Release microsphere containing insulin-like growth factor and macromolecular polylactic acid-glycolic acid copolymer and application of release microsphere in skin wounds

Abstract

The invention relates to the field of medicines, in particular to a release microsphere containing insulin-like growth factors and a macromolecular polylactic acid-glycolic acid copolymer and application of the release microsphere in skin wounds. The release microsphere (IGF-PLGA) comprises the following effective components: an insulin-like growth factor and a macromolecular polylactic acid-glycolic acid copolymer. The IGF-PLGA sustained release microspheres can slowly release IGF for a long time in vivo and in vitro; in an animal trapped flap model, compared with independent use of the IGF and IGF-PLGA sustained release microspheres, repair and healing of a mouse trapped flap can be remarkably promoted; animal skin flap tissue is extracted for HE and Masson dyeing, and it is found that the IGF-PLGA microsphere group can significantly promote granulation hyperplasia and collagen deposition; the IGF-PLGA sustained-release microsphere group can be used for effectively inhibiting the oxidative stress related protein expression of the skin flap tissue; the proliferation protein expression quantity of the IGF-PLGA group is higher than that of other groups.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a release microsphere containing insulin-like growth factor and a high-molecular weight polylactic acid-glycolic acid copolymer and application thereof in skin wounds. Background Art

[0002] A skin flap refers to a tissue block composed of skin and its attached subcutaneous tissue, including skin, subcutaneous tissue, deep tissue, blood supply tissue, etc. The perforator flap is a new type of skin flap that emerged in the late 1980s with the development of sophisticated microsurgical techniques and actual clinical needs. It is an axial flap that harvests skin and subcutaneous tissue with a small-diameter skin perforator blood vessel as the pedicle. It is flexible in design, causes little trauma to the donor site, and is easy to transplant. It has ushered in the era of small axial flaps, reduced damage to the appearance and function of the donor site, and improved the repair effect of the recipient site, making the application of skin flaps in surgical clinics more free and flexible, and realizing the transformation of skin flaps from "rough" to "refined" minimally invasive. As a result, tissue defect repair is no longer simply covering the wound surface, but can also reconstruct function and appearance, and is widely used in clinical surgical treatment. As an important technique used in microsurgery and orthopedics to cover large postoperative wounds, open wounds, skin defects and plastic surgery, flap technology has made great progress in surgical techniques, but the incidence of postoperative transplanted flap necrosis is still high, and the postoperative ischemic necrosis complication is currently a major problem that plagues clinicians. Studies have reported that the incidence of flap necrosis is 10%-15%, among which the necrosis rate of pedicled flaps is as high as 20.69%. Once the transplanted flap is ischemic and necrotized, the patient will face a second operation or even more serious consequences, which will not only increase the pain of the patient's reoperation, but also increase the patient's economic burden. Therefore, improving the survival rate and repair rate of transplanted flaps is the focus of current flap transplantation research and the main problem that needs to be solved. Summary of the invention

[0003] In view of the deficiencies of the prior art, the object of the present invention is to provide a release microsphere containing insulin-like growth factor and a high molecular weight polylactic acid-glycolic acid copolymer (IGF-PLGA release microsphere) and its application in skin wounds.

[0004] Insulin like growth factor (IGF) is a group of polypeptide substances with growth-promoting effects, which are widely distributed in various tissues and organs of the human body. As a broad-spectrum growth-promoting factor, the application of IGF in wound repair has been confirmed by literature, but there are few reports on its relationship with perforator flaps, and there is a lack of relevant literature research on the main molecular mechanisms and pathways involved. The inventors of this application explored the effect of IGF on perforator flaps and found that IGF growth factor has a short half-life, poor stability, and high storage requirements. It is easily decomposed by enzymes or bacteria under pathological or physiological conditions and leads to inactivation. When used on flap wounds, it is prone to leakage, decomposition and other risks. In order to solve this problem, the inventors of this application have found a biodegradable biomaterial polymer polylactic-co-glycolic acid copolymer (PLGA) that can effectively carry and deliver growth factors and combine it with insulin-like growth factor to form sustained-release microspheres, which not only have good sustained-release performance and safety, but also can promote angiogenesis in skin wounds and improve the survival rate and repair rate of transplanted flaps.

[0005] To achieve the purpose of the invention, the present invention adopts the following technical scheme: On the one hand, the present invention provides a release microsphere containing insulin-like growth factor (IGF) and high molecular weight polylactic acid-glycolic acid copolymer (PLGA), and the effective ingredients of the release microsphere include insulin-like growth factor and high molecular weight polylactic acid-glycolic acid copolymer.

[0006] In some embodiments of the present invention, in the release microspheres, the mass ratio of the insulin-like growth factor to the high molecular weight polylactic acid-glycolic acid copolymer is 250:1.

[0007] In some embodiments of the present invention, the preparation method of the release microspheres is as follows: the release microspheres of insulin-like growth factor and high molecular weight polylactic acid-glycolic acid copolymer are synthesized by the traditional W / O / W composite method: (1) the insulin-like growth factor is dissolved in a deionized solution, and the high molecular weight polylactic acid-glycolic acid copolymer is dissolved in dichloromethane; (2) the above two solutions are mixed and homogenized at 10000 rpm for 1 minute to promote emulsification, and the resulting single emulsion is rapidly emulsified at 400 rpm in ddH2O containing 1% w / v polyvinyl alcohol using a mechanical stirrer; (3) the emulsion is introduced into 200 ml of ddH2O containing 0.1% w / v methylcellulose solution, and stirred continuously for 4 hours, the microspheres are shaken, washed with water, and freeze-dried overnight to obtain the release microspheres containing insulin-like growth factor and high molecular weight polylactic acid-glycolic acid copolymer.

[0008] In another aspect, the present invention provides the use of the releasing microspheres in skin wounds.

[0009] In some embodiments of the present invention, the release microspheres are used in skin wounds, and the release microspheres promote the proliferation of skin flap granulation tissue and collagen synthesis.

[0010] In some embodiments of the present invention, the release microspheres promote angiogenesis in HUVEC cells.

[0011] In some embodiments of the present invention, in the skin wound, the released microspheres promote angiogenesis and improve the survival rate and repair rate of transplanted skin flaps by inhibiting oxidative stress and regulating the Ang / Tie signaling pathway.

[0012] In some embodiments of the present invention, the released microspheres can inhibit the oxidative stress response of the perforator flap wound surface and promote flap proliferation.

[0013] In some embodiments of the present invention, the releasing microspheres promote the viability and migration ability of 3T3 cells.

[0014] In some embodiments of the present invention, the releasing microspheres promote 3T3 cell proliferation and inhibit apoptosis; the releasing microspheres inhibit T-BHP-induced 3T3 cell oxidative stress; the microspheres promote 3T3 cell migration and HUVEC cell proliferation by inhibiting oxidative stress.

[0015] The beneficial effects of the present invention are:

[0016] IGF-PLGA sustained-release microspheres can release IGF for a long time in vivo and in vitro; in the animal perforator flap model, compared with the use of IGF alone, IGF-PLGA sustained-release microspheres can significantly promote the repair and healing of mouse perforator flaps; HE and Masson staining of animal flap tissues showed that compared with other groups, the IGF-PLGA microsphere group could significantly promote granulation proliferation and collagen deposition; western blot experiments showed that the IGF-PLGA sustained-release microsphere group could effectively inhibit the expression of oxidative stress-related proteins in flap tissues; immunofluorescence experiments of animal flap tissues showed that the expression of proliferation proteins in the IGF-PLGA group was higher than that in the other groups; after different stimulation treatments of human umbilical vein endothelial cells, it was found that the IGF-PLGA group could promote the growth and proliferation of endothelial cells.

[0017] This invention is based on the team's long-term accumulation of basic and translational medical research on skin trauma and regeneration in the field of orthopedic trauma. It is the first attempt to conduct research on IGF and perforator flaps at the in vivo and in vitro levels, aiming to clarify the relationship between IGF and vascular function repair, and analyze the relevant mechanisms and pathways of IGF intervention in the process of perforator flaps. The study will comprehensively use modern pharmacology, molecular biology, fluorescence imaging technology, gene silencing analysis and other technical means to fully explore the correlation between IGF and oxidative stress and Ang / Tie signaling pathways in the process of perforator flap repair, and finally evaluate the value of IGF as a potential flap treatment drug, providing exploratory research data references and a new theoretical system support for improving flap survival and repair rates after perforator flap surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 The characteristics and in vitro release experiment of IGF-PLGA microspheres. (A) Transmission electron microscopy results of IGF-PLGA microspheres. (B) In vitro release experiment of IGF-PLGA microspheres.

[0020] Figure 2 .IGF-PLGA microspheres accelerate mouse flap healing and promote granulation formation and collagen deposition. (A) Survival rate of mouse flaps in different groups. (B, C) Statistical graphs show the survival rate of flaps in different groups on days 3 and 9, and the data are reported as mean ± standard deviation. Compared with the flap group, *p<0.05, **p<0.01. (D) HE staining of different groups on day 7. (E) Masson staining of different groups on day 7.

[0021] Figure 3 .In the mouse model, IGF-PLGA microspheres inhibited flap oxidative stress and promoted flap tissue proliferation. (A) Immunofluorescence of HO-1 in flap tissues of different groups, scale bar = 20 μM. (B) WB results of HO-1 and Caspase 3 proteins in flap tissues of different groups. (C) PCNA immunofluorescence in flap tissues of different groups, scale bar = 20 μM. (D, E) Statistical graphs show the mean fluorescence intensity of HO-1 and PCNA. Experimental data are reported as mean ± standard deviation. (F, G) Statistical graphs show the protein expression levels of HO-1 and Caspase 3. Experimental data are reported as mean ± standard deviation. *p<0.05, **p<0.01.

[0022] Figure 4IGF-PLGA microspheres promote vascular proliferation in flap tissues through the Ang1 / Tie 2 pathway. (A) Immunointensity of CD31 in flap tissues of different groups, scale bar = 20 μM. (B) Western blot detection of CD31, VEGF, Ang 1 and Tie 2 protein expression in flap tissues of different groups. (C) Statistical graphs show the number of new blood vessels. Data are reported as mean ± SD. (D, E, F, G) Statistical graphs show the protein expression levels of CD31, VEGF, Ang 1 and Tie 2. Data are reported as mean ± SD. *p<0.05, **p<0.01.

[0023] Figure 5 IGF-PLGA microspheres promote the activity and migration ability of 3T3 cells. (A, B) The stimulation concentrations of T-BHP and IGF-1 were selected for the CCK8 experiment. (C) The T-BHP concentration in different groups of the CCK8 experiment was 200μM, and the IGF-1 concentration was 100ng / ml. (D) LDH release test in different groups. The concentration of T-BHP was 200μM, and the concentration of IGF-1 was 100ng / ml. (E) Scratch test in different groups, Bar=100×. (F) Statistical analysis of cell migration. Data are reported as mean ± standard deviation. Compared with the T-BHP group, *p<0.05, **p<0.01.

[0024] Figure 6 IGF-PLGA microspheres promote 3T3 cell proliferation and inhibit apoptosis. (A, B) Immunofluorescence images of PCNA and Caspase 3 in different groups, scale bar = 5μM. (C) Western blot detection of Bax and Bcl-2 expression levels in each group. (D, E) Statistical graphs show the mean fluorescence intensity of PCNA and Caspase 3, (F, G) Statistical graphs show the expression of T-BHP under IGF-PLGA stimulation. Data are expressed as mean ± standard deviation. Experimental data are reported as mean ± standard deviation. Compared with the T-BHP group, *p<0.05, **p<0.01.

[0025] Figure 7IGF-PLGA microspheres inhibit T-BHP-induced oxidative stress in 3T3 cells. (A) WB results of HO-1, SOD1 and SOD2 in different groups. (B, C, D) Statistical graphs show the protein expression levels of HO-1, SOD1 and SOD2. Experimental data are reported as mean ± standard deviation. Compared with the T-BHP group, *p<0.05, **p<0.01. (E) Immunofluorescence images of HO-1 in different groups, scale bar = 20μM. (F) Statistical graphs show the average fluorescence intensity of HO-1. Experimental data are reported as mean ± standard deviation. Compared with the T-BHP group, *p<0.05, **p<0.01.

[0026] Figure 8 IGF-PLGA microspheres promote angiogenesis of HUVEC. (A) Western blot detection of VEGF and Ang 1, Tie 2 expression in different groups. (B, C, D) Statistical graphs show the protein expression levels of VEGF and Ang 1, Tie 2. Our experimental data are reported as mean ± standard deviation. Compared with the T-BHP group, *p<0.05, **p<0.01. (E) CD31 immunodiffusion in different groups, scale bar = 20μM. (F) Statistical graph shows the average diffusion intensity of CD31. (G) Representative images of tubular structures formed by HUVEC within 8 hours. (h) Tube number graph within 8 hours. Our experimental data are reported as mean ± standard deviation. Compared with the TBHP group, *p<0.05, **p<0.01.

[0027] Fig. 9 IGF-PLGA microspheres promote 3T3 cell migration and HUVEC cell proliferation by inhibiting oxidative stress. (A) Western blot of VEGF and HO-1 in different groups. (B, C) Statistical graphs show the protein expression levels of VEGF and HO-1. Our experimental data are reported as mean ± standard deviation. *p<0.05, **p<0.01. (D) Scratch assay in different groups, Bar=100×. (E) Statistical analysis of cell migration. Our experimental data are reported as mean ± standard deviation. *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0028] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0029] Example 1: Structure and properties of IGF-PLGA sustained-release microspheres

[0030] Experimental methods:

[0031] 1.1 The traditional W / O / W composite method was used to synthesize IGF-PLGA sustained-release microspheres.

[0032] Initially, IGF-1 (10 μg) was dissolved in 600 μl of deionized solution, while PLGA (40 mg) was dissolved in 2.5 ml of dichloromethane. The two solutions were then mixed and homogenized at 10,000 rpm for 1 minute to promote emulsification. The resulting single emulsion was rapidly emulsified at 400 rpm in ddH2O containing 1% w / v polyvinyl alcohol using a mechanical stirrer. Subsequently, the emulsion was introduced into 200 ml of ddH2O containing 0.1% w / v methylcellulose solution and stirred continuously for 4 hours. The role of the methylcellulose solution is to partially saturate the dichloromethane to extend the time for water to extract dichloromethane from the oil droplets and prevent the microspheres from hardening prematurely before the two polymer phases separate. The IGF-PLGA microspheres were shaken, washed with water, and freeze-dried overnight.

[0033] 1.2 IGF-1 release experiment

[0034] 2 mg IGF-PLGA microspheres (yes) were dissolved in 1 ml NaOH (1 mol / L) containing 1% SDS and stirred at room temperature for 24 hours, then the solution was centrifuged at 5000 rpm for 5 minutes, and the IGF content in the supernatant was analyzed using an IGF-1 kit. Encapsulation efficiency = M2 / M1×100 (M2 is the measured weight of protein in the microspheres; M1 is the total weight of protein used for microsphere manufacturing). 2 mg microspheres were weighed and placed in an EP tube, phosphate buffer solution (PBS, 100 μl) was used as a release medium, 0.1% bovine serum albumin (BSA) was used as a stabilizer, and 0.8% (W / V) mannitol was used as a wetting agent. The test tubes were shaken at a constant temperature of 37°C, and the supernatants were collected at 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days and 12 days, respectively. The release amount of IGF-1 was quantified using a kit.

[0035] 1.3 Transmission Electron Microscopy (TEM)

[0036] Fresh samples were immersed in a 2% mixed solution, followed by rinsing the samples 3 times with PBS buffer for 10 minutes each time, and then rinsed with 1% citric acid for 1 hour. After rinsing again with PBS buffer 3 times (10 minutes each time), a gradient alcohol dehydration process was performed: 50%, 70%, 80% and 90% alcohol for 10 minutes each, followed by rinsing 3 times in 100% alcohol for 10 minutes each. Rinse with ethanol and acetone in a 1:1 ratio for 10 minutes, and then re-wash with pure acetone for 10 minutes. Subsequently, soak with acetone and 812 resin in a 1:1 ratio for 2 hours, and then place the sample in RT resin overnight. The sample was embedded and left to stand for 4 hours. Ultrathin sections were prepared and observed and photographed using an electron microscope.

[0037] Experimental results:

[0038] Transmission electron microscopy results ( Figure 1 A) shows that IGF-PLGA microspheres are round with diameters between 2 and 4 μm. Protein loading analysis experiments show that about 62% of IGF-1 is loaded in the microspheres. In the in vitro release experiment of IGF-1, ( Figure 1 B) In the first 24 hours, IGF-PLGA microspheres did not release IGF-1 suddenly. Subsequently, the release of IGF-1 gradually increased, showing a significant acceleration at 7 days. It entered a plateau on the 10th day, and the release reached about 80% on 14 days.

[0039] Example 2: IGF-PLGA microspheres accelerate skin healing in mice, promote granulation formation and collagen deposition

[0040] Experimental methods:

[0041] 2.1 Establishment of flap model

[0042] Sixty male C57BL / 6 mice (8-12 weeks old, weighing 20-25 g) were enrolled. All mice were housed under standard conditions and had free access to food and water. Before surgery, anesthesia was induced using 1% sodium pentobarbital (50 mg / kg, ip). A tail flap (1.5 cm × 4.5 cm) was then cut from the deep subcutaneous fascia on the back of each mouse. Subsequently, the bilateral iliac arteries were exposed and cut, and then the flap was immediately closed using 4-0 non-absorbable sutures. The IGF-1 group received in situ IGF-1 treatment, the IGF-PLGA group received in situ IGF-PLGA sustained-release microspheres treatment, and the control group was given an equal amount of normal saline. On the 7th day after surgery, the mice were euthanized under excessive anesthesia, and tissue samples from the central area of ​​the flap were collected in time for further analysis. After the end of drug administration, the flap conditions of mice in different treatment groups were photographed and observed on days 0, 3, 7, and 14 after surgery, and the survival of the perforator flap was statistically analyzed using Image J software.

[0043] 2.2HE staining:

[0044] Slice preparation: The skin flap of the mouse was fixed with 4% paraformaldehyde, trimmed flat, dehydrated, paraffin-embedded, sliced, and dried. The slices were placed in glass jars containing xylene (I), xylene (II), a 1:1 mixture of xylene and anhydrous ethanol, anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol, 70% ethanol, and 50% ethanol, respectively, and left to stand for 3 minutes. Finally, rinse with running water for 3 minutes. Stain with hematoxylin stain for 20 minutes. Differentiation solution (0.1% hydrochloric acid alcohol solution) was used for differentiation for 30 seconds, and then rinsed with running water for 5 minutes. Placed in eosin stain for 2 minutes, and then soaked in tap water for 5 minutes. Dehydration and transparency: The slices were placed in glass jars containing 95% ethanol (I), 95% ethanol (II), 100% ethanol (I), 100% ethanol (II), a 1:1 mixture of xylene and anhydrous ethanol, xylene (I), and xylene (II), and left to stand for 1 minute. Sealing: Seal the slide with brown resin, store it or observe and photograph it under a microscope and analyze the results.

[0045] 2.3Masson staining:

[0046] Masson staining is a staining method that uses alkaline hematoxylin and acidic Ponceau red. Hematoxylin can make collagen fibers appear blue, and Ponceau red can make muscle fibers appear red. It is one of the staining methods used to show fibers and inflammatory factors in tissues. The specific method of Masson staining is as follows: dewax the paraffin sections conventionally and stain them with the prepared Weigert iron hematoxylin staining solution for 5min-10min. Differentiate with acidic ethanol differentiation solution for 5-15s, then wash with water. Use Masson blueing solution to return to blue for 3-5min, then rinse with water. Wash with distilled water for 1min and then stain with Ponceau red fuchsin staining solution for 5-10min. During the above operation, prepare the weak acid working solution in a ratio of distilled water: weak acid solution = 2:1, wash with the weak acid working solution for 1min, then wash with phosphomolybdic acid solution for 1-2min, then wash with the prepared weak acid working solution for 1min, and directly put it into the aniline blue staining solution for staining for 1-2min. Wash with the prepared weak acid working solution for 1min and then quickly dehydrate with 95% ethanol. Dehydrate with anhydrous ethanol three times, 5-10 seconds each time, clear with xylene three times, 1-2 minutes each time, and finally seal with neutral resin.

[0047] Experimental results:

[0048] To evaluate the effect of IGF-PLGA microspheres on flap healing, three treatments were used in flap model mice: saline, IGF-1 alone, and IGF-PLGA microspheres. Figure 2As shown in A, flap healing was significantly improved in the IGF-1 and IGF-PLGA groups compared with the control group. Notably, the IGF-PLGA microsphere group had the best flap healing and thus the highest flap closure rate among all groups ( Figure 2 B and C). Granulation tissue formation is the central stage of wound repair after skin or subcutaneous tissue injury. After 7 days, the flap tissue was histologically evaluated using HE staining ( Figure 2 D), the results showed that the flap group showed the thinnest granulation, while the IGF-PLGA microsphere group showed more continuous granulation. In addition, Masson staining revealed different degrees of collagen deposition during flap healing. Notably, the IGF-PLGA microsphere group showed the highest collagen deposition, while the flap group showed the lowest collagen deposition ( Figure 2 E).

[0049] Example 3: IGF-PLGA microspheres inhibit oxidative stress in flap tissue and promote flap proliferation

[0050] Experimental methods:

[0051] 3.1 Immunofluorescence

[0052] Collect cell slides and animal tissues, fix with 4% paraformaldehyde for 15 minutes, wash with PBS three times, 3 minutes each time; permeabilize with 0.5% Triton-100 at room temperature for 20 minutes, wash with PBS three times, 3 minutes each time; dry PBS, add normal goat serum to the slide, block at room temperature for 30 minutes, wash off the blocking solution with absorbent paper, do not wash, add enough diluted primary antibody (SOD2, SOD1, HO-1, NRF2, Ang, Tie, etc.) to each slide and put it in a wet box, incubate overnight at 4°C. Add fluorescent secondary antibody, wash the slide with PBS three times, 3 minutes each time, dry the excess liquid on the slide with absorbent paper, add diluted fluorescent secondary antibody, incubate at 20-37°C in a wet box for 1 hour, and wash with PBS three times, 3 minutes each time (from the addition of fluorescent secondary antibody, all subsequent operation steps should be carried out in a darker place as much as possible). Re-staining the nucleus: add DAPI and incubate in the dark for 5 minutes to stain the nucleus of the specimen, wash off the excess DAPI 4 times with PBS 5 minutes each time, dry the liquid on the slide with absorbent paper, seal the slide with sealing solution containing anti-fluorescence quencher, and then observe and collect images under a fluorescence microscope.

[0053] 3.2 Western blot experiment

[0054] Animal skin flaps were collected, proteins were extracted, and Western Blot experiments were performed. Protein separation by SDS-polyacrylamide gel electrophoresis: gel preparation, electrophoresis separation of proteins, transfer, blocking, incubation with primary antibodies (oxidative stress-related proteins: SOD2, SOD1, HO-1, NRF2, etc.; pathway-related proteins Ang, Tie, the dilution ratio was 1:1000), incubation with secondary antibodies (1:10000). Exposure: Wash with 3‰ TBST for 10min×3 times. ECL developer A and B were mixed in equal volumes, and the PVDF membrane was laid flat on the plastic wrap, facing up, and the developer was added, and then the PVDF membrane was wrapped with plastic wrap and reacted in the dark for 5min. Then the PVDF membrane was placed in a Tanon developer for development.

[0055] Experimental results:

[0056] In this experiment, immunofluorescence staining was used to evaluate the expression levels of anti-oxidative stress and proliferation proteins. The results of immunofluorescence staining for HO-1 were as follows: Figure 3 As shown in A, compared with normal mouse flap tissue, a significant decrease in green fluorescence was observed in the flap group. After IGF-1 and IGF-PLGA treatment, green fluorescence increased, which was significantly stronger in the IGF-PLGA group. Quantitative analysis confirmed these observations and showed statistical significance ( Figure 3 D). Similarly, the proliferation protein PCNA showed a similar trend to HO-1 ( Figure 3 C), there was a statistically significant difference in the quantitative analysis ( Figure 3 E). In addition, WB experiments were performed to evaluate the expression levels of the antioxidant stress protein HO-1 and the apoptotic protein Caspase 3. The results for HO-1 reflected those observed in immunofluorescence, while the apoptotic proteins showed the opposite trend ( Figure 3 B). There is a statistically significant difference between the two ( Figure 3 F and G ), In conclusion, IGF-PLGA promoted flap proliferation by effectively inhibiting oxidative stress in the mouse model.

[0057] Example 4: IGF-PLGA microspheres promote angiogenesis via the Ang1 / Tie2 pathway in a mouse model

[0058] According to the experimental method of Example 3, in order to further study the role of IGF-PLGA in promoting skin flap angiogenesis, the expression of CD31 in the skin flap was evaluated. Figure 4 As shown in A, the number of new blood vessels in the flap group was reduced compared with the control group. In contrast, both the IGF-1 and IGF-PLGA groups showed new blood vessel formation. Statistical analysis confirmed the significance of the difference in the number of new blood vessels ( Figure 4 C). Subsequently, WB experiment was performed on the flap tissue ( Figure 4B), indicating that the expression levels of CD31 and VEGF were increased in the IGF-1 and IGF-PLGA groups. Notably, the IGF-PLGA group showed more pronounced expression. In addition, exploration of the signaling pathways by which IGF-PLGA promoted vascular proliferation revealed upregulation of Ang1 and Tie2. Statistical analysis showed the significance of these results ( Figure 4 DG). These results indicate that IGF-PLGA promotes flap angiogenesis through the Ang1 / Tie2 signaling pathway.

[0059] Example 5: IGF-PLGA microspheres promote the viability and migration ability of 3T3 cells

[0060] Experimental methods:

[0061] 5.1 CCK8 Experiment

[0062] The 96-well plate seeded with cells was placed in a 37°C, 5% CO2 incubator overnight. After the cells adhered to the wall, the old culture medium was aspirated, and 100 μl of different stimulating drugs prepared with 1% serum culture medium were added to each well according to the grouping. The culture was continued in the incubator for 24 hours, and 10 μl of CCK8 solution was added to each well. The culture was continued in the incubator for 4 hours, and the absorbance (OD) value at 450 nm was measured with an enzyme marker.

[0063] 5.2 LDH release experiment

[0064] Cells were seeded onto 96-well plates, ensuring that the cell density did not exceed 90% of the tested cells. Once the cells adhered to the plate, the old medium was removed and the cells were washed with PBS. Subsequently, treatment drugs prepared in low serum medium were added.

[0065] 5.3 Scratch test

[0066] The cells were plated at 5×10 5 The cells were seeded in a 6-well plate at a density of 10 cells / mL and incubated overnight in an incubator at 37°C. The next day, a ruler-guided scratch was made using a 10 μl pipette tip. The pipette tip was kept in a vertical position without tilting. The cells were then washed three times with PBS to remove the detached cells produced by the scratch. Subsequently, serum-free medium was added and the cells were further cultured in an incubator. Observations were made over 24 hours and photographs were taken to record the changes.

[0067] Experimental results:

[0068] Next, we used T-BHP as a damage inducer to explore the effect of IGF-PLGA on fibroblast 3T3 cells, and determined the concentration of T-BHP by CCK8 assay ( Figure 5A). The results showed that cell viability decreased with increasing T-BHP concentration. At 200 μM T-BHP, 3T3 cell viability decreased to about 50%, which was selected as the stimulation concentration for subsequent experiments. Figure 5 B shows the promoting effect of IGF-1 on 3T3 cell activity, among which 100ng / ml has the strongest effect. Therefore, 100ng / ml of IGF-1 was selected for subsequent experiments. CCK8 experiments showed that T-BHP stimulation reduced cell activity, while treatment with IGF-1 and IGF-PLGA increased cell activity, with the latter showing a more powerful effect ( Figure 5 C). The corresponding LDH experimental results were similar to those of the CCK8 experiment, with the T-BHP group showing the highest cell death rate, while the IGF-PLGA group observed the lowest death rate ( Figure 5 D). In addition, the migration ability of 3T3 cells was evaluated ( Figure 5 E). At 24 hours, the control group showed the greatest migration toward the middle of the scratch, while T-BHP significantly inhibited cell migration. After stimulation with IGF-1 and IGF-PLGA, cell migration was enhanced, with the IGF-PLGA group showing the strongest migration ability, and statistical analysis confirmed these observations ( Figure 5 F).

[0069] Example 6: IGF-PLGA microspheres promote 3T3 cell proliferation and inhibit apoptosis

[0070] According to the experimental method of Example 5, in order to further clarify the effect of IGF-PLGA on the proliferation and apoptosis of fibroblasts 3T3, the fluorescence intensity of the proliferation protein PCNA and the apoptosis protein Caspase3 was evaluated by immunofluorescence. Figure 6 A shows that the green fluorescence of PCNA was significantly decreased after T-BHP stimulation compared with the control group. However, IGF-1 and IGF-PLGA stimulation significantly increased the green fluorescence, with the IGF-PLGA group showing the strongest effect, which was statistically significant ( Figure 6 D). Similarly, Caspase3 expression showed enhanced green fluorescence after T-BHP stimulation, while IGF-1 and IGF-PLGA treatment attenuated this fluorescence with statistical significance ( Figure 6 B and E). In addition, WB experiments evaluated the expression levels of apoptotic protein Bax and anti-apoptotic protein Bcl-2 ( Figure 6 C). T-BHP stimulation resulted in increased Bax expression and decreased Bcl-2 expression compared with the control group. However, IGF-1 and IGF-PLGA stimulation reversed these effects induced by T-BHP with statistical significance ( Figure 6 F and G). The above results indicate that IGF-PLGA can promote the proliferation of fibroblast 3T3 cells and inhibit apoptosis.

[0071] Example 7: IGF-PLGA microspheres inhibit T-BHP-induced oxidative stress in 3T3 cells

[0072] To further explore the mechanism by which IGF-PLGA promotes the proliferation of fibroblasts 3T3, Western blot was used to detect the expression levels of oxidative stress-related proteins. After T-BHP stimulation, the expression of HO-1, SOD1, and SOD2 was reduced compared with the control group. However, stimulation with IGF-1 and IGF-PLGA resulted in an upregulation of the expression levels of these proteins ( Figure 7 A). Statistical analysis showed the significance of these changes ( Figure 7 BD). Then, immunofluorescence analysis of HO-1 fluorescence intensity showed that the green fluorescence in the TBHP group was significantly reduced ( Figure 7 E). In contrast, both IGF-1 and IGF-PLGA stimulation resulted in enhanced green fluorescence, with the IGF-PLGA group showing the strongest effect, which was statistically significant ( Figure 7 F).

[0073] Example 8: IGF-PLGA microspheres promote angiogenesis in HUVEC cells

[0074] Experimental methods:

[0075] 8.1 In vitro tube formation test

[0076] 96-well plates were coated with pre-chilled Matrigel solution and then incubated at 37°C for 30 min to allow the gel to solidify. HUVEC cells were harvested, suspended in medium containing 2% FBS, and seeded onto Matrigel-coated wells at a density of 100 μl at 3 × 104 cells per well. The plates were pre-incubated at 37°C for 30 min to promote cell attachment to the Matrigel. Images capturing tubular structure formation were captured every 1 h for 8 h using a real-time cell recorder.

[0077] Experimental results:

[0078] In order to explore the angiogenic effect of IGF-PLGA microspheres, HUVEC cells were cultured in vitro. First, the expression levels of VEGF and pathway proteins Ang1 / Tie2 were detected by Western blot. The results showed that the expression levels of VEGF and pathway proteins Ang1 and Tie2 were decreased after T-BHP stimulation compared with the control group, but were upregulated after IGF-1 and IGF-PLGA stimulation ( Figure 8 A). Statistical analysis confirmed these changes ( Figure 8BD). Next, immunofluorescence analysis of CD31 expression showed that the green fluorescence in the T-BHP group was reduced compared with the control group, indicating a decrease in CD31 expression. In contrast, IGF-1 and IGF-PLGA stimulation enhanced CD31 green fluorescence, with the IGF-PLGA group showing the most significant effect ( Figure 8 E). Quantitative analysis supported these observations ( Figure 8 F). In addition, an in vitro tube formation assay was performed to evaluate the effect of IGF-PLGA on the tube formation ability of HUVEC cells. Figure 8 As shown in G, after 8 hours of stimulation, the tube-forming ability of HUVEC cells in the T-BHP group was significantly reduced compared with the control group. In contrast, both IGF and IGF-PLGA stimulation enhanced the tube-forming ability of HUVEC cells, with the IGF-PLGA group showing the most prominent effect. Subsequently, statistical analysis of the number of tubes showed statistical significance ( Figure 8 H).

[0079] Example 9: IGF-PLGA microspheres promote 3T3 cell migration and HUVEC cell proliferation by inhibiting oxidative stress

[0080] The correlation between oxidative stress, cell migration and angiogenesis was verified by adding HO-1 inhibitor ZnPP. First, HUVEC cells were used to study the association between oxidative stress and angiogenesis. Fig. 9 As shown in A, compared with the control group, the expression levels of HO-1 and VEGF in the T-BHP group were decreased, while the expression in the IGF-PLGA group was increased. After the addition of Znpp, the expression of HO-1 and VEGF was decreased. Statistical analysis showed significant differences ( Fig. 9 B and C). Next, the relationship between oxidative stress and the migration ability of 3T3 cells was evaluated. Fig. 9 D shows that the cell migration ability of the IGF-PLGA group was significantly stronger than that of the T-BHP group. However, after the addition of Znpp, the cell migration ability was significantly reduced, indicating a statistically significant difference ( Fig. 9 E). These results suggest that IGF-PLGA microspheres can promote cell migration and angiogenesis by inhibiting oxidative stress, while promoting proliferation and angiogenesis in flap tissue.

[0081] In summary, the present invention is the first to conduct a study on the correlation between exogenous IGF and perforator flaps, and to verify and evaluate its significance through multiple methods at the in vivo and in vitro levels. Insulin-like growth factor is a group of polypeptide substances with growth-promoting effects, which can be produced by tissues such as the liver, kidney, lung, heart, brain and intestine. Most of them are synthesized in the liver and then released into the blood to act on target cells to achieve the effects of promoting growth and regulating metabolism. It has been proven to be related to vascular regeneration in various diseases, but there are still few studies on its application in perforator flap surgery and its molecular mechanism and significance in the vascularization process of perforator flaps. The research team has had many years of research foundation on the growth factor IGF in wound repair injury treatment and mechanism convenience. Based on the previous work, the present invention uses the biotechnology platform where the research team is located to carry out dual-level in vivo and in vitro systematic research, creatively explore the significance of IGF in the survival of perforator flaps and the promotion of angiogenesis, and clarify the potential value of IGF in the flap treatment process and the development direction of future research. The present invention uses Western Bolt experiment, immunohistochemistry, immunofluorescence and gene silencing, microangiography and laser Doppler blood flow technology to verify the effect of IGF on angiogenesis in vitro and in vivo, and evaluate the significance and role of IGF at multiple levels. The present invention uses Western Bolt, immunofluorescence and gene silencing and knockout techniques to study the role of oxidative stress in regulating the Ang / Tie signaling pathway in the angiogenesis process in vitro and in vivo, and provide more sufficient data support for the function of IGF in promoting flap angiogenesis.

[0082] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A release microsphere containing insulin-like growth factor and high molecular weight polylactic acid-co-glycolic acid, characterized in that: The effective components of the released microspheres include insulin-like growth factor and high-molecular weight polylactic acid-glycolic acid copolymer.

2. The release microspheres according to claim 1, characterized in that In the release microspheres, the mass ratio of the insulin-like growth factor to the high molecular weight polylactic acid-glycolic acid copolymer is 250:

1.

3. The release microspheres according to claim 2, characterized in that The preparation method of the release microspheres comprises the following steps: synthesizing the release microspheres of insulin-like growth factor and high molecular weight polylactic acid-glycolic acid copolymer by a traditional W / O / W composite method: (1) dissolving insulin-like growth factor in deionized water and dissolving high molecular weight poly(lactic acid-co-glycolic acid) copolymer in dichloromethane; (2) the two solutions were mixed and homogenized at 10,000 rpm for 1 min to promote emulsification, and the resulting single emulsion was rapidly emulsified in ddH2O containing 1% w / v polyvinyl alcohol at 400 rpm using a mechanical stirrer; (3) The emulsion is introduced into 200 ml of ddH2O containing 0.1% w / v methylcellulose solution and stirred continuously for 4 hours. The microspheres are shaken evenly, washed with water, and freeze-dried overnight to obtain the release microspheres containing insulin-like growth factor and high molecular weight polylactic acid-glycolic acid copolymer.

4. Use of the releasing microspheres as described in any one of claims 1 to 3 in skin wounds.

5. The use according to claim 4, characterized in that The released microspheres promote the proliferation of flap granulation tissue and the synthesis of collagen.

6. The use according to claim 4, characterized in that The released microspheres promote angiogenesis in HUVEC cells.

7. The use according to claim 4, characterized in that In skin wounds, the released microspheres promote angiogenesis and improve the survival rate and repair rate of transplanted skin flaps by inhibiting oxidative stress and regulating the Ang / Tie signaling pathway.

8. The use according to claim 7, characterized in that The released microspheres can inhibit the oxidative stress response of the perforator flap wound surface and promote flap proliferation.

9. The use according to claim 4, characterized in that The released microspheres promote the viability and migration ability of 3T3 cells.

10. The use according to claim 4, characterized in that The released microspheres promote 3T3 cell proliferation and inhibit apoptosis; the released microspheres inhibit T-BHP-induced 3T3 cell oxidative stress; the microspheres promote 3T3 cell migration and HUVEC cell proliferation by inhibiting oxidative stress.

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