【作者】 黄国宝；

【导师】 霍然；

【作者基本信息】 山东大学 ， 外科学（专业学位）， 2014， 博士

【摘要】 研究背景：难愈皮肤慢性溃疡是指由创(烧)伤、各种感染、长期压迫、糖尿病、血管病变、神经病变和长期放疗等因素所引起的、经历很长时间不愈合的,皮肤、皮下组织甚至合并有肌肉、骨骼等组织的坏死及缺损,常伴有慢性感染发生。一般而言,老年人较青壮年多见,下肢较其他部位多发。随着我国迈入老龄化社会的步伐不断加快,难愈皮肤慢性溃疡的发病率越来越高。一项针对外科各专业住院患者的研究表明,因难愈皮肤慢性溃疡需要住院治疗的患者约占外科住院患者的1.5%-3.0%,而且就目前而言,这一类患者在很多医院中并没有得到集中有效的治疗,而是分散到各个科室中,譬如神经内科、心内科、呼吸科、肿瘤科、放疗科以及外科门诊等,所以统计起来有很大的困难,其实际发病率可能会更高。相当数量的难愈皮肤慢性溃疡患者,是继发于心脑血管疾病、糖尿病、老年病和放射治疗等,其病程漫长,治疗涉及很多方面,这既延误了患者原发病的治疗,又给患者带来了新的痛苦,严重影响了患者的生活质量,同时给患者的家庭带来沉重的经济负担与护理压力,是一个日益严重的社会问题。由于难愈皮肤慢性溃疡病因复杂、类型繁多,无疑给治疗带来了很大的困难。除根据患者的具体情况做一般的病因治疗外,彻底有效的清创和局部用药是非常重要的治疗手段。目前,对皮肤慢性溃疡局部处理的方法很多,如各类药物治疗、生物敷料、转基因治疗等方法,对于难愈皮肤慢性溃疡,一些保护剂、抗生素、维生素、微量元素制剂也已经被广泛应用,但这些药物促进创面愈合的作用还需要进一步的研究,使用方法还需要进一步的改进。激光、光照射等治疗方法对操作要求很高,普及有一定的困难。目前,对于大多数的难愈皮肤慢性溃疡患者,彻底治愈创面的方法仍然是外科手术,不过由于很多该疾病患者合并有比较严重的心脑血管病、糖尿病等内科疾病,手术禁忌症多见,这严重限制了手术的实施。在做好外科清创的基础上,如何选择一种切实有效的促进难愈皮肤慢性溃疡愈合的药物,并辅以适宜创面愈合的外用敷料,从而减轻患者痛苦,缩短病程,一直是人们不断探索的问题。粒细胞-巨噬细胞集落刺激因子(GM-CSF,granulocyte-macrophage colony stimulating factor)是复杂的细胞因子网络中的一员,其作用广泛。随着对GM-CSF基础研究和临床研究的不断进展,重组人粒细胞-巨噬细胞集落刺激因子已经广泛应用于临床治疗的各个领域,如肿瘤放疗、化疗以后,可以作为升高白细胞的药物；骨髓移植后,应用重组人粒细胞-巨噬细胞集落刺激因子可以促进其造血功能的恢复；以及可以用于各种原因引起的创伤的治疗、在临床上用于乙型肝炎和抗肿瘤的辅助治疗等。目前,GM-CSF用于各种类型的创面尤其是慢性皮肤溃疡创面的治疗,是近年来人们一直关注的话题。临床应用研究中发现,GM-CSF发挥作用需要一定的时间,而且该药物只有持续的作用于创面,其促进愈合的作用才能发挥到最大限度。同时,外用敷料如果能够在创面上形成一种比较湿润的环境,也有利于创面的更快愈合。藻酸盐敷料是一种新型的功能活性敷料,它可以持续有效地吸收创面的渗液,并在创伤表面形成一层稳定的网状凝胶,这保证了GM-CSF不会很快流失,能够作用更持久。同时,该凝胶能保持创面湿润并使创面同外界环境隔绝,在创面周围形成一个相对密闭的环境,加速肉芽组织及其毛细血管生长,促进创面的各种修复细胞增殖,加速创面的上皮化。藻酸盐敷料不粘于创面,在换药去除敷料时没有明显的疼痛,患者容易接受。目的：根据难愈皮肤慢性溃疡的创面特点,从外用药物促进慢性溃疡自我修复最终达到消灭皮肤缺损的目的出发,将藻酸盐敷料(液超妥)与人粒细胞-巨噬细胞刺激因子(hGM-CSF,金扶宁)联合应用于难愈皮肤慢性溃疡创面,评价其治疗创面和促进愈合的有效性,初步探讨其促进愈合的有关细胞生物学机制,研究两者之间的协同作用,并对患者作出的客观评价进行分析,确定其最佳应用方法。方法：以患者自愿为原则,选择2009年10月-2012年3月在本医院住院治疗的难愈皮肤慢性溃疡患者,主要包括压疮、静脉曲张性溃疡、糖尿病足等,纳入标准：年龄18-60岁,创面经常规清创及抗炎治疗1月以上未能愈合者,创面面积10-100cm2。排除标准：(1)严格控制血糖后空腹血糖仍>10.0mmol/L。(2)严重心功能不全(心功能不全Ⅲ级及以上)、严重肾功能不全(肾功能不全Ⅱ期及以上)、严重消耗性疾病(恶性肿瘤、肺结核、慢性萎缩性胃炎、系统性红斑狼疮)、严重营养不良、外周血管疾病患者,急性感染和急性代谢功能紊乱患者。(3)妊娠或哺乳期患者。(4)依从性达不到试验要求者。所有患者均被详细告知试验内容,签署知情同意书后方可进入试验。共有60例患者符合入选标准,其中男35例,女25例,年龄20-75岁。60例患者中压迫性溃疡(压疮)25例,静脉曲张性溃疡12例,糖尿病足23例。溃疡面积11-85cm2,平均17.2+8.0cm2。病程1-3.5个月,平均.(1.8±2.1)个月。溃疡深度均在皮下软组织层。按照随机分组的原则,将患者分为藻酸盐敷料与hGM-CSF联合治疗组(A组)、hGM-CSF治疗组(B组)、对照组(C组)。3组患者年龄、性别、病情、病程、溃疡面积比较,差异均无统计学意义。所有患者换药时,均去除外层敷料,用生理盐水冲洗清理创面,通过标准化临床路径对患者进行创面清除坏死组织,并用无菌棉球揩干。联合组将人粒细胞-巨噬细胞刺激因子(金扶宁,长春金赛药业有限责任公司,批准文号：国药准字$20080003,规格为100ugrhGM-CSF/10g/支,分子量：14KD)均匀涂于创面,外面以与创面同样大小的液超妥藻酸盐敷料(英国施乐辉公司,批准文号：进2005第2640100号)覆盖创面。hGM-CSF治疗组将金扶宁均匀涂于创面,外面以凡士林油纱覆盖创面。对照组则仅用凡士林纱布覆盖创面。治疗过程中注意无菌操作。3组患者用药前及用药7、14、21d观察创面渗出物、肉芽组织及上皮生长情况及感染等状况的改变,观察有无伤口周围皮肤刺激、过敏等情况的发生；用药7、14、21d计算各组患者的创面愈合面积,计算创面的愈合率；用药前及用药7、14、21d患者行创面疼痛视觉模拟评分法(VAS)评分;用药7、14d患者对治疗效果进行评价；用药前及用药7、14d从患者创面剪取肉芽组织标本,HE染色处理,在光学显微镜下,观察并计算各组创面肉芽组织中的毛细血管及成纤维细胞的数量。数据采用SPSS16.0软件进行分析。结果：1.用药14、21d,3组患者的创面色泽、肉芽组织生长情况做比较,差异有统计学意义,A组创面色泽鲜红,肉芽组织生长良好,优于B组和C组；B组优于C组。2.用药7、14、21d,A组的愈合率分别为9.3+5.5%、23.2士7.7%、56.1士7.5%,B组的愈合率分别为6.5士4.3%、15.3±4.9%、33.7士7.9%,C组的愈合率分别为2.3士4.2%、8.5士6.9%、20.9+8.8%。各个时相点的3组数值之间均做比较,差异有统计学意义。3.用药7d、14、21d,各组患者行创面疼痛视觉模拟评分,各个时相点的3组数值之间均做比较,差异有统计学意义。4.用药7、14d,各组患者对治疗的效果进行评价,各个时相点的3组数值之间均做比较,差异有统计学意义。5.用药7、14d,创面肉芽组织中毛细血管数比较,差异有统计学意义,A组优于B组和C组,B组优于C组。6.用药7、14d,创面肉芽组织中成纤维细胞数比较,差异有统计学意义,A组优于B组和C组,B组优于C组。结论：难愈皮肤慢性溃疡病因复杂、类型繁多,治疗难度大,目前对皮肤溃疡局部处理方法很多,如药物治疗、转基因治疗等方法。对难愈皮肤慢性溃疡治疗的药物和方法的探索,一直是研究的热点。hGM-CSF作为一个既有促创面愈合作用、又可全身应用的细胞因子,局部应用可以有效、安全的治疗如压疮、糖尿病足溃疡、静脉性溃疡等各种难愈皮肤慢性溃疡创面,促进创面愈合。藻酸盐敷料应用于难愈皮肤慢性溃疡创面,使伤口同外界隔绝,形成一个相对密闭的湿润的环境,可以使新生微血管的增生速度加快,同时提高表皮细胞的再生能力并使其移动加快,这对于促进创面愈合有重要意义。另外,藻酸盐敷料在换药时疼痛明显减轻,不易损伤新生组织,易被患者接受。藻酸盐敷料与hGM-CSF联合应用治疗难愈皮肤慢性溃疡,对减少创面渗液量、促进肉芽组织生长、加速再上皮化具有明显协同作用；同时,二者联合应用,可有效减轻创面疼痛,提高患者对治疗的满意度,可作为难愈皮肤慢性溃疡创面的有效的治疗手段之一。藻酸盐敷料与hGM-CSF联合应用治疗难愈皮肤慢性溃疡,可明显促进创面愈合,其机制可能与其促进难愈皮肤慢性溃疡创面毛细血管的生成与成纤维细胞的转化、增殖和迁移有关。更多还原

【Abstract】 Background:Refractory chronic skin ulcer is caused by the factors, such as injury, burn, infections, long oppressed, diabetes, vascular lesions, neuropathy, and long-term radiation. With a long time of wound, it has necrosis and defects of the skin, subcutaneous tissue, and even muscle, bone, complicated by chronic infection. In general, elderly adults suffer form this disease easier than the young, and lower limbs its high risk site.Refractory chronic skin ulcer is a clinical common disease. According to one research on more than30000cases of hospitalized patients in our country,1.5%-3.0%of the patients need hospital treatment. A large number of refractory chronic skin ulcer patients are secondary to diseases, diabetes, and radiation therapy, and scattered in internal medicine, radiation therapy or surgery outpatient service and related professional make a diagnosis and give treatment, which produced great difficulty in statistics, so the actual incidence of a disease is higher. The oppressive, diabetic, radioactive and other kinds of proportion of ulcer will increase accordingly.Refractory chronic skin ulcer belongs to chronic diseases. It has no immediate threat to the patient’s life, but due to the course of months or even years and ten years, so the disease seriously affect the rehabilitation of the primary disease and the quality of daily life, and increase the economic burden of patient’s family. In addition, if the wound infections spread, it can lead to complications such as sepsis, not only increasing the primary disease but also endangering the patient lives. Refractory chronic skin ulcer is complex and the type is various, which make it difficult to cure. In addition to the treatment on cause, it is very important to local drug treatments. At present, many local treatment of chronic skin ulcers, such as drug therapy, biological dressing, transgenic therapy, methods of traditional Chinese medicine treatment, but very satisfactory therapy and drug have not been found. Especially for refractory chronic skin ulcer, some protective agents, antibiotics, vitamins, trace elements, is widely used, but the role of these drugs promote wound healing is limited. Laser, light treatment methods, such as operation requirements is very high, excessive exposure to the sun is easy to cause cancer. For refractory chronic skin ulcer, most surgeons preferred surgical clinical surgical treatment to eliminate the wound, but a lot of these patients combined with serious cardiovascular internal medicine diseases, such as surgery contraindications which limits the implementation of surgery, and these methods to make a lot of patients psychologically difficult to accept surgery. With the development of molecular biology, especially the further study of various growth factors makes into a new stage wound repair, how to promote the wound repair for treatment of refractory chronic skin ulcer has been continuously explore problems.Granulocyte and macrophage colony stimulating factor (GM-CSF) as a member of the complex network of cytokines, has extensive role. With the continuous progress of basic research and clinical research on GM-CSF, recombinant human granulocyte macrophage colony stimulating factor has. been widely used in clinical treatment of various fields, such as tumor after radiotherapy and chemotherapy, can be used as the drugs increased leukocyte; Bone marrow transplantation, can be applied to promote the recovery of hematopoietic function;And can be used for the treatment of trauma due to a variety of causes,clinically used to aid in the treatment of hepatitis b and antitumor and so on. At present, the topic of hGM-CSF for all kinds of wounds, especially the treatment of chronic wounds, have been focused on in recent years.Alginate dressings polysaccharide is a kind of similar to cellulose, which cannot dissolve itself. Its raw material is extracted from seaweed algae protein acid, at the time of making dressing, it is converted into a calcium salt. Alginate has strong absorbency. According to research, it can absorb the equivalent of20times its own weight of the liquid, and can effectively control the drainage and extend the use time of dressing. Contacting with the wound, calcium ion replacement can wound drainage of sodium ions, and the alginate form a layer on the surface of the wound after reaction and stable network gel. The gel can keep wounds moist and non-stick wound, cut off from the outside environment, and form a closed around the wound, which can accelerate wound granulation tissue and blood capillary growth, promote wound repair of cell proliferation, and accelerate the epithelium of the wound.Objective:According to characteristics of refractory chronic skin ulcer, for eliminating skin defect ultimately, the alginate dressings and granulocyte-macrophage stimulating factor (hGM-CSF,) are applied in the treatment of refractory chronic skin ulcer, to evaluate the effectiveness of the treatment of wounds and promoting healing, discuss on its mechanism of promoting the healing of the cell biology studies the synergy, make objective evaluation on the patients, and determine the best application methods.Methods:On the principle of voluntary, patients are choosen in October2009-March2012in Jinan Central Hospital of hospitalized patients with painless refractory chronic skin ulcer, mainly including bedsore, varicose veins ulcers, diabetic foot, etc. The standard included in:aged18to60, wound by conventional debridement and anti-inflammatory treatment for more than1month to heal the trauma, and wound area10-100cm2. Exclusion criteria:(1) After strict control, fasting blood glucose>10.0mmol/L(2)Serious cardiac insufficiency (cardiac insufficiency III level and above), severe renal insufficiency(renal insufficiency II and above), serious wasting disease (malignant tumor, tuberculosis, chronic atrophic gastritis, and systemic lupus erythematosus), severe malnutrition, peripheral vascular disease patients, patients with acute infection and acute metabolic disorders.(3) the patients with pregnancy or lactation.(4) poor compliance.(5) the dying cannot achieve the experimenter. Patients signed informed consent rear accessibility test.A total of60patients Meet the inclusion criteria, including male35cases,25cases of female, aged20to75years old. The cases include25cases of bedsore (11cases of diabetes, cerebral infarction sequela14cases),12cases of varicose ulcer,23cases of diabetic foot. The ulcer area is11-85cm2, an average of17.2±8.0cm2. Course:1-3.5months (1.8±2.1) months on average. Depth of the ulcer were in subcutaneous soft tissue layer, and wound secretion before using this drug inspection were reported positive bacteria.According to the principle of random grouping, the patients were divided into alginate dressings and hGM-CSF combined treatment group (group A), hGM-CSF treatment group (group B)and the conventional treatment group (group C). The age, sex, disease, the course of the disease, ulcer area of comparison of three groups were no statistically significant difference.When dressing, the patients were removed the outer covering, cleaned up the wound with saline rinse, removed of necrotic tissue and wound through standardized clinical pathway, and wiped dry with sterile cotton ball and. Daily dressing for wound exudation larger, when ooze reduced day after treatment. Group A were applied with granulocyte-macrophage stimulating factor (hGM-CSF, changchun Kinsey pharmaceutical industry limited liability company, approval number:S20080003approved by the state and specifications for100ug rhGM-CSF/10g, molecular weight:14KD)in wound, covered with alginate dressings (British&company, the approval number:2005.2640100). Group B were applied with hGM-CSF on wound and covered the wound with petrolatum gauze. Group C were only covered with vaseline gauze on the wound. After the completion of each group were covered with sterile gauze bandaging appropriately. Pay attention to the aseptic operation treatment process. Three groups of patients with drug use and drug use before7th,14th,21th day observation wound exudate, granulation tissue and epithelial growth and the change of infection situation, observe whether the wound around the occurrence of skin irritation, allergic, and so on and so forth; The preparation before and7th,14th,21th day calculated between groups in patients with wound healing area, speed testing, statistical wound healing progress; The preparation before and line7th,14th day wound pain visual analogue scale (VAS) score method, The preparation before and7th,14th day patients to evaluate the curative effect; Drugs and drug use before7th,14th day to return and tissue samples, by HE staining and optical microscope each wound granulation tissue in the number of capillaries and fibroblasts. Data were used by SPSS16.0software for analysis.Results:1. At the14th and21th days, three group of patients with wound also compare the colour and lustre, granulation tissue growth rate, difference was statistically significant, group A bright red color and granulation tissue grew well, better than that of group B and group C; Group B is better than that of group C.2. At the7th,14th,21th days, the healing rate of group A were9.3±5.5%,23.2±7.7%and56.1±7.5%, the healing rate of group B were6.5±4.3%,15.3±4.9%and33.7±7.9%, the healing rate of group C were2.3±4.2%,8.5±6.9%and20.9±8.8%. The difference between three groups was statistically significant.3. At the7th,14th.21th days, the difference of each wound pain in patients with visual analogue scale between three groups was statistically significant.4. At the7th,14th days, evaluation of its effect for the treatment of patients of group A score were3.7±1.5,4.9±1.3; group B were2.4±1.2,3.8±2.0; group C were2.0±.1,2.6±1.5. The difference between three groups was statistically significant.5. At the7th,14th days, wound granulation tissue of the capillary number were compared and the difference was statistically significant. Group A is better than that of group B and group C, group B is better than that of group C.6. At the7th,14th days, wound granulation tissue into fiber cell number comparison were compared and the difference was statistically significant, group A is better than that of group B and group C, group B is better than that of group C.Conclusion:The treatment of refractory chronic skin ulcer disease, due to the complex and the various type, is difficult. At present, the treatment on the refractory chronic skin ulcer, such as medication, transgenic therapy, Chinese medicine treatment methods, is various The drugs and methods on refractory chronic skin ulcer, has always been a research hot spot.Granulocyte-macrophage stimulating factor not only has effect on promoting wound healing, as a systemic application of cytokines, but also can; become an effective and safe treatment on refractory chronic skin ulcer caused by a variety of reasons, such as deep burn wounds, venous ulcers, diabetic ulcer, stress ulcer, burn remnant wounds, etc.Alginate dressings used in refractory chronic skin ulcer, form a relatively closed environment on the wound, which can accelerate the new capillary hyperplasia, keep the moist environment, enhances regeneration ability of skin cells, accelerate the epidermal cells, and promote wound healing. In addition, the alginate dressings during dressing change significantly reduce pain, which is not easy to damage the new organization, so it is easily accepted by patients.That alginate dressings and hGM-CSF joint application in treatment of painless chronic skin ulcer, can decrease the drainage of the wound, promote the growth of granulation tissue, and accelerate again epithelial change; At the same time, the two joint application, can effectively reduce the wound pain and improve patient satisfaction, can become an effective treatment of refractory chronic skin ulcer.Alginate dressings and hGM-CSF joint application in treatment of painless chronic skin ulcer, can obviously promote wound healing. Its mechanism to promote refractory chronic skin ulcer wound has relation to capillary generation and fibroblast proliferation and migration.更多还原