脂肪源性干细胞复合抗菌药物SYNO1对小型猪背部创面愈合影响的研究

发布时间：2018-09-05 08:18

【摘要】：创伤引起的皮肤全层或部分缺损是临床上常见的疾病,伤口由于感染或机体自身修复障碍(糖尿病)等原因造成延迟愈合,不仅给患者造成痛苦,而且增加了医疗资源的消耗。在急性的皮肤创面愈合过程中,各个阶段如炎症反应期、血管生成期、上皮化期和组织重塑期有序发生,但是在难治性创面愈合过程中,这些创面愈合的顺序被打乱从而导致持续炎症引起损害。而关于创伤治疗,一方面,要着重控制全身影响疾病愈合的因素,如感染、糖尿病和慢性消耗性疾病;另一方面,要控制局部的感染和无菌性炎症,同时尽早覆盖创面,促进创面愈合。创面愈合评估也包含两个方面,一个是创面愈合的速度,与伤口的胶原纤维的增值速度、毛细血管的长入,上皮爬行速度都有很大关系;另一方面是伤口愈合的质量,包括胶原纤维生长的有序化,是否有效连接,以及胶原细胞的接触抑制机制是否完善,以减少瘢痕增生。如何在治疗上选择一种新型的方法,能够同时兼顾到以上的伤口修复步骤的全面调控,成为目前研究的重点领域。近年来,在生物工程技术领域,利用具有自身分化潜能的成体组织来源的干细胞来治疗皮肤创面,取得了很大进展。其中,脂肪源性干细胞(ASCs)以其取材方便,应用安全,较其他组织来源的干细胞更具有临床应用价值。ASCs作为成体多能干细胞一种,同样可以分化成中胚层来源的各种细胞,如:向上皮细胞分化,加快伤口上皮化,促进创面愈合;ASCs还可以通过旁分泌作用分泌的细胞因子激发组织再生过程。如:分泌的血管生成因子(vascular endothelial growth factor,VEGF)等细胞因子控制炎症发展,是促进皮肤创面愈合的理想的生物活性细胞。ASCs用于皮肤创面愈合已经展现出光明的前景,目前,ASCs已经被主要用于体外动物实验的研究,一些临床试验正处在初级阶段。在动物实验中,ASCs和其分泌的细胞因子已经显出对于慢性难治性伤口的治疗作用,但是其中的具体机制还有待进一步研究中。临床的研究,也顾虑到干细胞致癌风险而进展缓慢,但目前研究结果并没有显示ASCs具有致癌的证据,这一结论还需要长期的随访得到证实。课题的研究内容,有助于了解皮肤创面的愈合机制及明确ASCs是如何发挥作用的。同时课题也探索了ASCs的应用方式,毕竟如何高效、简便的应用ASCs进行皮肤创面的治疗,也是其临床普及的重要方面。课题进行了一系列体内和体外实验,特别是在体内实验中,以Yorkshire猪作为实验对象,构建不同种类的创伤模型,探索ASCs的有效应用方式,评估ASCs的治疗效果。另一方面,SYNO1是经美国药监局(FDA)批准的新型促进伤口愈合药物,通过抗感染等方式加速伤口愈合,提高愈合质量。研究显示,控制伤口的炎症反应对于加快伤口愈合和提高伤口愈合质量,起到很关键的作用。SYNO1在先期的体内和体外实验中的抗感染作用也被证实。课题通过一系列体内和体外实验,特别构建了Yorkshire猪背部MRSA感染伤口模型,进一步验证SYNO1的抗感染和促进伤口愈合的作用,为I期临床应用打下基础。课题第一部分实验选择以脂肪源性干细胞为治疗方法,在体外实验中,进一步明确ASCs的生理学特性,包括人和Yorkshire猪的ASCs的提取步骤、鉴别方法;以及干细胞的标记和示踪方法;还包括一系列涉及到临床应用的提取技术和培养技术;不同密度ASCs培养方法和不同传代的ASCs的生物学活性的比较等。在体内实验中,研究ASCs在Yorkshire猪背部皮肤创伤模型中发挥的作用,探索应用局部注射的方法,在伤口周围及皮下注射经过PKH26标记的ASCs,后期应用免疫组织化学、PKH26标记示踪、伤口愈合率和上皮化率检测等方法,评估伤口愈合的速度和质量。课题第二部分选择SYNO1为治疗方法,在体外实验中,研究SYNO1对耐药性金黄色葡萄球菌(MRSA)在培养皿中的作用效果,包括体外MRSA的培养、鉴别、特性评估和实验应用步骤;在体内实验中,研究SYNO1在Yorkshire猪皮肤创面MRSA感染模型中的治疗效果,应用伤口上皮化及愈合率的监测对比、免疫组织化学方法评估毛细血管生成和胶原纤维生长的速率和有序性,应用PCR方法在分子水平评估伤口愈合机制的变化。结果表明,ASCs、SYNO1等治疗方法能够促进Yorkshire猪背部创面的愈合、控制炎症感染、提高伤口愈合速率和愈合质量。其中,ASCs作为具有生物修复活性的干细胞,在体外培养中,课题利用流式细胞仪鉴别ASCs表面标记分子和其贴壁效应可以用来鉴别其来源;P2代ASCs显示出更高的增值效率和活性;5000/cm2的培养密度显示出最高的投入产出比例。自体ASCs在Yorkshire猪背部创面局部注射后,能够在创面周围聚集(PKH26标记及示踪结果),能够增加愈合伤口胶原纤维生成速度,增加毛细血管密度,从而促进伤口愈合,提高伤口愈合速度和质量。SYNO1能够在体外实验中,对培养皿中的MRSA菌群起到显著抑制作用;在体内实验中,实验组和对照组的伤口菌群数量检测结果来看,SYNO1能够抑制伤口表面的MRSA生长,控制感染伤口,促进创面愈合。在课题的研究中,完成了这些新型的治疗方法初步的探索工作,还需要进一步的动物实验和初期的临床试验来证实其有效性和安全性。就目前的结果来看,有望在进一步的临床产业化过程中推广和普及。
[Abstract]:Full-thickness or partial skin defect caused by wound is a common clinical disease. Delayed healing of wound due to infection or body self-repair disorder (diabetes) not only causes pain to patients, but also increases the consumption of medical resources. Adult, epithelial and tissue remodeling stages occur in an orderly manner, but in the healing process of refractory wounds, the order of healing of these wounds is disrupted leading to sustained inflammation and damage. The evaluation of wound healing also includes two aspects: one is the rate of wound healing, which is closely related to the increment of collagen fibers, the growth of capillaries, and the speed of epithelial creeping; the other is the quality of wound healing. In recent years, bioengineering has become the focus of research in the field of bioengineering. In the field of technology, great progress has been made in the use of stem cells derived from adult tissues with self-differentiation potential to treat skin wounds. Adipose-derived stem cells (ASCs) have more clinical application value than stem cells derived from other tissues because of their convenience in obtaining materials and safety in application. Differentiation into a variety of mesodermal cells, such as: epithelial cells, accelerate wound epithelialization, promote wound healing; ASCs can also stimulate tissue regeneration by paracrine cytokines. For example, secretion of vascular endothelial growth factor (VEGF) and other cytokines control the development of inflammation, is to promote. ASCs have shown bright prospects for skin wound healing. At present, ASCs have been mainly used in animal experiments in vitro, and some clinical trials are at an early stage. In animal experiments, ASCs and cytokines secreted by ASCs have been shown to be effective against chronic refractory injuries. Clinical research, which also takes into account the risk of stem cell carcinogenesis, has progressed slowly. However, there is no evidence that ASCs are carcinogenic. This conclusion needs to be confirmed by long-term follow-up. The mechanism of healing and how ASCs plays a role is also clear. At the same time, the topic also explores the application of ASCs. After all, how to treat skin wounds with high efficiency and simple ASCs is also an important aspect of its clinical popularization. * a series of in vivo and in vitro experiments have been carried out, especially in vivo experiments, with Yorkshire pigs as the real cases. On the other hand, SYNO1 is a new wound healing drug approved by the U.S. Drug Administration (FDA), which accelerates wound healing and improves healing quality through anti-infection and other ways. Studies have shown that controlling inflammation in wounds is important for healing. Fast wound healing and improving the quality of wound healing play a key role. The anti infective effect of.SYNO1 in early and in vivo and in vitro experiments has also been confirmed. Through a series of in vivo and in vitro experiments, * a specially constructed Yorkshire pig's back MRSA infected wound model is further validated for SYNO1's anti infection and wound healing. The first part of the experiment chooses adipose derived stem cells as the treatment method. In vitro experiments, we further clarify the physiological characteristics of ASCs, including the extraction steps and identification methods of ASCs from human and Yorkshire pigs *, as well as the labeling and tracing methods of stem cells, and a series of clinical trials involving I. In vivo experiments, we studied the role of ASCs in the skin injury models of Yorkshire pigs, and explored the use of local injection methods to inject PKH26 labeled ASCs around and around the wound in ASCs * ASCs. Methods of immunohistochemistry, PKH26 labeling, wound healing rate and epithelialization rate were used to evaluate the speed and quality of wound healing. Identification, characteristic evaluation and experimental application steps; in vivo experiments, the therapeutic effect of SYNO1 on Yorkshire * pig skin wound MRSA infection model was studied, and the rate and order of capillary formation and collagen fiber growth were evaluated by immunohistochemical staining and comparison of wound epithelialization and healing rate. The PCR method was applied to the molecule. The results showed that ASCs, SYNO1 and other treatments could promote wound healing in Yorkshire pigs, control inflammatory * infection, improve wound healing rate and healing quality. ASCs, as a stem cell with bioremediation activity, was used in vitro culture to identify ASCs surface by flow cytometry. Marker molecules and their adherence effects can be used to identify their sources; P2 generation ASCs showed higher value-added efficiency and activity; 5000/cm2 culture density showed the highest proportion of input and output. Autologous ASCs could accumulate around the wound (PKH26 labeling and tracer results) after local injection of Yorkshire pig's back wound, which could increase healing injury. SYNO1 can inhibit MRSA microflora in culture dishes in vitro, and SYNO1 can inhibit wound microflora in vitro. In vivo, SYNO1 can inhibit wound microflora in both experimental and control groups. Surface MRSA growth, control infection wounds, promote wound healing. In this study, we have completed the preliminary exploration of these new treatment methods, but also need further animal experiments and initial clinical trials to confirm its effectiveness and safety. Popularization and popularization.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641

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