubator at 37 ℃ and with 5% carbon dioxide concentration for culture observation, changing the culture solution after culturing for 24h, removing the cells which are not attached to the wall, and supplementing a culture medium containing 1% of double antibody for continuous culture observation. The culture medium used was an adipose-derived stem cell serum medium (
CatNo:BSM0002)。
(4) Cell passage: subculturing when the primary cultured cells reach 80% fusion degree, removing culture medium, washing with normal saline, digesting with pancreatin with mass fraction of 0.125% (pancreatin digestion time is no more than 2min), diluting with large amount of normal saline after cell rounding and falling off, centrifuging at 300g for 10min, discarding supernatant, re-suspending cells with normal saline, counting cells, and counting according to 2 × 106The amount of each vial was inoculated into a cell culture flask (T75 flask was used), and no double antibody was added to the medium for subculture. The culture medium used was an adipose-derived stem cell serum medium (
CatNo: BSM0002), which will be used in the subsequent treatment process.
S2: pre-culturing the adipose-derived stem cells: adipose stem cells were inoculated in three batches:
in the first batch: number of cells inoculated per flask: 9X 106A T175 culture bottle, 3 bottles of inoculated cells, 40 ml/bottle of cell culture system, complete culture medium used for cell culture is phenol red-free complete culture medium,adding a primula maximowiczii flower extract solution into the culture medium, wherein the volume ratio of the primula maximowiczii flower extract solution to the phenol red-free complete culture medium is 1:30, and the concentration of the primula maximowiczii flower extract in the primula maximowiczii flower extract solution is 500 mg/L;
and (3) second batch: number of cells inoculated per flask: 6X 106Inoculating 3 bottles of cells in a T175 culture bottle, wherein the cell culture system is 40 ml/bottle, the complete culture medium used for cell culture is phenol red-free complete culture medium, a primula maximowiczii extract solution is added into the culture medium, the volume ratio of the primula maximowiczii extract solution to the phenol red-free complete culture medium is 1:25, and the concentration of the primula maximowiczii extract in the primula maximowiczii extract solution is 500 mg/L;
and (3) third batch: number of cells inoculated per flask: 3X 106And inoculating 3 bottles of cells in a T175 culture bottle, wherein the cell culture system is 40 ml/bottle, the complete culture medium used for cell culture is phenol red-free complete culture medium, a nopalin extract solution is added into the culture medium, the volume ratio of the nopalin extract solution to the phenol red-free complete culture medium is 1:20, and the concentration of the nopalin extract in the nopalin extract solution is 500 mg/L.
After the inoculation of the cells is finished, the cells are placed in a carbon dioxide incubator for culture at the temperature of 37 ℃ and the volume concentration of carbon dioxide of 5 percent, and the three batches of cells are cultured.
S3: starvation culture:
when the cell fusion degree of the cells of the first batch reaches 70%, removing the culture medium, washing the cells with physiological saline for 2 times, performing starvation culture with 40ml of compound electrolyte injection (Shanxi Shadongtai Sheng) in each bottle, and culturing and observing in an incubator at 37 ℃ and 5% carbon dioxide concentration. And collecting cell culture supernatant when the cell fusion degree reaches 95%, centrifuging for 8min under the condition of 400g to remove precipitates, and taking centrifuged supernatant (namely supernatant I) and placing the centrifuged supernatant at 4 ℃ for later use.
When the cell confluence of the second batch of cells is 70%, removing the culture medium, washing the cells with physiological saline for 2 times, adding the supernatant I into each bottle, and culturing and observing in an incubator at 37 ℃ and 5% carbon dioxide concentration. And when the cell fusion degree reaches 95%, collecting cell culture supernatant, centrifuging for 8min under the condition of 400g to remove precipitates, and taking the centrifuged supernatant (namely supernatant II) to be placed at 4 ℃ for later use.
When the cell fusion degree of the cells of the third batch reaches 70%, removing the culture medium, washing the cells with normal saline for 2 times, adding the supernatant II into each bottle, and culturing and observing in an incubator at 37 ℃ and 5% carbon dioxide concentration. And when the cell fusion degree reaches 95%, collecting cell culture supernatant, centrifuging for 8min under the condition of 400g to remove precipitates, and taking the centrifuged supernatant (namely supernatant III) and placing at 4 ℃ for later use. Since three replicates were set up, supernatant iii was in triplicate.
In this protocol, the nopal flower extract was prepared as follows:
the milled primula maximowiczii flower buds are extracted by using 80% ethanol solution, 200ml ethanol solution is used for every 100g primula maximowiczii flower buds, the extraction is carried out for 3h at 70 ℃, and then the filtrate is filtered and reserved. The same amount of ethanol solution (200 ml per 100g of nopaline flower buds) was added again to the solid mass, extracted at 70 ℃ for 3h and then filtered to retain the filtrate. Extracting for 3 times, and mixing the 3 filtrates to obtain extractive solution. And concentrating the extracting solution by using a rotary evaporator to obtain an extract, wherein the extract is the crude extract. Dispersing the crude extract in water, adding 3ml water into each g of crude extract, dissolving completely, and filtering to remove solid phase to obtain water solution of crude extract. Then purifying the crude extract water solution by using macroporous resin, wherein the specific method comprises the following steps: and (3) loading the crude extract aqueous solution into a macroporous resin D101, standing for 1h, eluting by using water with the volume of 1 time of that of the column, and discarding the eluent. Eluting with 3 times of 40% ethanol solution, and retaining eluate, wherein the eluate is the target eluate. Concentrating the target eluent by using a rotary evaporator, and then performing freeze-drying to obtain dry powder, wherein the dry powder is the primula maximowiczii flower extract. The nopal flower extract was dissolved in sterile water to obtain a nopal flower extract solution, and the nopal flower extract solution was filter sterilized using a 0.22 μm microfiltration membrane.
Example 2
This example is basically the same as example 1 except that in S1, the concentration of the diabody in the culture medium was 0.5% by volume; the temperature of enzymolysis digestion treatment is 35 ℃, and the duration is 80 min; trypsin solution was used instead of collagenase type i solution. In S2, the mass concentration of the annatto flower extract solution is 400 mg/L.
Example 3
This example is basically the same as example 1 except that, in S1, the concentration of the diabody in the culture medium was 1.5% by volume; the temperature of enzymolysis digestion treatment is 40 ℃, and the duration is 40 min. In S2, the mass concentration of the annatto flower extract solution is 300 mg/L.
Example 4
This example is basically the same as example 1 except that, in S1, the concentration of the diabody in the culture medium was 0.5% by volume; the temperature of enzymolysis digestion treatment is 40 ℃, and the duration is 60 min. In S2, the mass concentration of the annatto flower extract solution is 300 mg/L.
Comparative example 1 and comparative example 2 are substantially the same as in example 1, except that umbilical cord mesenchymal stem cells were replaced with P1-generation umbilical cord mesenchymal stem cells; in S2, the phenol red-free complete medium was not supplemented with the nopal flower extract solution.
Comparative example 3 and comparative example 4 are substantially the same as example 1 except that in S2, no annatto extract solution was used in the phenol red-free complete medium.
Comparative examples 5 to 8 are substantially the same as example 1 except that umbilical cord mesenchymal stem cells were replaced with P1-generation umbilical cord mesenchymal stem cells.
Comparative example 9 and comparative example 10 are substantially the same as in example 1, except that umbilical cord mesenchymal stem cells were replaced with P3-generation umbilical cord mesenchymal stem cells; in S2, the phenol red-free complete medium was not supplemented with the nopal flower extract solution.
Comparative example 11 and comparative example 12 are substantially the same as in example 1, except that adipose-derived mesenchymal stem cells were replaced with P3-generation adipose-derived mesenchymal stem cells; in S2, the phenol red-free complete medium was not supplemented with the nopal flower extract solution.
Comparative examples 13 to 16 are basically the same as example 1 except that umbilical cord mesenchymal stem cells were replaced with P3-generation umbilical cord mesenchymal stem cells.
Comparative examples 17 to 20 are substantially the same as example 1 except that adipose-derived mesenchymal stem cells were replaced with P3-generation adipose-derived mesenchymal stem cells.
Experimental example 1: total protein assay
Total protein content detection was performed on the supernatants III obtained in examples 1-3 and comparative examples 1-4 to determine the expression amount of secreted protein by a conventional biuret reagent method using crystallized bovine serum albumin as a standard to draw a standard curve, absorbance of the samples to be tested was detected by a spectrophotometer, and the protein content in each sample was calculated from the standard curve. The results are shown in Table 1. From the experimental results, it was found that examples 1 to 4 contained a large amount of secreted proteins in the supernatant iii obtained by starvation culture using P1-generation adipose-derived stem cells and preculture using the nopaline flower extract. The adipose-derived stem cells have high secretory activity, and the P1 generation adipose-derived stem cells are pretreated by using the nopaline flower extract, so that the expression of secretory proteins can be increased.
The umbilical cord mesenchymal stem cells of P1 generation which were not pre-cultured using the nopaline extract were used in comparative example 1 and comparative example 2, and the adipose stem cells of P1 generation which were not pre-cultured using the nopaline extract were used in comparative example 3 and comparative example 4, and it can be seen from the test data that: the activity of secreting the umbilical cord mesenchymal stem cells of P1 generation without preculture by using the nopaline flower extract is lower than that of the adipose-derived stem cells of P1 generation, and less secreted protein is obtained. The results of measuring the amount of secreted proteins in the P1 umbilical cord mesenchymal stem cells pre-cultured with the nopaline flower extract in comparative examples 5 to 8, and in the P1 adipose-derived stem cells pre-cultured with the nopaline flower extract in examples, show that: the protein secretion amount of the P1 generation umbilical cord mesenchymal stem cells pre-cultured by using the nopaline flower extract is far less than that of the P1 generation adipose-derived stem cells, and the protein secretion function of the P1 generation umbilical cord mesenchymal stem cells is not as good as that of the P1 generation adipose-derived stem cells.
Comparative examples 9 and 10 used umbilical cord mesenchymal stem cells of generation P3 without preculture using the nopaline extract, and comparative examples 11 and 12 used adipose mesenchymal stem cells of generation P3 without preculture using the nopaline extract, and it can be seen from the test data that: the activity of secreting the umbilical cord mesenchymal stem cells of P3 generation without preculture by using the nopaline flower extract is lower than that of the adipose-derived stem cells of P3 generation, and less secreted protein is obtained. The results of measuring the amount of secreted proteins of the umbilical cord mesenchymal stem cells of P3 generation pre-cultured using the nopaline flower extract in comparative examples 13 to 16 and the adipose mesenchymal stem cells of P3 generation pre-cultured using the nopaline flower extract in comparative examples 17 to 20 show that: the protein secretion amount of the P3 generation umbilical cord mesenchymal stem cells cultured by using the nopaline flower extract is less than that of the P3 generation adipose mesenchymal stem cells, and the protein secretion function of the P3 generation umbilical cord mesenchymal stem cells is not as good as that of the P3 generation adipose mesenchymal stem cells. And the protein secretion amount of the adipose-derived stem cells or adipose-derived mesenchymal stem cells is more than that of the umbilical cord mesenchymal stem cells of the generations P1 and P3, so that the secretion function is stronger.
Table 1: total protein content assay results for supernatants III of examples 1-4, comparative examples 1-8
Table 2: total protein content measurement results of supernatant III in comparative examples 9 to 20
Experimental example 2: experiment of biological Activity of secreted protein
The secretory protein can realize the repair of damaged tissues through anti-inflammation, the cells are prevented from being damaged by oxidative stress induced by hydrogen peroxide, and the secretory protein of the adipose-derived stem cells has the effects of resisting oxidation and regulating the redox state of the cell environment. The supernatants III of examples 1-3, comparative examples 11, 12, 3 and 1 were tested for anti-inflammatory effect using the assay inhibiting the activation of the mouse macrophage cell line RAW 264.7. This experiment was set with 5 replicates for a blank control group (no secreted protein and no lipopolysaccharide induction), a positive control group (lipopolysaccharide induction and no secreted protein), and an experimental group (7 groups total, lipopolysaccharide induction using supernatants iii from examples 1-3, comparative examples 11, 12, 3 and 1, respectively). For the blank control, the mouse RAW264.7 macrophage cell line was inoculated in DMEM-containing culture medium and cultured for 33 h. For the positive control, the mouse RAW264.7 macrophage cell line was inoculated in DMEM-containing culture medium, cultured for 24h, and then the cells were incubated with 1 μ g/ml (final concentration) of lipopolysaccharide for 8h to induce inflammatory response of RAW264.7 macrophages. For the experimental group, the mouse RAW264.7 macrophage strain was inoculated in DMEM-containing culture solution, after 24 hours of culture, the cells were treated with supernatant III for 1 hour (ensuring that the final concentration of the secreted protein in the culture system was 50g/L, and the amount of the secreted protein was calculated as the amount of total protein), and then the cells were incubated with 1. mu.g/ml (final concentration) of lipopolysaccharide for 8 hours, to induce inflammatory response of RAW264.7 macrophages. After the treatment of each group is finished, taking the culture supernatant of RAW264.7 macrophage to carry out ELISA detection, and measuring the expression level of IL-1 beta and IL-18 in the supernatant. The results of the experiment are shown in table 3. After being acted by lipopolysaccharide, RAW264.7 macrophage induces the secretion increase of IL-1 beta and IL-18 in cells, which indicates the generation of inflammation. In the experimental group treated by the adipose-derived stem cell secretory protein, the secretion amounts of IL-1 beta and IL-18 are reduced to different degrees, which indicates that the adipose-derived stem cell secretory protein has anti-inflammatory effect. Experimental results show that the anti-inflammatory activity of the secretory protein of the adipose-derived stem cells in examples 1-3 is high, and the increase of the secretion amounts of IL-1 beta and IL-18 caused by lipopolysaccharide can be well inhibited. Comparative examples 11 and 12 used secreted proteins of adipose-derived mesenchymal stem cells, and the anti-inflammatory activity was inferior to that of the proteins of examples 1 to 3. Comparative example 3 no activation of adipose-derived stem cells with the nopaline extract, although the amount of protein secretion was reduced, it still had good anti-inflammatory activity. Comparative example 1 used P1 generation umbilical cord mesenchymal stem cells for induction of secreted proteins, and the obtained secreted proteins had lower activity.
Table 3: biological Activity test results for secreted proteins (mean. + -. SE)
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (6)
1. A method for obtaining an adipose-derived stem cell secretory protein comprises the following steps:
s1: obtaining adipose-derived stem cells; obtaining a matrix blood vessel component from adipose tissue, separating the matrix blood vessel component to obtain primary adipose-derived stem cells, and performing primary subculture on the primary adipose-derived stem cells to obtain adipose-derived stem cells;
s2: pre-culturing the adipose-derived stem cells: inoculating the adipose-derived stem cells obtained in S1 in a phenol red-free complete culture medium, and pre-culturing the adipose-derived stem cells to obtain activated adipose-derived stem cells;
s3: starvation culture of adipose-derived stem cells: removing the phenol red-free complete culture medium of the activated adipose-derived stem cells, washing the activated adipose-derived stem cells with physiological saline, and performing starvation culture on the activated adipose-derived stem cells with compound electrolyte injection; collecting supernatant I after the starvation culture is finished;
adding a nopal flower extract solution to the phenol red free complete medium in S2; the volume ratio of the cochineal flower extract solution to the phenol red-free complete culture medium is 1 (20-30); the carmine extract solution is prepared by the following method: leaching a primula maximowiczii flower bud by using an ethanol solution to obtain an extracting solution, and concentrating and drying the extracting solution to obtain a crude extract; dispersing the crude extract in water, filtering to remove solid phase to obtain water solution of the crude extract; then purifying the crude extract water solution by using macroporous resin to obtain a target eluent, and concentrating and evaporating the target eluent to dryness to obtain a nopal blume extract; the nopaline extract is dispersed in water to obtain a nopaline extract solution, wherein the concentration of the nopaline extract solution is 300-500 mg/L.
2. The method for obtaining the secretory protein of the adipose stem cells of claim 1, wherein the stromal vascular fraction is obtained from adipose tissue in S1 as follows: cleaning and shearing adipose tissues, adding an enzyme solution into the adipose tissues, and carrying out enzymolysis and digestion treatment on the adipose tissues; stopping enzymolysis digestion by using an adipose-derived stem cell culture medium after enzymolysis digestion treatment, wherein the volume of the adipose-derived stem cell culture medium is 5-10 times of that of the enzyme solution; then filtered through a 100 μm cell strainer to obtain a stromal vascular fraction.
3. The method for obtaining the protein secreted from the adipose-derived stem cells of claim 2, wherein in S1, the enzyme solution is collagenase type I solution or trypsin solution; in the collagenase I solution, the concentration of collagenase I is 2-3 mg/ml; in the trypsin solution, the concentration of trypsin is 2-3 mg/ml; the temperature of enzymolysis and digestion treatment is 35-40 deg.C, and the duration is 40-80 min.
4. The method for obtaining the secretory protein of the adipose-derived stem cells of claim 3, wherein the primary adipose-derived stem cells are isolated from stromal vascular fraction in S1 by: inoculating the matrix blood vessel component into a fat stem cell culture medium containing double antibodies, and culturing the matrix blood vessel component; after the culture is finished, removing nonadherent cells to obtain adherent primary adipose-derived stem cells; the volume concentration of the double antibody in the adipose-derived stem cell culture medium is 0.5-1.5%.
5. The method for obtaining the secretory protein of the adipose-derived stem cell of claim 4, wherein the step of S3 further comprises the step of obtaining the supernatant II: taking the activated adipose-derived stem cells obtained in S2, removing the phenol red-free complete medium, and washing the activated adipose-derived stem cells with physiological saline; adding the supernatant I into the activated adipose-derived stem cells, and carrying out starvation culture on the activated adipose-derived stem cells; and collecting the supernatant II after the starvation culture is finished.
6. The method for obtaining the secretory protein of the adipose-derived stem cell of claim 5, wherein the step of S3 further comprises the step of obtaining the supernatant III: taking the activated adipose-derived stem cells obtained in S2, removing the phenol red-free complete medium, and washing the activated adipose-derived stem cells with physiological saline; adding the supernatant II into the activated adipose-derived stem cells, and carrying out starvation culture on the activated adipose-derived stem cells; and collecting the supernatant III after the starvation culture is finished.
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