Ertabaklar Hatice, Çalışkan Serçin Özlem, Kolli Bala, Ertuğ Sema, Özbilgin Ahmet, Malatyalı Erdoğan, Chang Kwang Poo
Adnan Menderes University, Faculty of Medicine, Department of Parasitology, Aydın, Turkey.
Uşak University, Faculty of Medicine, Department of Biophysics, Uşak, Turkey.
Mikrobiyol Bul. 2019 Apr;53(2):213-223. doi: 10.5578/mb.67473.
Cutaneous leishmaniasis (CL) is a parasitic disease transmitted by vector sand flies Phlebotomus and Lutzomyia. This disease is characterized by long time non-healing skin lesions, and caused by Leishmania species. CL is the most common infection in Eastern and Southeastern Anatolia in Turkey and L.tropica is known as the main agent of the disease. Number of cases is increasing in our country in time because of malnutrition, migration, travel, low socioeconomic level and ecological changes. For the treatment, the pentavalent antimonials are often used as intralesionally for many years, and it was reported that resistant cases have increased in recent years. New treatment methods and anti-Leishmanial activity of new agents have been investigated because of side effects, resistance development and toxic reactions of the present drugs. These studies are first carried out in vitro and afterwards with in vivo experimental animal models. Reporter gene technology has been used to investigate a variety of purposes like biological events in microorganisms and the efficacy and resistance of drugs in recent years. The major areas that green fluorescent protein (gfp) used are that they can be incorporated into different genes to determine the amount of expression of these genes in different organisms and can be used as markers in living cells. Especially gfp gene, which encodes the green fluorescent protein, is widely used nowadays. Gene-based assays have several advantages like being easy to follow-up, inexpensive and have improved biosecurity. The aim of the present study was to perform the transfection of L.tropica with "enhanced gfp (egfp)" and in vitro usefulness of gfp-transfectants as a drug screening model in comparison to the conventional methods. Promastigotes of L.tropica were transfected with p6.5/egfp by electroporation and selected for tunicamycin-resistance as previously described. L.tropica promastigotes transfected with gfp and in vitro effect of meglumine animoniate was assessed using different methods such as fluorescence microscopy, fluorometer and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide) assay. The use of gfp-transfected Leishmania strains was found more rapid and more sensitive by fluorescent microscopy and fluorometry than conventional assays for the evaluation of potential anti-leishmanial agents. Consequently, stable gfp-transfected Leishmania species will be used in vitro and in vivo for screening of anti-leishmanial drugs and vaccine development as well as for understanding the biology of the host-parasite interactions at the cellular level. As a result ot this study, gfp transfected model using a Turkish L.tropica isolate was established to be used in further studies.
皮肤利什曼病（CL）是一种由媒介白蛉属和罗蛉属沙蝇传播的寄生虫病。该疾病的特征是皮肤损伤长期不愈合，由利什曼原虫属引起。CL是土耳其东安纳托利亚和东南安纳托利亚最常见的感染疾病，热带利什曼原虫被认为是该病的主要病原体。由于营养不良、移民、旅行、社会经济水平低下和生态变化，我国的病例数一直在增加。多年来，五价锑剂常被用于皮损内注射治疗，据报道近年来耐药病例有所增加。由于现有药物的副作用、耐药性发展和毒性反应，人们对新的治疗方法和新型抗利什曼原虫药物的活性进行了研究。这些研究首先在体外进行，然后在体内实验动物模型中进行。近年来，报告基因技术已被用于研究多种目的，如微生物中的生物学事件以及药物的疗效和耐药性。绿色荧光蛋白（gfp）的主要应用领域是，它可以整合到不同基因中，以确定这些基因在不同生物体中的表达量，并可作为活细胞中的标记物。特别是编码绿色荧光蛋白的gfp基因，如今被广泛使用。基于基因的检测方法具有易于跟踪、成本低廉和生物安全性提高等优点。本研究的目的是用“增强型gfp（egfp）”对热带利什曼原虫进行转染，并与传统方法相比，评估gfp转染体在体外作为药物筛选模型的实用性。通过电穿孔法用p6.5/egfp对热带利什曼原虫前鞭毛体进行转染，并如前所述选择对衣霉素具有抗性的细胞。使用荧光显微镜、荧光计和XTT（2,3-双-（2-甲氧基-4-硝基-5-磺基苯基）-2H-四唑-5-羧基苯胺）测定法等不同方法评估了转染gfp的热带利什曼原虫前鞭毛体和葡甲胺锑酸盐的体外作用。通过荧光显微镜和荧光测定法发现，与评估潜在抗利什曼原虫药物的传统检测方法相比，使用gfp转染的利什曼原虫菌株更快速、更灵敏。因此，稳定转染gfp的利什曼原虫物种将在体外和体内用于抗利什曼原虫药物的筛选和疫苗开发，以及在细胞水平上了解宿主-寄生虫相互作用的生物学机制。作为本研究的结果，建立了一个使用土耳其热带利什曼原虫分离株的gfp转染模型，用于进一步的研究。
