中药五谷虫对感染创面抗菌作用的临床与分子机制研究
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摘要
慢性难治性感染创面临床常见,如脊髓损伤截瘫患者压疮溃烂和感染,慢性化脓性骨髓炎的感染创面,下肢静脉性溃疡和糖尿病晚期下肢溃疡创面等。这些感染创面的特点是:病程长,溃疡创面经久不愈;多为混合感染,常规治疗方法疗效差;创面的坏死组织界限不清,外科清创术治疗困难,严重影响患者健康和生活质量。特别是由于抗生素的滥用,细菌对抗生素的耐药情况日益严重,从而降低了抗生素对创面感染的抗菌功效,使这类创面治疗更加困难。因此,如何有效地治疗慢性难治性感染创面是外科领域一直没有解决的难题之一。
传统中药材“五谷虫”,又名蛆虫,为丽蝇科丝光绿蝇、大头金蝇的幼虫。《本草求源》、《本草便读》均记载:该虫性寒无毒,可搽敷外用,能治疗臁疮等感染性外科疾病。为临床治疗慢性难治性感染创面提供了一种新的思路。但是中医学对五谷虫治疗感染创面缺乏系统的研究,其抗菌机制还不十分清楚。近年来,Sherman R.A.等国外学者发现,五谷虫治疗感染创面,能有效地促进创面愈合,抵抗细菌感染。但目前研究仍主要集中于临床治疗病例报道,缺乏与五谷虫抗菌作用相关的临床与分子生物学的系统研究。
本课题采用中西医结合的方法,对五谷虫对感染创面抗菌作用的临床与分子机制进行系统地研究。旨在(1)探索系统的五谷虫培育和消毒技术,为临床和基础研究提供材料来源;(2)分析五谷虫的抗菌物质,观察其抗菌活性,确定其对感染创面起到生物学作用的有效成份,为进一步对其进行药效、药理学和“基因工程”制剂生产研究奠定理论基础;(3)探讨五谷虫对感染创面抗菌与促进愈合作用的机理,为五谷虫临床应用提供理论依据。
本课题研究分三部分:(1)医用五谷虫制备及其粗提物的抗菌活性研究;(2)五谷虫抗菌物质的纯化鉴定及其分子结构的实验研究;(3)五谷虫对创面的抗菌与促进愈合作用研究。
第一部分医用五谷虫制备及其粗提物的抗菌活性研究
1.医用五谷虫的制备技术研究
本研究在室内常温环境下,采用无菌饲料对五谷虫主要虫种“丝光绿蝇幼虫”进行人工饲养、繁殖传代,对虫卵、虫体进行“双重消毒”处理。研究发现:丝光绿蝇的幼虫能在室内环境下人工饲养、稳定传代,可以作为五谷虫临床与基础实验的材料来源。虫卵、虫体“双重消毒”的方法能有效地对五谷虫进行消毒处理,减少五谷虫在饲养和使用过程中受到医源性感染的机会。
2.五谷虫粗提物对标准菌株的抗菌活性研究
本研究采用离心分离方法获得五谷虫分泌物和虫体组织匀浆粗提物。分别以金黄色葡萄球菌、大肠埃希氏菌标准菌株作为指示菌,对五谷虫粗提物进行琼脂平板法抗菌试验,用SDS-PAGE对粗提物进行电泳分析。结果显示:五谷虫粗提物组的抑菌环直径明显大于对照组,统计学有显著性差异(P<0.01)。研究发现:五谷虫分泌物和虫体组织匀浆粗提物中均含有同样有效的抗菌物质,对金黄色葡萄球菌和大肠杆菌均有抗菌作用。该抗菌物质以分子量较小的有机成分为主。
3.五谷虫对感染创面致病细菌的抗菌作用研究
本研究以糖尿病足溃疡创面上分离培养的金黄色葡萄球菌为指示菌,提取五谷虫粗提物,对粗提物采用平板法做抗菌实验,头孢哌酮和生理盐水做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究发现:五谷虫对金黄色葡萄球菌(一般致病菌)具有抗菌作用。
以糖尿病足溃疡创面上分离培养的铜绿假单胞菌(绿脓杆菌)为指示菌,提取五谷虫粗提物,对粗提物采用平板法做抗菌实验,头孢哌酮和生理盐水做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究发现:五谷虫对绿脓杆菌(特殊致病菌)具有抗菌作用。
本实验以脊髓损伤后截瘫患者压疮感染创面上分离培养的金黄色葡萄球菌为指示菌,提取经过指示菌针刺感染诱导后的五谷虫粗提物作为实验组,采用平板法做抗菌实验,未经感染诱导的五谷虫粗提物和头孢哌酮做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究说明:通过针刺感染诱导的外源性刺激能增强五谷虫抗菌物质的抗菌能力。
第二部分五谷虫抗菌物质的活性鉴定、分离纯化及其分子结构的实验研究
1.浊度法检测五谷虫分泌物抗菌活性的实验研究
本研究以金黄色葡萄球菌标准菌株为指示菌,将五谷虫分泌物粗提物与指示菌共同培养。用酶标仪测量培养液的光密度值,绘制光密度—时间曲线。用考马斯亮蓝法检测并调整分泌物中的蛋白质含量,采用浊度法检测不同蛋白质浓度分泌物样品的抗菌活性。结果显示:五谷虫粗提物与金黄色葡萄球菌共同培养21小时,金黄色葡萄球菌的吸收光密度OD值随着时间的增加而降低,对照液中金黄色葡萄球菌的吸光密度OD值随着时间的增加而增加,与对照液相比较,五谷虫粗提物对于金黄色葡萄球菌的生长产生了明显的抑制作用。抗菌活性随着分泌物中蛋白含量的变化而出现变化,0.8mg/ml、0.4mg/ml蛋白含量的分泌物抑菌效果明显,0.2mg/ml、0.1mg/ml蛋白含量的分泌物样品液无明显抑菌效果。实验说明:蛋白质浓度的调整会引起五谷虫抗菌物质抗菌活性的相应变化。
2.五谷虫分泌物抗菌活性物质的鉴定分析
本研究将五谷虫分泌物样品液经过DEAE离子交换层析,收集洗脱液和淋洗液,将洗脱液和淋洗液分别与金黄色葡萄球菌液于37℃温箱共同培养21小时,浊度法检测抗菌活性。SDS-PAGE技术对五谷虫分泌物样品液、洗脱液和淋洗液进行电泳,行考马斯亮兰染色,完成抗菌物质的鉴定分析。结果显示:淋洗液的OD值随着时间的增加而增加;洗脱液的OD值明显低于淋洗液的OD值,洗脱液对于金黄色葡萄球菌的生长产生了明显的抑制作用。五谷虫分泌物样品液、洗脱液经过SDS—PAGE电泳可见相似的蛋白质成分条带,淋洗液未见明显的蛋白质条带显示。实验发现:五谷虫分泌物中的抗菌活性物质是蛋白质类物质,其等电点小于8;DEAE离子交换层析可以作为分泌物中抗菌物质的分离提纯方法。
3.五谷虫抗菌活性物质的纯化研究
本研究通过离子交换和凝胶过滤两步层析对五谷虫分泌物中的抗菌物质进行分离提纯,在提纯过程中用浊度法检测洗脱液的抗菌活性,将洗脱液进行编号收集,进行SDS-PAGE与银染,寻找并获得分泌物中抗菌物质的蛋白质条带。结果显示:离子交换层析后,五谷虫分泌物中的蛋白质成分被完全洗脱下来,进入到编号的收集管洗脱液中,第33管洗脱液的蛋白质成分具有明显的抗菌活性;对第33管洗脱液进行凝胶过滤层析,层析后的洗脱液进入到编号收集管中,第13、14、15管洗脱液具有抗菌活性,其他收集管的洗脱液无抗菌活性;第13、14、15管洗脱液均显示出一条清晰的蛋白条带而且分子量相同,分子量小于14.4KDa。实验证实:离子交换层析和凝胶过滤层析能够对五谷虫分泌物中的抗菌蛋白进行有效地分离、纯化。
4.五谷虫抗菌活性物质的分子结构及生物信息学研究
本实验将经过层析纯化和SDS-PAGE而获得的五谷虫分泌物的抗菌蛋白条带切下,用胰蛋白酶水解。采用纳升级电喷雾质谱技术对酶解后抗菌蛋白产生的肽片段进行分子结构与生物信息学研究,寻找与该抗菌蛋白相关的同源性物质,获得该抗菌蛋白的生物学信息。结果显示:抗菌蛋白中存在ENAGEDPGLAR、LGKDAVEDLESVGK、DAVEDLESVGK共3个肽片段的氨基酸序列信息;经Bioworks软件检索NCBInr数据库,搜索同源性蛋白质信息,共得到三种蛋白质:dermcidin preproprotein [Homo sapiens](人源汗腺抗菌肽前原蛋白);dermcidin preproprotein [Macaca mulatta](猕猴源汗腺抗菌肽前原蛋白);protein phosphatase 1 inhib.(蛋白磷酸酶1抑制蛋白)。研究发现:五谷虫分泌物的抗菌蛋白是与Dermcidin preproprotein具有一定同源性的抗菌肽类物质。
第三部分五谷虫对创面的抗菌与促进愈合作用研究
1.五谷虫对DM大鼠溃疡创面的抗菌与愈合作用
本研究采用付小兵DM大鼠溃疡创面动物模型,将五谷虫分泌物外敷于实验组创面,通过组织形态学和免疫组化的方法研究五谷虫分泌物对DM大鼠溃疡组织中碱性成纤维细胞生长因子(bFGF)和结缔组织生长因子(CTGF)表达的影响。结果显示:五谷虫分泌物能提高DM大鼠溃疡创面中bFGF和CTGF的表达,其表达水平明显高于对照组,统计学分析有显著性差异(P<0.01),实验说明五谷虫分泌物具有促进创面愈合的作用。
取实验组和对照组溃疡创面分泌物作细菌培养,记录创面细菌感染情况。结果显示:对照组金葡菌感染率为60%,实验组溃疡创面一般菌培养结果为阴性,未见致病细菌生长。实验发现:五谷虫分泌物具有预防创面细菌感染的作用。
2.五谷虫对感染创面抗菌与生物清创作用的临床研究
本研究对6例糖尿病足部溃疡患者的感染创面、5例脊髓损伤后截瘫患者的压疮创面运用活体五谷虫进行治疗,取创面分泌物作菌培养,记录培养结果,观察创面愈合情况。结果显示:创面分泌物细菌培养逐渐由阳性转为阴性,平均时间11.2天;感染创面脓性分泌物及坏死组织逐渐减少,新鲜肉芽组织形成,平均时间2.6天;创面逐渐缩小,获得良好愈合,平均时间25.6天。实验证实:五谷虫可以有效地抵抗创面的细菌感染,吞食坏死组织而对健康组织无破坏,五谷虫“生物清创术”是治疗慢性感染创面的安全而有效的方法。
The number of patients suffering from chronic infective wounds is on the rise. Those wounds are attributable to diseases such as pressure ulcer after spinal cord injury (SCI), chronic osteomyelitis and infective wounds caused by diabetes mellitus and peripheral vascular disease. Those diseases pose a significant impact on the health care system, because of chronicity of care required, long term treatment and bad healing effects.Those infective wounds are difficult to be treated with common debridement and operation.
How to control the infection caused by bacterium is the key point to cure infective wound. Antibacterial drugs posed a significant impact on such kinds of diseases in the past time. Recently, the problem of the rising incidence of antibiotic resistance became serious. Antibiotic resistant bacteria could damage the wound quickly. As a result of the prevalence of antibiotic resistant bacteria, antibacterial function of common antibiotics on the infective wound was declined dominantly. At present, it is a problem to control the bacterial infection and hasten the process of infective wound healing in the field of modern surgery.
Chinese traditional medicine“WuGuChong”is the larvae of Lucilia sericata, Chrysomyia megacephala or other relative Calliphoridae insects.“Ben Cao Qiu Yuan”and“Ben Cao Bian Du”which are Chinese traditional books record that the pharmic character of the larvae is cool and innoxious, it is a medicine for external application to cure surgical infection diseases such as carbuncle and sore. The application of WuGuChong could offer a new idea to treat infective wound effectively, but antibacterial mechanism of WuGuChong could not be achieved from Chinese traditional medicine. Recently, doctors of western country such as Sherman R.A. began to do clinical studies of WuGuChong for infective wound treatment and found that WuGuChong could be used for cleaning and disinfection of chronic wounds that are sloughy, necrotic, and infected. Nowadays, the researches are mainly focused on case reports and treatment skills. The biomolecular and clinical study about antibacterial function is lacking.
Under the guidance of the theory of integrated traditional and western medicine, we would do the clinical and biomolecular research of antibacterial function of Chinese medicine WuGuChong on infective wound. The destinations of the project are as follows: 1. Achieve the systemic rearing and disinfection skills of WuGuChong, offer the materials for clinical and basic research; 2. Achieve the purified antibacterial substance of WuGuChong, get the character of antibacterial activity, confirm the effective antibacterial component on infective wound, realize the molecular structural and biological information, the project will lay the theoretical foundation for farther pharmacological research and gene engineering preparation of WuGuChong; 3. Discuss the antibacterial and healing mechanism of WuGuChong on infective wound, get the scientific application skills for clinical treatment.
The idiographic study contents as follows: Part 1.The preparation study of medical WuGuChong and the antibacterial activity study of primary purification; Part 2. Analysis and identification of antibacterial activity of WuGuChong, separation and purification of effective antibacterial substance and molecular structure study.Part 3.The study of antibacterial and promoting healing function about WuGuChong on infective wound.
Part1. The preparation study of Medical WuGuChong and the antibacterial activity study of primary purification
1. The preparation study of Medical WuGuChong
We investigated the skills of rearing and disinfection for the larvae of Lucilia sericata which was the mostly species of WuGuChong. WuGuChong was propagated and reared with artificial asepsis diets in laboratory, eggs and larvaes were disinfected with“double steps disinfection”. The results showed that Lucilia sericata could be reared in doors and the generation was stable. The eggs and larvaes were disinfected with“double steps disinfection”, all eggs and larvae were alive, none larvae were dead because of disinfection. No bacteria and virus were found from the the homogenization of the larvae. The experiment approved that the larvae of lucilia sericata could be reared with artificial skill under the asepsis environment indoors. The larvae of lucilia sericata could be used as the materials for the clinical and experimental research.
2. Antibacterial activity study of primary purification of WuGuChong on standard bacterium
We studied the antibacterial activity and biological characters of secretion and hemolymph from the primary purification of WuGuChong. Primary purification of hemolymph and secretion of the larvae were achieved with centrifugation, the antibacterial activity was tested with disc method, took use of standard Escherichia coli and Staphylococcus aureus as the indicator organisms. SDS-PAGE analysis was admitted for the primary purification of hemolymph and secretion. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). The experiment approved that not only the primary purification of hemolymph but also the primary purification of secretion had the same broad antibacterial and sterilizing activity to both E. coli and S. aureu. Main antibacterial substance of WuGuChong was some kind of organic component (smaller molecular weight).
3. The antibacterial function of WuGuChong against harmful bacterium from infective wound
In this study, we used S. aureus as the indicator organisms which from the infective wounds of clinical DM cases, the primary purification of WuGuChong was collected, the antibacterial activity was tested with disc method, and then recorded the antibacterial effect, cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to common pathopoiesis bacterium of infective wound.
Using Pseudemonas aeruginosa as the indicator organisms which from the infective wound of clinical DM cases, the primary purification of WuGuChong was collected , the antibacterial activity was tested with disc method , and then recorded the antibacterial effect , cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to special pathopoiesis bacterium of infective wound.
In the study, the hemolymph of the larvae which was induced by the bacterium with pricking was collected, the hemolymph of the asepsis larvae which was not induced by the bacteria and Cefoperazone Sodium as the contrast groups, Staphylococcus aureus as the indicator organisms which from the infective wound of pressure ulcer after spinal cord injury. The antibacterial activity was tested with disc method, the antibacterial effect was observed. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01).The experiment approved that the antibacterial substance of WuGuChong was some native component which was not induced by external stimulation. External stimulation could enhance the antibacterial activity.
Part2. Analysis and identification of antibacterial activity of WuGuChong,separation and purification of effective antibacterial substance and molecular structure study.
1. The study of Turbidometric (TB) assay for antibacterial activity of WuGuChong secretion
In the experiment, we used standard Staphylococcus aureus as the indicator organisms. The secretion of WuGuChong and the indicator organisms were cultured together, measurement of Turbidometric (TB) assay was taken: collected the culture fluid (sample fluid and contrast fluid), measured the OD value on different time with ELIASA.OD-time curve was protracted. Checked and adjusted the protein content of the sample fluid with Commassie Blue, measured the antibacterial activity of sample fluid with different protein concentration. Total culture time was 21 hours, the OD value of sample fluid was declined with the time going on, the OD value of contrast fluid was increased with the time going on, evident antibacterial function was found in the sample fluid. Dominant antibacterial activity was found on the protein concentration of 0.8mg/ml and 0.4mg/ml, no dominant antibacterial activity was found on the protein concentration of 0.2mg/ml and 0.1mg/ml.The results approved that antibacterial activity in the secretion of WuGuChong was changed with the changing of protein concentration.
2. Analysis and identification of antibacterial substance of WuGuChong
In this experiment, we collected the secretion of WuGuChong, purified the secretion with DEAE-Sepharose ion exchange, and then collected the gradient elution and the rinse fluid. Not only elution but also rinse fluid was cultured with S. aureus at 37℃for 21 hours. Antibacterial assay was tested and confirmed for measurement of Turbidometric (TB) assay. The sample of secretion, elution and rinse fluid were checked by SDS-PAGE. The results showed that evident antibacterial activity was found in the elution fluid. Similar protein bandages were shown in both secretion and elution, no protein bandage was found in the sample of rinse fluid. The proteins of the secretion of larvae were eluted completely. The experiment approved that the antibacterial material of the secretion of WuGuChong was some kind of protein (PI<8), DEAE-sepharose ion exchange chromatography was elected as primary method and its gradient elution condition was optimized.
3. Purification research of antibacterial substance of WuGuChong
And then, we investigated the purifying effects of DEAE-Sepharose ion exchange chromatography and gel filtration chromatography for antibacterial substance of the secretion from WuGuChong. Antibacterial assay was tested with measurement of Turbidometric (TB) assay. The gradient elution was collected into serial number tubes in the course of chromatography. The gradient elution into serial number tubes was checked by SDS-PAGE and silver stain, searched the objective antibacterial protein, achieved the objective protein bandage. The results showed that protein contents of tube 33 had dominant antibacterial after ion exchange chromatography. The elution of tube 33 was purified with Superdex-75 gel filtration, antibacterial activity was found in 13, 14, 15 tube of the elution, no significant activity was found in other tubes. The results of SDS-PAGE and silver stain of the elution in tube 13-15 showed that there was a clear protein bandage (MW<14.4KDa), no other protein bandage was found in the elution of tube 13-15. The experiment approved that DEAE-Sepharose ion exchange followed by Superdex-75 gel filtration could purify the antibacterial substance effectively.
4. Molecular structure and biological informatics study of antibacterial substance of WuGuChong
In the study, the objective protein bandage was sliced and dealed with Trypsin, the fragments after zymohydrolysis were achieved. The nano-ESI-MS/MS instrument was used to assay the fragments. We did the study of molecular structure and biological informatics of antibacterial substance of WuGuChong with mass spectrum, searched the homologous substance and achieved effective biological information of the antibacterial substance. The results showed that three fragments were found as follows: ENAGEDPGLAR; LGKDAVEDLESVGK; DAVEDLESVGK. Antibacterial protein of the secretion of WuGuChong contained the information of those three fragments. Checked NCBInr database with Bioworks software, achieved the information of homologous proteins: dermcidin preproprotein [Homo sapiens]; dermcidin preproprotein [Macaca mulatta]; protein phosphatase 1 inhib. After identification and analysis of biological informatics, the experiment approved that the antibacterial substance of the secretion of WuGuChong was a kind of antibacterial peptide, there was a higher degree of similarity existed between WuGuChong and Dermcidin preproprotein.
Part 3. The study of antibacterial and promoting healing function about WuGuChong on infective wound
1. The antibacterial and healing function of WuGuChong on ulcer wound of DM rat
The“Fu Xiaobing”model of ulcer wound of DM rats was made. The ulcer wound of DM rats in maggot secretion group spread maggot secretions, but no secretion on ulcer wound was found in control group. Morphological and immunohistological detection were used to observe the expression of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) in ulcer tissue. It was found that the secretion of WuGuChong could elevate the expressions of bFGF and CTFG in ulcers of DM rats , the expression level was higher than that in contrast group (P<0.01), WuGuChong could promote the wound’s healing.
In this study, the secretion from the wound of experimental and contrast group was checked with bacterial culture, the results showed that no Staphylococcus aureus infection was seen in experimental group, Staphylococcus aureus infection occurred in contrast group, the rate of Staphylococcus aureus infection was 60%. The experiment approved that the secretion of WuGuChong could prevent bacterial infection on wound.
2. The clinical research about antibacterium and biological debridement of WuGuChong on infective wound
In the clinical research, WuGuChong therapeutics was used to repair different infective wounds, the cases as follows: 6 cases suffered from foot ulcer of diabetes mellitus, 5 cases suffered from pressure ulcer after spinal cord injury. The changes of wound surface were observed, bacterial culture was tested. The research showed that negative results were found in the bacterial culture(average 11.2days), the suppurative and necrotic tissue in the wound was declined, and a great amount fresh granulation tissue was generated(average 2.6 days), ideal wound healing occurred in all cases(average 25.6days). The clinical research approved that WuGuChong could resist bacterial infection of wounds, the biological debridement therapy of WuGuChong was valuable and safe in the debridement of intractable chronic wounds. WuGuChong could reach necrotic tissue in all areas of the wound and clean minutes areas without damaging healthy tissue.
传统中药材“五谷虫”,又名蛆虫,为丽蝇科丝光绿蝇、大头金蝇的幼虫。《本草求源》、《本草便读》均记载:该虫性寒无毒,可搽敷外用,能治疗臁疮等感染性外科疾病。为临床治疗慢性难治性感染创面提供了一种新的思路。但是中医学对五谷虫治疗感染创面缺乏系统的研究,其抗菌机制还不十分清楚。近年来,Sherman R.A.等国外学者发现,五谷虫治疗感染创面,能有效地促进创面愈合,抵抗细菌感染。但目前研究仍主要集中于临床治疗病例报道,缺乏与五谷虫抗菌作用相关的临床与分子生物学的系统研究。
本课题采用中西医结合的方法,对五谷虫对感染创面抗菌作用的临床与分子机制进行系统地研究。旨在(1)探索系统的五谷虫培育和消毒技术,为临床和基础研究提供材料来源;(2)分析五谷虫的抗菌物质,观察其抗菌活性,确定其对感染创面起到生物学作用的有效成份,为进一步对其进行药效、药理学和“基因工程”制剂生产研究奠定理论基础;(3)探讨五谷虫对感染创面抗菌与促进愈合作用的机理,为五谷虫临床应用提供理论依据。
本课题研究分三部分:(1)医用五谷虫制备及其粗提物的抗菌活性研究;(2)五谷虫抗菌物质的纯化鉴定及其分子结构的实验研究;(3)五谷虫对创面的抗菌与促进愈合作用研究。
第一部分医用五谷虫制备及其粗提物的抗菌活性研究
1.医用五谷虫的制备技术研究
本研究在室内常温环境下,采用无菌饲料对五谷虫主要虫种“丝光绿蝇幼虫”进行人工饲养、繁殖传代,对虫卵、虫体进行“双重消毒”处理。研究发现:丝光绿蝇的幼虫能在室内环境下人工饲养、稳定传代,可以作为五谷虫临床与基础实验的材料来源。虫卵、虫体“双重消毒”的方法能有效地对五谷虫进行消毒处理,减少五谷虫在饲养和使用过程中受到医源性感染的机会。
2.五谷虫粗提物对标准菌株的抗菌活性研究
本研究采用离心分离方法获得五谷虫分泌物和虫体组织匀浆粗提物。分别以金黄色葡萄球菌、大肠埃希氏菌标准菌株作为指示菌,对五谷虫粗提物进行琼脂平板法抗菌试验,用SDS-PAGE对粗提物进行电泳分析。结果显示:五谷虫粗提物组的抑菌环直径明显大于对照组,统计学有显著性差异(P<0.01)。研究发现:五谷虫分泌物和虫体组织匀浆粗提物中均含有同样有效的抗菌物质,对金黄色葡萄球菌和大肠杆菌均有抗菌作用。该抗菌物质以分子量较小的有机成分为主。
3.五谷虫对感染创面致病细菌的抗菌作用研究
本研究以糖尿病足溃疡创面上分离培养的金黄色葡萄球菌为指示菌,提取五谷虫粗提物,对粗提物采用平板法做抗菌实验,头孢哌酮和生理盐水做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究发现:五谷虫对金黄色葡萄球菌(一般致病菌)具有抗菌作用。
以糖尿病足溃疡创面上分离培养的铜绿假单胞菌(绿脓杆菌)为指示菌,提取五谷虫粗提物,对粗提物采用平板法做抗菌实验,头孢哌酮和生理盐水做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究发现:五谷虫对绿脓杆菌(特殊致病菌)具有抗菌作用。
本实验以脊髓损伤后截瘫患者压疮感染创面上分离培养的金黄色葡萄球菌为指示菌,提取经过指示菌针刺感染诱导后的五谷虫粗提物作为实验组,采用平板法做抗菌实验,未经感染诱导的五谷虫粗提物和头孢哌酮做对照。结果显示:实验组的抑菌环直径明显大于对照组,统计学分析有显著性差异(P<0.01)。研究说明:通过针刺感染诱导的外源性刺激能增强五谷虫抗菌物质的抗菌能力。
第二部分五谷虫抗菌物质的活性鉴定、分离纯化及其分子结构的实验研究
1.浊度法检测五谷虫分泌物抗菌活性的实验研究
本研究以金黄色葡萄球菌标准菌株为指示菌,将五谷虫分泌物粗提物与指示菌共同培养。用酶标仪测量培养液的光密度值,绘制光密度—时间曲线。用考马斯亮蓝法检测并调整分泌物中的蛋白质含量,采用浊度法检测不同蛋白质浓度分泌物样品的抗菌活性。结果显示:五谷虫粗提物与金黄色葡萄球菌共同培养21小时,金黄色葡萄球菌的吸收光密度OD值随着时间的增加而降低,对照液中金黄色葡萄球菌的吸光密度OD值随着时间的增加而增加,与对照液相比较,五谷虫粗提物对于金黄色葡萄球菌的生长产生了明显的抑制作用。抗菌活性随着分泌物中蛋白含量的变化而出现变化,0.8mg/ml、0.4mg/ml蛋白含量的分泌物抑菌效果明显,0.2mg/ml、0.1mg/ml蛋白含量的分泌物样品液无明显抑菌效果。实验说明:蛋白质浓度的调整会引起五谷虫抗菌物质抗菌活性的相应变化。
2.五谷虫分泌物抗菌活性物质的鉴定分析
本研究将五谷虫分泌物样品液经过DEAE离子交换层析,收集洗脱液和淋洗液,将洗脱液和淋洗液分别与金黄色葡萄球菌液于37℃温箱共同培养21小时,浊度法检测抗菌活性。SDS-PAGE技术对五谷虫分泌物样品液、洗脱液和淋洗液进行电泳,行考马斯亮兰染色,完成抗菌物质的鉴定分析。结果显示:淋洗液的OD值随着时间的增加而增加;洗脱液的OD值明显低于淋洗液的OD值,洗脱液对于金黄色葡萄球菌的生长产生了明显的抑制作用。五谷虫分泌物样品液、洗脱液经过SDS—PAGE电泳可见相似的蛋白质成分条带,淋洗液未见明显的蛋白质条带显示。实验发现:五谷虫分泌物中的抗菌活性物质是蛋白质类物质,其等电点小于8;DEAE离子交换层析可以作为分泌物中抗菌物质的分离提纯方法。
3.五谷虫抗菌活性物质的纯化研究
本研究通过离子交换和凝胶过滤两步层析对五谷虫分泌物中的抗菌物质进行分离提纯,在提纯过程中用浊度法检测洗脱液的抗菌活性,将洗脱液进行编号收集,进行SDS-PAGE与银染,寻找并获得分泌物中抗菌物质的蛋白质条带。结果显示:离子交换层析后,五谷虫分泌物中的蛋白质成分被完全洗脱下来,进入到编号的收集管洗脱液中,第33管洗脱液的蛋白质成分具有明显的抗菌活性;对第33管洗脱液进行凝胶过滤层析,层析后的洗脱液进入到编号收集管中,第13、14、15管洗脱液具有抗菌活性,其他收集管的洗脱液无抗菌活性;第13、14、15管洗脱液均显示出一条清晰的蛋白条带而且分子量相同,分子量小于14.4KDa。实验证实:离子交换层析和凝胶过滤层析能够对五谷虫分泌物中的抗菌蛋白进行有效地分离、纯化。
4.五谷虫抗菌活性物质的分子结构及生物信息学研究
本实验将经过层析纯化和SDS-PAGE而获得的五谷虫分泌物的抗菌蛋白条带切下,用胰蛋白酶水解。采用纳升级电喷雾质谱技术对酶解后抗菌蛋白产生的肽片段进行分子结构与生物信息学研究,寻找与该抗菌蛋白相关的同源性物质,获得该抗菌蛋白的生物学信息。结果显示:抗菌蛋白中存在ENAGEDPGLAR、LGKDAVEDLESVGK、DAVEDLESVGK共3个肽片段的氨基酸序列信息;经Bioworks软件检索NCBInr数据库,搜索同源性蛋白质信息,共得到三种蛋白质:dermcidin preproprotein [Homo sapiens](人源汗腺抗菌肽前原蛋白);dermcidin preproprotein [Macaca mulatta](猕猴源汗腺抗菌肽前原蛋白);protein phosphatase 1 inhib.(蛋白磷酸酶1抑制蛋白)。研究发现:五谷虫分泌物的抗菌蛋白是与Dermcidin preproprotein具有一定同源性的抗菌肽类物质。
第三部分五谷虫对创面的抗菌与促进愈合作用研究
1.五谷虫对DM大鼠溃疡创面的抗菌与愈合作用
本研究采用付小兵DM大鼠溃疡创面动物模型,将五谷虫分泌物外敷于实验组创面,通过组织形态学和免疫组化的方法研究五谷虫分泌物对DM大鼠溃疡组织中碱性成纤维细胞生长因子(bFGF)和结缔组织生长因子(CTGF)表达的影响。结果显示:五谷虫分泌物能提高DM大鼠溃疡创面中bFGF和CTGF的表达,其表达水平明显高于对照组,统计学分析有显著性差异(P<0.01),实验说明五谷虫分泌物具有促进创面愈合的作用。
取实验组和对照组溃疡创面分泌物作细菌培养,记录创面细菌感染情况。结果显示:对照组金葡菌感染率为60%,实验组溃疡创面一般菌培养结果为阴性,未见致病细菌生长。实验发现:五谷虫分泌物具有预防创面细菌感染的作用。
2.五谷虫对感染创面抗菌与生物清创作用的临床研究
本研究对6例糖尿病足部溃疡患者的感染创面、5例脊髓损伤后截瘫患者的压疮创面运用活体五谷虫进行治疗,取创面分泌物作菌培养,记录培养结果,观察创面愈合情况。结果显示:创面分泌物细菌培养逐渐由阳性转为阴性,平均时间11.2天;感染创面脓性分泌物及坏死组织逐渐减少,新鲜肉芽组织形成,平均时间2.6天;创面逐渐缩小,获得良好愈合,平均时间25.6天。实验证实:五谷虫可以有效地抵抗创面的细菌感染,吞食坏死组织而对健康组织无破坏,五谷虫“生物清创术”是治疗慢性感染创面的安全而有效的方法。
The number of patients suffering from chronic infective wounds is on the rise. Those wounds are attributable to diseases such as pressure ulcer after spinal cord injury (SCI), chronic osteomyelitis and infective wounds caused by diabetes mellitus and peripheral vascular disease. Those diseases pose a significant impact on the health care system, because of chronicity of care required, long term treatment and bad healing effects.Those infective wounds are difficult to be treated with common debridement and operation.
How to control the infection caused by bacterium is the key point to cure infective wound. Antibacterial drugs posed a significant impact on such kinds of diseases in the past time. Recently, the problem of the rising incidence of antibiotic resistance became serious. Antibiotic resistant bacteria could damage the wound quickly. As a result of the prevalence of antibiotic resistant bacteria, antibacterial function of common antibiotics on the infective wound was declined dominantly. At present, it is a problem to control the bacterial infection and hasten the process of infective wound healing in the field of modern surgery.
Chinese traditional medicine“WuGuChong”is the larvae of Lucilia sericata, Chrysomyia megacephala or other relative Calliphoridae insects.“Ben Cao Qiu Yuan”and“Ben Cao Bian Du”which are Chinese traditional books record that the pharmic character of the larvae is cool and innoxious, it is a medicine for external application to cure surgical infection diseases such as carbuncle and sore. The application of WuGuChong could offer a new idea to treat infective wound effectively, but antibacterial mechanism of WuGuChong could not be achieved from Chinese traditional medicine. Recently, doctors of western country such as Sherman R.A. began to do clinical studies of WuGuChong for infective wound treatment and found that WuGuChong could be used for cleaning and disinfection of chronic wounds that are sloughy, necrotic, and infected. Nowadays, the researches are mainly focused on case reports and treatment skills. The biomolecular and clinical study about antibacterial function is lacking.
Under the guidance of the theory of integrated traditional and western medicine, we would do the clinical and biomolecular research of antibacterial function of Chinese medicine WuGuChong on infective wound. The destinations of the project are as follows: 1. Achieve the systemic rearing and disinfection skills of WuGuChong, offer the materials for clinical and basic research; 2. Achieve the purified antibacterial substance of WuGuChong, get the character of antibacterial activity, confirm the effective antibacterial component on infective wound, realize the molecular structural and biological information, the project will lay the theoretical foundation for farther pharmacological research and gene engineering preparation of WuGuChong; 3. Discuss the antibacterial and healing mechanism of WuGuChong on infective wound, get the scientific application skills for clinical treatment.
The idiographic study contents as follows: Part 1.The preparation study of medical WuGuChong and the antibacterial activity study of primary purification; Part 2. Analysis and identification of antibacterial activity of WuGuChong, separation and purification of effective antibacterial substance and molecular structure study.Part 3.The study of antibacterial and promoting healing function about WuGuChong on infective wound.
Part1. The preparation study of Medical WuGuChong and the antibacterial activity study of primary purification
1. The preparation study of Medical WuGuChong
We investigated the skills of rearing and disinfection for the larvae of Lucilia sericata which was the mostly species of WuGuChong. WuGuChong was propagated and reared with artificial asepsis diets in laboratory, eggs and larvaes were disinfected with“double steps disinfection”. The results showed that Lucilia sericata could be reared in doors and the generation was stable. The eggs and larvaes were disinfected with“double steps disinfection”, all eggs and larvae were alive, none larvae were dead because of disinfection. No bacteria and virus were found from the the homogenization of the larvae. The experiment approved that the larvae of lucilia sericata could be reared with artificial skill under the asepsis environment indoors. The larvae of lucilia sericata could be used as the materials for the clinical and experimental research.
2. Antibacterial activity study of primary purification of WuGuChong on standard bacterium
We studied the antibacterial activity and biological characters of secretion and hemolymph from the primary purification of WuGuChong. Primary purification of hemolymph and secretion of the larvae were achieved with centrifugation, the antibacterial activity was tested with disc method, took use of standard Escherichia coli and Staphylococcus aureus as the indicator organisms. SDS-PAGE analysis was admitted for the primary purification of hemolymph and secretion. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). The experiment approved that not only the primary purification of hemolymph but also the primary purification of secretion had the same broad antibacterial and sterilizing activity to both E. coli and S. aureu. Main antibacterial substance of WuGuChong was some kind of organic component (smaller molecular weight).
3. The antibacterial function of WuGuChong against harmful bacterium from infective wound
In this study, we used S. aureus as the indicator organisms which from the infective wounds of clinical DM cases, the primary purification of WuGuChong was collected, the antibacterial activity was tested with disc method, and then recorded the antibacterial effect, cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to common pathopoiesis bacterium of infective wound.
Using Pseudemonas aeruginosa as the indicator organisms which from the infective wound of clinical DM cases, the primary purification of WuGuChong was collected , the antibacterial activity was tested with disc method , and then recorded the antibacterial effect , cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to special pathopoiesis bacterium of infective wound.
In the study, the hemolymph of the larvae which was induced by the bacterium with pricking was collected, the hemolymph of the asepsis larvae which was not induced by the bacteria and Cefoperazone Sodium as the contrast groups, Staphylococcus aureus as the indicator organisms which from the infective wound of pressure ulcer after spinal cord injury. The antibacterial activity was tested with disc method, the antibacterial effect was observed. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01).The experiment approved that the antibacterial substance of WuGuChong was some native component which was not induced by external stimulation. External stimulation could enhance the antibacterial activity.
Part2. Analysis and identification of antibacterial activity of WuGuChong,separation and purification of effective antibacterial substance and molecular structure study.
1. The study of Turbidometric (TB) assay for antibacterial activity of WuGuChong secretion
In the experiment, we used standard Staphylococcus aureus as the indicator organisms. The secretion of WuGuChong and the indicator organisms were cultured together, measurement of Turbidometric (TB) assay was taken: collected the culture fluid (sample fluid and contrast fluid), measured the OD value on different time with ELIASA.OD-time curve was protracted. Checked and adjusted the protein content of the sample fluid with Commassie Blue, measured the antibacterial activity of sample fluid with different protein concentration. Total culture time was 21 hours, the OD value of sample fluid was declined with the time going on, the OD value of contrast fluid was increased with the time going on, evident antibacterial function was found in the sample fluid. Dominant antibacterial activity was found on the protein concentration of 0.8mg/ml and 0.4mg/ml, no dominant antibacterial activity was found on the protein concentration of 0.2mg/ml and 0.1mg/ml.The results approved that antibacterial activity in the secretion of WuGuChong was changed with the changing of protein concentration.
2. Analysis and identification of antibacterial substance of WuGuChong
In this experiment, we collected the secretion of WuGuChong, purified the secretion with DEAE-Sepharose ion exchange, and then collected the gradient elution and the rinse fluid. Not only elution but also rinse fluid was cultured with S. aureus at 37℃for 21 hours. Antibacterial assay was tested and confirmed for measurement of Turbidometric (TB) assay. The sample of secretion, elution and rinse fluid were checked by SDS-PAGE. The results showed that evident antibacterial activity was found in the elution fluid. Similar protein bandages were shown in both secretion and elution, no protein bandage was found in the sample of rinse fluid. The proteins of the secretion of larvae were eluted completely. The experiment approved that the antibacterial material of the secretion of WuGuChong was some kind of protein (PI<8), DEAE-sepharose ion exchange chromatography was elected as primary method and its gradient elution condition was optimized.
3. Purification research of antibacterial substance of WuGuChong
And then, we investigated the purifying effects of DEAE-Sepharose ion exchange chromatography and gel filtration chromatography for antibacterial substance of the secretion from WuGuChong. Antibacterial assay was tested with measurement of Turbidometric (TB) assay. The gradient elution was collected into serial number tubes in the course of chromatography. The gradient elution into serial number tubes was checked by SDS-PAGE and silver stain, searched the objective antibacterial protein, achieved the objective protein bandage. The results showed that protein contents of tube 33 had dominant antibacterial after ion exchange chromatography. The elution of tube 33 was purified with Superdex-75 gel filtration, antibacterial activity was found in 13, 14, 15 tube of the elution, no significant activity was found in other tubes. The results of SDS-PAGE and silver stain of the elution in tube 13-15 showed that there was a clear protein bandage (MW<14.4KDa), no other protein bandage was found in the elution of tube 13-15. The experiment approved that DEAE-Sepharose ion exchange followed by Superdex-75 gel filtration could purify the antibacterial substance effectively.
4. Molecular structure and biological informatics study of antibacterial substance of WuGuChong
In the study, the objective protein bandage was sliced and dealed with Trypsin, the fragments after zymohydrolysis were achieved. The nano-ESI-MS/MS instrument was used to assay the fragments. We did the study of molecular structure and biological informatics of antibacterial substance of WuGuChong with mass spectrum, searched the homologous substance and achieved effective biological information of the antibacterial substance. The results showed that three fragments were found as follows: ENAGEDPGLAR; LGKDAVEDLESVGK; DAVEDLESVGK. Antibacterial protein of the secretion of WuGuChong contained the information of those three fragments. Checked NCBInr database with Bioworks software, achieved the information of homologous proteins: dermcidin preproprotein [Homo sapiens]; dermcidin preproprotein [Macaca mulatta]; protein phosphatase 1 inhib. After identification and analysis of biological informatics, the experiment approved that the antibacterial substance of the secretion of WuGuChong was a kind of antibacterial peptide, there was a higher degree of similarity existed between WuGuChong and Dermcidin preproprotein.
Part 3. The study of antibacterial and promoting healing function about WuGuChong on infective wound
1. The antibacterial and healing function of WuGuChong on ulcer wound of DM rat
The“Fu Xiaobing”model of ulcer wound of DM rats was made. The ulcer wound of DM rats in maggot secretion group spread maggot secretions, but no secretion on ulcer wound was found in control group. Morphological and immunohistological detection were used to observe the expression of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) in ulcer tissue. It was found that the secretion of WuGuChong could elevate the expressions of bFGF and CTFG in ulcers of DM rats , the expression level was higher than that in contrast group (P<0.01), WuGuChong could promote the wound’s healing.
In this study, the secretion from the wound of experimental and contrast group was checked with bacterial culture, the results showed that no Staphylococcus aureus infection was seen in experimental group, Staphylococcus aureus infection occurred in contrast group, the rate of Staphylococcus aureus infection was 60%. The experiment approved that the secretion of WuGuChong could prevent bacterial infection on wound.
2. The clinical research about antibacterium and biological debridement of WuGuChong on infective wound
In the clinical research, WuGuChong therapeutics was used to repair different infective wounds, the cases as follows: 6 cases suffered from foot ulcer of diabetes mellitus, 5 cases suffered from pressure ulcer after spinal cord injury. The changes of wound surface were observed, bacterial culture was tested. The research showed that negative results were found in the bacterial culture(average 11.2days), the suppurative and necrotic tissue in the wound was declined, and a great amount fresh granulation tissue was generated(average 2.6 days), ideal wound healing occurred in all cases(average 25.6days). The clinical research approved that WuGuChong could resist bacterial infection of wounds, the biological debridement therapy of WuGuChong was valuable and safe in the debridement of intractable chronic wounds. WuGuChong could reach necrotic tissue in all areas of the wound and clean minutes areas without damaging healthy tissue.
引文
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