Wortmannilatone F与G31P联合用药对旋毛虫抑杀作用和抗囊包形成与纤维化的实验研究

发布时间：2018-06-14 14:02

本文选题：旋毛虫病 + Wortmannilatone　；参考：《大连医科大学》2017年硕士论文

【摘要】：实验目的:旋毛虫病是一种对人类健康危害较大的寄生虫病,本病呈世界性分布,也是我国重点防治的一种食源性寄生虫病。目前国内外有关旋毛虫病的实验研究主要集中于病原学和免疫学,而针对旋毛虫病药物防治方面的研究较少。阿苯达唑和苯并咪唑是当前临床上应用最广的治疗旋毛虫病药物,该类药物非我国自主产权,且有一定的副作用,同时又有产生耐药性的趋向,因此开发具有我国自主产权的抑杀旋毛虫的药物,无论从临床上应用于防治旋毛虫病的实际工作方面,还是对我国社会经济发展方面都有着重要的意义。Wortmannilactone F是一种从邬氏黄丝曲菌(Talaromyces.Wortmannii)分离得到的新型化合物,它可以抑制蠕虫线粒体内膜上的电子传递,为NADH-延胡索酸还原酶抑制剂。我们的前期实验已证明该药对旋毛虫成虫具有明显的抑杀作用。G31P是一种趋化因子的竞争性抑制剂,能阻断IL8以及其他趋化因子与CXCR1/2结合,从而使得趋化因子失去正常的生物学效应,阻止多种炎症细胞的浸润,减轻病灶周围的炎症反应。在探讨防治旋毛虫病的临床用药时,除应选用能直接有效地抑杀幼虫,以防止其引发的机械损害和免疫病理损伤的药物外,还要考虑到能有效地阻止囊包形成、囊包外胶原纤维的增生和囊壁周围血管的新生,防止由于囊包形成和纤维化给病灶周围组织器官造成继发性损害,以达到有效地防治旋毛虫感染(病)的目的。本实验旨在探讨Wortmannilatone F与G31P联合用药对旋毛虫的抑杀作用和对囊包形成与纤维化的影响。实验方法:1.建立旋毛虫感染动物模型和实验分组:(1)建立模型:取32只BALB/c雄性小鼠,其中8只作为阴性对照组,其余24只小鼠全部感染旋毛虫囊包,囊包接种数为150±5个/鼠,建立旋毛虫病小鼠实验动物模型。囊包感染接种过程见参考文献[1]。(2)实验分组:将感染旋毛虫的24只小鼠,随机分为三个组:Wortmannilactone F治疗组、Wortmannilactone F与G31P联合治疗组(简称联合治疗组)、阳性模型组(Model)。未感染的8只作为阴性对照组(Control)。四组小鼠饲养条件相同。2.实验方法:在小鼠实验感染旋毛虫囊包10天后,Wortmannilactone F和G31P联合治疗组小鼠皮下注射G31P,500μg/kg,隔日注射1次,连续7次。同时给小鼠口服Wortmannilactone F,200mg/kg,每日1次,连续灌胃3天;Wortmannilactone F治疗组小鼠在感染旋毛虫10天后,口服Wortmannilactone F,200mg/kg,每日1次,连续灌胃3天,同时隔日皮下注射等量的生理盐水。阳性模型组和阴性对照组小鼠,用等量生理盐水代替Wortmannilactone F和G31P。停药后1天,将所有实验小鼠麻醉处死,无菌解剖小鼠,收集膈肌组织等进行药效的实验观察。3.实验观察指标:(1)抑杀旋毛虫幼虫观察:收集4组小鼠膈肌组织采用HE染色法,计数膈肌组织内旋毛虫囊包量并比较囊包大小,目的是观察Wortmannilactone F和G31P对旋毛虫囊包的联合抑杀疗效。(2)旋毛虫囊包形态学观察:对4组小鼠膈肌组织采用HE染色法,在光镜下观察药物对膈肌组织内旋毛虫囊包形态的影响、宿主在囊包外周产生的免疫病理反应,以及通过电子显微镜观察旋毛虫囊包形成的完整性和其内幼虫虫体发育等情况。(3)采用免疫组织化学染色检测FSP1的表达,以观察G31P对膈肌组织囊包外周成纤维细胞积聚数量和旋毛虫囊包形成完整性的影响。(4)采用Masson染色法检测旋毛虫囊包纤维化的程度,以观察G31P对膈肌组织囊包外周胶原纤维含量的影响,进而探讨药物治疗后对旋毛虫囊包形成完整性的影响。(5)应用免疫荧光染色检测各组小鼠膈肌组织中v WF因子的表达量,探讨在旋毛虫病鼠膈肌组织中囊包形成过程中,联合用药对其周围新生血管形成的影响。结果与讨论:1、联合用药对旋毛虫幼虫的抑杀效果观察(1)Wortmannilactone F治疗组和联合治疗组小鼠经药物作用后,在镜下计数膈肌组织中虫体囊包数量分别为11.5±2.1、6.4±1.2,减虫率分别达到58.0%、76.6%,进行统计学比较具有显著性差异(P0.01)。这两个实验组分别与阳性模型组(囊包数为27.4±2.5)相比较,,也具有显著性差异(P0.01)。(2)在光镜下观察阳性模型组小鼠感染的旋毛虫囊包内幼虫虫体形态,发现虫体完整,表皮光滑,完好地盘曲在宿主的营养细胞内,其外可见纤维化囊包。在电镜下观察,阳性模型组肌幼虫角质层光滑平坦,每隔等长距离有大小相等的横纹。而Wortmannilactone F治疗组和联合用药治疗组旋毛虫囊包内幼虫的虫体形态结构发生明显的变化,光镜下可见虫体小且僵直,似发育不良;在电镜下,可见肌幼虫角质层凹凸不平,出现不同程度的波折起伏,横纹大小不一,间距不齐形态学病理改变。2、免疫组化与Masson染色法观察联合用药对旋毛虫囊包的影响(1)免疫组织化学染色显示在阴性对照组中,小鼠膈肌中FSP1阳性的成纤维细胞极少,而在阳性模型组中,大量的成纤维细胞分布于幼虫周围。Wortmannilactone F治疗组和联合治疗组与阳性模型组相比较,小鼠膈肌囊包外周成纤维细胞数量显著减少(P0.01),统计学分析两个治疗组之间也有明显的统计学差异(P0.01)。(2)Masson染色法检测囊包外周胶原纤维含量显示,阴性对照组小鼠膈肌中因无旋毛虫感染,膈肌组织没有胶原纤维的聚集。而在阳性模型组和Wortmannilactone F治疗组小鼠膈肌中,发现大量的胶原纤维增生;然而在联合治疗组中胶原增生受到明显抑制。(3)应用Smart V3500软件测量3个实验组囊包形成面积:阳性模型组和Wortmannilactone F治疗组镜下小鼠膈肌中旋毛虫囊包面积分别为4445.1±402.2(μm2)、4332.2±493.7(μm2),两者统计学差异不明显(P0.05),而联合用药治疗组为1949.6±346.6(μm2),与前两组比较有明显的差异(P0.01)。3、免疫荧光染色检测联合用药对囊包新生血管的影响(1)在阴性对照组小鼠膈肌中旋毛虫囊包周围,视野中极少见到v WF阳性的血管内皮细胞(vascular endothelial cell,VEC),而在旋毛虫感染影响下,大量的血管内皮细胞在阳性模型组和Wortmannilactone F治疗组膈肌幼虫周围增生;经联合用药治疗后,血管内皮细胞数量明显减少。(2)应用Image Process plus 6.0软件分析平均每个高倍视野下血管内皮细胞占所有有核细胞的比例,阳性模型组和Wortmannilactone F治疗组分别为6.7±1.1%、6.2±1.2%,两者无显著差异(P0.05);而联合用药治疗组为2.2±0.4%,与前两组比较有显著性差异(P0.01)。结论:1、Wortmannilactone F具有明显的抑杀旋毛虫幼虫作用,与G31P联合用药比单独用药能更有效杀伤囊包内幼虫,明显减少膈肌组织中囊包形成的数量。2、联合用药对旋毛虫囊包内幼虫的体表结构产生一定的破坏作用。3、联合用药(主要是G31P的影响)抑制囊包外周血管网的新生,减少囊包内幼虫的营养来源,导致幼虫发育不良。4、联合用药抑制成纤维细胞的聚集,影响旋毛虫囊包发育的完整性和纤维化,进而抑制纤维化对宿主组织产生继发性二次损伤。
[Abstract]:Objective: Trichinella Trichinella is a kind of parasitic disease which is very harmful to human health. This disease is distributed worldwide. It is also a major food borne parasitic disease in China. At present, the domestic and foreign experimental studies on Trichinella mainly focus on etiology and immunology, but there are few studies on the prevention and control of Trichinella. Albendazole and benzimidazole are the most widely used drugs for the treatment of Trichinella spiralis, which are independent of property rights in China, and have certain side effects, and also have a tendency to produce resistance. Therefore, the drug of Trichinella suppressor, which has independent property rights in our country, is developed, which is applied to the practice of the prevention and control of Trichinella spiralis in clinical practice. In the field of work, it is still of great significance to the social and economic development of our country..Wortmannilactone F is a new compound derived from Talaromyces.Wortmannii. It can inhibit the electron transfer on the endocardium of the worm mitochondria, and it is the inhibitor of the NADH- of the fumaric acid reductase. It is proved that the drug has an obvious inhibitory effect on the adult of Trichinella spiralis.G31P, a competitive inhibitor of chemokines. It can block the combination of IL8 and other chemokines with CXCR1/2, thus making chemokines lose normal biological effects, prevent the infiltration of many inflammatory cells and reduce the inflammatory reaction around the focus. In the clinical use of caterpillar disease, besides the drugs that can suppress the larvae directly and effectively in order to prevent the mechanical damage and immune pathological damage caused by them, it is also necessary to consider the ability to effectively prevent the formation of the capsule, the proliferation of the collagen fibers outside the capsule and the angiogenesis around the wall of the capsule, and prevent the formation and fibrosis of the cyst around the focus group. The effect of Wortmannilatone F and G31P on the inhibition of Trichinella spiralis and the effect on the formation and fibrosis of Trichinella spiralis and the effect on the formation and fibrosis of Trichinella Trichinella were investigated in this experiment. 1. the animal model and experimental grouping of Trichinella were established: (1) establish a model: 32 BA LB/c male mice, 8 of them were negative control group, the other 24 mice were all infected with Trichinella Trichinella, and the number of inoculation was 150 + 5 / mice. The inoculation process of cysts was established in [1]. (2) experimental group: 24 mice infected with caterpillar were randomly divided into three groups: Wortmannilactone F treatment group, Wortmannilactone F and G31P combined treatment group (combined treatment group), positive model group (Model). 8 uninfected patients were negative control group (Control). The four group of mice feeding conditions same.2. test method: 10 days after the infection of Trichinella Trichinella in mice, Wortmannilactone F and G31P combined treatment group mice subcutaneous injection G31P 500 mu g/kg was injected 1 times every other day for 7 times. At the same time, the mice were given Wortmannilactone F, 200mg/kg, 1 times a day for 3 days. The mice in the Wortmannilactone F treatment group were given Wortmannilactone F, 200mg/kg, 1 times a day for 3 days after the infection of Trichinella Trichinella, and the same quantity of normal saline was injected subcutaneously every other day, and the positive model group was subcutaneously injected every other day. And the negative control group, 1 days after the withdrawal of Wortmannilactone F and G31P., all the experimental mice were killed, the mice were aseptic and the phrenic muscle tissue was collected, and the experimental observation indexes of.3. experiment were observed: (1) the larvae of Trichinella Trichinella were observed: the 4 groups of mice were collected by HE staining and counted. The volume of Trichinella spiralis in the diaphragm and the size of the cyst was compared to observe the combined inhibitory effect of Wortmannilactone F and G31P on the cysts of Trichinella Trichinella. (2) the morphological observation of Trichinella Trichinella capsule: the effect of the drug on the cysts of Trichinella spiralis in the diaphragm of the 4 groups of mice was observed under the light microscope. The immunological reaction of the periphery of the envelope, as well as the integrity of the cyst formation of Trichinella spiralis and the development of the larvae in the larvae were observed by electron microscopy. (3) immunohistochemical staining was used to detect the expression of FSP1, in order to observe the effect of G31P on the amount of fine cell accumulation in the peripheral peripheral fibroblasts and the formation integrity of Trichinella Trichinella in the diaphragmatic tissue. (4) the Masson staining method was used to detect the degree of Trichinella cystic fibrosis, in order to observe the effect of G31P on the content of collagen fibrils in the outer circumference of the diaphragmatic tissue, and to explore the effect of drug treatment on the integrity of the cysts of Trichinella spiralis. (5) the expression of V WF factor in the diaphragm of each group was detected by immunofluorescence staining. The effect of combined use of drugs on the formation of neovascularization around it during the capsule formation in the diaphragm of Trichinella Trichinella. Results and discussion: 1, the effect of combined use of drugs on the inhibition of Trichinella spiralis larvae (1) Wortmannilactone F treatment group and combined treatment group after drug action, the number of insect body sac in the diaphragm was counted under the microscope. The rate of worm reduction was 11.5 + 2.1,6.4 + 1.2, and the rate of worm reduction reached 58%, 76.6% respectively. There was a significant difference in statistical comparison (P0.01). The two experimental groups were compared with the positive model group (27.4 + 2.5), and also had significant difference (P0.01). (2) the larvae of Trichinella Trichinella infected by the positive model group were observed under the light microscope. It was found that the insect body was intact, the epidermis was smooth and the fibrotic sac was visible in the host's nutrient cells. Under the electron microscope, the cuticle of the muscle larvae of the positive model group was smooth and flat, and the size of the transverse grain was equal at every length. The Wortmannilactone F treatment group and the combined treatment group of Trichinella Trichinella were in the capsule larva. The morphologic structure of the insect body was obviously changed. Under the light microscope, the insect body was small and rigid, and it seemed to be dysplasia. Under the electron microscope, the cuticle of the muscle larvae was uneven, there were different degrees of wave break, the size of the transverse lines was different, the distance was uneven,.2, and the combined use of immunization and Masson staining to observe the cysts of Trichinella spiralis. Influence (1) immunohistochemical staining showed that in the negative control group, there were few FSP1 positive fibroblasts in the diaphragmatic muscles of the mice, while in the positive model group, a large number of fibroblasts were distributed in the.Wortmannilactone F treatment group and the combined treatment group with the positive model group, and the number of peripheral fibroblasts in the diaphragmatic capsule of mice was compared. Significant decrease (P0.01), statistically significant difference between the two treatment groups (P0.01). (2) the content of collagen fibrils in the outer circumference of the capsule was detected by Masson staining, and there was no Trichinella infection in the diaphragm of the negative control group and no aggregation of collagen fibrils in the diaphragmatic tissue. In the positive model group and the Wortmannilactone F treatment group, there was no aggregation of collagen fibrils in the diaphragm. In the diaphragm of the mice, a large number of collagen fibrils were found. However, the collagen proliferation was significantly inhibited in the combined treatment group. (3) Smart V3500 software was used to measure the area of the capsule formation in 3 experimental groups: the envelope area of the diaphragm in the diaphragm of the positive model group and the Wortmannilactone F group was 4445.1 + 402.2 (mu m2), 4332.2 +. 493.7 (M2), the statistical difference was not obvious (P0.05), but the combined treatment group was 1949.6 + 346.6 (mu m2), and there was a significant difference (P0.01) with the former two groups (P0.01).3. The effect of the immunofluorescence staining on the neovascularization of the cystic neovascularization (1) was around the cysts of Trichinella spiralis in the diaphragm of the negative control group, and the V WF was rarely seen in the field of vision. Vascular endothelial cell (VEC), and under the influence of Trichinella Trichinella infection, a large number of vascular endothelial cells proliferated around the phrenic larva in the positive model group and Wortmannilactone F treatment group, and the number of vascular endothelial cells decreased significantly after combined treatment. (2) the average of Image Process plus 6 software was used to analyze the average number of vascular endothelial cells. The ratio of vascular endothelial cells to all nucleated cells under high magnification was 6.7 + 1.1% and 6.2 + 1.2% in Wortmannilactone F treatment group, and there was no significant difference (P0.05), while the combined treatment group was 2.2 + 0.4%, compared with the first two groups (P0.01). Conclusion: 1, Wortmannilactone F had obvious suppression. The effect of Trichinella Trichinella larvae, combined with G31P, can more effectively kill the larva in the capsule than the single drug, and reduce the amount of.2 in the capsule formation in the diaphragm. The combination of drugs has a certain destruction effect on the body surface structure of the larvae of Trichinella spiralis, and the combination of drugs (mainly the effect of G31P) inhibits the newborn of the peripheral vascular network of the capsule. Reducing the nutrient source of the larva in the capsule, causing the larval dysplasia.4, the combination of drugs to inhibit the aggregation of fibroblasts, influence the integrity and fibrosis of the development of Trichinella Trichinella, and then inhibit the secondary two damage to the host tissue.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R532.14

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