阅读说明：本技术 一种治疗糖尿病皮肤溃疡的间充质干细胞制剂及其制备方法 (Mesenchymal stem cell preparation for treating diabetic skin ulcer and preparation method thereof ) 是由 黄军就 焦义仁 于 2021-10-11 设计创作，主要内容包括：本发明提供了一种治疗糖尿病皮肤溃疡的间充质干细胞制剂及其制备方法。该间充质干细胞制剂包含水胶体凝胶和间充质干细胞；所述水胶体凝胶包括羧甲基纤维素钠和果胶,所述羧甲基纤维素钠和果胶的摩尔比为80～100：1。本发明的间充质干细胞制剂采用特定的水胶体凝胶搭载间充质干细胞,有效延长了间充质干细胞在伤口部位的驻留时间,促进间充质干细胞的植入,且显著提高了糖尿病皮肤溃疡的伤口愈合率,有效促进皮肤组织再生、促进新血管生成和调节巨噬细胞极化,表明本发明的间充质干细胞制剂适用于糖尿病皮肤溃疡的预防和/或治疗。(The invention provides a mesenchymal stem cell preparation for treating diabetic skin ulcer and a preparation method thereof. The mesenchymal stem cell preparation comprises hydrocolloid gel and mesenchymal stem cells; the hydrocolloid gel comprises sodium carboxymethyl cellulose and pectin, wherein the molar ratio of the sodium carboxymethyl cellulose to the pectin is 80-100: 1. the mesenchymal stem cell preparation disclosed by the invention adopts the specific hydrocolloid gel to carry the mesenchymal stem cells, so that the residence time of the mesenchymal stem cells in a wound part is effectively prolonged, the implantation of the mesenchymal stem cells is promoted, the wound healing rate of the diabetic skin ulcer is obviously improved, the regeneration of skin tissues is effectively promoted, the generation of new blood vessels is promoted, and the polarization of macrophages is regulated.)

1. A mesenchymal stem cell preparation, comprising a hydrocolloid gel and mesenchymal stem cells; the hydrocolloid gel comprises sodium carboxymethyl cellulose and pectin, wherein the molar ratio of the sodium carboxymethyl cellulose to the pectin is 80-100: 1.

2. the mesenchymal stem cell preparation according to claim 1, wherein the molar ratio of the sodium carboxymethylcellulose to the pectin is 85-95: 1.

3. the mesenchymal stem cell preparation of claim 2, wherein the molar ratio of sodium carboxymethylcellulose to pectin is 90: 1.

4. the mesenchymal stem cell preparation of any one of claims 1 to 3, wherein the mesenchymal stem cells comprise umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells or menstrual blood mesenchymal stem cells.

5. The mesenchymal stem cell preparation according to any one of claims 1 to 3, which is prepared by a method comprising: the hydrocolloid gel is diluted with phosphate buffered saline, normal saline or complete medium, and then loaded with mesenchymal stem cells.

6. The mesenchymal stem cell preparation according to claim 5, wherein the dilution is to dilute the hydrocolloid gel to 5-10 times the volume.

7. The mesenchymal stem cell preparation of claim 5, wherein the mesenchymal stem cells are present at a concentration of 5 x 10⁶～2×10⁷one/mL.

8. Use of the mesenchymal stem cell preparation of any claim 1 to 7 in the preparation of a product for preventing and/or treating diabetic skin ulcer.

9. The use of claim 8, wherein the prevention and/or treatment of diabetic skin ulcers comprises one or more of the promotion of wound healing, the promotion of skin tissue regeneration, the promotion of neovascularization, or the modulation of macrophage polarization.

10. The use according to claim 8 or 9, wherein the prevention and/or treatment of diabetic skin ulcers comprises one or more of increased dermal layer thickness, increased number of neogenetic hair follicles, increased collagen fiber deposition, decreased scar width, decreased number of pro-inflammatory macrophages, increased number of anti-inflammatory macrophages, increased proportion of total cellular proliferation or increased number of neovascular vessels.

Technical Field

The invention belongs to the technical field of medicines. More particularly, relates to a mesenchymal stem cell preparation for treating diabetic skin ulcer and a preparation method thereof.

Background

Diabetic skin ulcers (DCUs) refer to lower limb infections, ulcer formation and deep tissue destruction caused by neuropathy and various peripheral vascular diseases of varying degrees due to long-term poor blood glucose control in Diabetic patients. Diabetic skin ulcers are ulcers and gangrene that occur in diabetic patients due to microcirculatory disturbance caused by neurovascular lesions, as a result of the combined action of neuropathy, vasculopathy and topical infection. The body of a diabetic is in a high-sugar environment for a long time, so that the endogenous nitric oxide is insufficient, and the function of white blood cells is deficient in the high-sugar environment, so that the immune response is insufficient. Meanwhile, lack of new blood vessels, intractable bacterial infection of wound surfaces, and new stimulation or poor differentiation of skin dermal cells are all reasons for the difficulty in healing DCU. Diabetic skin ulcers frequently and repeatedly attack, are easy to form stubborn and difficult-to-heal ulcers, are easy to develop deep after improper treatment, even cause disability and amputation, and seriously threaten the health and the life quality of patients. The traditional methods of drug therapy, vascular bypass surgery, percutaneous intravascular angioplasty, stent implantation and the like have certain limitations in the treatment of diabetic skin ulcer, which are reflected in poor effect, strong side effect, patient discomfort, induction of chronic inflammation and the like, so that the clinical application of the treatment methods is limited. Currently, there is still no clinically effective means for treating DCU.

Mesenchymal Stem Cells (MSCs) are derived from adult stem cells of early developmental mesodermal stromal tissue or ectodermal neuroepithelium and neural crest. MSCs are a heterogeneous population of cells with multi-lineage differentiation potential. In vivo, mesenchymal stem cells can differentiate into other cells to participate in tissue injury repair, and can promote tissue regeneration through paracrine action. Patent CN104248644A provides a composite preparation containing temperature-sensitive hydrogel and bone marrow mesenchymal stem cells, which can promote the repair of diabetic skin wounds, but the effect is not very ideal, and the average unhealed area of the wounds after use is still as high as about 25%.

Disclosure of Invention

Aiming at the defects of the existing method for treating the diabetic skin ulcer, the invention aims to provide the mesenchymal stem cell preparation for treating the diabetic skin ulcer and the preparation method thereof, and provides a new medicinal preparation for preventing and/or treating the diabetic skin ulcer.

The invention aims at providing a mesenchymal stem cell preparation.

The invention also aims to provide application of the mesenchymal stem cell preparation in preparing a product for preventing and/or treating diabetic skin ulcer.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a mesenchymal stem cell preparation, which comprises a hydrocolloid gel and mesenchymal stem cells; the hydrocolloid gel comprises sodium carboxymethyl cellulose and pectin, wherein the molar ratio of the sodium carboxymethyl cellulose to the pectin is 80-100: 1.

preferably, the molar ratio of the sodium carboxymethyl cellulose to the pectin is 85-95: 1.

most preferably, the molar ratio of sodium carboxymethylcellulose to pectin is 90: see example 1.

Because the reduction of cell activity in the transplantation process of the mesenchymal stem cells causes that the treatment effect of the mesenchymal stem cells on the diabetic skin ulcer is gradually weakened along with the passage of time, the treatment effect is worsened, and in addition, the treatment efficiency of the mesenchymal stem cells on the diabetic skin ulcer is also seriously influenced by factors such as poor survival capability of the mesenchymal stem cells, short residence time at a wound, low implantation rate of the cells, easy loss and the like, therefore, the specific hydrocolloid Gel (Hydroactive Gel) is used for carrying the mesenchymal stem cells, the residence time of the mesenchymal stem cells at the wound can be effectively prolonged, the activity of the cells is kept for a long time, the adverse effect of cell loss and low implantation rate on treatment is prevented, and the hydrocolloid Gel adopts natural materials which have extremely low toxicity, cannot cause cell death, and cannot bring additional influence to the wound.

Preferably, the mesenchymal stem cell comprises an umbilical cord mesenchymal stem cell, an adipose mesenchymal stem cell, a bone marrow mesenchymal stem cell, a placental mesenchymal stem cell, a dental pulp mesenchymal stem cell or a menstrual blood mesenchymal stem cell.

Most preferably, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell, see example 1.

Umbilical cord Wharton's jelly mesenchymal stem cells, abbreviated as Umbilical cord mesenchymal stem cells (WJMSCs), are mesenchymal stem cells obtained by separating from the viscous connective tissue of the Umbilical cord. Compared with mesenchymal stem cells from other sources, WJMSCs have the following characteristics suitable for clinical application: 1) the WJMSCs have wide sources, non-invasive collection and no risk, and no ethical dispute exists; 2) WJMSCs have stronger in-vitro amplification capacity, compared with mesenchymal stem cells, WJMSCs have higher cell colony forming frequency and more population doubling times, and the number of MSCs obtained by one umbilical cord is approximately equal to that of MSCs obtained by 5000mL bone marrow of an adult; 3) the WJMSCs have stronger pluripotency and self-renewal capacity, are also maintained at an earlier embryonic stage due to being derived from fetal tissues, and retain partial early embryonic stem cell markers and endoderm markers; 4) WJMSCs have strong immunoregulatory ability, and high-level expression of tolerance molecules such as IL-10, TGF-beta and HLA-G, immunoregulatory proteins such as CD200 and CD273 and cytokines such as IL-1 beta and IL-8 by the WJMSCs, so that the WJMSCs have lower immunogenicity and higher immunosuppressive activity.

Preferably, the preparation method comprises the following steps: the hydrocolloid gel is diluted with phosphate buffered saline, normal saline or complete medium, and then loaded with mesenchymal stem cells.

According to specific needs, pharmaceutically or physiologically acceptable substances can be added to adjust the temperature of the hydrogel (hydrogel) to the clinical temperature, and the substances can be selected from macromolecular substances such as polyethylene glycol (PEG) with different molecular weights or micromolecular substances such as vitamin C.

Further preferably, the dilution is to dilute the hydrocolloid gel to 5-10 times by volume.

Most preferably, the dilution is to dilute the hydrocolloid gel to 5 volumes, see example 1.

Preferably, the concentration of the mesenchymal stem cells is 5 × 10⁶～2×10⁷one/mL.

Most preferably, the concentration is 2 × 10⁷Per mL, see example 3.

The invention constructs a clinically common diabetic skin ulcer model, namely a skin wound model of a type II diabetic rat, transplants a plurality of groups of medicines into the wound, and the result shows that the mesenchymal stem cells of a mesenchymal stem cell preparation group have the longest residence time on the wound, good implantation effect, the best overall wound healing promotion effect, the largest number of newly-born hair follicles, the largest thickness of a dermis layer, the smallest width of a scar, the largest deposition of collagen in skin tissue, the smallest number of proinflammatory M1 macrophages (CD86 positive) in the dermis layer, the largest number of anti-inflammatory M2 macrophages (CD163 positive), the best proliferation of Ki-67 positive cells and the best regeneration effect of CD31 positive blood vessels, thereby showing that the mesenchymal stem cell preparation can obviously improve the wound healing rate of the diabetic skin ulcer, obviously promote the regeneration of the skin tissue and the generation of new blood vessels, and effectively regulate the polarization of the macrophages, is suitable for preventing and/or treating diabetic skin ulcer. Therefore, the application of the mesenchymal stem cell preparation in the preparation of products for preventing and/or treating diabetic skin ulcer is within the protection scope of the present invention.

Preferably, the preventing and/or treating of the diabetic skin ulcer comprises one or more of promoting wound healing, promoting skin tissue regeneration, promoting neovascularization, or modulating macrophage polarization.

Preferably, the prevention and/or treatment of diabetic skin ulcers comprises one or more of increasing dermal layer thickness, increasing number of neogenetic hair follicles, increasing collagen fiber deposition, decreasing scar width, decreasing number of pro-inflammatory macrophages, increasing number of anti-inflammatory macrophages, increasing proportion of total cell proliferation, or increasing number of neovascular vessels.

The invention has the following beneficial effects:

the mesenchymal stem cell preparation disclosed by the invention adopts the specific hydrocolloid gel to carry the mesenchymal stem cells, so that the residence time of the mesenchymal stem cells in a wound part is effectively prolonged, the implantation of the mesenchymal stem cells is promoted, the wound healing rate of the diabetic skin ulcer is obviously improved, the regeneration of skin tissues is effectively promoted, the generation of new blood vessels is promoted, and the polarization of macrophages is regulated.

Drawings

FIG. 1A shows the results of a cell survival assay; FIG. 1B shows the results of cell viability assays.

FIG. 2A is a morphological diagram of a Hydroactive Gel; FIG. 2B is a scanning electron micrograph.

FIG. 3A is a cell morphology map; FIG. 3B shows the survival assay results of WJMSCs; FIG. 3C is survival rate of WJMSCs; FIG. 3D shows the results of activity assays for WJMSCs; FIG. 3E shows the results of the proliferation potency assay of WJMSCs; FIG. 3F is a statistical result of the ratio of proliferating cells.

FIG. 4A shows the results of detecting apoptosis in WJMSCs; FIG. 4B shows the results of detecting the expression of apoptosis-and anti-apoptosis-related genes in WJMSCs; FIG. 4C shows the mitochondrial distribution and damage profile of WJMSCs.

FIG. 5A is a schematic diagram of the construction of a skin wound model and cell therapy for type II diabetic rats; FIG. 5B is the result of a wound healing situation; FIG. 5C is a statistical result of wound healing rate on day 7; figure 5D is a statistical result of wound healing rate on day 14.

FIG. 6A is the H & E staining results; FIG. 6B is a statistical result of the number of new hair follicles; fig. 6C is a statistical result of the dermis layer thickness; fig. 6D is a statistical result of scar width.

FIG. 7 shows Masson trichrome staining results.

FIG. 8A is a test of CD86 positive M1 macrophages conversion; FIG. 8B shows the results of the detection of the transformation of CD 163-positive M2 macrophages; FIG. 8C shows the results of measuring the proliferation of Ki-67-positive cells; FIG. 8D shows the detection of the new formation of CD 31-positive blood vessels.

FIG. 9A shows the results of measuring the distribution of EGFP-positive WJMSCs in skin wounds 24h after transplantation; FIG. 9B is the result of examining the distribution of EGFP-positive WJMSCs on skin wounds after 72h transplantation; FIG. 9C is a quantitative statistic of the number of EGFP-positive cells in FIG. 9A; fig. 9D is a quantitative statistic of the EGFP-positive cell number in fig. 9B.

Wherein, Hydroactive Gel represents hydrocolloid Gel, WJMSCs represents umbilical cord mesenchymal stem cells, and Hydroactive Gel + WJMSCs represents mesenchymal stem cell preparation obtained by loading umbilical cord mesenchymal stem cells on the hydrocolloid Gel.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation of mesenchymal Stem cell preparation

Mixing sodium carboxymethylcellulose and pectin according to a ratio of 90: 1 to obtain 1mL of hydrocolloid Gel (Hydroactive Gel), diluting the Hydroactive Gel to 5 times and 10 times of volume respectively by using a complete culture medium of WJMSCs, and carrying umbilical cord mesenchymal stem cells (the concentration is 5 multiplied by 10)⁶～2×10⁷one/mL) to obtain the mesenchymal stem cell preparation. Was used in the following experiments.

Example 2 Effect of Hydroactive Gel on WJMSCs

(1) Influence of Hydroactive Gel diluted in different proportions on WJMSCs

By Live/Dead^TMCell Imaging Kit detects the survival condition of WJMSCs in the mesenchymal stem Cell preparation (figure 1A), and the result shows that the survival condition of WJMSCs has no significant change under the condition of 5-fold and 10-fold dilution ratio of Hydroactive Gel; CCK-8 is used for detecting the cell activity of the WJMSCs in the single WJMSCs and the mesenchymal stem cell preparation respectively (figure 1B), and the result shows that the cell activity of the WJMSCs is not changed obviously under the dilution ratio of 5 times and 10 times of Hydroactive Gel. The description shows that the Hydroactive Gel is diluted to 5-10 times of the volume, has no obvious influence on the survival condition and the cell activity of the WJMSCs when the WJMSCs are carried, has no toxicity on the WJMSCs, and is suitable for carrying the WJMSCs. In addition, compared with the 10-fold volume of diluted Hydroactive Gel, the dilution multiple of the Hydroactive Gel is 5, and the preparation method of the mesenchymal stem cell is obtained by loading the mesenchymal stem cell with the Hydroactive GelThe agent has higher viscosity, and is more beneficial to prolonging the residence time of the WJMSCs at the wound site.

The Hydroactive Gel used in the following experiments was 5-fold volume diluted Hydroactive Gel prepared by the method of example 1, and the mesenchymal stem cell preparations used were 5-fold volume diluted Hydroactive Gel prepared by the method of example 1 and loaded with WJMSCs.

(2) Wrapping condition of Hydroactive Gel on WJMSCs

The morphology of the Hydroactive Gel (FIG. 2A) was observed under a microscope at different temperatures (4 ℃ and 37 ℃), and the results show that the Hydroactive Gel at different temperatures has little change, is in a Gel state, and has certain viscosity. And respectively scanning the Hydroactive Gel and the mesenchymal stem cell preparation by using a scanning electron microscope to obtain an electron microscope scanning image shown in figure 2B, wherein the WJMSCs can be completely wrapped by the Hydroactive Gel and effectively attached to pores of the Hydroactive Gel.

(3) Effect of Hydroactive Gel on survival rate, cell activity and proliferation capacity of WJMSCs

The WJMSCs and the mesenchymal stem cell preparation are respectively cultured for 24h, the cell morphology graph in the figure 3A is obtained by microscope bright field shooting, and it can be seen that the two groups of cells have no significant difference in morphology, are fibrous and grow adherently, which indicates that the WJMSCs are carried by using Hydroactive Gel, and have no significant influence on the cell morphology of the WJMSCs.

By Live/Dead^TMCell Imaging Kit detects the survival conditions of individual WJMSCs and WJMSCs in mesenchymal stem Cell preparations (figure 3B), and counts the survival conditions to obtain the survival rate of figure 3C, which shows that the survival rate of WJMSCs is not significantly influenced by carrying the WJMSCs by using Hydroactive Gel.

CCK-8 was used to detect the cell activity of WJMSCs alone and in mesenchymal stem cell preparations (fig. 3D), and the results showed that the cell activity of WJMSCs in mesenchymal stem cell preparations was slightly lower than that of WJMSCs alone 24h after cell inoculation; at 48h post-inoculation, there was no significant difference in the cellular activities of WJMSCs in the mesenchymal stem cell preparation compared to WJMSCs alone.

The proliferation capacities of the individual WJMSCs and the WJMSCs in the mesenchymal stem cell preparation are detected by using an EdU kit (figure 3E), the number of the EdU positive cells in the two groups is counted, and a proportion statistical result of the proliferation cells in the figure 3F is calculated, wherein the result shows that the proliferation capacities of the two groups have no obvious difference, which indicates that the WJMSCs are carried by using Hydroactive Gel, and the proliferation capacities of the WJMSCs are not obviously influenced.

(4) Effect of Hydroactive Gel on apoptosis and mitochondria of WJMSCs

TUENL immunofluorescence is used for detecting the apoptosis conditions of the WJMSCs in the single WJMSCs and the mesenchymal stem cell preparation (figure 4A), and the results show that the apoptosis conditions of the two cells tend to be consistent, which shows that the apoptosis of the WJMSCs is not obviously influenced by carrying the WJMSCs by using Hydroactive Gel.

qRT-PCR is used for detecting the apoptosis of the WJMSCs and the expression of anti-apoptosis related genes (BAX, BCL2 and AVEN) of the WJMSCs in the mesenchymal stem cell preparation (figure 4B), and the result shows that the expression of the apoptosis related genes and the anti-apoptosis related genes of the two groups of cells has no obvious difference, which indicates that the WJMSCs are carried by Hydroactive Gel, and the WJMSCs have no obvious influence on the apoptosis related genes and the anti-apoptosis related genes.

MitoTracker immunofluorescence is used for detecting the mitochondrial distribution and damage conditions of WJMSCs in single WJMSCs and mesenchymal stem cell preparations (figure 4C), and the results show that the mitochondrial forms and the distribution of two groups of cells have no significant difference, which shows that the loading of the WJMSCs by using Hydroactive Gel has no significant influence on the mitochondrial distribution of the WJMSCs and does not damage the mitochondria of the WJMSCs.

In conclusion, the Hydroactive Gel can effectively wrap the mesenchymal stem cells, has no toxicity to the mesenchymal stem cells, does not influence the morphology, survival rate, cell activity, proliferation capacity, apoptosis and anti-apoptosis of the mesenchymal stem cells, and the mitochondrial morphology and distribution, and is suitable for carrying the mesenchymal stem cells to prepare the mesenchymal stem cell preparation.

Example 3 therapeutic Effect of mesenchymal Stem cell preparations on diabetic skin ulcers

(1) Effect of mesenchymal Stem cell preparation on healing of skin wound

SD rats were bred on a high-sugar, high-fat diet for 4 weeks, and type II diabetic rats were constructed by intraperitoneal injection of Streptozotocin (STZ)Model, construction of skin wound model and schematic cell therapy of type II diabetic rats are shown in FIG. 5A, PBS, Hydroactive Gel, WJMSCs (number 2 × 10) are added at week 6⁶Individual), mesenchymal stem cell preparation (number of WJMSCs 2X 10)⁶2 x 10 of the total⁷one/mL) and WJMSCs (number 1X 10)⁷Individual) were transplanted to the skin wounds of diabetic rats, and then wound healing was examined on 5 th, 7 th, 10 th and 14 th days after transplantation, respectively, and the overall results are shown in fig. 5B, in which the wound healing rate on 7 th day is shown in fig. 5C and the wound healing rate on 14 th day is shown in fig. 5D.

As can be seen from fig. 5B, the wounds in each group improved to a different extent over time, but none of the wounds improved as compared to the mesenchymal stem cell preparation group; FIG. 5C shows that 7 days after drug transplantation in each group, the non-healed wound area was 71.28% + -7.24 in PBS group, 64.82% + -5.71 in Hydroactive Gel group, WJMSCs (2X 10)⁶Individual) group of 67.42% + -1.48, mesenchymal stem cell preparation group of 43.84% + -2.11, WJMSCs (1 × 10)⁷Individual) group of the mesenchymal stem cell preparations had an unhealed wound area of 58.24% ± 2.60, and the wound healing effect of the mesenchymal stem cell preparation group was found to be the best and significantly better than that of the other groups; FIG. 5D shows that the unhealed wound area of PBS group was 48.57% + -5.07, that of Hydroactive Gel group was 45.16% + -4.43, and that of WJMSCs (2X 10) 14 days after drug transplantation in each group⁶Individual) group of 40.72% + -1.34, mesenchymal stem cell preparation group of 7.65% + -1.50, WJMSCs (1X 10)⁷Individual) group of the mesenchymal stem cell preparations had an unhealed wound area of 37.98% ± 2.68, and it was found that the mesenchymal stem cell preparation group had the best wound healing promoting effect, which was significantly superior to the other groups. Fig. 5B-5D all demonstrate that mesenchymal stem cell preparations can significantly improve the wound healing rate of diabetic skin ulcers.

(2) Effect of mesenchymal Stem cell preparation on wound skin tissue regeneration

Skin tissue samples of wounds 14 days after drug transplantation of each group of diabetic rats of example 3(1) were collected, fixed, dehydrated, embedded and sectioned, and then subjected to H & E staining (FIG. 6A) and Masson trichrome staining (FIG. 7), respectively, to examine the skin tissue regeneration of different groups of diabetic rats.

Fig. 6A is analyzed to obtain the statistical result of the number of the new hair follicles in each group of skin tissues (fig. 6B), the statistical result of the thickness of the dermis layer in each group of skin tissues (fig. 6C), and the statistical result of the width of the scar of each group of skin tissues (fig. 6D), and it can be seen from fig. 6B to 6D that the number of the new hair follicles in the mesenchymal stem cell preparation group is the largest, the thickness of the dermis layer is the thickest, the width of the scar is the smallest, and there is a significant difference from each other group. As can be seen from fig. 7, the mesenchymal stem cell preparation group had the darkest color, i.e., the skin tissue deposited the most collagen.

Therefore, the mesenchymal stem cell preparation can obviously increase the thickness of the dermis layer, increase the number of the new hair follicles, reduce the width of scars and increase the deposition amount of collagen fibers, so the mesenchymal stem cell preparation can obviously promote the regeneration of skin tissues.

(3) Effect of mesenchymal stem cell preparation on macrophage transformation and angiogenesis at wound

In the same manner as in example 3(1), the method for constructing the type ii diabetic rat skin wound model includes transplanting PBS, Hydroactive Gel, WJMSCs, and mesenchymal stem cell preparation to the diabetic rat skin wound, and performing immunohistochemical detection on each group of skin tissue samples to obtain the transformation status of CD 86-positive M1 macrophages in each group of skin tissue of fig. 8A, the transformation status of CD 163-positive M2 macrophages in each group of skin tissue of fig. 8B, the proliferation status of Ki-67-positive cells in each group of skin tissue of fig. 8C, and the new formation status of CD 31-positive blood vessels in each group of skin tissue of fig. 8D.

As can be seen from fig. 8A, the pro-inflammatory M1 macrophages (CD86 positive) were the least abundant in the dermal layer of the mesenchymal stem cell preparation group; as can be seen from fig. 8B, the anti-inflammatory M2 macrophages (CD163 positive) were the most abundant in the dermal layer of the mesenchymal stem cell preparation group; as can be seen from fig. 8C, the number of Ki-67 positive cells was the greatest in the mesenchymal stem cell preparation group; as can be seen from fig. 8D, the number of CD 31-positive angiogenesis was the greatest in the mesenchymal stem cell preparation group. Therefore, the mesenchymal stem cell preparation can effectively reduce the number of proinflammatory macrophages, increase the number of anti-inflammatory macrophages, increase the total cell proliferation proportion and increase the number of new blood vessels.

In conclusion, the mesenchymal stem cell preparation can obviously improve the wound healing rate of the diabetic skin ulcer, can obviously promote the regeneration of skin tissues and the generation of new blood vessels, effectively regulates the polarization of macrophages, and is suitable for preventing and/or treating the diabetic skin ulcer.

Example 4 Implantation and retention of mesenchymal Stem cells in diabetic skin ulcer wounds

The same as example 3(1) in the method for constructing the skin wound model of the type ii diabetic rat, the WJMSCs overexpressing EGFP and the mesenchymal stem cell preparation are respectively transplanted to the skin wound of the diabetic rat, after 24h and 72h of transplantation, each group of skin tissue samples are fixed, dehydrated and sliced, and then respectively subjected to tissue immunofluorescence detection, so as to respectively obtain the distribution conditions of the EGFP-positive WJMSCs at the skin wound of fig. 9A and 9B, and the quantity of the EGFP-positive cells in fig. 9A is quantitatively counted to obtain fig. 9C, and the quantity of the EGFP-positive cells in fig. 9B is quantitatively counted to obtain fig. 9D.

As can be seen from fig. 9A to 9D, after the drugs were transplanted for 24h and 72h, the number of EGFP positive cells in the mesenchymal stem cell preparation group was the largest and was significantly higher than that in the WJMSCs group, which indicates that the use of Hydroactive Gel to carry the WJMSCs effectively prolongs the residence time of the WJMSCs on the wound, so that the WJMSCs are not easily lost at the diabetic skin ulcer wound, and further promotes the implantation of the WJMSCs, which is beneficial for the treatment of diabetic skin ulcer.

In conclusion, the mesenchymal stem cell preparation disclosed by the invention adopts the specific hydrocolloid gel to carry the mesenchymal stem cells, so that the residence time of the mesenchymal stem cells in the wound part is effectively prolonged, the implantation of WJMSCs is promoted, the wound healing rate of diabetic skin ulcer is remarkably improved, the regeneration of skin tissues is effectively promoted, the generation of new blood vessels is promoted, and the polarization of macrophages is regulated, and the mesenchymal stem cell preparation disclosed by the invention is suitable for preventing and/or treating the diabetic skin ulcer.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.