组织工程皮肤试剂盒的研发及在治疗犬小孢子菌感染中的应用

发布时间：2018-10-31 18:03

【摘要】：基于已有研究,发现皮肤真菌患病犬样本中94.29%的犬为单一真菌种类病原体。考虑到犬小孢子菌的高感染率及存在动物到人的传染风险,本研究选取犬小孢子菌感染犬作为研究对象。从新兽药的临床前研究减少动物的使用量和从众多可供使用的药物中筛选疗效更好的药物这2个因素出发,本研究采用个体犬分离的真菌感染特制的组织工程皮肤试剂盒,以检测待使用的外用抗真菌药物,探索在治疗犬小孢子菌感染中的应用。其中,为了对试剂盒的结构特别是角质层结构的稳定性的反复测试及探索一套组织工程皮肤试剂盒的运输培养基,本研究还对组织工程皮肤的表皮层结构和运输培养基进行了标准化。1 组织工程皮肤的构建及试剂盒组装利用培养的犬皮肤成纤维细胞和角质形成细胞能够成功构建出组织工程皮肤。经切片和HE染色检测显示,成纤维细胞与自分泌细胞外基质的复合物经过处理并未残存活细胞成分,角质形成细胞紧密贴附于成纤维细胞与自分泌细胞外基质的复合物表面,并增殖分化为多层细胞成分,具有与犬天然皮肤类似的表皮层微结构,包括角质层、颗粒层和基底层。2 组织工程皮肤试剂盒表皮层结构的标准化为了对组织工程皮肤的表皮层标准化,通过定量的方式计算表皮层屏障功能总积分,试验组为8分,对照组为6分。组织工程皮肤的等级评定为:试验组"优秀",对照组"良好",显然试验组在屏障功能上优于对照组。3 组织工程皮肤试剂盒运输培养基的标准化为了对组织工程皮肤试剂盒的运输条件特别是运输时营养供给标准化,通过定量的方法计算出运输后的组织工程皮肤屏障功能总积分7分,等级评定为“优秀",与对照组无显著差异。4试剂盒在治疗犬小孢子菌感染中的应用从犬患处分离真菌,经过纯化培养和鉴定培养筛选出犬小孢子菌,并将菌接种于预先制备好的组织工程皮肤检测试剂盒孵育6h,之后对试验组和对照组进行组织学染色和流式细胞仪计数分析,结果显示药物作用试验组,盐酸特比奈芬、硝酸咪康唑和环吡酮胺组抗感染药物阻断效率AIDE平均值分别为:87.45%、72.50%和69.14%。样本犬用药前临床症状积分为2,动物治疗试验显示,盐酸特比奈芬组在3w时临床症状积分为0,临床症状积分下降指数SSRI已达100%并持续至4w;硝酸咪康唑和环吡酮胺组4w时临床症状积分为1和0,对应的SSRI分别为50%、100%。
[Abstract]:Based on previous studies, 94.29% of canines with skin fungi were found to be single fungal pathogens. Considering the high infection rate of microsporozoa canis and the risk of animal to human infection, this study selected the canine microsporozoa infection dog as the research object. Based on the two factors of reducing the use of new veterinary drugs in preclinical studies and screening more effective drugs from many available drugs, a tissue engineering skin kit specially designed for fungal infection in dogs was used in this study. To detect the external antifungal drugs to be used and to explore the application in the treatment of canine microsporozoa infection. Among them, in order to repeatedly test the structure of the kit, especially the stability of the cuticle structure, and to explore a set of transport medium for tissue engineering skin test kit, The structure of the epidermis and the transport medium of tissue engineered skin were standardized. 1 the construction of tissue engineered skin and the assembly of the kit could successfully use the cultured canine skin fibroblasts and keratinocytes Tissue engineered skin was constructed. The results of sections and HE staining showed that the complex of fibroblast and extracellular matrix (ECM) did not remain alive after treatment, and the keratinocytes were closely attached to the surface of the complex between fibroblast and extracellular matrix (ECM). They proliferate and differentiate into multilayer cellular components with epidermal microstructures similar to those of canine natural skin, including the cuticle. In order to standardize the surface of tissue engineering skin, the total integral of the barrier function of the epidermis was calculated by quantitative method, and the test group was 8 points. The control group was 6 points. The grade of tissue engineering skin was "excellent" in the test group and "good" in the control group. Obviously, the test group was superior to the control group in barrier function. 3 the transport medium of tissue engineered skin kit was standardized in order to standardize the transport conditions of tissue engineering skin kit, especially the nutrition supply during transportation. The total score of skin barrier function of tissue engineering after transportation was calculated by quantitative method, which was rated as "excellent", and there was no significant difference between the control group and the control group. 4 the application of the kit in the treatment of microsporozoa infection in dogs was used to isolate fungi from the affected areas of dogs. Microsporozoa canis was screened by purified culture and identified and incubated with tissue engineering skin detection kit for 6 hours. Then histological staining and flow cytometry analysis were performed on the experimental group and control group. The results showed that the average value of AIDE of anti-infective drugs in the experimental group, terbinafine hydrochloride group, miconazole nitrate group and cyclopyranone amine group was 87.4575% and 69.14%, respectively. The clinical symptom score of the dogs before treatment was 2. The clinical symptom score of terbinafine hydrochloride group was 0 at 3w, and the decrease index of clinical symptom score (SSRI) reached 100% and lasted until 4 ws. In miconazole nitrate and cyclopyramidine group, the scores of clinical symptoms were 1 and 0 at 4 weeks, and the corresponding SSRI were 50% and 100%, respectively.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.292

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