本文選題：旋毛蟲病 + Wortmannilatone��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：實驗?zāi)康?旋毛蟲病是一種對人類健康危害較大的寄生蟲病,本病呈世界性分布,也是我國重點防治的一種食源性寄生蟲病。目前國內(nèi)外有關(guān)旋毛蟲病的實驗研究主要集中于病原學(xué)和免疫學(xué),而針對旋毛蟲病藥物防治方面的研究較少。阿苯達唑和苯并咪唑是當(dāng)前臨床上應(yīng)用最廣的治療旋毛蟲病藥物,該類藥物非我國自主產(chǎn)權(quán),且有一定的副作用,同時又有產(chǎn)生耐藥性的趨向,因此開發(fā)具有我國自主產(chǎn)權(quán)的抑殺旋毛蟲的藥物,無論從臨床上應(yīng)用于防治旋毛蟲病的實際工作方面,還是對我國社會經(jīng)濟發(fā)展方面都有著重要的意義。Wortmannilactone F是一種從鄔氏黃絲曲菌(Talaromyces.Wortmannii)分離得到的新型化合物,它可以抑制蠕蟲線粒體內(nèi)膜上的電子傳遞,為NADH-延胡索酸還原酶抑制劑。我們的前期實驗已證明該藥對旋毛蟲成蟲具有明顯的抑殺作用。G31P是一種趨化因子的競爭性抑制劑,能阻斷IL8以及其他趨化因子與CXCR1/2結(jié)合,從而使得趨化因子失去正常的生物學(xué)效應(yīng),阻止多種炎癥細(xì)胞的浸潤,減輕病灶周圍的炎癥反應(yīng)。在探討防治旋毛蟲病的臨床用藥時,除應(yīng)選用能直接有效地抑殺幼蟲,以防止其引發(fā)的機械損害和免疫病理損傷的藥物外,還要考慮到能有效地阻止囊包形成、囊包外膠原纖維的增生和囊壁周圍血管的新生,防止由于囊包形成和纖維化給病灶周圍組織器官造成繼發(fā)性損害,以達到有效地防治旋毛蟲感染(病)的目的。本實驗旨在探討Wortmannilatone F與G31P聯(lián)合用藥對旋毛蟲的抑殺作用和對囊包形成與纖維化的影響。實驗方法:1.建立旋毛蟲感染動物模型和實驗分組:(1)建立模型:取32只BALB/c雄性小鼠,其中8只作為陰性對照組,其余24只小鼠全部感染旋毛蟲囊包,囊包接種數(shù)為150±5個/鼠,建立旋毛蟲病小鼠實驗動物模型。囊包感染接種過程見參考文獻[1]。(2)實驗分組:將感染旋毛蟲的24只小鼠,隨機分為三個組:Wortmannilactone F治療組、Wortmannilactone F與G31P聯(lián)合治療組(簡稱聯(lián)合治療組)、陽性模型組(Model)。未感染的8只作為陰性對照組(Control)。四組小鼠飼養(yǎng)條件相同。2.實驗方法:在小鼠實驗感染旋毛蟲囊包10天后,Wortmannilactone F和G31P聯(lián)合治療組小鼠皮下注射G31P,500μg/kg,隔日注射1次,連續(xù)7次。同時給小鼠口服Wortmannilactone F,200mg/kg,每日1次,連續(xù)灌胃3天;Wortmannilactone F治療組小鼠在感染旋毛蟲10天后,口服Wortmannilactone F,200mg/kg,每日1次,連續(xù)灌胃3天,同時隔日皮下注射等量的生理鹽水。陽性模型組和陰性對照組小鼠,用等量生理鹽水代替Wortmannilactone F和G31P。停藥后1天,將所有實驗小鼠麻醉處死,無菌解剖小鼠,收集膈肌組織等進行藥效的實驗觀察。3.實驗觀察指標(biāo):(1)抑殺旋毛蟲幼蟲觀察:收集4組小鼠膈肌組織采用HE染色法,計數(shù)膈肌組織內(nèi)旋毛蟲囊包量并比較囊包大小,目的是觀察Wortmannilactone F和G31P對旋毛蟲囊包的聯(lián)合抑殺療效。(2)旋毛蟲囊包形態(tài)學(xué)觀察:對4組小鼠膈肌組織采用HE染色法,在光鏡下觀察藥物對膈肌組織內(nèi)旋毛蟲囊包形態(tài)的影響、宿主在囊包外周產(chǎn)生的免疫病理反應(yīng),以及通過電子顯微鏡觀察旋毛蟲囊包形成的完整性和其內(nèi)幼蟲蟲體發(fā)育等情況。(3)采用免疫組織化學(xué)染色檢測FSP1的表達,以觀察G31P對膈肌組織囊包外周成纖維細(xì)胞積聚數(shù)量和旋毛蟲囊包形成完整性的影響。(4)采用Masson染色法檢測旋毛蟲囊包纖維化的程度,以觀察G31P對膈肌組織囊包外周膠原纖維含量的影響,進而探討藥物治療后對旋毛蟲囊包形成完整性的影響。(5)應(yīng)用免疫熒光染色檢測各組小鼠膈肌組織中v WF因子的表達量,探討在旋毛蟲病鼠膈肌組織中囊包形成過程中,聯(lián)合用藥對其周圍新生血管形成的影響。結(jié)果與討論:1、聯(lián)合用藥對旋毛蟲幼蟲的抑殺效果觀察(1)Wortmannilactone F治療組和聯(lián)合治療組小鼠經(jīng)藥物作用后,在鏡下計數(shù)膈肌組織中蟲體囊包數(shù)量分別為11.5±2.1、6.4±1.2,減蟲率分別達到58.0%、76.6%,進行統(tǒng)計學(xué)比較具有顯著性差異(P0.01)。這兩個實驗組分別與陽性模型組(囊包數(shù)為27.4±2.5)相比較,,也具有顯著性差異(P0.01)。(2)在光鏡下觀察陽性模型組小鼠感染的旋毛蟲囊包內(nèi)幼蟲蟲體形態(tài),發(fā)現(xiàn)蟲體完整,表皮光滑,完好地盤曲在宿主的營養(yǎng)細(xì)胞內(nèi),其外可見纖維化囊包。在電鏡下觀察,陽性模型組肌幼蟲角質(zhì)層光滑平坦,每隔等長距離有大小相等的橫紋。而Wortmannilactone F治療組和聯(lián)合用藥治療組旋毛蟲囊包內(nèi)幼蟲的蟲體形態(tài)結(jié)構(gòu)發(fā)生明顯的變化,光鏡下可見蟲體小且僵直,似發(fā)育不良;在電鏡下,可見肌幼蟲角質(zhì)層凹凸不平,出現(xiàn)不同程度的波折起伏,橫紋大小不一,間距不齊形態(tài)學(xué)病理改變。2、免疫組化與Masson染色法觀察聯(lián)合用藥對旋毛蟲囊包的影響(1)免疫組織化學(xué)染色顯示在陰性對照組中,小鼠膈肌中FSP1陽性的成纖維細(xì)胞極少,而在陽性模型組中,大量的成纖維細(xì)胞分布于幼蟲周圍。Wortmannilactone F治療組和聯(lián)合治療組與陽性模型組相比較,小鼠膈肌囊包外周成纖維細(xì)胞數(shù)量顯著減少(P0.01),統(tǒng)計學(xué)分析兩個治療組之間也有明顯的統(tǒng)計學(xué)差異(P0.01)。(2)Masson染色法檢測囊包外周膠原纖維含量顯示,陰性對照組小鼠膈肌中因無旋毛蟲感染,膈肌組織沒有膠原纖維的聚集。而在陽性模型組和Wortmannilactone F治療組小鼠膈肌中,發(fā)現(xiàn)大量的膠原纖維增生;然而在聯(lián)合治療組中膠原增生受到明顯抑制。(3)應(yīng)用Smart V3500軟件測量3個實驗組囊包形成面積:陽性模型組和Wortmannilactone F治療組鏡下小鼠膈肌中旋毛蟲囊包面積分別為4445.1±402.2(μm2)、4332.2±493.7(μm2),兩者統(tǒng)計學(xué)差異不明顯(P0.05),而聯(lián)合用藥治療組為1949.6±346.6(μm2),與前兩組比較有明顯的差異(P0.01)。3、免疫熒光染色檢測聯(lián)合用藥對囊包新生血管的影響(1)在陰性對照組小鼠膈肌中旋毛蟲囊包周圍,視野中極少見到v WF陽性的血管內(nèi)皮細(xì)胞(vascular endothelial cell,VEC),而在旋毛蟲感染影響下,大量的血管內(nèi)皮細(xì)胞在陽性模型組和Wortmannilactone F治療組膈肌幼蟲周圍增生;經(jīng)聯(lián)合用藥治療后,血管內(nèi)皮細(xì)胞數(shù)量明顯減少。(2)應(yīng)用Image Process plus 6.0軟件分析平均每個高倍視野下血管內(nèi)皮細(xì)胞占所有有核細(xì)胞的比例,陽性模型組和Wortmannilactone F治療組分別為6.7±1.1%、6.2±1.2%,兩者無顯著差異(P0.05);而聯(lián)合用藥治療組為2.2±0.4%,與前兩組比較有顯著性差異(P0.01)。結(jié)論:1、Wortmannilactone F具有明顯的抑殺旋毛蟲幼蟲作用,與G31P聯(lián)合用藥比單獨用藥能更有效殺傷囊包內(nèi)幼蟲,明顯減少膈肌組織中囊包形成的數(shù)量。2、聯(lián)合用藥對旋毛蟲囊包內(nèi)幼蟲的體表結(jié)構(gòu)產(chǎn)生一定的破壞作用。3、聯(lián)合用藥(主要是G31P的影響)抑制囊包外周血管網(wǎng)的新生,減少囊包內(nèi)幼蟲的營養(yǎng)來源,導(dǎo)致幼蟲發(fā)育不良。4、聯(lián)合用藥抑制成纖維細(xì)胞的聚集,影響旋毛蟲囊包發(fā)育的完整性和纖維化,進而抑制纖維化對宿主組織產(chǎn)生繼發(fā)性二次損傷。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R532.14

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