CN111494423B - Stem cell composition, application thereof and stem cell dropping liquid - Google Patents
Stem cell composition, application thereof and stem cell dropping liquid Download PDFInfo
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Abstract
The invention provides a stem cell composition, application thereof and a stem cell dropping liquid, belonging to the technical field of skin trauma repair, wherein the stem cell composition comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells; the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5. The stem cell composition provided by the invention can heal skin trauma, and the healing time is only 13 days.
Description
Technical Field
The invention belongs to the technical field of skin trauma repair, and particularly relates to a stem cell composition, application thereof and stem cell drops.
Background
Large-area skin defects caused by wounds, burns, chronic ulcers and the like often form wound surfaces which are difficult to heal, and risks such as necrosis, infection and the like are brought due to slow healing, so that the health and the life quality of patients are seriously affected. Wound repair is a complex and ordered biological process, including a hemostasis stage, an inflammation stage, a proliferation stage, a remodeling stage and other continuous processes, and relates to participation and mutual coordination of various cells, cytokines and growth factors, wherein factors such as hypoxia, necrosis, infection, advanced age and the like can delay wound healing or make wound healing difficult, so that the problem to be solved in the field of tissue repair is to achieve reconstruction and functional repair. In the prior art, human bone marrow mesenchymal stem cells and adipose mesenchymal stem cells are mostly focused to induce wound healing.
Disclosure of Invention
In view of the above, the present invention aims to provide a stem cell composition, an application thereof and a stem cell drop solution, wherein the stem cell composition provided by the present invention can heal skin trauma without obvious scar hyperplasia phenomenon during healing.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a stem cell composition, which comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells;
the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5.
Preferably, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 1: 1.
The invention also provides application of the stem cell composition in the technical scheme in preparation of a medicine for healing skin wounds.
Preferably, the stem cell composition is suspended in a phosphate buffer to obtain the drug.
Preferably, the number of the human umbilical cord mesenchymal stem cells in the medicament is 0.5-1.5 multiplied by 107One per ml.
Preferably, the number of the human umbilical vascular endothelial cells in the medicament is 0.5-1.5 multiplied by 107One per ml.
Preferably, the pH of the phosphate buffer is 7.4.
The invention also provides application of the stem cell composition in the technical scheme in preparation of a medicament for accelerating growth of an epidermal layer and/or a dermal layer of a new skin.
The invention also provides a stem cell dropping liquid, which comprises the stem cell composition and the phosphate buffer solution.
Preferably, the human umbilical cord in the stem cell dropThe number of the mesenchymal stem cells is 0.5-1.5 multiplied by 107Each per ml, the number of the human umbilical vascular endothelial cells is 0.5-1.5 multiplied by 107One per ml.
The invention provides a stem cell composition, which comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells; the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5.
The invention has the advantages that:
the stem cell composition provided by the invention can be used for healing skin trauma after 13 days, the growth of epidermal layers and dermal layers of newly-born skin is accelerated during the healing period, no obvious scar hyperplasia phenomenon exists at the healing later stage, no scar is left on the wound, and the growth speed of hair follicles is higher; the two stem cells are derived from the umbilical cord, so that the method has the advantages of abundant sources, noninvasive materials, low immunogenicity, avoidance of ethical disputes and the like, and meanwhile, the bones and the fat have material-drawing wounds, limited sources and use limitations (autografting).
Human umbilical cord mesenchymal stem cells (HUC-MSC, hereinafter abbreviated as MSC) have strong proliferation ability and multiple differentiation ability, and have the ability of regenerating tissues such as skin, cartilage and bone. The human umbilical cord mesenchymal stem cells maintain extracellular matrix (ECM) by producing cytokines and growth factors to regulate an immune system and promote wound repair and tissue regeneration cells, and the MSC and the secreted cytokines play a remarkable role in promoting the generation of dermis, endothelium and the like.
The human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells (HUVEC) have mutual promotion effect. HUVECs increased the proportion of MSC myogenic phenotype and co-culture of HUC-MSCs with HUVECs allowed angiogenesis within 24 h. However, single culture of HUVECs in co-culture conditioned medium failed to induce the formation of cell aggregate structures, indicating that HUVECs alone were not sufficient to support vascular stabilization and to provide nutrients for wound healing. Therefore, differentiation and paracrine actions of HUC-MSC by itself combined with angiogenesis promotion by HUVEC can accelerate wound healing more effectively.
Drawings
FIG. 1 shows the wound healing of mice in different groups at different times after surgery;
FIG. 2 is a histological observation of the wound surface of mice at days 3,6 and 13 after surgery (scale bar 100. mu.M) in different groups.
Detailed Description
The invention provides a stem cell composition, which comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells; the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5. In the present invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is preferably 1: 1. The source of the human umbilical cord mesenchymal stem cells and the human umbilical cord vascular endothelial cells is not specially limited, and the human umbilical cord mesenchymal stem cells and the human umbilical cord vascular endothelial cells are obtained by adopting conventional commercial products or a conventional method for obtaining the two stem cells.
The invention also provides application of the stem cell composition in the technical scheme in preparation of a medicine for healing skin wounds. The stem cell composition is preferably suspended in a phosphate buffer solution to obtain the medicament. In the invention, the number of the human umbilical cord mesenchymal stem cells in the medicament is preferably 0.5-1.5 multiplied by 107One/ml, more preferably 1X 107One per ml. In the invention, the number of the human umbilical vascular endothelial cells in the medicament is preferably 0.5-1.5 multiplied by 107One/ml, more preferably 1X 107One per ml. In the invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells in the medicament is preferably 0.5-1.5: 0.5-1.5, and more preferably 1 × 107One per ml. In the invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells in the medicament is preferably 0.5-1.5: 0.5-1.5, and more preferably 1: 1. In the present invention, the pH of the phosphate buffer is preferably 7.4.
The invention also provides application of the stem cell composition in the technical scheme in preparation of a medicament for accelerating growth of an epidermal layer and/or a dermal layer of a new skin. The stem cell composition is preferably suspended in a phosphate buffer solution to obtain the medicament. In the present invention, theThe number of the human umbilical cord mesenchymal stem cells in the medicament is preferably 0.5-1.5 multiplied by 107One/ml, more preferably 1X 107One per ml. In the invention, the number of the human umbilical vascular endothelial cells in the medicament is preferably 0.5-1.5 multiplied by 107One/ml, more preferably 1X 107One per ml. In the invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells in the medicament is preferably 0.5-1.5: 0.5-1.5, and more preferably 1: 1. In the invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells in the medicament is preferably 0.5-1.5: 0.5-1.5, and more preferably 1: 1. In the present invention, the pH of the phosphate buffer is preferably 7.4.
The invention also provides a stem cell dropping liquid, which comprises the stem cell composition and the phosphate buffer solution. In the invention, the number of the human umbilical cord mesenchymal stem cells in the stem cell dropping liquid is preferably 0.5-1.5 × 107One/ml, more preferably 1X 107Per ml; the number of the human umbilical cord vascular endothelial cells is preferably 0.5-1.5 multiplied by 107One/ml, more preferably 1X 107One per ml. In the invention, the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells in the stem cell dropping liquid is preferably 0.5-1.5: 0.5-1.5, and more preferably 1: 1. In the present invention, the pH of the phosphate buffer is preferably 7.4. The method for preparing the stem cell drop of the present invention is not particularly limited, and the stem cell composition may be suspended in a phosphate buffer. The application method of the stem cell dropping liquid is not particularly limited, and the application method of the dropping liquid for promoting the healing of the skin wound can be adopted.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of clinical-grade HUC-MSC preparation:
1.1 primary clinical-grade HUC-MSC isolation:
1.1.1 examination of the experiment environment and the operation of the instruments and equipment before the start of the experiment. Wiping and disinfecting the operation table top by 75% ethanol solution, and wiping and disinfecting instruments and supplies by high pressure or alcohol. Preparing the required articles for the experiment.
1.1.2 both ends of umbilical cord tissue were ligated, soaked in a physiological saline bottle containing double antibody (cat # 15140122, Gibco), and transported to the laboratory at 2-8 ℃. Opening the bottle in an ultraclean working counter, discarding the preservation solution in the bottle, adding physiological saline (only by immersing umbilical cord) into the bottle, and washing by shaking.
1.1.3 after discarding the saline, the umbilical cord was poured into a sterile 100mm petri dish and the surface of the umbilical cord was sprayed with a 75% ethanol solution. Cleaning the umbilical cord sprayed with alcohol for 2-3 times by using normal saline, adding the normal saline into a culture dish, dividing the umbilical cord into 3-4 cm sections by using sterile scissors, clamping the center of the umbilical cord by using forceps, pushing the umbilical cord towards two ends, and washing off blood.
1.1.4 the cleaned umbilical cord is placed in a sterile 100mm petri dish containing physiological saline, the umbilical vein is found, the umbilical cord is blunt stripped from the side of the vein, and the vein is removed. Umbilical cord mesenchyme (Fahrenheit gelatin) is separated, the separated umbilical cord mesenchyme is put into a culture medium in time, and the umbilical arteries (2) are removed.
1.1.5 heating a 50mL centrifuge tube, cutting half, placing the separated tissue mass, and cutting with sterile scissors. After being cut into pieces, the tissues are evenly paved in a T-75 cell culture bottle.
1.1.6 placing T-75 obtained in 1.1.5 into an incubator (37 ℃, 5% CO)2) After culturing for 4-6 h, 6mL of a culture medium containing 5% of UltraGRo-Advanced (cat #: HPCFDCGL50, Helios) in alpha-MEM medium (cat No.: c12571500BT, Gibco).
1.2 Primary clinical-grade HUC-MSC passage
And 1.2.1 observing the periphery of the tissue block by a microscope, replacing the culture medium every 5 days, and climbing out cells around the tissue block for about 8-10 days. When the cell fusion degree reaches 80%, the tissue block is discarded by gentle tapping, and 5mL of physiological saline is added to wash away residual culture medium in the bottle.
1.2.2 after discarding the physiological saline, 2mL of Tryple-Express (cat # 12604021, Gibco) was added, left to stand at room temperature or 37 ℃ for digestion for 2min until the cells were observed to be clear and round by an inverted microscope, and gently shaken and floated.
1.2.3 digestion of the cells added 6mL of physiological saline to stop digestion, after washing the residual cells on the lower wall of the flask, the liquid was transferred to a centrifuge tube and centrifuged at 1500rpm for 5 min. The supernatant was discarded, and 20mL of α -MEM medium containing 5% ultraGRo-Advanced was added to the pellet to prepare a cell suspension. Counting 20 μ L, inoculating to culture flask at 150 ten thousand/T-175, placing into incubator under 5% CO2And culturing at 37 ℃.
1.3 clinical-grade HUC-MSC passage
1.3.1 the state of the cells in the T-175 cell culture flask was observed with an inverted microscope, and the passaging operation was carried out when the degree of cell confluence reached 80%.
1.3.2 open the T-175 cell culture flask in the clean bench top where the cells are to be passaged and discard the medium in the flask. 10mL of physiological saline was added to wash out the residual medium in the flask. After the physiological saline is discarded, 4mL of Tryple-Express is added, and the mixture is kept stand and digested for 2min at room temperature or 37 ℃ until the cells are observed to be transparent and round by an inverted microscope and are gently shaken and floated.
1.3.3 digestion of the cells added to 12mL of saline to stop digestion, after washing the residual cells on the lower wall of the flask, the liquid was transferred to a centrifuge tube and centrifuged at 1500rpm for 5 min.
1.3.4 discard the supernatant, add 20mL of complete medium to the pellet and resuspend to make a cell suspension. After counting 20. mu.L, the cells were inoculated into culture flasks at a rate of 150 ten thousand/T-175. Placing into an incubator under 5% CO2And culturing at 37 ℃.
1.4 clinical HUC-MSC cryopreservation
1.4.1 the state of the cells in the T-175 cell culture flask was observed by an inverted microscope, and the freezing operation was carried out when the degree of cell fusion reached 80%. The T-175 cell culture flask in which the cells were to be passaged was opened in a clean bench top and the medium in the flask was discarded. 10mL of physiological saline was added to wash out the residual medium in the flask.
1.4.2 after physiological saline is discarded, 4mL of Tryple-Express is added, standing and digestion is carried out for 2min at room temperature or 37 ℃ until the cells are observed to be transparent and round by an inverted microscope, and the cells are gently shaken and floated. The digested cells are added with 12mL of normal saline to stop digestion, and after residual cells on the lower bottle wall are washed, the liquid is transferred to a centrifuge tube and centrifuged at 1500rpm for 5 min.
1.4.3 discard the supernatant, add 20mL of physiological saline to the pellet, and make a cell suspension. 20 μ L of the suspension was counted and centrifuged at 1500rpm for 5min to discard the supernatant. Adding frozen stock solution (Cat: 07930, STEM CELL TECHNOLOGIES) with freezing density of 300 ten thousand/mL, and packaging the CELL frozen stock solution into freezing tubes. Placing into a programmed cooling box, storing at-80 deg.C overnight, and transferring to liquid nitrogen for storage.
Note: all cell preparation is completed in GMP laboratory of east Hospital of Shanghai city, and the cell reaches clinical grade use standard, and is qualified after quality detection in GMP laboratory quality inspection center. The frozen system enters a biological sample warehouse of eastern hospitals in Shanghai city for storage through related warehousing procedures.
1.5 preparation of HUC-MSC suspension
1.5.1 taking out P3 generation cells from a sample bank for recovery, wherein the specific recovery steps are as follows: cells were thawed rapidly with an automated cell resuscitator, the cell cryopreserved mixture was diluted 1:9 with 5% UltraGRo-Advanced α -MEM preheated to 37 ℃, centrifuged 100g, the supernatant was discarded after 5min, the cells were resuspended in 5% UltraGRo-Advanced α -MEM and inoculated into T175 flasks (P4), and when 80% of the cells had confluent, passaged to 2T 175 flasks (P5) according to the above passage procedure for culture.
1.5.2 when the cell growth converged to 80%, adding Tryple-Express, standing and digesting at 37 ℃ for 3min until the cells are observed to be transparent and round by an inverted microscope, slightly shaking and floating, adding 5% UltraGRo-Advanced alpha-MEM culture medium to terminate digestion, centrifuging for 100g and 5min, discarding the supernatant, resuspending in PBS and counting (trypan blue staining method), taking 1 × 107Individual HUC-MSCs were resuspended in 0.5mL PBS (in 1.5mL EP tubes) on ice for use;
2. preparation of clinical-grade HUVEC formulation:
2.1 Primary clinical-grade HUVEC isolation
2.1.1 examination of the experiment environment and the operation of the instruments and equipment before the start of the experiment. Wiping and disinfecting the operation table top by 75% ethanol solution, and wiping and disinfecting instruments and supplies by high pressure or alcohol. Preparing the required articles for the experiment.
2.1.2 ligature both ends of umbilical cord tissue, soaking in physiological saline bottle containing double antibody, and transporting to laboratory at 2-8 deg.C. Opening the bottle in an ultraclean working counter, discarding the preservation solution in the bottle, adding physiological saline (only by immersing umbilical cord) into the bottle, and washing by shaking.
2.1.3 after discarding the saline, the umbilical cord was poured into a sterile 100mm petri dish and the surface of the umbilical cord was sprayed with a 75% ethanol solution. Cleaning the umbilical cord sprayed with alcohol for 2-3 times by using normal saline, adding the normal saline into a culture dish, dividing the umbilical cord into 3-4 cm sections by using sterile scissors, clamping the center of the umbilical cord by using forceps, pushing the umbilical cord towards two ends, and washing off blood.
2.1.4 put the cleaned umbilical cord into a sterile 100mm culture dish containing normal saline, find the umbilical vein, blunt-peel the umbilical cord from the side of the vein, and remove the vein and put into the culture dish. Umbilical cord mesenchyme (Fahrenheit), umbilical artery (2) was removed and placed in a petri dish.
2.1.5 vessels in the petri dish were flushed three more times with saline and 5ml syringe, respectively, to remove intravascular blood. One end of the vein or artery obtained in the 2.1.4 step was clamped with a vascular clamp, collagenase (cat # 17100017, Gibco) was injected into the vessel with a 5mL syringe until the vessel was significantly swollen, and the other end was clamped with a vascular clamp. Cover the petri dish and put 5% CO2And digesting for 15min in a cell culture box at 37 ℃.
2.1.6 digested vessels were washed 3 times with saline and 5mL syringe, the washed liquid was collected into a 50mL centrifuge tube and centrifuged at 1500rpm for 5 min. 10mL of complete ECM medium (cat. No.: 1001, Sciencell) was added to the pellet and resuspended to prepare a cell suspension. And planting one hole of a six-hole plate according to a blood vessel of 4-5 cm.
2.2 Primary clinical-grade HUVEC passage
2.2.1 microscope observation of six well plates, medium change every 3 days. When the cell fusion degree reaches 80%, 5mL of physiological saline is added to wash out residual culture medium in the bottle.
2.2.2 after discarding the physiological saline, adding 1ml of Cryplel-Express, standing and digesting for 2min at room temperature or 37 ℃ until the cells are observed to be transparent and round by an inverted microscope, and slightly shaking and floating.
2.2.3 the digested cells were stopped by adding 5mL of physiological saline, and after washing the residual cells on the bottom wall of the flask, the liquid was transferred to a centrifuge tube and centrifuged at 1500rpm for 5 min.
2.2.4 discard the supernatant and inoculate a six-well plate in a ratio of 1: 3. Placing into an incubator under 5% CO2And culturing at 37 ℃.
2.3 clinical HUVEC passage
2.3.1 the state of the cells in the six-well plate cell culture flask was observed by an inverted microscope, and the subculture operation was carried out when the degree of cell fusion reached 80%. Six-well plates of cells to be passaged were opened in a clean bench top and the medium in the flask was discarded. 10mL of physiological saline was added to wash out the residual medium in the flask. The remaining passaging steps can be performed as described above with reference to 1.2.
2.3.2 cryopreservation of umbilical vascular endothelial cells the procedure was carried out as described in 1.4 above.
2.4 preparation of HUVEC suspension
2.4.1 taking out P3 generation cells from the sample bank for recovery, wherein the specific recovery steps are as follows: rapidly thawing the cells by using an automatic cell resuscitator, diluting the frozen cell mixed solution and an ECM complete culture solution containing ECM preheated to 37 ℃ in advance at a ratio of 1:9, centrifuging for 100g, discarding supernatant after 5min, resuspending the cells by using the ECM complete culture solution, inoculating the cells into a T175 flask (P4 generation), and carrying out passage to 2T 175 flasks (P5 generation) for culture according to the passage steps when the cell growth is 80 percent converged.
2.4.2 when the cell growth converges to 80%, adding Tryple-Express, standing and digesting at 37 deg.C for 3min until the cell is observed to be transparent and round by an inverted microscope, gently shaking and floating, adding ECM complete culture medium to stop digestion, centrifuging for 100g and 5min, discarding supernatant, suspending in PBS and counting (trypan blue staining method), taking 1 × 107Individual HUVECs were resuspended in 0.5mL PBS (in 1.5mL EP tubes) and placed on ice until use.
Example 2
Preparation of stem cell preparation drops helpful for healing skin trauma:
1X 10 prepared in example 17HUC-MSC (0.5mL PBS) and 1X 107HUVEC (0.5mL PBS) were mixed well, dispensed into each tube 0.1mL and placed in icePreserving at the temperature of upper or 4 ℃ for standby, and obtaining stem cell product dropping liquid.
Example 3
First, experiment method
1. 1.4X 10 of the suspension were obtained as described in example 1(1.5 step preparation of HUC-MSC suspension)7Individual P5 HUC-MSCs were resuspended in 0.7mL PBS (in 1.5mL EP tubes) and placed on ice for use;
2. 7X 10 of the suspension were obtained as in example 1(2.4 preparation of a suspension of HUVEC)6Individual P5 HUVECs were resuspended in 0.7mL PBS (in 1.5mL EP tubes) on ice until ready for use;
3. the experiment was divided into 3 treatment groups: PBS, MSC and MSC + VEC groups. PBS group 0.1mL PBS per tube, MSC group 1X 10 per tube6MSC/0.1mL PBS, 1X 10 per tube for MSC + VEC group6A MSC and 1 x 106VECs/0.1 mL PBS, and each group for 7 repeat. The cell mixture prepared in steps 1 and 2 is divided into groups.
4. Animal experiments were performed in a clean bench of an animal house, and the operating table was wiped with a 75% ethanol solution for sterilization before the start of the experiment, and the ophthalmic scissors and forceps were autoclaved.
5. 27 male Balb/c mice at 4 weeks of age were randomized into 3 groups: PBS group, MSC group and MSC + VEC group. 5% chloral hydrate (0.15 mL/mouse) is injected into the abdominal cavity of each mouse for anesthesia, the back hair is removed, the hair removal paste is used for removing the clean impure hair, and a cotton swab is used for smearing 75% ethanol solution for wiping and disinfection.
6. Using eye scissors and tweezers sterilized at high temperature and high pressure to cut out a whole skin injury wound surface with the diameter of 1.0cm, deep fascia and stop bleeding by using sterile gauze on the back of a mouse.
7. After the molding is successful, a ruler is added for photographing and recording, 3 prepared groups of dropping liquid are taken out from ice, the mixed liquid containing the cell preparation is gently mixed by an insulin injection needle, and 0.1mL of uniform dropping liquid is sucked in groups and waits for 3min at the wound (in order to enable the wound to better absorb the cell mixed liquid).
8. Wrapping with 3M transparent plaster, and breeding in single cage. Wound healing photographing records are respectively carried out 3 days, 6 days and 13 days after operation. Each group was randomly sacrificed 3 mice at days 3,6 and 13, respectively, and neonatal skin tissue from the back was fixed with 4% paraformaldehyde and HE stained.
9. Wound area was counted using Image J software, Graphpad prism software was used for data statistics, the statistical data were expressed as mean + -SE, one-way ANOVA was used for inter-group analysis, and differences of P <0.5 were considered statistically significant.
Second, experimental results
1. Comparison of the effects of different groups on the wound healing Rate
The wounds on days 1, 3,6 and 13 were recorded by photography in the experiment to visually show the difference in wound healing between the different groups. As can be seen from figure 1, the MSC + VEC group healed most rapidly with increasing days, and at day 13, the MSC + VEC group healed substantially completely compared to the other two groups of wounds, and no scar hyperplasia was seen.
The wound area was counted by using Image J software, and the wound area was counted on days 1, 3,6 and 13 as in table 1. On day 3 post-surgery, the wound area was significantly reduced in the MSC + VEC group (p <0.05vs. pbs or MSC group); from this data, the MSC + VEC treated group recovered faster, with the wound area decreasing to 58.31% of the PBS group on post-operative day 3, while the MSC group wound area was 83.22% of the PBS group. On day 6 post-surgery, the wound area was decreasing in the MSC group (p <0.001vs. pbs group), but the wound area was still lowest in the MSC + VEC group (p <0.05vs. MSC group). On day 13 post-surgery, the wound area of the MSC + VEC group was 11.56% of that of the PBS group, and the wound area of the MSC group was 35.81% of that of the PBS group.
TABLE 1 wound area (unit: cm) at surgery 1, 3,6 and 13d for different groups2)
| | 3d | 6d | 13d | |||
| PBS | 0.9883±0.05598 | 0.9822±0.06116 | 0.8855±0.07250 | 0.3085±0.02950 | ||
| MCS | 0.9452±0.04816 | 0.8174±0.02060* | 0.4523±0.01018*** | 0.1105±0.0075*** | ||
| MCS+VEC | 0.9694±0.1039 | 0.5727±0.07812***# | 0.3320±0.04098***# | 0.03567±0.001764***# |
Note: data in table are the wound area of the day, p <0.01vs. pbs group; MSC group # p <0.5, # # p <0.001vs
2. Histological comparison of wound-surface neogenesis skin of different groups
HE staining results (fig. 2) show: on day 3 post-surgery, the neogenetic epidermis was thicker in the MSC + VEC group, more microvascular formation was visible in the dermis, microvessels were also present in the MSC group, whereas PBS epidermis was thinner, microvessels were fewer and skin level was unclear. On day 6 after surgery, the microvasculature of the dermis layer of the MSC + VEC group was further increased, the dermis layer was thickened, the skin level was more obvious, the epidermis and the dermis layer of the MSC group were thinner than those of the MSC + VEC group, and the growth of the PBS neogenetic skin was still slow. On day 13 after surgery, compared with the MSC group, the MSC + VEC group had clear skin levels, thicker epidermis and dermis layers, and more hair follicle hyperplasia, while the PBS group gradually appeared microvessels, but the neogenetic skin level was not evident.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. Use of a stem cell composition in the preparation of a medicament for promoting healing of a skin wound; the stem cell composition comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells;
the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5.
2. Use of a stem cell composition for the preparation of a medicament for accelerating the growth of epidermal and dermal layers of neonatal skin; the stem cell composition comprises human umbilical cord mesenchymal stem cells and human umbilical cord vascular endothelial cells;
the number ratio of the human umbilical cord mesenchymal stem cells to the human umbilical cord vascular endothelial cells is 0.5-1.5: 0.5-1.5.
3. The use according to claim 1 or 2, wherein the ratio of the number of human umbilical cord mesenchymal stem cells to the number of human umbilical cord vascular endothelial cells is 1: 1.
4. The use according to claim 1 or 2, wherein the stem cell composition is suspended in a phosphate buffer to obtain the medicament.
5. The use of claim 4, wherein the amount of human umbilical cord mesenchymal stem cells in the medicament is 0.5-1.5 x 107One per ml.
6. The use of claim 4, wherein the amount of human umbilical cord vascular endothelial cells in the medicament is 0.5 to 1.5 x 107One per ml.
7. Use according to claim 4, wherein the phosphate buffer has a pH of 7.4.
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