WO2016037388A1 - 促进伤口愈合的制剂及其制备方法及使用方法 - Google Patents

促进伤口愈合的制剂及其制备方法及使用方法 Download PDF

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WO2016037388A1
WO2016037388A1 PCT/CN2014/087374 CN2014087374W WO2016037388A1 WO 2016037388 A1 WO2016037388 A1 WO 2016037388A1 CN 2014087374 W CN2014087374 W CN 2014087374W WO 2016037388 A1 WO2016037388 A1 WO 2016037388A1
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preparation
wound healing
endothelial
endothelial progenitor
cells
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French (fr)
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洪挺祯
蓝传清
陆家祺
刘承叡
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洪挺祯
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides

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  • the invention relates to a pharmaceutical preparation, in particular to a preparation for promoting wound healing, a preparation method thereof and a use method.
  • Diabetes is a common chronic disease in the world. It is a disease in which organisms change the way glucose (sugar) is converted into energy. The reason is that the body can't make enough insulin, or can't effectively use the insulin made in the body to convert sugar. Diabetes is prone to a variety of complications, one of which is vascular disease. Hyperglycemia hardens the cell membrane of red blood cells, causing blood flow to reach the wound tissue through the tiny blood vessels. Moreover, hemoglobin in diabetic patients is not easy to release oxygen, resulting in hypoxia and nutrient deficiency in wound tissue. In addition, hyperglycemia also affects the proliferation of fibroblasts and epithelial cells. Therefore, the wounds of diabetic patients tend to stagnate in the inflammatory phase and evolve into chronic ulcers.
  • hyperbaric oxygen therapy treatments for wound healing in diabetic patients include hyperbaric oxygen therapy, silver-containing activated carbon fiber dressings, and human platelet-derived growth factor (PDGF) gels.
  • the hyperbaric oxygen treatment system places the patient in a hyperbaric chamber, pressurizes the chamber and maintains it at 2.5 atmospheres of absolute pressure, allowing the person to absorb pure oxygen through the oxygen mask contained on the head to increase the oxygen concentration in the blood, thereby improving Tissue hypoxia, promote wound healing, and enhance the bactericidal ability of white blood cells.
  • this method may cause pressure damage to the lungs, eardrums, and sinuses due to high pressure, convulsions due to high concentrations of oxygen, and changes in vision due to swelling of the lens after treatment.
  • the silver-containing activated carbon fiber dressing is composed of a PET non-woven fabric, a silver-containing activated carbon fiber cloth, and a polyethylene (PE) film three-layer structure.
  • the activated carbon fiber itself can be adsorbed to absorb blood and water and bacteria.
  • silver ions can destroy the cell membrane and nucleus of bacteria, and achieve the effect of sterilization.
  • the function of far-infrared rays of activated carbon fibers has the effect of promoting blood circulation and accelerating metabolism, thereby shortening the time for wound healing.
  • this product still takes a long time to heal the wounds of diabetes.
  • the human platelet growth factor gel which contains a recombinant human platelet-derived growth factor (rh-PDGF), promotes wound healing by the growth factor contained therein, but is expensive.
  • the object of the present invention is to provide a preparation for promoting wound healing, a preparation method thereof and a use method thereof, which can prepare a preparation with accelerated wound healing by a simple and relatively low-cost preparation method, and the preparation can be anytime and anywhere Apply to patients.
  • the present invention provides a formulation for promoting wound healing, including a cell culture medium, which is a transdermal endothelial progenitor cell, which is a technique for solving the problems of the prior art. It is cultured and formed, and the transformed endothelial progenitor cells are formed by transducing microRNA (L)-7-7 to an endothelial progenitor cell.
  • a cell culture medium which is a transdermal endothelial progenitor cell, which is a technique for solving the problems of the prior art. It is cultured and formed, and the transformed endothelial progenitor cells are formed by transducing microRNA (L)-7-7 to an endothelial progenitor cell.
  • a formulation in which the cell culture medium is an endothelial basal medium (EBM) prior to culturing endothelial progenitor cells.
  • EBM endothelial basal medium
  • a formulation is provided, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient.
  • a formulation for promoting wound healing is provided, the excipient being monoglycerol.
  • a preparation method for a preparation for promoting wound healing comprising the following steps: a separation step: separating mononuclear cells from human whole blood, and The nucleus cell is seeded on a human-fibronectin-coated plate to obtain endothelial progenitor cells; the transgenic step: transfecting the tiny ribonucleotide let-7g to endothelial progenitor cells to obtain Transduced endothelial progenitor cells; and a culture step: the transduced endothelial progenitor cells are cultured for a predetermined period of time to obtain a preparation for promoting wound healing.
  • a preparation method in which a cultured medium of transformed endothelial precursor cells is cultured in an endothelial growth medium-2 (EGM-2). After 7 days, the endothelial cell growth medium was replaced with endothelial cell basal medium on the 7th day, followed by continuous culture for 2 days to collect endothelial cell basal medium to obtain a preparation for promoting wound healing.
  • EMM-2 endothelial growth medium-2
  • a method of preparation is provided, the predetermined time being 6 to 10 days.
  • a method of preparation in which mononuclear cells are isolated from human whole blood by a gradient gradient Histopaque-1077 by density gradient centrifugation.
  • Another technical means employed by the present invention to solve the problems of the prior art is to provide a method of promoting wound healing comprising: administering a therapeutically effective amount of a formulation to a wound of an organism.
  • a method of promoting wound healing is provided, the organism being a patient having diabetes.
  • endothelial progenitor cells and microRNAs are specifically selected by the technical means employed in the present invention.
  • Let-7g is used together to prepare a formulation that promotes wound healing. It is due to the ability of endothelial progenitor cells to differentiate into vascular endothelial cells to repair incomplete vascular endothelial layers and promote angiogenesis.
  • let-7g has been found in the past to protect vascular endothelial cells, treat and prevent diseases such as hardening of the arteries.
  • endothelial progenitor cells release factor-to-medium with accelerated wound healing.
  • the medium not only has a good effect of healing wounds, but also has a simple preparation method and is not expensive in time and cost, and therefore can be generally applied to patients whose wounds are not easily healed, such as diabetes, without increasing the number of patients. Economic pressure. Further, the preparation of the present invention is used by directly applying the preparation to the wound of a patient, and the operation method is simple and can be used anytime and anywhere.
  • FIG. 1 is a flow chart showing a formulation for promoting wound healing in accordance with an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing wound construction of a diabetic mouse in an experiment for promoting wound healing in an experiment for testing wound healing performance of a diabetic mouse according to an embodiment of the present invention.
  • Fig. 3 is a view showing the actual image of the degree of wound healing of each group of diabetic mice in an experiment for testing the wound healing performance of diabetic mice according to an embodiment of the present invention.
  • FIG. 4 is a line graph showing the trend of wound healing rate of each group of diabetic mice in an experiment for testing wound healing in a mouse according to an embodiment of the present invention.
  • a preparation for promoting wound healing comprising a cell culture medium formed by culturing a transformed endothelial progenitor cell and transfected
  • the plastid endothelial progenitor cells are formed by transfecting microRNA miRNA-7-7 into an endothelial progenitor cell.
  • the cell culture medium is an endothelial basal medium (EBM) for culturing endothelial progenitor cells before culturing the endothelial precursor cells.
  • EBM endothelial basal medium
  • the preparation of the present invention further comprises at least one pharmaceutically acceptable carrier, diluent or excipient for the purpose of improving the stability of the wound healing promoting formulation of the present invention, its wide range of applications, or reducing its irritation.
  • a glycerin is added to the formulation to convert the otherwise liquid formulation to a semi-fluid by the viscosity of the glycerin to facilitate application of the formulation to the topical application.
  • a method for preparing a preparation for promoting wound healing comprises the following steps:
  • Step S1 mononuclear cells are isolated from human whole blood, and mononuclear cells are seeded on a human-fibronectin-coated plate to obtain endothelial progenitor cells ( Step S1);
  • step S2 transfecting the tiny ribonucleotide let-7g to endothelial progenitor cells to obtain transfected endothelial progenitor cells
  • mononuclear cells were isolated from human whole blood by gradient gradient centrifugation by a gradient agent Histopaque-1077 to obtain endothelial progenitor cells.
  • 10 ml of the gradient agent Histopaque-1077 and 10 ml of human whole blood were uniformly mixed in a 50 c.c. centrifuge tube, and then centrifuged at 700 g for 30 minutes, and centrifuged to collect a mononuclear cell containing a high concentration. Buffy-coat layer.
  • the brownish yellow layer was suspended in 30 ml of DMEM (Dulbecco's Modified Eagle Medium), centrifuged at a rate of 250 g for 10 minutes, the supernatant was discarded and the cell pellet was retained, and the cell pellet was suspended in DMEM. The cells were centrifuged, and this step was repeated 2 to 3 times to obtain a mononuclear cell mass. Finally, the mononuclear cell pellet was back dissolved in Endothelial Cell Growth Medium-2 (EGM-2) and seeded with human fibronectin at a density of 3 ⁇ 10 6 cells/ml. The well plate is used to obtain endothelial progenitor cells.
  • DMEM Dynamic Eagle Medium
  • the transfer step 75 ng of let-7g was diluted in 100 ⁇ l of the medium and shaken to uniformly mix. Next, 3 ⁇ l of the transesterified liquid was added to the diluted let-7 g, uniformly mixed, and placed in an environment of 15 to 25 ° C for 5 to 10 minutes to form a transfer complex. The transgenic complex was added to the endothelial precursor cells, and after standing for 3 hours, the medium was further added to complete the transformation.
  • transgenic let-7g of endothelial progenitor cells were cultured in endothelial cell growth medium for 7 days, and on day 7 the endothelial cell growth medium was replaced with endothelial cell basal medium, followed by continuous culture for 2 days.
  • a preparation for promoting wound healing is obtained by collecting endothelial cell basal medium.
  • the cultured endothelial progenitor cells can be cultured for 6 to 10 days.
  • the method for promoting wound healing of the present invention for promoting wound healing is a method of administering a therapeutically effective amount of a preparation or a preparation containing a carrier, a diluent or an excipient to a wound of an organism, wherein the organism may be A patient with diabetes.
  • the following is an experiment in which the wound healing promoting preparation of the present invention is used for treating diabetic mice.
  • the main processes of this experiment included the construction of an animal model of diabetes, the construction of a wound model of a diabetic animal, the healing of a wound in a diabetic animal, and statistical analysis.
  • streptozotocin has a damaging effect on islet ⁇ cells, and mice in C57BL/6Jk are induced to develop diabetes.
  • streptozotocin was injected at a dose of 50 mg/kg in the peritoneum of 8 to 12 weeks of mice for 7 consecutive days, and the blood glucose concentration of the mice was controlled to be 150 to 200 mg/dL.
  • EBM mice were induced to have diabetes
  • let-7g(-)-EPC 7 were classified as let-7g(+)-EPC.
  • a total of 3 groups were grouped for the next experiment.
  • the EBM group treats wounds of diabetic mice with endothelial cell basal medium
  • the let-7g(-)-EPC group treats wounds of diabetic mice with medium cultured with endothelial progenitor cells
  • let-7g The (+)-EPC group was used to treat wounds of diabetic mice using a culture medium in which endothelial progenitor cells transformed with let-7 g were cultured.
  • the hairy part of the diabetic mouse whose part of the back is to be wounded is shaved and disinfected.
  • Two 6-mm symmetrical and deep-to-skin (Panniculus carnosus) wounds were created on the back of the mouse using a 6-mm puncture biopsy tool.
  • the annular silicon splint was placed and secured to the periphery of the wound and covered with a occlusive dressing on the wound surface.
  • endothelial cell basal medium 50 tl of endothelial cell basal medium, medium for culturing endothelial progenitor cells, and culture medium for transdermal let-7 g of endothelial progenitor cells are respectively 1 with glycerol.
  • a ratio of 1 was mixed, and the above three media were used to treat the right wound of the EBM group, the let-7g(-)-EPC-CM group, and the let-7g(+)-EPC-CM group.
  • the left wound was treated without any treatment, and it was naturally healed as a control group, and the control group was used to compare the rate of wound healing with the experimental group.
  • the frequency of treatment is daily, and the experiment is carried out until the wound is completely healed or other factors are over.
  • the wound healing-promoting preparation of the present invention was used for the wound healing of diabetic mice until the day when the wound healing degree of each group was significantly different, so the experiment was stopped before the time when the experiment was expected to be stopped.
  • wound residual rate will The daily wound area is divided by the original wound area.
  • the wound healing rate was reduced by 100% of the wound residual rate.
  • the difference in wound healing rate is the absolute value of the wound healing rate of the control group and the experimental group.
  • Table 2 below shows the wound healing rates of the three control groups comparing the EBM group, the let-7g(-)-EPC-CM group, and the let-7g(+)-EPC-CM group by the Kruskal-Wallis assay. As well as comparing the wound healing rates of the three experimental groups, it was found that there were significant differences. The reason is that although the wound of the control group is subjected to natural healing without any treatment, a part of the medium applied in the experimental group can still be absorbed into the body through the peripheral circulation, thereby affecting the wound healing in the distant (control group). .

Abstract

一种促进伤口愈合的制剂，包括细胞培养基，细胞培养基将经转质的内皮前躯细胞予以培养而形成，且经转质的内皮前驱细胞为将微小核醣核酸let-7g转质至一内皮前驱细胞而形成，促进伤口愈合的制剂的制备方法包括分离步骤、转质步骤、及培养步骤，分离步骤为自人类全血分离出单核球细胞，并将单核球细胞种在涂布有人类纤维连接蛋白的孔盘（human-fibronectin-coated plate）以得到内皮前躯细胞，转质步骤为将微小核醣核酸let-7g转质至内皮前驱细胞，以得到经转质的内皮前驱细胞，培养步骤为将经转质的内皮前驱细胞予以培养一预定时间而得到促进伤口愈合的制剂。

Description

促进伤口愈合的制剂及其制备方法及使用方法 技术领域
本发明相关于一种医药制剂，特别是相关于一种促进伤口愈合的制剂及其制备方法及使用方法。
背景技术
糖尿病为全球常见的慢性疾病，是一种生物体将葡萄糖(糖类)转换成能量的方式出现变化的疾病。原因为身体无法制造足够的胰岛素，或是无法有效利用体内制造的胰岛素以进行糖类的转换。糖尿病易引发多种并发症，其中之一为血管病变。高血糖使红血球的细胞膜变硬，导致血流不易通过微细血管而到达伤口组织。且，糖尿病患体内的血红素亦不易释放出氧气，而造成伤口组织缺氧以及营养素不足。此外，高血糖亦会影响纤维母细胞以及上皮细胞的增生，因此，糖尿病患的伤口容易停滞在发炎期，而演变成慢性溃疡。
目前相关于糖尿病患者伤口愈合的治疗方式主要包括高压氧治疗、含银活性碳纤维敷料、以及人类血小板衍生生长因子(platelet-derived growth factor，PDGF)凝胶。高压氧治疗系将病患置于高压舱内，舱内加压并维持在2.5绝对大气压下，使人经由其头上所载的氧气面罩吸取纯氧气，以提高血中含氧浓度，进而改善组织缺氧、促进伤口愈合、以及增强白血球的杀菌能力。然而此方式可能因高压造成肺部、耳膜、以及鼻窦的压力伤害、因高浓度氧气而造成抽搐、以及治疗后因水晶体变肿而导致视力改变。再者，此治疗方式对于病患极为不便，因病患无法自行于家中操作。含银活性碳纤维敷料系由PET不织布、含银活性碳纤维布、以及聚乙烯(polyethylene，PE)膜三层结构所组成。当敷料接触伤口渗液时，可藉由活性碳纤维本身良好的吸附性，以吸附血水渗液及细菌。同时，银离子可破坏细菌的细胞膜及细胞核，而达到杀菌的效果。此外，活性碳纤维的远红外线的功能具有促进血液循环以及加速新陈代谢的功效，进而缩短伤口愈合的时间。然而，此产品对于糖尿病患伤口的愈合仍需花费较长的时间。且，将纳米银粒均匀镀设于活性碳纤维布上，是一件繁复且加工不易的制作工序，加上纳米银粒的造价并不便宜，因而导致此产品的成本提高，从而降低商业竞争力。另外，人类血小板生长因子凝胶，含有基因重组的人类血小板源生长因子(Recombinate human platelet-derived growth factor，rh-PDGF)，藉由其所含的生长因子以促进伤口愈合，然而其费用昂贵。
发明内容
本发明的目的即在于提供一种促进伤口愈合的制剂及其制备方法及使用方法，能藉由简易且成本相对较低的制备方法以制备出具有加速伤口愈合的制剂，且此制剂能随时随地应用于病患身上。
本发明为解决习知技术的问题所采用的技术手段提供一种促进伤口愈合的制剂，包括细胞培养基(cell culture medium)，细胞培养基将经转质的内皮前躯细胞(endothelial progenitor cell)予以培养而形成，且经转质的内皮前驱细胞为将微小核醣核酸(microRNA)let-7g转质至一内皮前驱细胞而形成。
在本发明的一实施例中系提供一种制剂，细胞培养基于培养内皮前躯细胞之前系为内皮细胞基础培养基(endothelial basal medium，EBM)。
在本发明的一实施例中系提供一种制剂，还包括至少一种医药学上可接受的载剂、稀释剂或赋形剂。
在本发明的一实施例中系提供一种促进伤口愈合的制剂，赋形剂为一甘油。
本发明为解决习知技术的问题所采用的另一技术手段系提供一种促进伤口愈合的制剂的制备方法，包括以下步骤：分离步骤：自人类全血分离出单核球细胞，并将单核球细胞种在涂布有人类纤维连接蛋白的孔盘(human-fibronectin-coated plate)以得到内皮前躯细胞；转质步骤：将微小核醣核酸let-7g转质至内皮前驱细胞，以得到经转质的内皮前驱细胞；以及培养步骤：将经转质的内皮前驱细胞予以培养一预定时间而得到促进伤口愈合的制剂。
在本发明的一实施例中系提供一种制备方法，于培养步骤中，将经转质的内皮前驱细胞的培养系为培养于内皮细胞生长培养基(endothelial growth medium-2，EGM-2)7天，并于第7天将内皮细胞生长培养基更换为内皮细胞基础培养基，接着持续培养2天以收集内皮细胞基础培养基而得到促进伤口愈合的制剂。
在本发明的一实施例中系提供一种制备方法，预定时间为6至10天。
在本发明的一实施例中系提供一种制备方法，分离步骤中，藉由梯度剂Histopaque-1077以密度梯度离心法自人类全血中分离出单核球细胞。
本发明为解决习知技术的问题所采用的另一技术手段系提供一种促进伤口愈合的方法，包括：将治疗有效量的制剂投予至一生物体的伤口。
在本发明的一实施例中系提供一种促进伤口愈合的方法，生物体为一患有糖尿病的患者。
经由本发明所采用的技术手段，在本发明中，特别选用内皮前驱细胞以及微小核糖核酸 let-7g以共同制备出促进伤口愈合的制剂。其系因内皮前驱细胞具有分化成血管内皮细胞，以修复不完整的血管内皮层，以及促使血管新生的能力。而微小核醣核酸中let-7g则在过往研究发现其可保护血管内皮细胞，治疗及预防血管硬化等疾病。
藉由将let-7g转质入内皮前驱细胞，以调控内皮前驱细胞的表现，并培养此经转质let-7g的内皮前驱细胞，使内皮前驱细胞释放出具有加速伤口愈合的因子至培养基中。此培养基不仅具有良好的治愈伤口的效果，且，制备方法简单，所费的时间及成本皆不高，因此可普遍地应用于伤口不易愈合的患者，例如糖尿病患，而不增加病患于经济上的压力。此外，本发明的制剂的使用方法为直接将制剂施用于病患的伤口处，操作方式简便而可随时随地使用。
附图说明
图1为显示根据本发明的一实施例的一促进伤口愈合的制剂的流程图。
图2为显示根据本发明的实施例的一促进伤口愈合的制剂用于一测试糖尿病小鼠伤口愈合表现的实验中，糖尿病小鼠的伤口建构的示意图。
图3为显示根据本发明的实施例的促进伤口愈合的制剂用于测试糖尿病小鼠伤口愈合表现的实验中，各组糖尿病小鼠伤口愈合程度的实际影像图。
图4为显示根据本发明的实施例的促进伤口愈合的制剂用于测试糖尿病小鼠伤口愈合表现的实验中，各组糖尿病小鼠伤口愈合速率的趋势的折线图。
符号说明
S1  分离步骤
S2  转质步骤
S3  培养步骤
具体实施方式
以下根据图1，而说明本发明的实施方式。该说明并非为限制本发明的实施方式，而为本发明的实施例的一种。
依据本发明的一实施例的一促进伤口愈合的制剂，包括细胞培养基(cell culture medium)，细胞培养基将经转质的内皮前躯细胞(endothelial progenitor cell)予以培养而形成，且经转质的内皮前驱细胞为将微小核醣核酸(microRNA)let-7g转质至一内皮前驱细胞而形成。
详细而言，细胞培养基于培养内皮前躯细胞之前为内皮细胞基础培养基(endothelial basal medium，EBM)，用以特别培养内皮前躯细胞。
为了使本发明的促进伤口愈合的制剂稳定性佳、应用范围广泛、或减少其刺激性等因素，本发明的制剂还包括至少一种医药学上可接受的载剂、稀释剂或赋形剂。举例而言，将一甘油添加至制剂中，藉由甘油的黏性使得原本为液态的制剂转变为半流体，以便于本制剂应用在外敷的用途上。
在本实施例中，促进伤口愈合的制剂的制备方法，包括以下步骤：
(1)分离步骤：自人类全血分离出单核球细胞，并将单核球细胞种在涂布有人类纤维连接蛋白的孔盘(human-fibronectin-coated plate)以得到内皮前躯细胞(步骤S1)；
(2)转质步骤：将微小核醣核酸let-7g转质至内皮前驱细胞，以得到经转质的内皮前驱细胞(步骤S2)；以及
(3)培养步骤：将经转质的内皮前驱细胞予以培养一预定时间而得到促进伤口愈合的制剂(步骤S3)。
详细而言，在本实施例中系藉由梯度剂Histopaque-1077以密度梯度离心法自人类全血中分离出单核球细胞以得到内皮前躯细胞。将10ml的梯度剂Histopaque-1077以及10ml的人类全血加入50c.c.的离心管中均匀混合后，以700g的离心速度将其离心30分钟，离心后收集含有高浓度的单核球细胞的棕黄层(buffy-coat layer)。接着，将棕黄层悬浮于30ml的DMEM(Dulbecco′s Modified Eagle Medium)中，以250g的离心速度将其离心10分钟，舍弃上清液且保留细胞团块，再将细胞团块悬浮于DMEM中并予以离心，并重复此步骤2～3次以得到单核球细胞团块。最后，将单核球细胞团块回溶于内皮细胞生长培养基-2(Endothelial Cell Growth Medium-2，EGM-2)，以3x106cells/ml的密度种在涂布有人类纤维连接蛋白的孔盘以得到内皮前躯细胞。
于转质步骤中，将75ng的let-7g稀释在100μl的培养基中，并将之震荡以均匀地混合。接着，加入3μl的转质液至经稀释的let-7g，均匀混合后置于15～25℃的环境下5～10分钟以形成转质复合物。将转质复合物加到内皮前躯细胞，静置3小时后，再加入培养基以完成转质。
于培养步骤中，将经转质let-7g的内皮前驱细胞培养于内皮细胞生长培养基7天，并于第7天将内皮细胞生长培养基更换为内皮细胞基础培养基，接着持续培养2天以收集内皮细胞基础培养基而得到促进伤口愈合的制剂。当然，本发明不以此为限。根据细胞状况、转质 效率或实验上等需求，经转质的内皮前驱细胞的培养时间可为6至10天。
本发明的促进伤口愈合的制剂用于促进伤口愈合的方法为将治疗有效量的制剂或经添加载剂、稀释剂或赋形剂的制剂投予至一生物体的伤口，其中，生物体可为一患有糖尿病的患者。
以下为将本发明的促进伤口愈合的制剂用于治疗糖尿病小鼠的实验。此实验的主要流程系包括糖尿病动物模型的建构、糖尿病动物伤口模型的建构、糖尿病动物伤口的治愈、以及统计分析。
首先，利用链脲佐菌素(streptozotocin，STZ)具有对胰岛β细胞的破坏作用，诱发C57BL/6Jk的小鼠其糖尿病的产生。在本实施例中，将链脲佐菌素以50mg/kg的剂量注射于8至12周的小鼠腹膜内连续7天，并控制小鼠的血糖浓度在150～200mg/dL。20只小鼠经诱发糖尿病后，其中6只小鼠归为EBM组、7只小鼠归为let-7g(-)-EPC组、以及7只小鼠归为let-7g(+)-EPC组总共3个组别以进行接下来的实验。详细而言，EBM组为以内皮细胞基础培养基处理糖尿小鼠的伤口；let-7g(-)-EPC组为利用经培养内皮前躯细胞的培养基处理糖尿病小鼠的伤口；let-7g(+)-EPC组则为利用经培养经转质let-7g的内皮前躯细胞的培养基处理糖尿病小鼠的伤口。
请同时参照图2，糖尿病小鼠的模型建构完后，糖尿病小鼠其背部欲形成伤口的部位的毛系为被剃掉，并予以消毒。利用6-mm的穿刺活体细胞工具于小鼠背部制造出两个左右对称且深度达肉层(Panniculus carnosus)的伤口。将环状硅片夹板放置并固定于伤口外围，并以封闭敷料(occlusive dressing)覆盖于伤口表面。
糖尿病小鼠的伤口建立完成后，分别将50tl的内皮细胞基础培养基、经培养内皮前躯细胞的培养基、以及经培养经转质let-7g的内皮前躯细胞的培养基与甘油以1∶1的比例混合，并将上述三种培养基分别用以处理EBM组、let-7g(-)-EPC-CM组、以及let-7g(+)-EPC-CM组的小鼠的右边伤口而作为实验组，左边的伤口则未经任何处理，使之自然愈合而作为控制组，控制组系用以与实验组相比较其伤口愈合的速率。处理频率为每天，实验的进行直至伤口完全愈合或其它因素而结束。
本发明的促进伤口愈合的制剂用于糖尿病小鼠伤口的实验进行至9天各组的伤口愈合程度已有显著性差异，故在所预期停止实验的时间前即停止本实验。
本实验系藉由数字影像器材每天拍摄小鼠伤口区域的影像，并利用image-J分析系统计算伤口区域的像素以得到伤口残余率、伤口愈合速率、及伤口愈合速率差。伤口残余率为将 每日的伤口面积除以原始伤口面积。伤口愈合速率为以100％减掉伤口残余率。伤口愈合速率差为控制组与实验组其伤口愈合速率相减的绝对值。
如图3所示为实际拍摄各组小鼠伤口愈合状况的图片，可观察到let-7g(+)-EPC-CM组中的实验组其小鼠伤口愈合程度最为良好。
如下表1所示，其为利用McNemar检定以个别分析三个不同组别其控制组及实验组的伤口愈合速率，可得知无论是EBM组、let-7g(-)-EPC-CM组、或是let-7g(+)-EPC-CM组的实验组，相较于控制组皆有较高的伤口愈合速率。
【表1】
表1.在每个群组中伤口愈合速率的McNemar检定
Figure PCTCN2014087374-appb-000001
SPSS统计软件分析，P＜0.05具有显著性差异
下表2所示为藉由Kruskal-Wallis检定以比较EBM组、let-7g(-)-EPC-CM组、以及let-7g(+)-EPC-CM组的三个控制组的伤口愈合速率以及比较其三个实验组的伤口愈合速率，可得知其皆有显著性差异。其系因虽然控制组的伤口未经任何处理而采用自然愈合方式，然而于实验组所施用的培养基，其一部分仍可经由周边循环吸收到体内，进而影响远处(控制组)的伤口愈合。
【表2】
表2.群组之间伤口愈合速率的Kruskal-Wallis检定
Figure PCTCN2014087374-appb-000002
SPSS统计软件，P＜0.05具有显著性差异
为了进一步比较EBM组、let-7g(-)-EPC-CM组、与let-7g(+)-EPC-CM组的治愈效果，下表3～6为藉由Kruskal-Wallis检定及Mann-Whitney(M-W U)检定以分析三个组别以及三个组别中两两为一组的伤口愈合速率差。同时搭配图4所示的EBM组、let-7g(-)-EPC-CM组、以及let-7g(+)-EPC-CM组的伤口愈合速率差的趋势，可得知let-7g(+)-EPC-CM组的糖尿病小鼠其伤口治愈效果最为良好，而EBM组则为最差。亦即，经培养经转质let-7g的内皮前驱细胞的培养液具有最佳促进伤口愈合的效果。
【表3】
表3.伤口愈合速率差的Kruskal-Wallis检定
Figure PCTCN2014087374-appb-000003
SPSS统计软件，P＜0.05具有显著性差异
【表4】
表4.EBM与let7g(-)EPC-CM相比伤口愈合速率差的M-W U检定
Figure PCTCN2014087374-appb-000004
SPSS统计软件，P＜0.05具有显著性差异
【表5】
表5.EBM与let7g(+)EPC-CM相比伤口愈合速率差的M-W U检定
Figure PCTCN2014087374-appb-000005
SPSS统计软件，P＜0.05具有显著性差异
【表6】
表6.let7g(-)EPC-CM与let7g(+)EPC-CM相比伤口愈合速率差的M-W U检定
Figure PCTCN2014087374-appb-000006
SPSS统计软件，P＜0.05具有显著性差异
以上的叙述以及说明仅为本发明的较佳实施例的说明，对于此项技术具有通常知识者当可依据所界定申请专利范围以及上述的说明而作其它的修改，然而此些修改仍应是为本发明的发明精神而在本发明的权利范围中。

Claims (10)

  1. 一种促进伤口愈合的制剂，其特征在于，其包含细胞培养基(cell culture medium)，该细胞培养基系将经转质的内皮前躯细胞(endothelial progenitor cell)予以培养而形成，且该转质的内皮前驱细胞系为将微小核醣核酸(microRNA)let-7g转质至一内皮前驱细胞而形成。
  2. 如权利要求1所述的制剂，其特征在于，该细胞培养基于培养该内皮前躯细胞之前系为内皮细胞基础培养基(endothelial basal medium，EBM)。
  3. 如权利要求1所述的制剂，其特征在于，还包括至少一种医药学上可接受的载剂、稀释剂或赋形剂。
  4. 如权利要求3所述的制剂，其特征在于，该赋形剂系为一甘油。
  5. 一种促进伤口愈合的制剂的制备方法，其特征在于，其包含以下步骤：
    分离步骤：自人类全血分离出单核球细胞，并将该单核球细胞种在涂布有人类纤维连接蛋白的孔盘(human-fibronectin-coated plate)以得到内皮前躯细胞；
    转质步骤：将微小核醣核酸let-7g转质至该内皮前驱细胞，以得到经转质的内皮前驱细胞；以及
    培养步骤：将经转质的该内皮前驱细胞予以培养一预定时间而得到该促进伤口愈合的制剂。
  6. 如权利要求5所述的制备方法，其特征在于，于培养步骤中，将经转质的该内皮前驱细胞的培养系为培养于内皮细胞生长培养基(endothelial growth medium-2，EGM-2)7天，并于第7天将该内皮细胞生长培养基更换为内皮细胞基础培养基，接着持续培养2天以收集该内皮细胞基础培养基而得到该促进伤口愈合的制剂。
  7. 如权利要求5所述的制备方法，其特征在于，该预定时间系为6至10天。
  8. 如权利要求5所述的制备方法，其特征在于，于该分离步骤中，系藉由梯度剂Histopaque-1077以密度梯度离心法自该人类全血中分离出该单核球细胞。
  9. 一种促进伤口愈合的方法，其特征在于，其包含：将治疗有效量的如权利要求1所述的制剂或如权利要求3所述的制剂投予至一生物体的伤口。
  10. 如权利要求9所述的方法，其特征在于，该生物体系为一患有糖尿病的患者。
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