CN114404452A

CN114404452A - Preparation for promoting angiogenesis and preparation method and application thereof

Info

Publication number: CN114404452A
Application number: CN202210088102.8A
Authority: CN
Inventors: 莫朝晖; 罗寓芳; 莫嘉可; 蒋斯维; 梁芳
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-04-29

Abstract

The invention discloses a preparation for promoting angiogenesis and a preparation method and application thereof, and active components of the preparation comprise Mesenchymal Stem Cells (MSCs), Endothelial Colony Forming Cells (ECFCs) and extracellular matrix. On the basis of the mixed cell preparation, the extracellular matrix hyaluronic acid is added, and the proliferation and migration of the mixed cells can be promoted, so that the activity of the cells can be improved to a great extent, and the problem of low cell transplantation efficiency is solved; furthermore, the extracellular matrix hyaluronic acid can promote the differentiation function of MSCs-ECFCs mixed cells through a CD44/139-5p channel, enhance angiogenesis and realize the application of the extracellular matrix hyaluronic acid in promoting the angiogenesis capacity in vivo. The invention is prepared by combining mixed cells of extracellular matrix, promotes the regeneration of blood vessels, improves the recovery of blood flow of ischemic diseases, provides an angiogenesis inducing preparation for the ischemic diseases, and provides a new method for peripheral vascular diseases, ischemic heart diseases and the like.

Description

Preparation for promoting angiogenesis and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation for promoting angiogenesis, and a preparation method and application thereof.

Background

Peripheral arterial disease is characterized by ischemia of the lower extremities due to arterial stenosis and the accumulation of atherosclerotic plaques. Currently, more than 2 million people worldwide suffer from peripheral artery disease, with PAD incidence predicted to double by 2050. Critical limb ischemia is the most critical clinical manifestation of peripheral arterial disease, usually manifested by pain at rest, unhealed ulcers and necrosis of tissue with gangrene. Advanced age, smoking, hypercholesterolemia, and diabetes are risk factors for the development of peripheral arterial disease. About 25% of patients with critical limb ischemia will die within 1 year, and another 30% will undergo amputation. At present, the curative effect of internal medicine treatment mainly aiming at resisting platelet aggregation, resisting coagulation, improving circulation and relieving pain is very limited, and the requirement for exploring a novel treatment technology is very urgent. Related research reports that stem cell transplantation can effectively establish a vascular bed, improve blood supply of lower limbs, improve amputation rate and gangrene rate, improve survival rate of patients and provide a new idea for treating diabetes lower limb ischemia.

Mesenchymal Stem Cells (MSCs) are stem cells derived from early-stage mesoderm or neuroectoderm, have self-renewal, multipotential differentiation potential and immunoregulation functions, have low immunogenicity and high proliferation capacity, and have unique advantages in application of biological tissue engineering. The MSCs can play an immunoregulation function at local inflammation parts, improve local tissue microenvironment, secrete angiogenesis factors, chemotactic factors and the like to promote angiogenesis. More importantly, MSCs can be transformed into a variety of cell lines, and related studies indicate that MSCs can differentiate into perivascular cells and directly participate in the process of angiogenesis. Endothelial Colony Forming Cells (ECFCs) are derived from umbilical cords and peripheral blood, are a fine subset separated and identified from Endothelial cells, have the characteristic of forming Endothelial cell precursor cells, and have better clonal expansion capacity. In addition to secreting pro-angiogenic factors, ECFCs have the ability to directly promote vascular repair and neogenesis, which can differentiate into mature endothelial cells and integrate with the supporting cells into the three-dimensional structure of the blood vessel. Because of the commonality and complementarity of MSCs and ECFCs in the angiogenic process, relevant studies have demonstrated that ECFCs-MSCs combination therapy can promote angiogenesis more effectively than single cell therapy.

However, cell transplantation efficiency remains an issue to be solved. At present, the stem cell transplantation mainly adopts an intravenous injection or local administration mode, and most of transplanted cells input by veins are dispersed in circulation or phagocytized by macrophages and the like; in the migration process of local stem cell transplantation, the local stem cell transplantation can be damaged by inflammatory factors released by local tissues, the cell activity is reduced, and the cell number is reduced, which all cause low cell transplantation efficiency. In order to effectively improve the homing capacity of stem cells, related researches have conducted intervention on stem cells in an in vitro culture process through an enzyme-linked modification or gene modification mode, but the mode is complex and is applied to the problem of great safety of patients.

Disclosure of Invention

The invention aims to provide a preparation for promoting angiogenesis with good cell activity and high cell transplantation efficiency, and a preparation method and application thereof.

The preparation for promoting the regeneration of blood vessels comprises the active ingredients of Mesenchymal Stem Cells (MSCs), Endothelial Colony Forming Cells (ECFCs) and extracellular matrix.

The extracellular matrix is hyaluronic acid; the total concentration of the mesenchymal stem cells and the endothelial clone forming cells in the preparation is (0.4-6.0) x10⁷Per mL; the number ratio of mesenchymal stem cells to endothelial colony forming cells is (2 to up to4) (1-3), the concentration of extracellular matrix in the preparation is 0.5-1 mg/mL.

Preferably, the number ratio of the mesenchymal stem cells to the endothelial clone forming cells is 3: 2.

The preparation also comprises one of PBS with pH 7.4, complete culture medium and matrigel.

The preparation method of the preparation for promoting the regeneration of blood vessels comprises the following steps:

culturing the mesenchymal stem cells and the endothelial clone forming cells in a culture medium; dissolving extracellular matrix in one of PBS at pH 7.4, complete medium and matrigel; then adding mesenchymal stem cells and endothelial clone to form cells according to the total concentration and proportion of the cells to obtain a preparation;

or culturing the endothelial clone forming cells of the mesenchymal stem cells in a culture medium, and adding the extracellular matrix into the cultured cells to the required concentration after culturing the cells to the required concentration and proportion to obtain the preparation.

In the step 1), the mesenchymal stem cells are derived from umbilical cords and autologous or allogeneic other various tissue sources; the endothelial colony forming cells are derived from umbilical cord blood and autologous or allogeneic peripheral blood sources.

The preparation for promoting the regeneration of blood vessels is applied to the medicines for treating ischemic vascular diseases and the regeneration of related tissues.

The invention has the beneficial effects that: on the basis of the mixed cell preparation, the extracellular matrix hyaluronic acid is added, and the proliferation and migration of the mixed cells can be promoted, so that the activity of the cells can be improved to a great extent, and the problem of low cell transplantation efficiency is solved; furthermore, the extracellular matrix hyaluronic acid can promote the differentiation function of MSCs-ECFCs mixed cells through a CD44/139-5p channel, enhance angiogenesis and realize the application of the extracellular matrix hyaluronic acid in promoting the angiogenesis capacity in vivo. The invention is prepared by combining mixed cells of extracellular matrix, promotes the regeneration of blood vessels, improves the recovery of blood flow of ischemic diseases, provides an angiogenesis inducing preparation for the ischemic diseases, and provides a new method for peripheral vascular diseases, ischemic heart diseases and the like. The invention also provides a new method for optimizing the curative effect of stem cell transplantation and provides a signal path for promoting angiogenesis of stem cells.

Drawings

FIG. 1 Effect of different concentrations of hyaluronic acid in example 1 on the proliferation and migration capacity of MSCs and ECFCs, respectively; wherein: a to C are the effects on ECFCs; d to F are the influence of MSCs.

FIG. 2 Effect of different concentrations of hyaluronic acid in example 1 on the proliferation and migration capacity of mixed cells of MSCs and ECFCs.

FIG. 3 hyaluronic acid promotes angiogenesis of MSC-ECFC in example 2A: a schematic diagram of an experimental flow; statistical map of the proportion of CD31 positive cells；C, a blood vessel number statistical chart; d, a blood vessel caliber statistical chart; e, dyeing the meat eye by using the matrigel suppository and HE; f, CD31 staining.

FIG. 4 HA-MSC-ECFC in example 3 promotes revascularization and restoration of blood perfusion in ischemic limb models: a: a schematic diagram of an experimental flow; b, hyaluronic acid is combined with cell injection to treat ischemia lower limb blood flow perfusion; c, staining of murine CD31 in tissues D, shown by fluorescent double-stained sections of murine CD31(mCD31) and human CD31(hCD 31); f, the change of the skin temperature of each group; g, gangrene of each group of limbs; h: changes in blood flow to each group; and I, counting the blood vessel density.

FIG. 5 HA promotes angiogenesis of MSC-ECFC mixed cells in example 4 involving the CD44/miR-139-5p pathway; a, expression of miR-139-5p in each group, B: and (5) detecting each group of western blot.

FIG. 6 in vitro validation of HA enhancement of MSC-ECFC function by activation of CD44 to inhibit miR-139-5p expression in example 5; HA intervenes the expression of MSC group CD 44; HA intervenes in the expression of CD44 in the ECFC group; c: HA intervenes in the expression of MSC-ECFC group CD 44; e, expressing each group of miR-139-5 p; change in miR-139-5p expression after upregulation of CD 44G: carrying out flow detection on CD31 positive cells, wherein the change H of miR-139-5p expression after the CD44 is reduced; expression of CD31 positive cell and CD31 negative cell miR-139-5 p.

FIG. 7 inhibition or enhancement of proliferation and migration function of MSC-ECFC mixed cells following miR-139-5p mimetic or inhibitor transfection in MSC-ECFC in example 5; A-C, detecting the cell migration ability by healing the scratch; D-E change in CD31 following miR-139-5p mimic transfection in MSC-ECFC.

Detailed Description

The PBS solution in the present invention is a phosphate buffer solution with pH 7.4.

Example 1 hyaluronic acid enhances the function of MSCs, ECFCs

1) Culturing MSCs extracted from umbilical cords or bone marrow and ECFCs extracted from umbilical cord blood or peripheral blood in a 6-well plate until the cell fusion degree is 80%, adding complete culture medium containing no hyaluronic acid and hyaluronic acid concentration of 0.5mg/ml and 1mg/ml, taking pictures under microscope, and culturing in incubator; after 24 hours, the scratch healing condition is observed by photographing again, and the cell migration capacity is determined.

The effect of hyaluronic acid on the migratory capacity of ECFCs in endothelial colony-forming cells is shown in FIGS. 1A and B, which show that hyaluronic acid promotes the migration of ECFCs at concentrations of 0.5mg/ml and 1 mg/ml.

The effect of hyaluronic acid on the migration ability of mesenchymal stem cells MSCs is shown in fig. 1D and F, which shows that hyaluronic acid enhances migration of MSCs at both 0.5mg/ml and 1mg/ml concentrations.

2) Culturing Mesenchymal Stem Cells (MSCs) and Endothelial Colony Forming Cells (ECFCs) in a 96-well plate until the cell fusion degree is 60-70%, adding a complete culture medium without hyaluronic acid and a complete culture medium containing 0.5mg/ml or 1mg/ml HA, culturing in an incubator for further culturing, taking out the 96-well plate after 24 hours, adding 100ul CCK8 reagent into each well, and culturing in the incubator for 2 hours. The 96-well plate was taken out, and the absorbance at 450nm was measured by a spectrophotometer to confirm the cell proliferation ability.

The effect of hyaluronic acid on the proliferation potency of ECFCs on endothelial colony-forming cells is shown in FIG. 1C, which shows that two concentrations of hyaluronic acid have no significant effect on the proliferation potency of ECFCs.

The effect of hyaluronic acid on the proliferation potency of mesenchymal stem cell MSCs is shown in fig. 1F, which shows that both concentrations of hyaluronic acid enhance the proliferation potency of ECFCs.

3) Mixing MSCs and ECFCs according to the proportion of 3:2 of the number of cells, adding the mixture into a culture dish for culture, culturing the cell fusion degree to 80% in a 6-well plate, adding complete culture media which do not contain hyaluronic acid and contain hyaluronic acid with the concentration of 0.5mg/ml and 1mg/ml into the mixture, photographing again to observe the healing condition of the scratch after 6 hours, 12 hours and 18 hours respectively, and carrying out the proliferation capacity test according to the test method of the proliferation capacity (in the step 2); the proliferation and migration capacity of the MSC-ECFC mixed cells are determined.

The results of the proliferation and migration ability of the mixed cells by hyaluronic acid are shown in fig. 2G to I, and it is understood from the graphs that two concentrations of hyaluronic acid can significantly enhance the proliferation and migration ability of the MSC-ECFC mixed cells.

Example 2: hyaluronic acid promotes angiogenesis of MSC-ECFC

It has been reported that MSCs-ECFCs mixed cells have enhanced angiogenic ability compared to single cells, and this experiment compares the effect of a hyaluronic acid-mixed cell preparation, mixed cells, and hyaluronic acid in promoting angiogenesis in combination with single cells.

Mixed cell MSC and ECFC were mixed at a ratio of 3:2, followed by mixed cell concentration of 5X 10⁶Adding each/ml of the mixture into matrigel to obtain matrigel (MSC-ECFC) containing mixed cells;

mixed cell MSC and ECFC were mixed at a ratio of 3:2, followed by mixed cell concentration of 5X 10⁶The mixed cells were added to matrigel at one ml, and HA was added thereto to a concentration of 0.5mg/ml, to obtain a mixture containing 0.5mg/ml HA + cells (HA-MSC-ECFC).

Culturing single cell MSC, then according to the cell concentration of 5 × 10⁶MSC cells were added to the matrigel and HA was added thereto to a concentration of 0.5mg/ml, to give a mixture containing 0.5mg/ml HA + MSC. According to a cell concentration of 5X 10⁶Each/ml MSC was added to the matrigel to obtain matrigel containing MSC.

Culturing single cell ECFC, and obtaining the cell concentration of 510⁶ECFC was added to the matrigel at one ml and HA was added thereto to a concentration of 0.5mg/ml to give a solution containing 0.5mg/ml HA + ECFC. According to a cell concentration of 5X 10⁶ECFC was added to the matrigel at one ml to give a matrigel containing ECFC.

500ul of PBS-added matrigel (PBS) containing 5X 10⁶Matrigel (MSC) containing 5X 10 MSCs/ml⁶Matrigel with ECFC per ml (ECFC), matrigel with mixed cells (MSC-ECFC), HA + mixed cells with 0.5mg/ml (HA-MSC-ECFC), HA + MSC with 0.5mg/ml (HA-MSC) and HA + ECFC with 0.5mg/ml (HA-ECFC) were injected into the abdominal subcutaneous tissue of nude mice (the flow is shown in FIG. 3A), the experimental animals were sacrificed after 14 days, the matrigel tissue was removed, and gross observations, H, and&e staining and immunohistochemical staining of human CD 31.

The results are shown in FIG. 3, which shows that: FIGS. 3B, C and D show that the ratio of CD31 positive cells, the number of blood vessels and the vessel diameter size in the matrigel of HA-MSC-ECFC group are significantly higher than those of MSC, ECFC, MSC-ECFC, HA-ECFC and HA-MSC groups; in FIG. 3E, the HA-MSC-ECFC stroma is stained red and the HA-MSC-ECFC group perfusion is significantly increased compared to MSC, ECFC, MSC-ECFC, HA-ECFC, and HA-MSC; in fig. 3E, F, the HA-MSC-ECFC treated group showed a significant increase in vascular density and larger vascular diameter compared to MSC-ECFC, indicating that hyaluronic acid promotes angiogenesis of MSCs and ECFCs, whereas angiogenesis was more pronounced with the HA-MSC-ECFC preparation.

Example 3: HA-MSC-ECFC promotes revascularization and restoration of blood perfusion in ischemic limb models

1) Selecting SPF level nude mice, and breeding animals under specific conditions according to the stipulation of animal ethics treatment guidance opinion. After 1% pentobarbital is injected into the abdominal cavity of a male nude mouse with the body weight of 10 weeks for anesthesia, the left femoral artery is ligated by 8-0 silk thread, and is incised and excised by an electric condenser. The overlying skin was closed with 5-0 silk suture. After waking up, the mice returned to their cages without restriction of food intake.

2) Post-operative HLI model mice were randomly divided into 4 groups. Group A was injected intramuscularly with 100ul PBS, group B was injected intramuscularly with 100ul hyaluronic acid solution (PBS) at a concentration of 0.5mg/ml, and group C was injected intramuscularly with 100ul solution containing 5X 10⁷MSC-ECFC Mixed cells (5X 10 cells) per ml⁶Wherein the MSC: ECFC ═ 3:2 in PBS) mixed cells, and group D was injected with 100ul of hyaluronic acid-MSC-ECFC mixed preparation (hyaluronic acid final concentration 0.5mg/ml, mixed cells 5 × 10)⁶MSC: ECFC ═ 3:2, solution in PBS);

real-time microcirculation imaging analysis was performed on

days

0, 7 and 14 post-ischemia using a PeriCam Perfusion Speckle Imager (PSI) based on laser speckle contrast analysis technique; activity indices were evaluated on

days

0, 7 and 14 after ischemia (activity scoring criteria: mice placed on iron cages to drag the tail-response-affected side feet were completely immobilized for 3 points, feet were slightly active but not bent at the palms and toes for 2 points, only bent at the palms and not moved at the toes for 1 point, and bent at the palms/toes and developed resistance for 0 point); skin temperature was measured on

days

0, 3, 7, 14 after ischemia; experimental animals were sacrificed on day 14 post-ischemia, and the revascularization was confirmed by fluorescence double staining of the ischemic lateral gastrocnemius muscle tissue, vole CD31(mCD31) and human CD31(hCD31) (see fig. 4A for the procedure).

The results of the injection of the different solutions are shown in fig. 4, from which it can be seen that: the mixed cell injection (MSC-ECFC) can obviously improve the blood perfusion recovery of the lower limb at the ischemic side, and the HA-MSC-ECFC mixed preparation (HA-MSC-ECFC) can more rapidly recover the blood perfusion (figure 4-B, F), and is close to the preoperative perfusion level on the 7 th day after the operation; in addition, HA-MSC-ECFC injection accelerated recovery of limb mobility and skin temperature (FIG. 4-G, H). Immunofluorescent staining of murine CD31(mCD31) in fig. 4 showed that the ischemia muscle angiogenesis was significantly higher for MSC-ECFC injection than for control and hyaluronic acid groups and for HA-MSC-ECFC injection than for mixed cell groups (fig. 4C & D, I & J). HA-MSC-ECFC group ischemic muscle mouse CD31(mCD31) and human CD31(hCD31) fluorescence double-stained sections showed that human cells can integrate into host cells to form blood vessels (fig. 4E).

Example 4: HA promotes angiogenesis of MSC-ECFC mixed cells and involves CD44/miR-139-5p pathway

Using the method of example 3, tissue was harvested at day 14 to detect CD44, miR-139-5p expression, and the results are shown in FIG. 5, which shows that: the injection of MSC-ECFC inhibited miR-139-5p expression, HA-MSC-ECFC further inhibited miR-139-5p expression than MSC-ECFC (FIG. 5A), activated CD44 expression, and up-regulated VEGF, PDGF (FIG. 5B). Since CD44 is a receptor for HA, VEGF, PDGF are likely downstream of miR-139-5p, and thus HA promotes angiogenesis of MSC-ECFC mixed cells, likely involving the CD44/miR-139-5p pathway.

Example 5: in vitro verification that HA enhances MSC-ECFC function by activating CD44 to inhibit miR-139-5p expression

To confirm that HA inhibits miR-139-5p expression by activating CD44, we tested in vitro HA treatment on ECFC or MSC-ECFC, and the results are shown in FIG. 6, which shows that: increased expression of CD44 (FIGS. 6A-D), but post-expression inhibition of miR-139-5p (FIG. 6E); although CD44 was enhanced by HA-treated MSCs, miR-139-5p expression had no significant effect ((FIG. 6E); we subsequently showed significant inhibition of miR-139-5p expression after overexpression of CD44 on ECFC-transfected CD44 plasmid (figure 6F), whereas depletion of HA after knockdown of CD44 with si-RNA inhibited miR-139-5p in ECFC (FIG. 6G), therefore, CD44 is a negative regulator of miR-139-5p in ECFC, and HA inhibits miR-139-5p expression by activating CD44 furthermore, we found that the population of CD31 positive cells in mixed cells was increased after HA treatment of MSC-ECFC (FIG. 6H), and miR-139-5p expression was significantly reduced in the CD31 positive cell population (fig. 6I) — suggesting that HA promotes endothelial cell activity in mixed cells and simultaneously reduces overall miR-139-5p expression.

Isolation of CD31+ and CD31 "cells by flow cytometry showed HA increased the ratio of CD31+ cells in the mixed cells, the results of which are shown in figure 7: and after the miR-139-5p mimic or the inhibitor is transfected in the MSC-ECFC, the proliferation and migration functions of the MSC-ECFC mixed cells are inhibited or enhanced, and the miR-139-5p mimic is transfected to reduce the proportion of CD31+ cells in the mixed cells (figure 7).

Claims

1. A preparation for promoting angiogenesis, characterized in that the active ingredients comprise mesenchymal stem cells, endothelial colony forming cells and extracellular matrix.

2. The agent for promoting angiogenesis according to claim 1, wherein the extracellular matrix is hyaluronic acid; mesenchymal stemThe total concentration of the cells and endothelial colony forming cells in the preparation is (0.4-6.0) × 10⁷Per mL; the number ratio of the mesenchymal stem cells to the endothelial clone forming cells is (2-4): 1-3, and the concentration of the extracellular matrix in the preparation is 0.5-1 mg/mL.

3. The angiogenesis promoting agent according to claim 2, wherein the number ratio of the mesenchymal stem cells to the endothelial colony forming cells is 3: 2.

4. The formulation for promoting angiogenesis of claim 1, wherein the formulation further comprises one of PBS (pH 7.4), complete medium and matrigel.

5. The method for preparing the agent for promoting angiogenesis according to any one of claims 1 to 4, comprising the steps of:

or culturing the mesenchymal stem cells and the endothelial clone forming cells in a culture medium, culturing to the required concentration and proportion, and adding the extracellular matrix to the required concentration to obtain the preparation.

6. The method for preparing the preparation for promoting angiogenesis according to any one of claim 5, wherein in the step 1), the mesenchymal stem cells are derived from umbilical cord and other various tissue sources, such as autologous or allogeneic sources; the endothelial colony forming cells are derived from umbilical cord blood and autologous or allogeneic peripheral blood sources.

7. Use of the agent for promoting angiogenesis according to claim 1 as a medicament for treating ischemic vascular diseases and regeneration of related tissues.