旋毛虫感染致小鼠寄生部位组织细胞凋亡的研究

发布时间：2018-01-05 08:30

本文关键词：旋毛虫感染致小鼠寄生部位组织细胞凋亡的研究　出处：《承德医学院》2013年硕士论文　论文类型：学位论文

【摘要】：目的：建立河南猪株旋毛虫实验感染昆明小鼠动物模型,拟通过病理学和分子生物学实验手段检测旋毛虫感染小鼠模型不同时间小肠组织和肌肉组织的细胞凋亡情况,探讨旋毛虫感染状态下宿主被寄生部位组织细胞凋亡的发生机理,以选择性的促进或抑制细胞凋亡的发生,为预防、治疗旋毛虫病、杀灭旋毛虫提供实验基础。方法：将80只雄性健康小鼠随机分为A组(成虫期)和B组(幼虫期),每组40只。A组随机分为感染旋毛虫3、5、7d组及正常对照组,每组10只；B组随机分为感染旋毛虫3、4、5w组及正常对照组,每组10只。每组小鼠各感染旋毛虫幼虫200条,对照组正常饲养。A组小鼠分别于感染后3、5、7d取其小肠组织,B组小鼠分别于感染后3、4、5w取其肌肉组织。常规病理切片,苏木素-伊红(HE)染色法观察小鼠小肠和肌肉组织的病理学变化,Western-blot法检测不同感染时期小肠和肌肉组织中Caspase-3蛋白表达水平的变化,原位末端标记法(TUNEL法)检测小鼠小肠和肌肉组织的细胞凋亡指数。运用病理学和分子生物学方法对旋毛虫成虫引起小肠组织,幼虫引起肌肉组织的细胞凋亡情况进行综合评价。结果：1成虫期：1.1 HE染色结果：正常对照组小鼠小肠组织未见任何病理变化,小肠绒毛结构正常,黏膜层无改变,黏膜上皮无变化,固有膜正常；感染3d组小肠黏膜层上皮细胞水肿不明显,黏膜层稍见出血,固有膜空泡状改变不明显；感染5d组小肠黏膜层上皮细胞可见轻微水肿,黏膜层肠腺可见明显出血,炎细胞较少,固有膜明显空泡状改变；感染7d组小肠黏膜层大量炎性细胞浸润并伴有大量出血,局部黏膜上皮细胞水肿,固有膜空泡状改变更加显著,肠绒毛缺损明显。1.2 Western-blot结果：正常对照组小鼠Caspase-3蛋白平均灰度值为(0.79+0.03),旋毛虫成虫发育过程中,Caspase-3蛋白平均灰度比值随着旋毛虫感染时间的延长明显增加(P0.05)。感染3、5、7d组的各个灰度值分别为(0.86+0.03)、(1.04+0.02)、(1.34+0.02)。与正常对照组相比,感染旋毛虫3、5、7d组Caspase-3蛋白平均灰度比值明显增加(P0.05),3、5、7d组组间两两比较亦具有差异性(P0.05)。1.3 TUNEL法结果：正常对照组小肠黏膜层可见零星散在的,细胞核呈深棕色或棕黄色的凋亡细胞,细胞凋亡指数为(0.08+0.02)%,随着感染时间的增长,细胞凋亡指数明显增加,旋毛虫感染3、5、7d组细胞凋亡指数分别为(0.26+0.03)%、(0.38±±0.03)%和(0.73+0.09)%。与正常对照组相比(P0.05),3、5、7d组组间两两比较均具有显著性差异(P0.05)。2幼虫期：2.1 HE染色结果：正常对照组小鼠肌肉组织未见任何病理学变化,肌纤维粗细均匀,排列规律整齐,随着感染时间延长至第3w、4w,HE染色结果表现为肌纤维变性肿胀、粗细不均匀、排列紊乱、横纹消失,呈网状结构,虫体所在部位肌浆溶解,虫体附近的肌细胞坏死、崩解,肌间质水肿及炎性细胞浸润,炎性细胞主要以淋巴细胞、大单核细胞和嗜酸性粒细胞为主。感染第5w,幼虫周围形成囊包,随着幼虫长大、卷曲,寄生部位肌组织逐渐增大呈纺锤状,形成梭形肌腔包绕虫体,急性炎症逐渐消退。2.2 Western-blot结果：正常对照组小鼠Caspase-3蛋白平均灰度值为(0.23±0.02),旋毛虫幼虫发育过程中,感染第3、4w,Caspase-3蛋白平均灰度比值随着旋毛虫感染时间的延长明显增加,分别为(0.31+0.02)和(0.54+0.02)。感染第5w,幼虫周围囊包形成,急性严重消退,Caspase-3蛋白平均灰度值明显下降为(0.36+0.03)。与正常对照组相比,感染旋毛虫3、4、5w组Caspase-3蛋白平均灰度比值明显增加(P0.05),3、4、5w组组间两两比较亦具有显著性(P0.05)。2.3 TUNEL法结果：正常对照组小鼠肌肉组织中可见零星散落的,细胞核呈深棕色或棕黄色的凋亡细胞,即基本无表达,其细胞凋亡指数为(0.12±±0.03)%,随着感染时间增长,细胞凋亡指数明显增加,旋毛虫感染3、4、5w组细胞凋亡指数分别为(0.32+0.05)%、(0.68+0.03)%和(0.47+0.05)%。与正常对照组相比(P0.05),3、4、5w组组间两两比较均具有显著性差异(P0.05)。结论：1旋毛虫与宿主在相互作用过程中存在细胞凋现象,旋毛虫感染所致宿主寄生部位组织细胞凋亡与病理损伤存在一定的关系。2旋毛虫成虫侵袭小鼠肠道可诱导宿主小肠组织发生细胞凋亡,在一定时间内,随着感染时间延长,组织凋亡水平亦增加,感染第7d达到凋亡的高峰。3旋毛虫幼虫寄生于小鼠肌肉组织可诱导宿主肌肉组织发生细胞凋亡,且组织凋亡水平与感染时间存在密切的相关性,感染3、4w细胞凋亡水平明显上升,到达第5w,此时囊包形成,细胞凋亡水平下降。
[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.

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R383.15

【参考文献】