CN113249316A - Preparation method and application of umbilical cord/placenta mesenchymal stem cell-derived exosome - Google Patents

Abstract

A preparation method and application of umbilical cord/placenta mesenchymal stem cell-derived exosome belong to the field of bioengineering, and the preparation method comprises the following steps: 1. the separation culture and identification of the placenta mesenchymal stem cells specifically comprise (1) the separation culture of UC/PL-MSCs, (2) the detection of the surface antigen expression of the UC/PL-MSCs, (3) the identification of the induced differentiation potential of the placenta mesenchymal stem cells, and (4) the identification of the induced differentiation potential of adipogenic cells; and step 2, preparing the UC/PL-MSCs source exosome. The preparation method of the placenta mesenchymal stem cell-derived exosome provided by the invention is simple and easy to operate, and the prepared placenta mesenchymal stem cell-derived exosome is applied to the aspects of anti-aging and tissue repair.

Description

Preparation method and application of umbilical cord/placenta mesenchymal stem cell-derived exosome

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a preparation method and application of an umbilical cord/placenta mesenchymal stem cell-derived exosome.

Background

Although the medical level of perinatal medicine and neonatal intensive care has been greatly improved, hypoxic ischemic encephalopathy caused by birth asphyxia is still one of the main causes of neonatal death, and many surviving neonates are accompanied by permanent nervous system sequelae such as cerebral palsy, mental retardation, learning disorder, depressive 30286, hearing and vision impairment and the like. In recent years, a great deal of research proves that stem cells can bring a new strategy for treating neonatal brain injury, Mesenchymal Stem Cells (MSCs) are a class of stem cells with self-renewal capacity and multi-directional differentiation potential, have the characteristic of low immunogenicity, are considered as the transplantation sources with the most treatment potential, and have been applied to the clinical test stage of treating severe intracerebroventricular hemorrhage of premature infants, but the treatment mechanism of the cells is not clear. Previous studies suggest that MSCs can migrate to damaged tissues and differentiate into precursor cells with therapeutic potential, thereby exerting a therapeutic effect. However, recent studies have found that transplantation of MSC-derived culture media can exert therapeutic effects similar to stem cell transplantation, and further studies have shown that the brain injury protective effect of culture media may be derived from the paracrine effect of stem cells. The exosome, as a bioactive substance secreted by the MSC, becomes a research hotspot in recent years. Exosomes are vesicles with the diameter of 30-100 nm released by fusion of cell multivesicular bodies and cytoplasmic membranes, and are originally considered as transportation tools of cell metabolites. The intensive research finds that the exosome is composed of a double-layer lipid membrane, contains a large amount of bioactive substances such as protein, lipid and nucleic acid, and can be used as an intercellular communication tool to participate in complex pathophysiological processes such as immune regulation, vascular proliferation, nerve function repair and the like. The exosome has the advantages of long half-life period, low immunogenicity, good stability, capability of passing through a blood brain barrier and the like, and has great potential in the treatment of brain injury. Research reports that the treatment of the stem cells by using exosome alone instead of the stem cells also plays similar biological functions and can even play better therapeutic action. Therefore, MSC-derived exosomes are expected to become emerging strategies for treatment of neonatal brain injury.

The exosome can be adhered to the surface of a receptor cell in a ligand-receptor interaction mode, and the exosome can also be released into a target cell through endocytosis uptake of the receptor cell or direct fusion of a vesicle and a cell membrane to play a role in intercellular communication, so that the biological behavior of the target cell is regulated. The production of exosomes and the binding process to target cells are shown in figure 1.

As shown in fig. 1, the cell membrane is invaginated to form an early endosome, the cell releases particulate matter into the early endosome, the early endosome further matures to form a late endosome, the late endosome is fused with a specific part of the cell membrane to form a bud bubble, and the exosome in the bubble is released out of the cell; exosomes may adhere to the surface of a receptor cell by way of ligand-receptor interactions, or the exosome contents may be released into the target cell by receptor endocytic uptake or direct fusion of vesicles and cell membrane.

Exosomes contain not only proteins and lipids, but also a large amount of nucleic acids. Since the first identification of exosomes containing mRNA and miRNA in 2007, thousands of mRNA and miRNA have been identified in exosomes, and the presence of other specific nucleic acids such as siRNA, mtDNA, tRNA, double-stranded DNA, and the like has been found. The research on miRNA is clear. miRNA is small non-coding RNA, and is combined with complementary sequence of target mRNA to degrade target mRNA, so that translation is stopped and gene is silenced, gene expression is influenced in biological processes of cell differentiation, proliferation, apoptosis and the like, for example, miR-125a can promote angiogenesis, and miRNA-19a can inhibit apoptosis. It is noted that exosomes can be produced by almost all eukaryotic cells, the composition and composition of their contents depending on their parent cell source, and are influenced by the extracellular microenvironment. For example, when MSC is subjected to hypoxia-ischemia treatment, the level of miR-133b and miR-22 in exosome is increased, and the miRNA can also promote neural restoration by regulating extracellular microenvironment. Indicating that information can be fed back between the MSC and the external environment through exosomes.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of umbilical cord/placenta mesenchymal stem cell-derived exosome and application thereof in the aspect of anti-aging.

The preparation method of the umbilical cord/placenta mesenchymal stem cell-derived exosome comprises the following steps:

step 1, separating, culturing and identifying umbilical cord/placenta mesenchymal stem cells UC/PL-MSCs (MSCs)

Step 1.1, isolation and culture of UC/PL-MSCs

Preparing human umbilical cord/placenta, cutting umbilical cord/placenta tissue into meat paste, digesting with 100U/mL collagenase type I and 1.5 μ g/mL DNase I in LG-DMEM solution, filtering with cell screen, and centrifuging. Resuspending the cells, collecting the cells, inoculating into T25 flask, and culturing at 37 deg.C with 5% CO₂Culturing in an incubator. Until the primary cell fusion degree reaches 70-80%, digesting and passaging by using 0.25% pancreatin. The growth and morphology of the cells were observed with an inverted microscope.

The following steps 1.2-1.4 are not in sequence;

step 1.2, detecting the surface antigen expression of UC/PL-MSCs

Taking UC/PL-MSCs growing to the 4 th generation, digesting with 0.25% pancreatin, resuspending cells, adjusting cell concentration, adding rabbit anti-mouse CD29, CD44, CD105, CD45, CD34 and isotype control, and incubating. Washed with buffer, centrifuged and the supernatant discarded. After buffer solution is added into each tube, goat anti-rabbit fluorescent secondary antibody is added into each tube respectively, and incubation is carried out. Washing with a buffer solution, centrifuging, discarding supernatant, resuspending cells, and detecting the UC/PL-MSCs surface antigen expression.

Step 1.3, identification of differentiation potential induced by umbilical cord/placenta mesenchymal stem cells

And (3) inoculating UC/PL-MSCs growing to the 4 th generation into a six-hole plate coated with 0.1% gelatin, and adding umbilical cord/placenta mesenchymal stem cells to induce the culture medium when the cell fusion degree reaches 60-70%. After fixing with 4% paraformaldehyde solution, adding alizarin red dye solution for dyeing. The effect of cell staining was observed.

Step 1.4, adipogenic cell induced differentiation potential identification

And (3) inoculating UC/PL-MSCs growing to the 4 th generation in a six-hole plate, adding an EGF (epidermal growth factor) solution and a bFGF (basic fibroblast growth factor) solution of an umbilical cord/placenta mesenchymal stem cell adipogenic induced differentiation culture medium for alternate action when the cell fusion degree reaches 100%, and dyeing with an oil red O staining solution after fixing with a 4% paraformaldehyde solution. The effect of cell staining was observed.

Step 2, preparation of UC/PL-MSCs source exosome

(a) And collecting the 4 th generation UC/PL-MSCs, re-suspending the cells, and sequentially passing the UC/PL-MSCs suspension through a track etching membrane (Whatman) with the pore diameter of 10 mu m, 5 mu m and 1 mu m. Centrifuging the cell suspension, filtering, and resuspending the precipitate to obtain exosome, and storing at-80 deg.C for a long time;

or

(b) Growing to 4 th generation UC/PL-MSCs about 1 × 10⁷1 × 10⁶Inoculating the cells into T25 cell bottle at a density of about 50%, washing with PBS, adding LG-DMEM medium without fetal calf serum, 37 deg.C, and 5% CO₂And continuing culturing in the incubator. Collecting cell supernatant, centrifuging, filtering, ultracentrifuging, and resuspending the obtained exosome precipitate with PBS for long-term storage at-80 ℃.

The mesenchymal stem cell exosome shows treatment advantages for nerve tissue repair and regeneration, and experiments show that when the exosome derived from umbilical cord/placenta mesenchymal stem cells is injected into the vitreous body of a mouse with damaged optic nerve, the exosome can migrate to the ganglion cell layer from the vitreous body, and the survival and axon regeneration of receptor cells (retinal ganglion cells) are promoted. In a mouse skin incision model, after an exosome is injected intravenously, the exosome can be recruited to a soft tissue wound area, and through the property of changing fiber cells, the formation of collagen I and III is promoted at the early stage of wound healing, and the expression of the collagen is inhibited at the late stage so as to inhibit scar formation and finally promote the healing of skin wounds.

Experimental research suggests that exosome can be used as a biological tool, can be applied to whole-body anti-aging, local anti-aging and repair and skin anti-aging and repair, and provides a new treatment strategy for anti-aging and tissue repair through health product drinking, intravenous injection, joint injection, intramuscular injection, epidermis smearing and infiltration and the like. In regenerative therapy, exosomes can avoid some defects (immune rejection, ethical problems and the like) of stem cell therapy, and have the advantages of high stability, easiness in storage, no need of proliferation, convenience in quantitative use, recruitment to a damaged part and the like; exosomes, because they contain multiple proteins and RNAs, have higher safety and greater tissue regeneration potential than a single cytokine.

The invention has the beneficial effects that:

the invention provides a preparation method of an umbilical cord/placenta mesenchymal stem cell-derived exosome, which is simple and easy to operate. The prepared umbilical cord/placenta mesenchymal stem cell-derived exosome can be applied to the aspects of anti-aging and tissue repair.

Drawings

FIG. 1 production of exosomes and binding to target cells.

Detailed Description

Examples

A preparation method of umbilical cord/placenta mesenchymal stem cell-derived exosome comprises the following steps:

step 1, separating, culturing and identifying umbilical cord/placenta mesenchymal stem cells (UC/PL-MSCs)

Reagent: collagenase type i (Invitro-gen), dnase i (Sigma), LG-DMEM medium, fetal bovine serum and 0.25% trypsin (Gibco), umbilical cord/placental mesenchymal stem cell adipogenic differentiation medium kit and umbilical cord/placental mesenchymal stem cell differentiation induction medium kit (Cyagen), rabbit anti-mouse CD63, CD9, CD81, Tsg101, β -actin primary antibodies were purchased from Abcam, HRP-labeled goat anti-rabbit IgG secondary antibodies (Bioworld), ECL luminescence (merckmill), track etched membranes of 10 μm, 5 μm, 1 μm (Whatman).

The method specifically comprises the following steps:

step 1.1, isolation and culture of UC/PL-MSCs

Preparing human umbilical cord/placenta, cutting umbilical cord/placenta tissue into meat paste, adding 100mL LG-DMEM solution containing 100U/mL I-type collagenase and 1.5 μ g/mL DNase I, standing at 37 deg.C and 5% CO₂Digesting in the incubator for 1.5 h. After 1.5h, adding a proper amount of LG-DMEM medium containing 10% fetal bovine serum by volume fraction, filtering by using a 100-micron cell screen, and centrifuging for 5min at 1000 r/min. Resuspending the cells in LG-DMEM medium containing 20% fetal calf serum and 100U/mL streptomycin by volume,at about 4X 10⁶Cell density of (2) cells were seeded into T25 flasks at 37 ℃ with 5% CO₂Culturing in an incubator. After 72h of culture, the solution is changed for the first time, and then the solution is changed every 3d until the primary cell fusion degree reaches 80%, the cells are digested by 0.25% pancreatin and passaged according to the ratio of 1: 3. The growth and morphology of the cells were observed with an inverted microscope.

Step 1.2, detecting the surface antigen expression of UC/PL-MSCs

Taking the 4 th generation UC/PL-MSCs with good growth state, digesting with 0.25% pancreatin, re-suspending the cells with flow cell buffer (PBS containing volume fraction of 1% BSA), and adjusting the cell concentration to 3 × 10⁵Rabbit anti-mouse CD29, CD44, CD105, CD45, CD34 and isotype control were added to each tube and incubated at 4 ℃ for 30 min. After incubation, the cell was washed 2 times with flow cytometric buffer and centrifuged to discard the supernatant. After 100 mul of flow cytometry buffer solution is added into each tube, goat anti-rabbit fluorescent secondary antibody is added into each tube respectively, and the tubes are incubated for 30min at 4 ℃ in a dark place. And (3) washing for 2 times by using a flow cytometry buffer after incubation, centrifuging to remove a supernatant, and detecting the UC/PL-MSCs surface antigen expression by using a flow cytometer after the flow cytometry buffer is used for resuspending cells.

Step 1.3, identification of differentiation potential induced by umbilical cord/placenta mesenchymal stem cells

Taking the 4 th generation UC/PL-MSCs with good growth state at 2 × 10⁴cells/cm²The cells are inoculated in a six-well plate coated with 0.1% of gelatin, and when the cell fusion degree reaches 70%, umbilical cord/placenta mesenchymal stem cells are added for induction culture medium. After the induction for 21d, fixing for 30min by 4% paraformaldehyde solution, and adding alizarin red dye solution for dyeing for 5 min. The staining effect of the cells was observed under an inverted fluorescence microscope.

Step 1.4, adipogenic cell induced differentiation potential identification

Taking the 4 th generation UC/PL-MSCs with good growth state at 2 × 10⁴cells/cm²The cell density of the cells is inoculated in a six-hole plate, when the cell fusion degree reaches 100 percent, the umbilical cord/placenta mesenchymal stem cell adipogenic induced differentiation culture medium EGF liquid and bFGF liquid are added for alternate action for 4 times (21d), and then oil red O staining solution is used for staining for 30min after 4 percent paraformaldehyde solution is fixed. Observation under an inverted fluorescent microscopeCell staining effect.

Step 2, preparation of UC/PL-MSCs source exosome

(a) Collecting UC/PL-MSCs of generation 4 by about 1 × 10⁷Cells were resuspended in 1mL PBS. The UC/PL-MSCs suspension was sequentially passed through a track-etched membrane (Whatman) with a pore size of 10 μm, 5 μm, 1 μm 6 times using an AvaniExtruder. The cell suspension was then centrifuged at 2000g for 10min and the supernatant was centrifuged at 16500g for 20 min. The supernatant was collected, filtered through a 0.22 μm filter, and ultracentrifuged at 100000g for 1 hour. The supernatant was discarded and the pellet was resuspended in 100. mu.L PBS, which was the exosome. Storing at-80 deg.C for a long time.

Or

(b) About 1X 10 times of the 4 th generation UC/PL-MSCs with good growth condition⁷1 × 10⁶Inoculating the cells into T25 cell bottle at a density of about 50%, washing with PBS for 3 times, adding LG-DMEM medium without fetal calf serum, 37 deg.C, and 5% CO₂And continuing culturing in the incubator. After 24h, approximately 60mL of cell supernatant was collected. Centrifuging at 300g for 10min at 4 deg.C, centrifuging the supernatant at 4 deg.C for 10min at 1000g, centrifuging the supernatant at 4 deg.C for 30min at 10000g, filtering the supernatant with 0.22 μm filter, ultracentrifuging at 100000g for 2h, and resuspending the obtained exosome precipitate with 100 μ LPBS at-80 deg.C for long-term storage.

Step 4, identifying UC/PL-MSCs

Under an inverted microscope, the UC/PL-MSCs can be observed to have more adherent cell growth after 48 hours, and the cells are mostly fusiform in shape with oval shape after passage and grow in vortex arrangement. With the increase of the number of passages, the cell morphology tends to be consistent, and the cell morphology of the 4 th generation is basically consistent and takes a long fusiform as a main part. Flow cytometry detection results show that UC/PL-MSCs obtained through separation culture highly express CD29, CD44 and CD105, the positive rate is higher than 99.0%, and hematopoietic stem cell surface antigens CD45 and CD34 are not expressed. The results show that UC/PL-MSCs obtained by separation and culture by an enzyme digestion method meet the 'ISCT mesenchymal stem cell standard' established by the International society for cell therapy.

Claims (7)

Translated fromChinese

1.一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，包括以下步骤：1. a preparation method of umbilical cord/placental mesenchymal stem cell-derived exosome, is characterized in that, comprises the following steps:步骤1、UC/PL-MSCs的分离培养与鉴定Step 1. Isolation, culture and identification of UC/PL-MSCs步骤1.1、UC/PL-MSCs的分离培养Step 1.1. Isolation and culture of UC/PL-MSCs准备人体脐带/胎盘，将脐带/胎盘组织剪成肉糜状，100U/mLⅠ型胶原酶、1.5μg/mL DNA酶Ⅰ的LG-DMEM溶液消化，用细胞筛网过滤，收集细胞接种，进行细胞培养，直至原代细胞融合度达到70％～80％时，消化传代，观察细胞生长状况和形态；Prepare human umbilical cord/placenta, cut the umbilical cord/placenta tissue into minced meat, digest with 100U/mL collagenase type I, 1.5 μg/mL DNase I in LG-DMEM solution, filter with a cell mesh, collect cells for seeding, and conduct cell culture , until the fusion degree of primary cells reaches 70% to 80%, digest and passage, and observe the growth status and morphology of cells;以下步骤1.2～1.4不分先后顺序；The following steps 1.2 to 1.4 are in no particular order;步骤1.2、检测UC/PL-MSCs的表面抗原表达Step 1.2. Detection of surface antigen expression of UC/PL-MSCs取生长至第4代UC/PL-MSCs，0.25％胰酶消化，重悬细胞，调节细胞浓度，加入兔抗小鼠CD29、CD44、CD105、CD45、CD34以及同型对照品，孵育，洗涤，离心弃上清，每管各加入缓冲液后，分别加入山羊抗兔荧光二抗，孵育，洗涤，离心弃上清，重悬细胞，检测UC/PL-MSCs表面抗原表达；Take UC/PL-MSCs grown to the fourth passage, digest with 0.25% trypsin, resuspend the cells, adjust the cell concentration, add rabbit anti-mouse CD29, CD44, CD105, CD45, CD34 and isotype controls, incubate, wash, and centrifuge Discard the supernatant, add buffer to each tube, add goat anti-rabbit fluorescent secondary antibody, incubate, wash, discard the supernatant by centrifugation, resuspend the cells, and detect the surface antigen expression of UC/PL-MSCs;步骤1.3、脐带/胎盘间充质干细胞诱导分化潜能鉴定Step 1.3. Identification of the differentiation potential of umbilical cord/placental mesenchymal stem cells取生长至第4代UC/PL-MSCs，接种在六孔板中，待细胞融合度达到60％～70％时，加入脐带/胎盘脐带间充质干细胞进行诱导培养基，加入茜素红染液染色，观察细胞染色效果；Take the UC/PL-MSCs grown to the fourth generation and inoculate them in a six-well plate. When the cell confluence reaches 60% to 70%, add umbilical cord/placental cord mesenchymal stem cells for induction medium, add alizarin red staining Liquid staining to observe the staining effect of cells;步骤1.4、成脂细胞诱导分化潜能鉴定Step 1.4. Identification of adipogenic differentiation potential取生长至第4代UC/PL-MSCs，接种在六孔板中，直至细胞融合度达到100％时，加入脐带/胎盘间充质干细胞成脂诱导分化培养基EGF液和bFGF液交替作用，油红O染液染色，观察细胞染色效果；Take the UC/PL-MSCs grown to the fourth generation and inoculate them in six-well plates until the cell confluence reaches 100%, then add the umbilical cord/placental mesenchymal stem cell adipogenic differentiation medium EGF solution and bFGF solution alternately. Oil red O staining solution was used to observe the staining effect of cells;步骤2、UC/PL-MSCs来源外泌体的制备Step 2. Preparation of UC/PL-MSCs-derived exosomes(a)收集第4代UC/PL-MSCs，重悬细胞，将UC/PL-MSCs悬液依次通过孔径为10μm、5μm、1μm的径迹蚀刻膜，随后将细胞悬液离心，过滤，重悬沉淀，即为外泌体；(a) Collect the fourth-generation UC/PL-MSCs, resuspend the cells, pass the UC/PL-MSCs suspension through track-etched membranes with pore sizes of 10 μm, 5 μm, and 1 μm in turn, then centrifuge the cell suspension, filter, and resuspend the cells. Suspended precipitates are exosomes;或者or(b)取生长至第4代UC/PL-MSCs，接种，待细胞融合度达到50％时，洗涤，加入无胎牛血清的LG-DMEM培养基，继续培养，收集细胞上清，离心，过滤，超速离心，得到的外泌体沉淀用PBS重悬，即为外泌体-。(b) Take UC/PL-MSCs grown to the fourth generation, inoculate, and when the cell confluence reaches 50%, wash, add LG-DMEM medium without fetal bovine serum, continue to culture, collect cell supernatant, centrifuge, Filtration, ultracentrifugation, and the obtained exosome pellet were resuspended in PBS, namely exosome-.2.根据权利要求1所述的一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，所述的步骤1.1中，采用100μm的细胞筛网过滤，以1000r/min离心5min，用含体积分数20％胎牛血清、100U/mL链青霉素的LG-DMEM培养基重悬细胞。2. The method for preparing umbilical cord/placental mesenchymal stem cell-derived exosomes according to claim 1, wherein in the step 1.1, a 100 μm cell mesh is used for filtration, and 1000 r/min is used for filtration. Centrifuge for 5 min, and resuspend the cells in LG-DMEM medium containing 20% fetal bovine serum and 100 U/mL streptomycin.3.根据权利要求1所述的一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，所述的步骤1.2中，用流式细胞缓冲液重悬细胞，所述的流式细胞缓冲液为含体积分数1％BSA的PBS。3. the preparation method of a kind of umbilical cord/placental mesenchymal stem cell-derived exosomes according to claim 1, is characterized in that, in described step 1.2, resuspend cells with flow cytometry buffer, described The flow cytometry buffer was PBS containing 1% BSA by volume.4.根据权利要求1所述的一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，所述的步骤2(a)中，细胞悬液以2000g的离心力离心10min，取上清，后以16500g离心力离心20min，留取上清，用0.22μm过滤器过滤上清后，以100000g离心力超速离心1h，弃去上清，用100μL PBS重悬沉淀。4. the preparation method of a kind of umbilical cord/placental mesenchymal stem cell-derived exosome according to claim 1, is characterized in that, in described step 2 (a), cell suspension is centrifuged 10min with the centrifugal force of 2000g , take the supernatant, and then centrifuge at 16500g for 20min, save the supernatant, filter the supernatant with a 0.22μm filter, ultracentrifuge at 100000g for 1h, discard the supernatant, and resuspend the pellet with 100μL PBS.5.根据权利要求1所述的一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，所述的步骤2(b)中，以1×10⁶个细胞的密度接种至T25细胞瓶中；4℃下以300g离心力离心10min，取上清4℃1000g离心力离心10min，随后取上清4℃下10000g离心力离心30min，用0.22μm滤器过滤上清后100000g离心力超速离心2h，得到的外泌体沉淀用100μL PBS重悬。5. The method for preparing umbilical cord/placental mesenchymal stem cell-derived exosomes according to claim 1, wherein in the step 2(b), a density of 1×10⁶ cells is used Inoculated into T25 cell flasks; centrifuged at 300g for 10min at 4°C, centrifuge the supernatant at 1000g at 4°C for 10min, then take the supernatant and centrifuge at 10,000g at 4°C for 30min, filter the supernatant with a 0.22 μm filter, and then centrifuge at 100,000g for ultracentrifugation 2h, the obtained exosome pellet was resuspended with 100 μL of PBS.6.根据权利要求1所述的一种脐带/胎盘间充质干细胞源性外泌体的制备方法，其特征在于，分离培养的UC/PL-MSCs高表达CD29、CD44、CD105，阳性率均高于99.0％，不表达造血干细胞表面抗原CD45和CD34。6. the preparation method of a kind of umbilical cord/placental mesenchymal stem cell-derived exosomes according to claim 1, it is characterized in that, the UC/PL-MSCs of isolation and culture highly express CD29, CD44, CD105, and the positive rate is all. Above 99.0%, the hematopoietic stem cell surface antigens CD45 and CD34 were not expressed.7.一种脐带/胎盘间充质干细胞源性外泌体的应用，其特征在于，所述的脐带/胎盘间充质干细胞源性外泌体由权利要求1-6任一项所述的脐带/胎盘间充质干细胞源性外泌体的制备方法制得，应用于抗衰老和组织修复方面。7. an application of umbilical cord/placental mesenchymal stem cell-derived exosome is characterized in that, described umbilical cord/placental mesenchymal stem cell-derived exosome is described in any one of claims 1-6 The preparation method of umbilical cord/placental mesenchymal stem cell-derived exosomes is used in anti-aging and tissue repair.

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