【摘要】：創(chuàng)傷引起的皮膚全層或部分缺損是臨床上常見的疾病,傷口由于感染或機體自身修復(fù)障礙(糖尿病)等原因造成延遲愈合,不僅給患者造成痛苦,而且增加了醫(yī)療資源的消耗。在急性的皮膚創(chuàng)面愈合過程中,各個階段如炎癥反應(yīng)期、血管生成期、上皮化期和組織重塑期有序發(fā)生,但是在難治性創(chuàng)面愈合過程中,這些創(chuàng)面愈合的順序被打亂從而導(dǎo)致持續(xù)炎癥引起損害。而關(guān)于創(chuàng)傷治療,一方面,要著重控制全身影響疾病愈合的因素,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無菌性炎癥,同時盡早覆蓋創(chuàng)面,促進創(chuàng)面愈合。創(chuàng)面愈合評估也包含兩個方面,一個是創(chuàng)面愈合的速度,與傷口的膠原纖維的增值速度、毛細血管的長入,上皮爬行速度都有很大關(guān)系;另一方面是傷口愈合的質(zhì)量,包括膠原纖維生長的有序化,是否有效連接,以及膠原細胞的接觸抑制機制是否完善,以減少瘢痕增生。如何在治療上選擇一種新型的方法,能夠同時兼顧到以上的傷口修復(fù)步驟的全面調(diào)控,成為目前研究的重點領(lǐng)域。近年來,在生物工程技術(shù)領(lǐng)域,利用具有自身分化潛能的成體組織來源的干細胞來治療皮膚創(chuàng)面,取得了很大進展。其中,脂肪源性干細胞(ASCs)以其取材方便,應(yīng)用安全,較其他組織來源的干細胞更具有臨床應(yīng)用價值。ASCs作為成體多能干細胞一種,同樣可以分化成中胚層來源的各種細胞,如:向上皮細胞分化,加快傷口上皮化,促進創(chuàng)面愈合;ASCs還可以通過旁分泌作用分泌的細胞因子激發(fā)組織再生過程。如:分泌的血管生成因子(vascular endothelial growth factor,VEGF)等細胞因子控制炎癥發(fā)展,是促進皮膚創(chuàng)面愈合的理想的生物活性細胞。ASCs用于皮膚創(chuàng)面愈合已經(jīng)展現(xiàn)出光明的前景,目前,ASCs已經(jīng)被主要用于體外動物實驗的研究,一些臨床試驗正處在初級階段。在動物實驗中,ASCs和其分泌的細胞因子已經(jīng)顯出對于慢性難治性傷口的治療作用,但是其中的具體機制還有待進一步研究中。臨床的研究,也顧慮到干細胞致癌風險而進展緩慢,但目前研究結(jié)果并沒有顯示ASCs具有致癌的證據(jù),這一結(jié)論還需要長期的隨訪得到證實。課題的研究內(nèi)容,有助于了解皮膚創(chuàng)面的愈合機制及明確ASCs是如何發(fā)揮作用的。同時課題也探索了ASCs的應(yīng)用方式,畢竟如何高效、簡便的應(yīng)用ASCs進行皮膚創(chuàng)面的治療,也是其臨床普及的重要方面。課題進行了一系列體內(nèi)和體外實驗,特別是在體內(nèi)實驗中,以Yorkshire豬作為實驗對象,構(gòu)建不同種類的創(chuàng)傷模型,探索ASCs的有效應(yīng)用方式,評估ASCs的治療效果。另一方面,SYNO1是經(jīng)美國藥監(jiān)局(FDA)批準的新型促進傷口愈合藥物,通過抗感染等方式加速傷口愈合,提高愈合質(zhì)量。研究顯示,控制傷口的炎癥反應(yīng)對于加快傷口愈合和提高傷口愈合質(zhì)量,起到很關(guān)鍵的作用。SYNO1在先期的體內(nèi)和體外實驗中的抗感染作用也被證實。課題通過一系列體內(nèi)和體外實驗,特別構(gòu)建了Yorkshire豬背部MRSA感染傷口模型,進一步驗證SYNO1的抗感染和促進傷口愈合的作用,為I期臨床應(yīng)用打下基礎(chǔ)。課題第一部分實驗選擇以脂肪源性干細胞為治療方法,在體外實驗中,進一步明確ASCs的生理學(xué)特性,包括人和Yorkshire豬的ASCs的提取步驟、鑒別方法;以及干細胞的標記和示蹤方法;還包括一系列涉及到臨床應(yīng)用的提取技術(shù)和培養(yǎng)技術(shù);不同密度ASCs培養(yǎng)方法和不同傳代的ASCs的生物學(xué)活性的比較等。在體內(nèi)實驗中,研究ASCs在Yorkshire豬背部皮膚創(chuàng)傷模型中發(fā)揮的作用,探索應(yīng)用局部注射的方法,在傷口周圍及皮下注射經(jīng)過PKH26標記的ASCs,后期應(yīng)用免疫組織化學(xué)、PKH26標記示蹤、傷口愈合率和上皮化率檢測等方法,評估傷口愈合的速度和質(zhì)量。課題第二部分選擇SYNO1為治療方法,在體外實驗中,研究SYNO1對耐藥性金黃色葡萄球菌(MRSA)在培養(yǎng)皿中的作用效果,包括體外MRSA的培養(yǎng)、鑒別、特性評估和實驗應(yīng)用步驟;在體內(nèi)實驗中,研究SYNO1在Yorkshire豬皮膚創(chuàng)面MRSA感染模型中的治療效果,應(yīng)用傷口上皮化及愈合率的監(jiān)測對比、免疫組織化學(xué)方法評估毛細血管生成和膠原纖維生長的速率和有序性,應(yīng)用PCR方法在分子水平評估傷口愈合機制的變化。結(jié)果表明,ASCs、SYNO1等治療方法能夠促進Yorkshire豬背部創(chuàng)面的愈合、控制炎癥感染、提高傷口愈合速率和愈合質(zhì)量。其中,ASCs作為具有生物修復(fù)活性的干細胞,在體外培養(yǎng)中,課題利用流式細胞儀鑒別ASCs表面標記分子和其貼壁效應(yīng)可以用來鑒別其來源;P2代ASCs顯示出更高的增值效率和活性;5000/cm2的培養(yǎng)密度顯示出最高的投入產(chǎn)出比例。自體ASCs在Yorkshire豬背部創(chuàng)面局部注射后,能夠在創(chuàng)面周圍聚集(PKH26標記及示蹤結(jié)果),能夠增加愈合傷口膠原纖維生成速度,增加毛細血管密度,從而促進傷口愈合,提高傷口愈合速度和質(zhì)量。SYNO1能夠在體外實驗中,對培養(yǎng)皿中的MRSA菌群起到顯著抑制作用;在體內(nèi)實驗中,實驗組和對照組的傷口菌群數(shù)量檢測結(jié)果來看,SYNO1能夠抑制傷口表面的MRSA生長,控制感染傷口,促進創(chuàng)面愈合。在課題的研究中,完成了這些新型的治療方法初步的探索工作,還需要進一步的動物實驗和初期的臨床試驗來證實其有效性和安全性。就目前的結(jié)果來看,有望在進一步的臨床產(chǎn)業(yè)化過程中推廣和普及。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R641

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