细胞自噬在重症急性胰腺炎中的表达及意义的研究

发布时间：2018-08-09 09:30

【摘要】：目的:通过研究重症急性胰腺炎动物模型中自噬的表达及变化情况,探讨自噬与重症急性胰腺炎的关系,为临床上重症急性胰腺炎的治疗提供潜在靶点。方法:将48只健康清洁级雄性SD大鼠,随机分配为两组,即对照组(SO)和急性坏死性胰腺炎组(ANP),每组24只大鼠。ANP组采用经胰胆管逆行注射5%牛磺胆酸钠方法制造急性坏死性胰腺炎动物模型。对照组仅翻动胰腺组织,不注射药物。每组大鼠按照3h、6h和12h三个时相点再分为3个亚组,每亚组8只大鼠,分别于手术造模后不同时间点收集血清及胰腺组织,血清用于淀粉酶的检测;胰腺组织一部分用4%甲醛固定,行HE染色和免疫组化测定LC3B、Beclin1及P62的表达;另一部分用2.5%戊二醛固定行电镜观察。结果:1、造模情况及胰腺组织HE染色病理学评分:(1)大体观察:SO组胰腺无结构异常,周围网膜清晰,未见腹水形成;ANP 3h亚组可见胰腺组织肿胀;6h亚组胰腺组织肿胀明显加重,可见胰腺组织有散在出血点,有少量腹水出现;12h亚组胰腺组织及周围网膜有片状出血性坏死,可见中等量血性腹水。(2)胰腺组织病理学评分:根据Schmidt评分标准:(1)ANP组与SO组对应时间点各亚组比较病理学评分具有显著性差异(P0.05);(2)ANP 12h亚组的病理学评分显著增加,且胰腺组织损伤程度亦增加,与ANP 3h亚组相比具有显著性差异(P0.05)。2、大鼠血清淀粉酶测定:(1)SO组的各亚组血清淀粉酶值均无明显差异(P0.05);(2)ANP组与SO组相比,对应时间点各亚组之间血清淀粉酶值显著升高,且有显著性差异(P0.05)。3、透射电镜观察胰腺组织:(1)SO组电镜下胰腺细胞核膜结构完整,胞质中细胞器的结构基本正常,少见自噬小体;(2)ANP 3h亚组可见有呈“C”形双层膜结构的自噬小体;6h亚组见线粒体明显肿胀,自噬小体增多,部分自噬小体与溶酶体融合;12亚组可见自噬小体及自噬溶酶体进一步增多,并存在溶酶体内降解内容物现象;4、免疫组化方法检测胰腺组织LC3B、Beclin1及P62:(1)SO组中LC3B有少量表达,各亚组间评分无明显差异(P0.05);ANP组相较SO组,各亚组LC3B评分均有明显增加(P0.05);ANP组中随造模时间的延长,LC3B阳性细胞表达增多,评分增加,且各亚组间评分具有明显差异(P0.05);(2)SO组Beclin1呈低表达,且各亚组间评分无明显差别(P0.05);与SO组比较,ANP组中各亚组Beclin1表达显著增加(P0.05);其中,ANP 6h亚组和12h亚组细胞胞浆着色明显变深,阳性细胞表达明显增加,与3h亚组比较均具有显著性差异(P0.05);(3)SO组P62有少量表达,各亚组间评分无明显差异(P0.05);与SO组比较,ANP组各亚组P62表达均有明显增高,具有显著性差异(P0.05);ANP组中各亚组间评分无明显差异(P0.05)。结论:1、采用5%牛磺胆酸钠经胰胆管逆行注射法,成功建立了急性坏死性胰腺炎(ANP)的动物模型;2、与SO组相比,ANP组中自噬的表达显著增加,并且自噬的表达随胰腺病理损伤程度的加重而增加;3、与SO组相比,ANP组中P62的表达显著增加,表明自噬的降解功能明显下降;4、自噬的功能受损可能参与到急性胰腺炎腺泡细胞损伤的过程中。
[Abstract]:Objective: To study the expression and changes of autophagy in the animal model of severe acute pancreatitis (SAP), and to explore the relationship between autophagy and severe acute pancreatitis (SAP) and provide potential targets for the treatment of severe acute pancreatitis. Methods: 48 healthy and clean male SD rats were randomly assigned to two groups, that is, the control group (SO) and the acute necrotizing pancreatitis. The.ANP group of 24 rats in each group was used to produce acute necrotizing pancreatitis by retrograde injection of 5% sodium taurocholate to the group of 24 rats. The control group only turned the pancreas tissue and did not injecting drugs. The rats in each group were divided into 3 subgroups according to the three phase points of 3H, 6H and 12h, and 8 rats in each subgroup were different at the same time after the operation. The serum and pancreatic tissue were collected at the interval, and the serum was used for the detection of amylase. The pancreas tissue was fixed with 4% formaldehyde, HE staining and immunohistochemistry were used to determine the expression of LC3B, Beclin1 and P62. The other part was fixed with 2.5% glutaraldehyde for electron microscopy. Results: 1, the modeling situation and the HE staining of pancreatic gland pathology score: (1) gross observation: SO group pancreas No abnormal structure of the glands, clear surrounding omentum, no ascites formation, ANP 3H subgroup can see the swelling of the pancreas tissue, 6h subgroup of pancreatic tissue swelling obviously aggravated, the pancreatic tissue has scattered bleeding point, a small amount of ascites appear, the pancreas tissue and peripheral omentum in the 12h subgroup are hemorrhagic necrotic, and the pancreatic tissue disease is visible. (2) pancreatic tissue disease Science score: according to the Schmidt score standard: (1) the comparative pathological score of each group in group ANP and group SO had significant difference (P0.05); (2) the pathological score of the ANP 12h subgroup increased significantly, and the degree of pancreatic tissue injury increased, and the ratio of the ANP 3H subgroup was significantly different (P0.05).2, the serum amylase determination in the rat: (1) SO. There was no significant difference in serum amylase between the subgroups of the group (P0.05). (2) compared with the SO group, the serum amylase value of the ANP group was significantly higher than that of the SO group, and there was a significant difference (P0.05).3. The transmission electron microscope was used to observe the pancreatic tissue: (1) the nuclear membrane structure of the pancreatic cells in the SO group was intact and the structure of the organelle in the cytoplasm was basically normal. See autophagic corpuscle; (2) ANP 3H subgroup can see autophagic body with "C" bilayer structure; 6h subgroup can see obvious mitochondria swelling, autophagosome increasing, partial autophagosome and lysosome fusion; the 12 subgroup can see autophagosome and autophagosome further increase, coexist in dissolve enzyme to degrade content phenomenon; 4, immunohistochemical method inspection There was a small amount of expression of LC3B in group LC3B, Beclin1 and P62: (1) SO group, and there was no significant difference between the sub groups (P0.05). The LC3B scores in each subgroup were significantly increased in the ANP group than in the SO group (P0.05), and the expression of the LC3B positive cells increased with the prolongation of the model time in the ANP group, and the score of the subgroups increased, and the scores of each subgroup were significantly different (2) Group Beclin1 showed low expression and no significant difference in scores between subgroups (P0.05). Compared with group SO, Beclin1 expression in each group of group ANP increased significantly (P0.05); in ANP 6h subgroup and 12h subgroup, the cytoplasmic staining was obviously deeper, the expression of positive cells increased obviously, and there was significant difference (P0.05) compared with the 3H subgroup (P0.05); (3) SO group had a small number of tables. There was no significant difference in scores between subgroups of each group (P0.05). Compared with group SO, the expression of P62 in each subgroup of group ANP was significantly higher (P0.05), and there was no significant difference between the sub groups in group ANP (P0.05). Conclusion: 1, the animal model of acute necrotizing pancreatitis (ANP) was successfully established by using 5% sodium taurocholate through the retrograde injection of pancreatic bile duct. 2, compared with group SO, the expression of autophagy increased significantly in group ANP, and the expression of autophagy increased with the severity of pancreatic pathological damage; 3, compared with group SO, the expression of P62 increased significantly in group ANP, indicating that the degradation function of autophagy decreased obviously; 4, the impairment of autophagy may be involved in the process of acinar cell injury in acute pancreatitis.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R576

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