本文關(guān)鍵詞：旋毛蟲感染致小鼠寄生部位組織細胞凋亡的研究　出處：《承德醫(yī)學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 旋毛蟲 成蟲 幼蟲 Caspase-3 TUNEL 凋亡指數(shù)

【摘要】：目的：建立河南豬株旋毛蟲實驗感染昆明小鼠動物模型,擬通過病理學(xué)和分子生物學(xué)實驗手段檢測旋毛蟲感染小鼠模型不同時間小腸組織和肌肉組織的細胞凋亡情況,探討旋毛蟲感染狀態(tài)下宿主被寄生部位組織細胞凋亡的發(fā)生機理,以選擇性的促進或抑制細胞凋亡的發(fā)生,為預(yù)防、治療旋毛蟲病、殺滅旋毛蟲提供實驗基礎(chǔ)。方法：將80只雄性健康小鼠隨機分為A組(成蟲期)和B組(幼蟲期),每組40只。A組隨機分為感染旋毛蟲3、5、7d組及正常對照組,每組10只；B組隨機分為感染旋毛蟲3、4、5w組及正常對照組,每組10只。每組小鼠各感染旋毛蟲幼蟲200條,對照組正常飼養(yǎng)。A組小鼠分別于感染后3、5、7d取其小腸組織,B組小鼠分別于感染后3、4、5w取其肌肉組織。常規(guī)病理切片,蘇木素-伊紅(HE)染色法觀察小鼠小腸和肌肉組織的病理學(xué)變化,Western-blot法檢測不同感染時期小腸和肌肉組織中Caspase-3蛋白表達水平的變化,原位末端標記法(TUNEL法)檢測小鼠小腸和肌肉組織的細胞凋亡指數(shù)。運用病理學(xué)和分子生物學(xué)方法對旋毛蟲成蟲引起小腸組織,幼蟲引起肌肉組織的細胞凋亡情況進行綜合評價。結(jié)果：1成蟲期：1.1 HE染色結(jié)果：正常對照組小鼠小腸組織未見任何病理變化,小腸絨毛結(jié)構(gòu)正常,黏膜層無改變,黏膜上皮無變化,固有膜正常；感染3d組小腸黏膜層上皮細胞水腫不明顯,黏膜層稍見出血,固有膜空泡狀改變不明顯；感染5d組小腸黏膜層上皮細胞可見輕微水腫,黏膜層腸腺可見明顯出血,炎細胞較少,固有膜明顯空泡狀改變；感染7d組小腸黏膜層大量炎性細胞浸潤并伴有大量出血,局部黏膜上皮細胞水腫,固有膜空泡狀改變更加顯著,腸絨毛缺損明顯。1.2 Western-blot結(jié)果：正常對照組小鼠Caspase-3蛋白平均灰度值為(0.79+0.03),旋毛蟲成蟲發(fā)育過程中,Caspase-3蛋白平均灰度比值隨著旋毛蟲感染時間的延長明顯增加(P0.05)。感染3、5、7d組的各個灰度值分別為(0.86+0.03)、(1.04+0.02)、(1.34+0.02)。與正常對照組相比,感染旋毛蟲3、5、7d組Caspase-3蛋白平均灰度比值明顯增加(P0.05),3、5、7d組組間兩兩比較亦具有差異性(P0.05)。1.3 TUNEL法結(jié)果：正常對照組小腸黏膜層可見零星散在的,細胞核呈深棕色或棕黃色的凋亡細胞,細胞凋亡指數(shù)為(0.08+0.02)%,隨著感染時間的增長,細胞凋亡指數(shù)明顯增加,旋毛蟲感染3、5、7d組細胞凋亡指數(shù)分別為(0.26+0.03)%、(0.38±±0.03)%和(0.73+0.09)%。與正常對照組相比(P0.05),3、5、7d組組間兩兩比較均具有顯著性差異(P0.05)。2幼蟲期：2.1 HE染色結(jié)果：正常對照組小鼠肌肉組織未見任何病理學(xué)變化,肌纖維粗細均勻,排列規(guī)律整齊,隨著感染時間延長至第3w、4w,HE染色結(jié)果表現(xiàn)為肌纖維變性腫脹、粗細不均勻、排列紊亂、橫紋消失,呈網(wǎng)狀結(jié)構(gòu),蟲體所在部位肌漿溶解,蟲體附近的肌細胞壞死、崩解,肌間質(zhì)水腫及炎性細胞浸潤,炎性細胞主要以淋巴細胞、大單核細胞和嗜酸性粒細胞為主。感染第5w,幼蟲周圍形成囊包,隨著幼蟲長大、卷曲,寄生部位肌組織逐漸增大呈紡錘狀,形成梭形肌腔包繞蟲體,急性炎癥逐漸消退。2.2 Western-blot結(jié)果：正常對照組小鼠Caspase-3蛋白平均灰度值為(0.23±0.02),旋毛蟲幼蟲發(fā)育過程中,感染第3、4w,Caspase-3蛋白平均灰度比值隨著旋毛蟲感染時間的延長明顯增加,分別為(0.31+0.02)和(0.54+0.02)。感染第5w,幼蟲周圍囊包形成,急性嚴重消退,Caspase-3蛋白平均灰度值明顯下降為(0.36+0.03)。與正常對照組相比,感染旋毛蟲3、4、5w組Caspase-3蛋白平均灰度比值明顯增加(P0.05),3、4、5w組組間兩兩比較亦具有顯著性(P0.05)。2.3 TUNEL法結(jié)果：正常對照組小鼠肌肉組織中可見零星散落的,細胞核呈深棕色或棕黃色的凋亡細胞,即基本無表達,其細胞凋亡指數(shù)為(0.12±±0.03)%,隨著感染時間增長,細胞凋亡指數(shù)明顯增加,旋毛蟲感染3、4、5w組細胞凋亡指數(shù)分別為(0.32+0.05)%、(0.68+0.03)%和(0.47+0.05)%。與正常對照組相比(P0.05),3、4、5w組組間兩兩比較均具有顯著性差異(P0.05)。結(jié)論：1旋毛蟲與宿主在相互作用過程中存在細胞凋現(xiàn)象,旋毛蟲感染所致宿主寄生部位組織細胞凋亡與病理損傷存在一定的關(guān)系。2旋毛蟲成蟲侵襲小鼠腸道可誘導(dǎo)宿主小腸組織發(fā)生細胞凋亡,在一定時間內(nèi),隨著感染時間延長,組織凋亡水平亦增加,感染第7d達到凋亡的高峰。3旋毛蟲幼蟲寄生于小鼠肌肉組織可誘導(dǎo)宿主肌肉組織發(fā)生細胞凋亡,且組織凋亡水平與感染時間存在密切的相關(guān)性,感染3、4w細胞凋亡水平明顯上升,到達第5w,此時囊包形成,細胞凋亡水平下降。
[Abstract]:Objective: to establish the Henan pig strains of Trichinella spiralis infection in Kunming experimental animal model of mice to apoptosis by pathology and molecular biology experimental methods to detect Trichinella infection in mice model of intestinal tissue at different time and muscle tissue, to investigate the state of Trichinella infection host is the occurrence mechanism of parasitic tissue apoptosis, to promote or inhibit the occurrence of selective. Cell apoptosis for the prevention, treatment of trichinosis, provide the experimental basis to kill Trichinella. Methods: 80 healthy male mice were randomly divided into group A (adult) and group B (larvae), 40 rats in each group were randomly divided into.A infected 3,5,7d group and normal control group, 10 rats in each group; B group divided into 3,4,5w infected group and normal control group, 10 rats in each group. Each group of mice infected with Trichinella spiralis larvae by 200, control group of normal feeding group.A mice were infected with 3,5, 7d take the small intestine of mice in group B were infected with 3,4,5w from the muscle tissue. The pathological section, hematoxylin eosin (HE) staining and pathological changes in small intestine muscle tissue was observed in mice, the expression of Caspase-3 protein detected by Western-blot infection period of intestine and muscle tissue, and in situ labeling method (TUNEL method) to detect the apoptosis index of mouse small intestine and muscle tissue. Using pathology and molecular biology methods caused by the small intestine tissue to induce apoptosis of adult Trichinella spiralis larvae, the muscle tissue in the comprehensive evaluation. Results: 1 adults: 1.1 HE staining: normal control group mice had no any pathological intestinal tissue changes in the structure of the intestinal villi of normal mucosa, no change, no change in the mucosal epithelium, lamina propria of normal intestinal mucosa; 3D infection group layer of epithelial cells of mucosa edema is not obvious The slight bleeding, membrane vacuole change is not obvious; group 5D infection of intestinal mucosal epithelial cell layer showed slight edema, mucosa of intestinal gland showed obvious bleeding, less inflammatory cells, lamina propria obvious vacuolization; 7d infection group of intestinal mucosal layer infiltration of inflammatory cells and associated with massive hemorrhage, edema of epithelial cells local, intrinsic membrane vacuole change more significantly, intestinal villi defect significantly.1.2 Western-blot results: normal control group, the average gray value of mouse Caspase-3 protein (0.79+0.03), the process of development of adult Trichinella spiralis, Caspase-3 egg white average gray level ratio increased with the increase of Trichinella spiralis infection time (P0.05). The gray values were 3,5,7d infection group for (0.86+0.03), (1.04+0.02), (1.34+0.02). Compared with the normal control group, 3,5,7d group, Caspase-3 protein of Trichinella spiralis infection significantly increased the average gray ratio (P0.05), 3,5,7d Group comparison between the 22 groups also has the difference (P0.05).1.3 TUNEL results: normal control group of intestinal mucosa shows sporadic in the nuclei of apoptotic cells deep brown or brown yellow, cell apoptosis index (0.08+0.02)%, with increased duration of infection, the apoptosis index increased significantly, apoptosis index group 3,5,7d were infected with Trichinella spiralis (0.26+0.03)% and (0.38 + 0.03)% and (0.73+0.09). Compared with the normal control group (P0.05), 22 groups of 3,5,7d were significant difference (P0.05).2 larvae: 2.1 HE staining: normal control group mice had no any pathological muscle tissue the changes of muscle fiber, uniform thickness, arrangement of the order, as the infection time is extended to 3W, 4W, HE staining results showed myofibrosis swelling, uneven thickness, disordered stripes disappear, reticular structure, body position of muscle Dissolution, muscle cell necrosis, the body near the disintegration of the muscle interstitial edema and inflammatory cell infiltration of inflammatory cells, mainly lymphocytes, mononuclear cells and eosinophils. The infection of 5W larvae formed around the capsule, as the larvae grow up, curl, muscle tissue gradually increased spindlelike parasitic site the formation, fusiform muscle cavity around the body, the acute inflammation subsided gradually.2.2 Western-blot results: in the normal control group the average gray value of Caspase-3 protein in mice (0.23 + 0.02), in the development process of Trichinella spiralis larvae, 3,4w infection, Caspase-3 protein average gray level ratio along with the prolonging of time of Trichinella spiralis infection increased significantly, respectively (0.31+0.02) and (0.54+0.02). The infection of 5W larvae around the sac formation, acute severe subside, the average gray value of Caspase-3 protein was decreased significantly (0.36+0.03). Compared with the normal control group, 3,4,5w group, Caspase-3 infected with Trichinella spiralis The average gray protein ratio were increased (P0.05), 22 groups of 3,4,5w also significantly (P0.05).2.3 TUNEL method results: normal control mice muscle tissue visible scattered, apoptotic cell nuclei were dark brown or brown yellow, which is basically no expression of the apoptosis index (0.12 + + 0.03)%, with the infection time growth, apoptosis index increased significantly, the apoptosis index in 3,4,5w group were infected with Trichinella spiralis (0.32+0.05)%, (0.68+0.03)% and (0.47+0.05). Compared with the normal control group (P0.05), 22 groups of 3,4,5w were significant difference (P0.05). Conclusion: 1 host cell apoptosis phenomenon of Trichinella spiralis and exist in the interaction process, caused by Trichinella infection host parasite tissue cell apoptosis and pathological damage. There is a certain relationship between.2 of Trichinella spiralis adult mice intestinal invasion can induce the host intestine group The fabric of apoptosis, in a certain period of time, with the infection time prolonged, apoptosis level also increased, the 7d reached the peak of apoptosis of infected.3 of Trichinella spiralis larvae are parasitic in muscle tissue can induce host muscle tissue apoptosis, and apoptosis level and infection time is dependent on the apoptosis of 3,4w cells significantly, infection rise, arrive at the 5W, at this time the sac formation, apoptosis decreased.

【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R383.15

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