WO2019041799A1 - 一种用于组织损伤修复的水凝胶及制备方法 - Google Patents

一种用于组织损伤修复的水凝胶及制备方法 Download PDF

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WO2019041799A1
WO2019041799A1 PCT/CN2018/080831 CN2018080831W WO2019041799A1 WO 2019041799 A1 WO2019041799 A1 WO 2019041799A1 CN 2018080831 W CN2018080831 W CN 2018080831W WO 2019041799 A1 WO2019041799 A1 WO 2019041799A1
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hydrogel
pge2
skin
solution
preparation
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French (fr)
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李宗金
张帅强
韩之波
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天津昂赛细胞基因工程有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

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  • the present invention relates to a hydrogel for tissue damage repair and a method of preparation.
  • PGE2 Prostaglandin E2
  • MSCs mesenchymal stem cells
  • the mechanism of action may be that PGE2 secreted by MSCs induces macrophage polarization to M2 type, promotes the secretion of IL-10, and then exerts anti-fibrosis and collagen deposition. Therefore, this led us to determine whether M2 type polarization can occur in macrophages under the stimulation of exogenous PGE2.
  • simple PGE2 has a short half-life and a short contact time with cells, which greatly reduces the value of PGE2 alone.
  • chitosan, collagen, fibrin and hyaluronic acid have good histocompatibility, biodegradability and rich biological activity.
  • Many types of scaffold materials based on these natural macromolecular compounds have shown important application value in the field of regenerative medicine. Therefore, the use of such biomaterials has the characteristics of a network structure, and the hydrogels formed thereon are coated.
  • Specific bioactive molecules to form bioactive hydrogels that slowly release specific growth factors, and the release of growth factors interacting with tissue cells can improve the local microenvironment and enhance the biological function of the cells, and Accelerate the repair and regeneration of the organization.
  • PGE2 hydrogels there are no reports of PGE2 hydrogels.
  • a second object of the present invention is to provide a method of preparing a hydrogel for tissue damage repair.
  • a third object of the present invention is to provide a use of a hydrogel for tissue damage repair in the preparation of a skin damage repair preparation.
  • a method for preparing a hydrogel for tissue damage repair comprising the steps of:
  • PGE2 powder is dissolved in a phosphate buffer solution to prepare a PGE2 solution having a concentration of 0.1 mg/ml to 5 mg/ml, and the PGE2 solution is added dropwise to the step at a mass ratio of 1:1-4.
  • the obtained hydrogel is stirred at 2-8 ° C for 3-5 hours; stored at 2-8 ° C or frozen at -18-22 ° C, which is a shorthand for prostaglandin E2.
  • the hydrogel material is preferably at least one of chitosan, collagen, hyaluronic acid, and fibrin.
  • the hydrogel material in the hydrogel has a mass content of from 0.01% to 5%.
  • a hydrogel prepared for tissue damage repair prepared by the above method.
  • hydrogel for tissue damage repair is used in the preparation of a skin damage repair preparation.
  • the hydrogel for tissue damage repair of the present invention can prolong the release time of PGE2 to some extent.
  • Direct treatment for tissue damage can promote the recovery of damaged skin histology and function. It can induce the macrophage of the injury site to inhibit the inflammatory response to M2 type polarization, accelerate the healing of the injury, and promote the angiogenesis of the injury site at an early stage.
  • the hydrogel of the present invention is non-cytotoxic and can be directly applied to the wound surface, is convenient to use, is inexpensive, and has an effective therapeutic effect.
  • the hydrogel of the invention can be applied to the damaged part of the skin to realize the slow release of PGE2, prolong the action time of PGE2 and the tissue cells in the injured part, and improve the microenvironment of the damaged part, thereby providing a new perspective for the repair and treatment of skin damage.
  • the present invention is a PGE2 hydrogel coated with chitosan, collagen, fibrin and hyaluronic acid.
  • the hydrogel has the characteristics of slowly releasing PGE2 and promoting the polarization of macrophages to M2 type, and also relates to such PGE2.
  • hydrogel can inhibit inflammation at the injury site, enhance the expression of angiogenic factors at the injury site, improve the angiogenesis of damaged skin, and promote the repair of skin damage.
  • Figure 1 shows the temperature stability of a hydrogel for tissue damage repair.
  • FIG. 2 The cells were treated with PGE2 and PGE2 hydrogels to check the level of PGE2 in culture medium at different time points. Among them, A was treated with PGE2 to check the level of PGE2 in culture medium at different time points. B was treated with PGE2 hydrogel to check the level of PGE2 in culture medium at different time points.
  • Figure 3 assesses the appropriate hydrogel concentration for tissue damage repair.
  • FIG. 4 shows that hydrogels for skin lesion repair can induce macrophage polarization to M2 in vitro;
  • A macrophages with PBS, chitosan hydrogel (CS hydrogel), PGE2, PGE2 After co-culture of hydrogel, LPS and IL-4 for 48 hours, the expression of CD206 and CD68 was detected by immunofluorescence.
  • B Quantitative analysis data for the A map. PGE2 hydrogel can significantly increase the expression of CD206.
  • IL-4 was used as a positive control to increase CD206 expression.
  • Figure 5 shows the effect of PGE2 hydrogel on the expression of macrophage inflammatory factors;
  • A treatment of macrophages with PBS, CS hydrogel, PGE2 and PGE2 hydrogel (CS+PGE2) for 48 hours, M2 Western detection of macrophage-associated gene IL-10 and M1 macrophage-associated gene IL-6.
  • B Quantitative data for IL-10 protein expression.
  • PGE2 hydrogel significantly promoted the expression of macrophage inflammatory factor IL-10.
  • C Quantitative data for IL-6 protein expression.
  • Figure 6 shows the therapeutic effect of PGE2 hydrogel on skin lesions.
  • A An animal model of resectable skin lesions was treated with PGE2 hydrogel, PGE2, CS hydrogel and PBS. The area of the lesion of the animal model was measured every 3 days.
  • B Quantitative analysis of the rate of wound healing in each group, showing that PGE2 hydrogel can significantly promote wound healing;
  • C skin tissue on the 7th and 14th day after treatment for HE staining.
  • Figure 7 shows that hydrogels for skin lesion repair enhance the anti-inflammatory ability of skin lesion sites, and molecular imaging traces the effect of PGE2 hydrogel on reactive oxygen species in skin lesion sites.
  • BLI was used to detect ROS in PGE2 hydrogels, PGE2, CS hydrogels, and lesion sites at different time points after PBS treatment.
  • B Quantitative analysis of BLI signals for different groups of ROS activity.
  • Figure 8 shows the hydrogel-induced macrophage-induced M2-type polarization in skin damage repair in vivo, and quantitative analysis of the number of CD206-positive cells on the 1st, 4th, and 7th day after treatment in each group.
  • Figure 9 Effect of molecular imaging tracer PGE2 hydrogel on VEGF expression levels in skin lesion sites.
  • A Expression of Vegfr2 was detected by bioluminescence on days 0, 4, 7, 10 and 14 after treatment in each group.
  • B Quantitative analysis of bioluminescent signals.
  • a method for preparing a hydrogel for tissue damage repair comprising the steps of:
  • PGE2 powder is dissolved in a neutral phosphate buffer solution to prepare a PGE2 solution having a concentration of 1 mg/ml, and the PGE2 solution is added dropwise to the step (1) at a mass ratio of 1:2.
  • the hydrogel was stirred at 6 ° C for 4 hours; at 6 ° C, the PGE 2 was abbreviated as prostaglandin E2.
  • This embodiment can also be stored at 2 ° C or at 8 ° C.
  • a method for preparing a hydrogel for tissue damage repair comprising the steps of:
  • PGE2 powder is dissolved in a neutral phosphate buffer solution to prepare a PGE2 solution having a concentration of 0.1 mg/ml, and the PGE2 solution is added dropwise to the step (1) at a mass ratio of 1:1.
  • the hydrogel was stirred at 2 ° C for 5 hours; at -18 ° C, the PGE 2 was abbreviated as prostaglandin E2.
  • a method for preparing a hydrogel for tissue damage repair comprising the steps of:
  • PGE2 powder is dissolved in a neutral phosphate buffer solution to prepare a PGE2 solution having a concentration of 5 mg/ml, and the PGE2 solution is added dropwise to the step (1) at a mass ratio of 1:4.
  • the mixture was stirred at 8 ° C for 3 hours; at -22 ° C, the PGE 2 was abbreviated as prostaglandin E2.
  • hydrogels for tissue damage repair are referred to as PGE2 hydrogels.
  • PGE2 hydrogel prepared in Example 1 stored at different temperatures (-80 ° C, -20 ° C, 0 ° C, 4 ° C and 37 ° C) was added to 96. Macrophages were cultured in wells and CCK-8 staining was performed 24 hours later. The cells were plated at a concentration of 3 x 10 4 /well (4 wells/group) (Fig. 1).
  • the specific dyeing method is as follows:
  • the time is set from 0.5h to 48h.
  • the cell culture supernatants of each group were collected by using PGE2, PGE2 hydrogel and cell culture for a certain period of time.
  • the microplate reader detects the absorbance at a wavelength of 412 nm.
  • the quantitative results of PGE2 are expressed in terms of mass concentration. The results showed that: (Fig. 2A) PGE2 decreased linearly in the concentration of the culture solution. (Fig. 2B)
  • the cells were treated with PGE2 hydrogel.
  • the concentration of PGE2 in the culture medium peaked at the 10th hour and then slowly decreased.
  • the maintenance of the effective concentration was significantly better than the direct use of PGE2.
  • Hydrogels used for tissue damage repair prolong the release time of PGE2.
  • peritoneal macrophages were exposed to different concentrations of PGE2 for 24 hours.
  • VEGF gene expression increased in a concentration-dependent manner and peaked at 1 ⁇ M.
  • the optimum PGE2 hydrogel concentration was 1 ⁇ mol/L (Fig. 3).
  • PGE2 hydrogel (prepared in Experimental Example 1) induces macrophage to M2 type polarization in vitro
  • Macrophages of mouse ascites were counted by screening, and inoculated into a 48-well plate of a prior glass plate at 1 ⁇ 10 4 .
  • the macrophages were pretreated under different conditions (LPS, IL-4, PBS, hydrogel alone, PGE2 alone and PGE2 hydrogel).
  • Triton X-100 prepared in PBS was permeabilized at room temperature for 15-20 min (the antigen expressed on the cell membrane was omitted).
  • M1/M2 related genes in macrophages of different condition pretreatments (LPS, IL-4, PBS, hydrogel alone, PGE2 solution and PGE2 hydrogel). It is shown that the expression of M1 marker gene (TNF- ⁇ , IL-6, iNOs, IL-1 ⁇ ) is most obvious in macrophage cells after LPS stimulation; M2 marker gene (CD206) after IL-4 stimulation in vitro , IL-10, IL-1ra, Arg-1) have the highest expression level; PBS-stimulated macrophages express M1/M2 related marker gene levels close to LPS-treated macrophages, macrophages stimulated by PGE2 alone The expression levels of related genes approached IL-4-treated macrophages, while the expression of macrophage-associated genes treated with PGE2 hydrogel was closer to M2 macrophages.
  • PGE2 hydrogel (prepared in Experimental Example 1) accelerates the healing of skin damage
  • Skin resection lesions were performed on 8-10 week old FVB male mice. Grouped according to the type of treatment the animals received (3 in each group): PBS group (supplied with skin lesions + injury site smeared with 20 ⁇ l PBS); hydrogel group alone (subjected to skin damage + injury site smeared 20 ⁇ l hydrogel) ); PGE2 alone (applying skin damage + injury site 20 microliters of PGE2 solution); PGE2 hydrogel group (substituting skin damage + injury site 20 microliters of PGE2 hydrogel).
  • mice were anesthetized by intraperitoneal injection of chloral hydrate (4%, 350 mg/kg), and the mice were fixed in a prone position on a surgical heating pad, and the shaving device was used to remove the back hair and the skin of the iodophor disinfection area;
  • the digital camera recorded 0, 1, 4, 7, 10, and 13 days postoperative wound healing.
  • Image-Pro Plus 6.0 image analysis software measured the wound area and calculated the wound healing rate.
  • Wound healing rate [(original wound area - current time point measurement area) / original wound area] ⁇ 100%
  • mice were selected from each group at 7 and 14 days after operation. After anesthesia, fresh skin tissue and a few normal skin around the wound were taken and fixed with 4% paraformaldehyde for 1 d, gradient dehydration, paraffin embedding, and 5 ⁇ m serial sections. Conventional hematoxylin-eosin staining, neutral gum seal, observation of neonatal skin pathological changes under light microscope (Fig. 6C): (C) HE staining of skin tissue on day 7 and day 14 after treatment, observation of wound The closing situation. PGE2 hydrogel can better promote the recovery of skin function.
  • PGE2 hydrogel prepared in Experimental Example 1 enhances the anti-inflammatory ability of skin lesion sites
  • mice were anesthetized by intraperitoneal injection of chloral hydrate (4%, 350 mg/kg).
  • mice were intraperitoneally injected with the firefly luciferase substrate Lumino (100 mg/kg).
  • the mouse was placed in a dark box of a small animal living imaging system, placed in a position (prone position), and the head was aligned with the vent in the dark box.
  • PGE2 hydrogel prepared in Experimental Example 1 induced macrophage polarization at the site of injury to M2
  • PGE2 hydrogel (prepared in Experimental Example 1) promotes angiogenesis at the injury site
  • VEGF-R2-Fluc transgenic mice were subjected to skin resection lesions and grouped according to the treatment.
  • BLI was used to monitor angiogenesis in real time, and each treatment was compared to promote VEGF-R2 gene expression (Fig. 9).
  • the results showed that the expression of VEGF-R2 gene in the injured site was significantly higher in the mice treated with PGE2 hydrogel than in the other animals (p ⁇ 0.05).
  • angiogenesis at the site of skin injury was evaluated by CD31 staining, and the number of angiogenesis in the PGE2 hydrogel treatment group was significantly higher than that in the other groups (p ⁇ 0.05) (Fig. 10).

Abstract

本发明公开了一种用于组织损伤修复的水凝胶及制备方法，其制备方法包括如下步骤：使用无菌蒸馏水溶解水凝胶材料，溶胀，获得水凝胶；将PGE2粉末溶于磷酸盐缓冲液中制成PGE2溶液，将PGE2溶液滴加到水凝胶中，在2-8℃条件下，搅拌3-5小时；保存或冻存，所述PGE2为前列腺素E2的简写。本发明的水凝胶能够一定程度的延长PGE2的释放时间，直接用于组织损伤的治疗，可以促进损伤皮肤组织学和功能学的恢复。可以诱导损伤位点巨噬细胞向M2型极化抑制炎症反应，加速了损伤的愈合，并在早期促进了损伤部位的血管新生。此外，本发明的水凝胶无细胞毒性，可直接涂抹于创伤表面，使用方便，价格低廉，并具有有效的治疗作用。

Description

一种用于组织损伤修复的水凝胶及制备方法 技术领域
本发明涉及一种用于组织损伤修复的水凝胶及制备方法。
背景技术
皮肤损伤(Skin Wound)已经成为一种严重的健康问题。创伤、烧伤、慢性溃疡等造成大面积皮肤缺损，常形成难以愈合的创面，不仅给患者造成痛苦，而且降低了生活质量。当前被证明有效的治疗方法(包括干细胞治疗和皮肤移植)存在医疗成本高等缺陷，其使用受到限制。前列腺素E2(Prostaglandin E2,PGE2)是一种重要的细胞生长和调节因子，其作用为扩张血管，增加器官血流量，同时具有免疫抑制和抗炎作用。并且有报道称间充质干细胞(Mesenchymal stem cells,MSCs)可以通过调控局部炎症反应而发挥促进皮肤愈合的作用。其中的作用机制可能是，MSCs分泌的PGE2诱导了巨噬细胞向M2型极化，促进了IL-10的分泌，进而发挥抗纤维化和减少胶原蛋白沉积的作用。因此，这使我们想确定在外源性PGE2的刺激下是否能使巨噬细胞发生M2型的极化。然而，单纯的PGE2半衰期短，与细胞的接触时间比较短，这一特性大大降低了PGE2的单独使用价值。
壳聚糖、胶原、纤维蛋白和透明质酸作为一类天然大分子化合物，具有较好的组织相容性、生物可降解性及丰富的生物学活性。以这些天然大分子化合物为基础的多种类型支架材料已经在再生医学领域显示出重要的应用价值，因此，利用这类生物材料具有网状结构的特征，将其形成的水凝胶包被上特定的生物活性分子，从而制成生物活性水凝胶，这种水凝胶能够缓慢释放特定生长因子，释放的生长因子与组织细胞相互作用可以改善局部微环境，增强细胞的生物学作用，并加快组织的修复和再生。目前，尚未有将含有PGE2水凝胶的报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种用于组织损伤修复的水凝胶。
本发明的第二个目的是提供一种用于组织损伤修复的水凝胶的制备方法。
本发明的第三个目的是提供一种用于组织损伤修复的水凝胶的在制备皮肤损伤修复制剂中的应用。
本发明的技术方案概述如下：
一种用于组织损伤修复的水凝胶的制备方法，包括如下步骤：
(1)使用无菌蒸馏水溶解水凝胶材料，2-8℃溶胀12-24小时，获得水凝胶；
(2)将PGE2粉末溶于磷酸盐缓冲液中制成浓度为0.1mg/ml-5mg/ml的PGE2溶液，按质量比为1:1-4的比例，将所述PGE2溶液滴加到步骤(1)获得的水凝胶中，在2-8℃条件下，搅拌3-5小时；2-8℃保存或在-18--22℃冻存，所述PGE2为前列腺素E2的简写。
所述水凝胶材料优选为壳聚糖、胶原、透明质酸和纤维蛋白中至少一种。
所述水凝胶中水凝胶材料的质量含量为0.01％-5％。
上述方法制备的用于组织损伤修复的水凝胶。
上述用于组织损伤修复的水凝胶在制备皮肤损伤修复制剂中的应用。
本发明的优点：
本发明的用于组织损伤修复的水凝胶能够一定程度的延长PGE2的释放时间。直接用于组织损伤的治疗，可以促进损伤皮肤组织学和功能学的恢复。可以诱导损伤位点巨噬细胞向M2型极化抑制炎症反应，加速了损伤的愈合，并在早期促进了损伤部位的血管新生。此外，本发明的水凝胶无细胞毒性，可直接涂抹于创伤表面，使用方便，价格低廉，并具有有效的治疗作用。
本发明的水凝胶，涂抹于皮肤损伤部位可以实现PGE2的缓慢释放，延长PGE2与损伤部位组织细胞的作用时间，改善损伤部位的微环境，从而为皮肤损伤的修复治疗提供新的视角。
本发明以壳聚糖、胶原、纤维蛋白和透明质酸包被的PGE2水凝胶，这类水凝胶具有缓慢释放PGE2和促进巨噬细胞向M2型极化的特性，还涉及这类PGE2水凝胶在以皮肤损伤为特征的疾病的局部涂抹治疗中，能够抑制了损伤部位炎症，增强损伤部位血管新生因子表达，改善损伤皮肤血管新生，促进皮肤损伤修复的作用。
附图说明
图1表示用于组织损伤修复的水凝胶的温度稳定性。
图2用PGE2、PGE2水凝胶处理细胞检查不同时间点培养液PGE2水平。其中A用PGE2处理细胞检查不同时间点培养液PGE2水平，B用PGE2水凝胶处理细胞检查不同时间点培养液PGE2水平。
图3评估适宜的用于组织损伤修复的水凝胶浓度。
图4表示用于皮肤损伤修复的水凝胶在体外能够诱导巨噬细胞向M2型极化；(A)将巨噬细胞与PBS、壳聚糖水凝胶(CS水凝胶)、PGE2、PGE2水凝胶、LPS和IL-4共培养48小时后，细胞免疫荧光检测CD206和CD68的表达。(B)对A图的定量分析数据。PGE2水凝胶能明显提高CD206的表达。IL-4作为提高CD206表达的阳性对照。
图5为PGE2水凝胶对巨噬细胞炎症因子表达的影响；(A)用PBS、CS水凝胶、PGE2和PGE2水凝胶(是CS+PGE2)处理巨噬细胞48小时后，M2型巨噬细胞相关基因IL-10和M1型巨噬细胞相关基因IL-6的Western检测。(B)IL-10蛋白表达的定量数据。PGE2水凝胶对巨噬细胞炎症因子IL-10表达具有明显的促进。(C)IL-6蛋白表达的定量数据。
图6为PGE2水凝胶对皮肤损伤的治疗作用，(A)用PGE2水凝胶、PGE2、CS水凝胶和PBS治疗切除性皮肤损伤动物模型。每3天测量动物模型的损伤部位面积。(B)定量分析各组伤口愈合的速度，显示PGE2水凝胶能明显促进伤口愈合；(C)取治疗后第7天和第14天皮肤组织进行HE染色。
图7表示用于皮肤损伤修复的水凝胶增强了皮肤损伤位点抗炎能力，分子影像示踪PGE2水凝胶对皮肤损伤部位活性氧的影响。(A)BLI检测PGE2水凝胶、PGE2、CS水凝胶和PBS治疗后不同时间点损伤位点的ROS。(B)BLI信号的定量分析不同组ROS活性的高低。
图8表示体内用于皮肤损伤修复的水凝胶诱导巨噬细胞向M2型极化，定量分析各组治疗后的第1、4、7天损伤位点CD206阳性细胞的数量。
图9分子影像示踪PGE2水凝胶对皮肤损伤部位VEGF表达水平的影响，(A)在各组治疗后的第0、4、7、10和14天，用生物发光检测Vegfr2的表达。(B)生物发光信号的定量分析。
图10PGE2水凝胶治疗后皮肤血管新生的评价。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
实施例1
一种用于组织损伤修复的水凝胶的制备方法，包括如下步骤：
(1)使用无菌蒸馏水溶解壳聚糖，6℃溶胀18小时，获得壳聚糖质量含量为1％的水凝胶；
(2)将PGE2粉末溶于中性磷酸盐缓冲液中制成浓度为1mg/ml的PGE2溶液，按质量比为1:2的比例，将所述PGE2溶液滴加到步骤(1)获得的水凝胶中，在6℃条件下，搅拌4小时；6℃保存，所述PGE2为前列腺素E2的简写。
本实施例也可以采用2℃保存或8℃保存。
实施例2
一种用于组织损伤修复的水凝胶的制备方法，包括如下步骤：
(1)使用无菌蒸馏水溶解水凝胶材料(质量比为1：1的胶原和透明质酸)，2℃溶胀24小时，获得水凝胶材料质量含量为0.01％的水凝胶；
(2)将PGE2粉末溶于中性磷酸盐缓冲液中制成浓度为0.1mg/ml的PGE2溶液，按质量比为1:1的比例，将所述PGE2溶液滴加到步骤(1)获得的水凝胶中，在2℃条件下，搅拌5小时；-18℃冻存，所述PGE2为前列腺素E2的简写。
实施例3
一种用于组织损伤修复的水凝胶的制备方法，包括如下步骤：
(1)使用无菌蒸馏水溶解纤维蛋白，8℃溶胀12小时，获得纤维蛋白质量含量为5％的水凝胶；
(2)将PGE2粉末溶于中性磷酸盐缓冲液中制成浓度为5mg/ml的PGE2溶液，按质量比为1:4的比例，将所述PGE2溶液滴加到步骤(1)获得的水凝胶中，在8℃条件下，搅拌3小时；-22℃冻存，所述PGE2为前列腺素E2的简写。
以下内容中，用于组织损伤修复的水凝胶简称PGE2水凝胶。
实验1：
PGE2水凝胶(实验例1制备)的特性
PGE2水凝胶的温度稳定性
为了评价温度对PGE2水凝胶稳定性的影响，将不同温度(-80℃、-20℃、0℃、4℃和37℃)条件下存放的PGE2水凝胶(实施例1制备)加入96孔板培养巨噬细胞，24h后进行CCK-8染色。细胞均按照3×10 4/孔的浓度铺盘(4孔/组)(图1)。
具体染色方法如下：
(1)配制CCK-8染色液(按照CCK-8原液:完全培养基＝1:9进行稀释)；
(2)吸出培养基；
(3)每孔加入100μl染色液，将孔板放入温箱(37℃)孵育4h；
(4)吸出上清(100μl)并转移到新的孔板中；
(5)使用酶标仪检测(吸光度)OD450值；
酶联免疫吸附试验检测PGE2释放(见图2)
时间设为0.5h至48h。分别用PGE2、PGE2水凝胶与细胞培养一定时间后收集各组细胞培养上清液。按照PGE2酶联免疫试剂盒说明书进行操作，酶标仪检测在波长412nm处的吸光度值。PGE2的定量结果用质量浓度表示。结果显示：(图2A)PGE2在培养液的浓度直线下降。(图2B)以PGE2水凝胶处理细胞，培养液中的PGE2浓度在第10小时达到峰值，然后缓慢下降，有效浓度的维持明显优于直接使用PGE2。
用于组织损伤修复的水凝胶延长了PGE2的释放时间。
PGE2水凝胶(实验例1制备)浓度的选择
(1)将不同浓度的PGE2水凝胶加入细胞培养液与巨噬细胞共同孵育；
(2)选择浓度分别为0，0.35，0.70，1.00，2.00和5.00μmol/L的PGE2水凝胶；
结果表明，将腹腔巨噬细胞暴露于不同浓度的PGE2中24小时。VEGF基因表达以浓度依赖性方式增加，在1μM达到峰值。最适的PGE2水凝胶浓度为1μmol/L(图3)。
实验2：
PGE2水凝胶(实验例1制备)体外诱导巨噬细胞向M2型极化
细胞爬片的免疫荧光染色
(1)提取小鼠腹水的巨噬细胞经筛选后计数，以1×10 4接种于事先玻璃片的48孔板。
(2)待细胞贴壁后，以不同的条件(LPS，IL-4，PBS，单独水凝胶，单独PGE2溶液和PGE2水凝胶)预处理巨噬细胞。
(3)将细胞置于培养箱中进行培养，48小时后弃上清，在培养板中将已爬好细胞的玻璃片用PBS浸洗3次。
(4)用4％的多聚甲醛固定爬片15min，PBS浸洗玻璃片3次。
(5)0.5％Triton X-100(PBS配制)室温通透15-20min(细胞膜上表达的抗原省略此步骤)。
(6)PBS浸洗玻璃片3次，每次3min，吸水纸吸干PBS，在玻璃片上滴加正常山羊血清，室温封闭30min。
(7)吸水纸吸掉封闭液，不洗，每张玻璃片上滴加足够量的稀释好的一抗并放入湿盒，4℃孵育过夜。
(8)加荧光二抗：PBS浸洗爬片3次，每次3-5min，吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗，湿盒中20-37℃避光孵育2h，PBS浸洗爬片3次，每次3min。
(9)复染核：滴加DAPI避光孵育5min，对标本进行染核，PBS洗3次洗去多余的DAPI。
(10)用吸水纸吸干爬片上的液体，用含抗荧光淬灭剂的封片液封片，然后在荧光显微镜下观察采集图像。
加入的LPS和IL-4作为阳性对照，应用的PGE2水凝胶浓度为1μmol/L。研究结果显示，PGE2水凝胶能够明显诱导巨噬细胞向M2型极化(图4)：(A)将巨噬细胞与PBS、CS水凝胶、PGE2、PGE2水凝胶、LPS和IL-4共培养48小时后，细胞免疫荧光检测CD206和CD68的表达。(B)对A图的定量分析数据。PGE2水凝胶能明显提高CD206的表达。IL-4作为提高CD206表达的阳性对照。
此外，使用实时定量RT-PCR检测不同条件预处理(LPS，IL-4，PBS，单独水凝胶，单独PGE2溶液和PGE2水凝胶)的巨噬细胞M1/M2相关基因的表达水平，结果显示：LPS刺激后，巨噬细胞细胞中M1型标记基因(TNF-α、IL-6，iNOs,IL-1β)的表达升高最为明显；IL-4体外刺激后，M2型标记基因(CD206,IL-10,IL-1ra,Arg-1)的表达水平最高；PBS刺激的巨噬细胞表达M1/M2相关标记基因水平接近于LPS处理的巨噬细胞，经单独PGE2刺激后的巨噬细胞相关基因表达水平趋近IL-4处理的巨噬细胞，而经PGE2水凝胶处理的巨噬细胞相关基因表达更接近M2型巨噬细胞。且用Western Blot针对IL-10(M2型巨噬细胞高表达)和IL-6(M1型巨噬细胞高表达)两种蛋白进行检测，结果两种蛋白的表达水平变化趋势与免疫荧光染色和RT-PCR结果一致(图5)：(A)用PBS、CS水凝胶、PGE2和PGE2水凝胶处理巨噬细胞48小时后，M2型巨噬细胞相关基因IL-10和M1型巨噬细胞相关基因IL-6的Western检测。(B)IL-10蛋白表达的定量数据。PGE2水凝胶对巨噬细胞炎症因子IL-10表达具有明显的促进。(C)IL-6蛋白表达 的定量数据。PGE2水凝胶对巨噬细胞炎症因子IL-6表达具有明显的抑制。
实验3：
PGE2水凝胶(实验例1制备)能够加速皮肤损伤的愈合
对8-10周龄FVB雄性小鼠进行皮肤切除损伤。根据动物接受治疗类型进行分组(每组3只)：PBS组(经受皮肤损伤+损伤位点涂抹20微升PBS)；单独水凝胶组(经受皮肤损伤+损伤位点涂抹20微升水凝胶)；单独PGE2组(经受皮肤损伤+损伤位点涂抹20微升PGE2溶液)；PGE2水凝胶组(经受皮肤损伤+损伤位点涂抹20微升PGE2水凝胶)。
切除性皮肤损伤模型
(1)高压消毒手术器械(剪刀、尖镊、微型血管夹、持针器)，使用医用酒精消毒手术台区域；
(2)经腹腔注射水合氯醛(4％，350mg/kg)麻醉小鼠，将小鼠以俯卧位固定于手术加热垫上，使用剃毛器去除背部毛发，碘伏消毒术区皮肤；
(3)用无菌眼科剪在其背部形成一直径约1cm皮肤全层损伤创面，深及筋膜，无菌纱布止血；
(4)将3毫米厚度的环状有机硅胶片用尼龙缝线缝合在伤口上放，以防止伤口收缩；(5)将小鼠置于加热垫上复温，待苏醒后放回饲养笼。
创面愈合率测定
术后0、1、4、7、10、13d数码相机记录各组创面愈合情况，Image-Pro Plus 6.0图像分析软件测量创面面积，计算创面愈合率。
创面愈合率＝[(原始创面面积－当前时间点测量面积)/原始创面面积]×100％
HE染色和创面愈合率测定结果均表明：在切除性皮肤损伤模型中，PGE2水凝胶能够加速创面的愈合(图6)：(A)用PGE2水凝胶、PGE2、CS水凝胶和PBS治疗切除性皮肤损伤动物模型。每3天测量动物模型的损伤部位面积。(B)定量分析各组伤口愈合的速度，显示PGE2水凝胶能明显促进伤口愈合。
HE染色
分别于术后7天和14天每组选取10只小鼠，麻醉后取创面新生皮肤组织及周围少许正常皮肤，用4％多聚甲醛固定1d，梯度脱水，石蜡包埋，5μm连续切片，常规苏木精-伊红染色，中性树胶封片，光学显微镜下观察新生皮肤病理学改变(图6C)：(C)取治疗后第7天和第14天皮肤组织进行HE染色，观察伤口的闭合情况。PGE2水凝胶能更好的促进皮肤功能的恢复。
实验4：
PGE2水凝胶(实验例1制备)增强了皮肤损伤位点抗炎能力
评价损伤位点ROS的含量
为了评价PGE2水凝胶能否增强损伤位点的抗炎能力，我们在小鼠皮肤切除性损伤后不同时间点，使用生物发光成像技术评价损伤位点ROS的含量(图7)：分子影像示踪PGE2水凝胶对皮肤损伤部位活性氧的影响。(A)BLI检测PGE2水凝胶、PGE2、CS水凝胶和PBS治疗后不同时间点损伤位点的ROS。(B)BLI信号的定量分析不同组ROS活性的高低。PGE2水凝胶能明显降低损伤部位的活性氧水平。
小动物活体生物发光成像
(1)经腹腔注射水合氯醛(4％，350毫克/千克)麻醉小鼠。
(2)向小鼠经腹腔注射萤火虫荧光素酶底物Lumino(100毫克/千克)。
(3)底物注射5分钟后，将小鼠放入小动物活体成像系统暗箱内，摆放好体位(俯卧位)，将其头部对准暗箱内出气孔。
(4)根据荧光信号强度调整曝光时间，一般为0.5-2分钟，连续采集图像直到信号强度开始下降。
(5)数据分析：使用Living Image软件测定每只小鼠的荧光信号强度，进行统计学分析。
实验5：
PGE2水凝胶(实验例1制备)诱导了损伤位点巨噬细胞向M2型极化
我们对治疗后小鼠皮肤冰冻切片进行免疫荧光染色，定量分析各组治疗后的第1、4、7天损伤位点CD206阳性细胞的数量(图8)。发现随着时间的变化，在1、4、7天表达M2型巨噬细胞表面分子标志CD206的巨噬细胞比例上升，且PGE2水凝胶治疗组明显高于其他组。
皮肤组织冰冻切片免疫染色
(1)将切片从-20℃冰箱中取出，室温放置30min；
(2)将切片放入(-20℃预冷30min的)丙酮原液中固定10min；
(3)将切片置于空气中干燥30min；
(4)使用PBS洗涤切片(5min，2次)；
(5)破膜：0.1％Triton X-100，室温，10min；PBS洗涤，5min，2次；
(6)血清封闭，4℃，45min；
(7)加一抗，4℃，过夜；
(8)次晨复温，1h；PBS洗涤，5min，4次；
(9)加二抗(避光)，室温，2h；PBS洗涤，5min，5次；
(10)DAPI封片，荧光显微镜下观察，拍照。
实验6：
PGE2水凝胶(实验例1制备)促进损伤部位血管新生作用
VEGF-R2转基因小鼠评价血管新生
为了评价PGE2水凝胶能否促进损伤部位血管新生作用，我们对VEGF-R2转基因小鼠进行皮肤切除性损伤处理(方法如前所述)。分组(每组3只)：PBS组(经受皮肤损伤+损伤位点涂抹20微升PBS)；单独水凝胶组(经受皮肤损伤+损伤位点涂抹20微升水凝胶)；单独PGE2组(经受皮肤损伤+损伤位点涂抹20微升PGE2溶液)；PGE2水凝胶组(经受皮肤损伤+损伤位点涂抹20微升PGE2水凝胶)。在损伤后不同时间点，使用BLI技术(方法如前所述)评价VEGF-R2基因的表达。
免疫荧光染色方法见实验6
为了阐明PGE2水凝胶促进损伤位点血管新生的机制，对表达VEGF-R2-Fluc转基因小鼠进行皮肤切除性损伤，并根据治疗方式分组。损伤后不同时间点，使用BLI技术实时监测血管生成，比较各治疗方式促进VEGF-R2基因表达的情况(图9)。结果显示，接受PGE2水凝胶治疗的小鼠，在损伤位点VEGF-R2基因的表达显著高于接受其他治疗的动物(p＜0.05)。损伤治疗后1，4，7天，利用CD31染色评价皮肤损伤位点血管新生情况，发现PGE2水凝胶治疗组血管新生数量显著高于其他组(p＜0.05)(图10)。
实验证明，实施例2和实施例3制备的PGE2水凝胶性质和效果与实施例1制备的PGE2水凝胶相似。

Claims (5)

  1. 一种用于组织损伤修复的水凝胶的制备方法，其特征是包括如下步骤：
    (1)使用无菌蒸馏水溶解水凝胶材料，2-8℃溶胀12-24小时，获得水凝胶；
    (2)将PGE2粉末溶于磷酸盐缓冲液中制成浓度为0.1mg/ml-5mg/ml的PGE2溶液，按质量比为1:1-4的比例，将所述PGE2溶液滴加到步骤(1)获得的水凝胶中，在2-8℃条件下，搅拌3-5小时；2-8℃保存或在-18--22℃冻存，所述PGE2为前列腺素E2的简写。
  2. 根据权利要求1所述的方法，其特征是所述水凝胶材料为壳聚糖、胶原、透明质酸和纤维蛋白中至少一种。
  3. 根据权利要求1或2所述的方法，其特征是所述水凝胶中水凝胶材料的质量含量为0.01％-5％。
  4. 权利要求1-3之一所述的方法制备的用于组织损伤修复的水凝胶。
  5. 权利要求4的用于组织损伤修复的水凝胶在制备皮肤损伤修复制剂中的应用。
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