Sept4和miR-454在日本血吸虫病小鼠肝纤维化组织中的表达及作用研究

发布时间：2018-05-04 10:17

  本文选题：Sept4 + mi　； 参考：《南通大学》2014年硕士论文

【摘要】：目的探索Sept4对日本血吸虫病小鼠慢性肝纤维化的作用;同时观察miR-454在活化的肝星状细胞HSCs和日本血吸虫诱导的肝纤维化组织中的表达变化及其对HSCs的影响和机制。方法1.ICR小鼠30只,适应性喂养1w后,随机分为3组,即PBS组(血吸虫感染+PBS处理组),Ad-GFP组(血吸虫感染+Ad-GFP处理组),Ad-Sept4组(血吸虫感染+Ad-Sept4处理组)。各组小鼠感染血吸虫,12w后经吡喹酮杀虫治疗3d(250mg/kg/24h),然后经尾静脉分别给各小鼠注射Ad-Sept4、Ad-GFP或PBS。于注射2w后(即感染约15w后)处死小鼠,取肝组织进行HE染色和天狼星红染色,观察肝纤维化组织的变化情况;用Western Blot和qRT-PCR技术检测肝纤维化相关基因的表达变化情况;用TUNEL观察凋亡情况。2.ICR小鼠16只,适应性喂养1w后,随机分为2组,即正常组(未感染)和感染组(血吸虫感染),感染组在感染日本血吸虫8w后处死小鼠,取肝脏组织以备用。应用qRT-PCR技术检测miR-454在肝纤维化组织中及活化的HSCs中的表达变化;用MTT、流式细胞术检测miR-454对HSCs细胞增殖和细胞周期的影响;应用Western Blot检测mi R-454对HSCs活化的影响。结果1.与Ad-GFP组相比,Ad-Sept4组的肝脏纤维化程度减轻;TUNEL阳性细胞数量增加;Sept4及cleaved-caspase-3表达上升;而α-SMA、pro-collα1(I)、TGF-β1和IL-6等表达下降。2.在肝纤维化组织中及活化的HSCs中miR-454的表达下降,α-SMA和Smad4表达上升;过表达mi R-454可以降低HSCs中的α-SMA和Smad4表达,但不能影响HSCs的增殖和细胞周期。3.在活化的HSCs中,miR-454可以抑制Smad4 3’UTR端的荧光素酶活性。结论1.Ad-Sept4对血吸虫病小鼠慢性肝纤维化具有抑制作用。2.miR-454在血吸虫病肝纤维化组织及活化HSCs中表达降低。3.过表达miR-454可以通过靶向Smad4 3’UTR区发挥抑制HSCs活化的作用。
[Abstract]:Objective to investigate the effect of Sept4 on chronic hepatic fibrosis in mice with schistosomiasis japonicum, and to observe the expression of miR-454 in activated hepatic stellate cells (HSCs) and Schistosoma japonicum induced hepatic fibrosis (HSCs). Methods Thirty 1.ICR mice were randomly divided into three groups after one week of adaptive feeding: PBS group (PBS treatment group) (Ad-GFP treatment group) and Ad-Sept4 group (Ad-Sept4 treatment group). The mice in each group were infected with Schistosoma japonicum for 12 weeks and treated with praziquantel for 3 days, 250 mg / kg / 24 h / h, and then each mouse was injected with Ad-Sept4Ad-GFP or PBSvia caudal vein respectively. The mice were killed 2 weeks after injection (that is, 15 weeks after infection), the liver tissues were stained with HE and Sirius red to observe the changes of liver fibrosis tissues, and the expression of genes related to liver fibrosis were detected by Western Blot and qRT-PCR techniques. After 1 week of adaptive feeding, 16 ICR mice were randomly divided into two groups: normal group (uninfected) and infected group (Schistosoma japonicum infection). The infected mice were killed after 8 weeks of infection with Schistosoma japonicum, and liver tissues were taken for reserve. QRT-PCR technique was used to detect the expression of miR-454 in liver fibrosis tissues and activated HSCs, MTT and flow cytometry were used to detect the effect of miR-454 on the proliferation and cell cycle of HSCs cells, and Western Blot was used to detect the effect of miR-454 on HSCs activation. Result 1. Compared with Ad-GFP group, the degree of hepatic fibrosis in Ad-Sept4 group reduced the number of Tunel positive cells increased, while the expression of 伪 -SMA-pro-coll 伪 1 and TGF- 尾 1 and IL-6 decreased .2.The expression of TGF- 尾 1 and TGF- 尾 1 were decreased in Ad-Sept4 group, and the expression of TGF- 尾 1 and IL-6 were decreased. The expression of 伪 -SMA and Smad4 increased in hepatic fibrosis tissues and activated HSCs, and the overexpression of miR-454 could decrease the expression of 伪 -SMA and Smad4 in HSCs, but it could not affect the proliferation and cell cycle of HSCs. In activated HSCs, miR-454 inhibited luciferase activity at the end of Smad4 3'UTR. Conclusion 1.Ad-Sept4 can inhibit chronic hepatic fibrosis in mice with schistosomiasis. 2. The expression of miR-454 in schistosomiasis liver fibrosis tissue and activated HSCs is decreased. Overexpression of miR-454 can inhibit the activation of HSCs by targeting the region of Smad4 3'UTR.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.21

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