本文選題：燒傷 + 微粒皮移植��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景創(chuàng)面修復(fù)是燒傷領(lǐng)域永恒的命題,尤其對(duì)于大面積燒傷患者,創(chuàng)面修復(fù)尤其關(guān)鍵,直接關(guān)系到患者的存活率及生存質(zhì)量。大面積創(chuàng)面引起機(jī)體應(yīng)激嚴(yán)重,消耗過(guò)度,并且容易引細(xì)菌定植、進(jìn)而引起全身感染,導(dǎo)致全身臟器損傷。盡早的覆蓋創(chuàng)面是燒傷患者治療的關(guān)鍵,因而創(chuàng)面覆蓋物的選擇與進(jìn)展是貫穿燒傷發(fā)展始終的,其中包括臨時(shí)覆蓋物與永久的覆蓋物。永久的覆蓋主要指真皮替代物和字體皮,大面積救治治療過(guò)程中應(yīng)用的主要植皮方式有:Meek植皮術(shù)、自異體混合植皮術(shù)、微粒皮移植術(shù)以及自體表皮細(xì)胞培養(yǎng)移植等,然而,任何一種植皮方式都需要合適的臨時(shí)覆蓋物來(lái)覆蓋間隙以及保證永久覆蓋物存活。臨時(shí)覆蓋物的發(fā)展也對(duì)創(chuàng)面修復(fù)起到了重要的作用,臨時(shí)覆蓋從傳統(tǒng)的紗布、凡士林紗布、陰離子輔料等到膠原膜或膠原支架覆蓋物,應(yīng)用越來(lái)越廣泛。因此,創(chuàng)面覆蓋物的更新發(fā)展,也是創(chuàng)面修復(fù)技術(shù)的發(fā)展的重要支持。在大面積燒傷封閉創(chuàng)面的過(guò)程中,各種植皮方式應(yīng)用各有利弊。其中,Meek植皮術(shù)多應(yīng)用于早起切痂后覆蓋創(chuàng)面,實(shí)現(xiàn)了擴(kuò)展比例可為1:3至1:9,并且實(shí)現(xiàn)了機(jī)械化,但是擴(kuò)展比例有限;自異體混含植皮術(shù),植皮存活率穩(wěn)定,但存在手術(shù)重復(fù)操作多,耗時(shí)長(zhǎng)等缺點(diǎn);自體表皮細(xì)胞培養(yǎng)移植,理論可擴(kuò)展1000倍,但是存在創(chuàng)面愈合質(zhì)量差的問(wèn)題;微粒皮移植術(shù)理論上可擴(kuò)展100倍,臨床應(yīng)用可擴(kuò)展10～20倍,但是存在覆蓋物選擇、方向性及均一性等臨床難題。本研究著眼于微粒皮移植,覆蓋物的選擇一直是影響微粒皮移植成活率的重要因素,活性高的異體皮是理想的微粒皮移植覆蓋物,但是異體皮來(lái)源受限往往限制了大面積燒傷患者的微粒皮移植開(kāi)展,因而,尋找一種可以模擬皮膚功能的覆蓋物對(duì)微粒皮移植十分關(guān)鍵。在諸多覆蓋物材料的發(fā)展中,其中在創(chuàng)面修復(fù)領(lǐng)域,膠原材料的使用越來(lái)越廣泛。膠原自身為創(chuàng)面修復(fù)過(guò)程中所需要的重要成分,并且在皮膚及創(chuàng)面修復(fù)中起重要作用。并且,膠原的獲取途徑越來(lái)越多,提取方式及加工方式越來(lái)越成熟。在創(chuàng)面修復(fù)方面,膠原的應(yīng)用也越來(lái)越廣泛,已有的產(chǎn)品包括真皮替代物及外用敷料。近年來(lái),魚乘源的膠原材料越來(lái)越多的應(yīng)用在醫(yī)學(xué)領(lǐng)域,其中包括止血材料及創(chuàng)面修復(fù)領(lǐng)域,如何利用魚膠原材料促進(jìn)創(chuàng)面愈合具有重大臨床意義。本研究旨在尋找一種可替代異體皮膚功能的生物敷料,結(jié)合魚膠原的應(yīng)用發(fā)展,構(gòu)建一種模擬皮膚結(jié)構(gòu)功能的雙層魚膠原膜,并將之應(yīng)用于微粒皮移植覆蓋物,為微粒皮移植提供一種來(lái)源廣泛的覆蓋物選擇,從而使微粒皮移植在大面積燒傷患者的創(chuàng)面修復(fù)中應(yīng)用更廣,創(chuàng)面修復(fù)效率更高。第一部分雙層魚膠原膜(FCM-B)的構(gòu)建研究目的:制備一種模擬皮膚結(jié)構(gòu),包含表皮真皮,并且在功能上模擬皮膚的雙層膠原敷料,外層可以模擬表皮致密并且韌性高具備保護(hù)及隔絕功能;內(nèi)層疏松柔軟,具備良好的貼附功能。用于創(chuàng)面可以達(dá)到吸收滲液,并且可以保護(hù)創(chuàng)面。研究方法:利用食用廢料的羅非魚魚皮與魚鱗,經(jīng)酸聯(lián)合酶消化提取技術(shù),提取并純化魚膠原。在不添加具有毒性的交聯(lián)劑的基礎(chǔ)上,應(yīng)用不同的交聯(lián)方法可以獲得理化特性差別明顯的膠原膜。利用脫水熱處理(DHT,dehydrothermaltreatment)交聯(lián)的方法得到外層魚膠原膜(FCM-O,Fish Collagen Membrane Outer-layer),利用真空冷凍干燥(FC,vacuum Frozen drying)技術(shù)得到內(nèi)層魚膠原膜(FCM-I,Fish Collagen Membrane Inner-layer)。將制備好的FCM-O提前鋪在制備FCM-I的模具底部,再次利用真空冷凍干燥技術(shù),制備成膜。研究結(jié)果:成功制備具有雙層結(jié)構(gòu),外層致密柔韌,內(nèi)層疏松柔軟的雙層魚膠原膜,從外觀上和質(zhì)地上與人皮膚具有相似性。第二部分雙層魚膠原膜(FCM-B)的特性研究研究目的:通過(guò)FCM-B的結(jié)構(gòu)特性及理化特性檢測(cè)來(lái)驗(yàn)證雙層魚膠原膜與皮膚結(jié)構(gòu)和功能上的相似性。研究方法:1.結(jié)構(gòu)觀察1.1組織學(xué)觀察利用FCM-B冰凍切片,行HE染色、Massion染色,觀察其結(jié)構(gòu)與膠原排列情況,并與正常皮膚組織切片做對(duì)比,觀察其結(jié)構(gòu)的相似性;1.2電鏡掃描觀察并對(duì)FCM-B進(jìn)行掃描電鏡掃描,觀察膠原膜的結(jié)構(gòu),觀察各層次的表征,并測(cè)量各層次的厚度。2.物理特性2.1力學(xué)性能取FCM-O,FCM-I及FCM-B啞鈴型樣本,利用Instron5567材料測(cè)試系統(tǒng)(Instron,USA),進(jìn)行力學(xué)測(cè)試,獲得楊氏模量、斷裂伸長(zhǎng)率及斷裂強(qiáng)度等指標(biāo),重復(fù)測(cè)量3遍。2.2 水蒸氣透過(guò)率(WVTR,Water Vapor Transmission Rate)取直徑3.5cm的FCM-O,FCM-I及FCM-B樣本,使用水蒸氣透過(guò)率檢測(cè)儀(W3/030,Labthink,中國(guó))獲得結(jié)果,測(cè)量重復(fù)3次。2.3吸收容量(C%)將已經(jīng)制備好的三種膠原膜用雙蒸水浸泡,在相同環(huán)境浸泡相同時(shí)間直至膠原膜充分吸收水分,通過(guò)測(cè)量浸泡前后膠原膜質(zhì)量變化計(jì)算C%。3.生物化學(xué)特性3.1生物可降解性將制備的FCM-B、FCM-O及FCM-I用PBS處理,浸泡前后的干重計(jì)算降解速率,并計(jì)算半量降解時(shí)間t1/2。3.2細(xì)胞毒性將FCM浸提液按一定濃度剃度加入等量人成纖維細(xì)胞懸液中,使用CCK-8試劑盒,通過(guò)檢測(cè)吸光度(OD)值,計(jì)算成纖維細(xì)胞的相對(duì)增值率。統(tǒng)計(jì)方法:FCM的各項(xiàng)指標(biāo)(計(jì)量資料)結(jié)果均用平均值±標(biāo)準(zhǔn)差表示;兩組值之間應(yīng)用t檢驗(yàn);多組組比較應(yīng)用oneway-ANOVA分析,實(shí)驗(yàn)組與各組間比較應(yīng)用SNK檢驗(yàn)。研究結(jié)果:1.結(jié)構(gòu)觀察組織學(xué)切片結(jié)果顯示FCM-B冰凍切片與正常皮膚組織切片在雙層結(jié)構(gòu)和膠原排列上具有結(jié)構(gòu)相似性;電鏡掃描結(jié)果顯示FCM-B在厚度與孔隙上上與皮膚表皮真皮具有相似性。2.物理學(xué)特性通過(guò)檢測(cè)測(cè)得FCM-I機(jī)械性能顯著FCM-O(p0.05);FCM-I的WVTR顯著高于FCM-O(p0.05),FCM-B的WTVR接近正常皮膚水平。FCM-I吸收容量顯著高于FCM-O(p0.05)。3.生物化學(xué)性能檢測(cè)體外降解時(shí)間T1/2(FCM-O)約為15天;T1/2(FCM-I)約為21天,FCM-O的生物降解時(shí)間明顯長(zhǎng)于FCM-I(p0.05)。細(xì)胞毒性試驗(yàn)表明FCM浸提液對(duì)人成纖維細(xì)胞增值率無(wú)顯著影響。第三部分雙層魚膠原膜(FCM-B)覆蓋SD大鼠微粒皮移植實(shí)驗(yàn)研究目的:為了驗(yàn)證雙層膠原膜在微粒皮移植過(guò)程中能起到異體皮的作用,能夠促進(jìn)微粒皮移植創(chuàng)面的愈合。研究方法:SD大鼠背部做直徑2cm的圓形全層皮膚缺損創(chuàng)面,根據(jù)1:10的比例將取下的皮膚取薄皮制備成微粒,回植在創(chuàng)面上,根據(jù)覆蓋物的不同分為空白對(duì)照組、異體皮覆蓋組及FCM-B、FCM-I、FCM-O覆蓋組,觀察不同覆蓋物對(duì)微粒皮移植存活的影響,計(jì)算創(chuàng)面愈合率及收縮率;留取組織標(biāo)本,分別進(jìn)行膠原(Massion)染色、HE染色,觀察表皮細(xì)胞生長(zhǎng)情況及創(chuàng)面的膠原分布情況。統(tǒng)計(jì)方法:多組組比較應(yīng)用oneway-ANOVA分析,實(shí)驗(yàn)組與各組間比較應(yīng)用SNK檢驗(yàn)。研究結(jié)果:實(shí)驗(yàn)早期發(fā)現(xiàn)FCM-I、FCM-O創(chuàng)面愈合不佳,在3周時(shí)FCM-B覆蓋組與異體皮覆蓋組的創(chuàng)面愈合率差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05),創(chuàng)面收縮率異體皮覆蓋組低于FCM-B(p0.05),組織學(xué)觀察發(fā)現(xiàn)上皮形成厚度FCM-B組大于異體皮覆蓋組(p0.05)。研究結(jié)論本研究構(gòu)建的雙層膠原膜,在結(jié)構(gòu)上與皮膚的表皮真皮具有高度的相似性,并且在理化特性上接近理想創(chuàng)面覆蓋物要求,通過(guò)動(dòng)物實(shí)驗(yàn)結(jié)果證實(shí),FCM-B替代同種異體皮作為微粒皮移植覆蓋物具有可能性。
[Abstract]:Background wound repair is an eternal topic in the field of burn. Especially for patients with large area burns, the repair of the wound is particularly critical, which is directly related to the survival rate and quality of life of the patients. Large area wounds cause severe stress, excessive consumption, and easy to induce bacterial colonization, and then cause systemic infection, resulting in systemic organ damage. Early coverage of the wound is the key to the treatment of burn patients, so the selection and progress of the wound cover is throughout the development of the burn, including the temporary cover and permanent cover. The permanent cover mainly refers to the dermis and the skin. The main skin grafting methods used in the process of large area treatment are: Meek skin grafting, self grafting. Allograft skin grafting, particle skin grafting and autologous epidermal cell culture, however, any kind of skin grafting method requires suitable temporary cover to cover the gap and guarantee the survival of the permanent cover. The development of the temporary cover also plays a vital role in the wound repair, temporarily covering from the traditional gauze, the petticic. Bayashi Sabu, the anionic excipients wait until the collagen membrane or the collagen scaffold covers more and more widely. Therefore, the renewal and development of the wound cover is also an important support for the development of the wound repair technology. In the process of large area burns, the application of various skin grafting methods has advantages and disadvantages. Among them, Meek skin grafting should be used for early eschar cutting. After covering the wound, the expansion ratio can be 1:3 to 1:9, and the mechanization is realized, but the expansion ratio is limited; the survival rate of skin grafting is stable from the allograft containing skin grafting, but there are many disadvantages of repeated operation and time consuming, and the theory of autoepidermal cell culture and transplantation can be extended 1000 times, but the quality of wound healing is poor. The particle skin grafting can be expanded 100 times in theory, and the clinical application can be expanded by 10~20 times, but there are clinical problems such as selection of cover, orientation and uniformity. This study focuses on the skin grafting of particles. The selection of cover has always been an important factor affecting the survival rate of particle skin transplantation, and the high active skin allograft is the ideal skin grafting. Cap, but the limitation of the source of allograft often restricts the skin grafting of large area burn patients. Therefore, it is critical to find a kind of skin graft that can simulate skin function. In the development of many covering materials, the use of collagen is more and more widely used in the field of wound repair. The important components of the wound repair process are important and play an important role in the repair of skin and wound. Moreover, more and more ways of obtaining collagen are obtained. The methods of extraction and processing are becoming more and more mature. In the field of wound repair, the application of collagen is becoming more and more extensive, and the existing products include dermal substitute and external dressing. The collagen material of fish multiplying source is more and more applied in the field of medicine, including the field of hemostat and wound repair. How to use the raw material of fish glue to promote the healing of the wound is of great clinical significance. This study aims to find a biological dressing that can replace the function of the skin allograft, and combine the application and development of fish collagen to construct a kind of simulated skin. The skin structure function of the double layer fish gum membrane, which is applied to the skin graft covering, provides a wide selection of cover for the skin grafting of particles, so that the skin grafting is more widely used and the wound healing efficiency is higher in the wound repair of the large area burned patients. The construction of the first part of the first part of the double layer fish glue film (FCM-B) Objective: to prepare a kind of skin structure, including epidermis dermis, and the double layer collagen dressings that mimic the skin in function. The outer layer can simulate the compact and high tenacity of the epidermis and have the function of protection and isolation; the inner layer is loose and soft and has good adhesion function. It can be used in the wound surface and can protect the wound and protect the wound. Methods: the fish collagen was extracted and purified by the acid combined enzyme digestion and extraction technology using the fish skin and fish scales of edible waste. On the basis of no toxic crosslinking agent, different cross-linking methods could be used to obtain the collagen membrane with obvious differences in physicochemical properties. The cross linking of DHT (dehydrothermaltreatment) was used. The original membrane (FCM-O, Fish Collagen Membrane Outer-layer) was obtained by means of vacuum freeze drying (FC, vacuum Frozen drying) technology to obtain the original film of inner layer of fish gum (FCM-I, Fish Collagen). Research results: the double layer fish glue original film with double structure, dense and flexible outer layer and soft and soft inner layer is similar to human skin in appearance and texture. The research aim of the characteristics of the second part double layer fish gelatin original film (FCM-B) is to verify the double layer of the original film by the structure and physicochemical properties of FCM-B Similar to skin structure and function. Research methods: 1. structure observation 1.1 histology observation using FCM-B frozen section, HE staining, Massion staining, observe the structure and collagen arrangement, and compare with normal skin tissue sections, observe the structure of the similarity, 1.2 scanning electron microscope and scanning electron microscope for FCM-B, Observe the structure of the collagen membrane, observe the characterization of various levels, and measure the physical properties of the thickness.2. of each level 2.1 mechanical properties of FCM-O, FCM-I and FCM-B dumbbell type samples, using the Instron5567 material test system (Instron, USA) to carry out mechanical testing, obtain the young's modulus, breaking elongation and fracture strength and so on, repeated measurement of the 3 times.2.2 water evaporation. The gas transmittance (WVTR, Water Vapor Transmission Rate) takes the FCM-O, FCM-I, and FCM-B samples of the diameter 3.5cm, and uses the water vapor transmittance detector (W3/030, Labthink, China) to obtain the results. The repeated 3 times.2.3 absorption capacity is measured to soak the prepared three kinds of collagen membranes in double water and soak the same time in the same environment until collagen. The membrane fully absorbs moisture and calculates the C%.3. biochemical properties 3.1 biodegradability by measuring the quality changes of the collagen membrane before and after soaking. The FCM-B, FCM-O and FCM-I are treated with PBS, the dry weight before and after soaking is calculated and the degradation time is calculated, and the t1/2.3.2 cytotoxicity of the half quantity is calculated by the FCM extract shaving at a certain concentration and adding the same amount of people. In fibroblast suspension, CCK-8 kit was used to calculate the relative value of fibroblasts by detecting the value of absorbance (OD). Statistical methods: the results of FCM were expressed with mean standard deviation; t test was used between the two groups; oneway-ANOVA analysis was used in multiple groups. The experimental group and each group should be compared. The results of SNK test. The results of the study: 1. the histological section of the structure observation showed that the frozen section of FCM-B and the normal skin tissue were similar to the structure of the double layer structure and the collagen arrangement, and the scanning electron microscope showed that the FCM-B was similar to the skin epidermis and the skin epidermis was similar to the skin epidermis. The physical properties of the skin epidermis were detected by the FCM-I machine. The performance of the weapon was significantly higher than that of FCM-O (P0.05), and the WVTR of FCM-I was significantly higher than that of FCM-O (P0.05). The WTVR of FCM-B was close to the normal skin level, and the.FCM-I absorption capacity was significantly higher than that of FCM-O (P0.05) for about 15 days of biodegradation in vitro. The experiment showed that the FCM extract had no significant effect on the rate of human fibroblast increment. Third part of the double layer fish gelatin original membrane (FCM-B) covered the experimental study of SD rat skin grafting. In order to verify the effect of the double collagen membrane on the skin graft in the process of particle skin transplantation, it can promote the healing of the skin graft. The method of research: SD A round full layer skin defect wound on the back of 2cm was made on the back of the rat. According to the 1:10 proportion, the skin of the skin was prepared to make the particles and replanted on the wound. According to the different coverage, the skin was divided into blank control group, the allograft skin cover group and the FCM-B, FCM-I, and FCM-O cover group. The effects of different cover on the survival of the skin graft were observed and the wound was calculated. The healing rate and the contraction rate of the tissue specimens were left with collagen (Massion) staining and HE staining. The growth of epidermal cells and the distribution of collagen in the wound were observed. Statistical methods were compared with oneway-ANOVA analysis in groups of groups. The experimental group and each group were compared with SNK. The results of the study were that FCM-I, FCM-O wound healing was not healed in the early stage of the experiment. There was no significant difference in the wound healing rate between the FCM-B group and the allogenic skin cover group at 3 weeks (P0.05). The wound shrinkage rate of the skin allograft group was lower than FCM-B (P0.05). The histological observation found that the epithelial thickness FCM-B group was larger than the allogeneic skin cover group (P0.05). Conclusion the double layer collagen membrane constructed in this study was structured with the skin. The epidermis of the epidermis is highly similar and close to the requirements of the ideal wound cover in physical and chemical properties. The results of animal experiments have proved that the replacement of the allogenic skin by FCM-B as a skin graft cover is possible.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R644

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相關(guān)期刊論文 前7條

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