IL-22对小鼠重症急性胰腺炎肾损伤保护作用的研究

发布时间：2018-05-14 14:26

本文选题：白细胞介素-22 + 重症急性胰腺炎　；参考：《山东大学》2017年硕士论文

【摘要】：目的本实验通过向小鼠腹腔注射高浓度左旋精氨酸建立重症急性胰腺炎动物模型,并用外源性白细胞介素-22(interleukin-22,IL-22)干预模型小鼠,观察模型小鼠胰腺及肾脏的损伤情况。探索重症急性胰腺炎相关肾损伤发病机制,同时研究外源性IL-22对左旋精氨酸诱导的重症急性胰腺炎相关肾损伤的保护作用及其相关的作用机制。方法选取48只健康雄性SPF级的BALB/c小鼠,随机分为4组(每组12只):正常对照组(NS组),重症急性胰腺炎模型组(SAP组),IL-22治疗组(IL-22组),PBS治疗对照组(PBS组)。SAP模型小鼠的造模方法为分2次腹腔内注射20%左旋精氨酸(4g/kg体质量,间隔1h);NS组在同样的时间节点腹腔内注入等量无菌生理盐水。IL-22组在造重症急性胰腺炎模型的同时分5次皮下注射重组小鼠IL-22(200ng/次,间隔24h),PBS组在相同时间点皮下注射等量的PBS溶液。观察并记下各组小鼠活动状态及存活状况。注射左旋精氨酸或生理盐水72h后,统计每组小鼠死亡率后采集小鼠全血,剖取胰腺和肾脏组织。光镜下观察胰腺和肾脏组织HE染色后的切片的病理学改变并分别给胰腺和肾的病理损伤程度评分;用全自动生化仪检测血清中淀粉酶(amylase,AMY)、肌酐(creatinine,Cr)、尿素氮(blood urea nitrogen,BUN)的水平。Western Blotting方法检测肾组织中总的信号转导与转录激活因子3(signal transducer and activator of transcription-3,STAT3)及磷酸化的 STAT3(p-STAT3)蛋白的表达量;RT-PCR法检测肾组织中IL-22RA1、Bcl-2和Bcl-XL的mRNA相对表达量。采用SPSS19.0进行统计学分析,计量资料分析采用t检验,统计量用均数±标准差(x±s)表示,死亡率分析采用fisher确切概率法,检验标准取α =0.05,P0.05为差异有统计学意义。结果光学显微镜下观察NS组小鼠HE染色的病理切片:胰腺组织无坏死及水肿,胰腺细胞排列规律;肾组织无明显病理损伤改变。与NS组相比较,左旋精氨酸诱导的SAP组和PBS组小鼠,胰腺细胞坏死,组织肿胀,大量炎性细胞浸润,肾脏皮质区部分肾小管管腔扩张,间隙增宽,血清AMY、Cr、BUN水平较其明显升高,统计学均有意义(P0.05)。相较于PBS组,IL-22组小鼠胰腺组织损伤明显减轻,肾组织切片无明显病理损伤,且血清中AMY、Cr、BUN水平有不同程度减低(P0.05),两组小鼠肾组织中STAT3蛋白总表达量无明显差异,但IL-22组肾脏中p-STAT3蛋白表达量增高,且RT-PCR检测IL-22RA1、Bcl-2及Bcl-XL的mRNA表达均有升高(P0.05)。IL-22组小鼠病死率为8.3%,PBS组小鼠病死率为41.7%,P=0.77,差异无统计学意义。结论腹腔注射高浓度左旋精氨酸可以建立小鼠重症急性胰腺炎相关性肾损伤模型。外源性IL-22对重症急性胰腺炎相关性肾损伤有保护作用,其相关机制可能为激活STAT3通路,上调下游抗凋亡基因的表达,维护胰腺及肾脏组织细胞活性及细胞膜稳定性,从而保护胰腺及肾脏功能。
[Abstract]:Objective to establish the animal model of severe acute pancreatitis (SAP) by intraperitoneal injection of high concentration of L-arginine, and to observe the pancreatic and renal injury of the model mice induced by exogenous interleukin-22 (IL-22). To explore the pathogenesis of renal injury associated with severe acute pancreatitis, and to study the protective effect of exogenous IL-22 on the renal injury associated with severe acute pancreatitis induced by L-arginine and its related mechanism. Methods 48 healthy male BALB/c mice of SPF grade were selected. They were randomly divided into 4 groups: normal control group (n = 12) and severe acute pancreatitis model group (n = 12), SAP group (n = 10) and control group (n = 12). The model of PBS group (n = 12) was established by intraperitoneal injection of 20% L-arginine (20% L-arginine 4g / kg) into abdominal cavity. At the same time, the NS group was intraperitoneally injected with the same amount of aseptic saline. IL-22 group was subcutaneously injected with recombinant IL-22(200ng/ 5 times while the model of severe acute pancreatitis was established, and the same amount of PBS solution was injected subcutaneously at the same time point in the control group. The activity and survival of each group were observed and recorded. After 72 hours of injection of L-arginine or normal saline, the whole blood of each group was collected and pancreas and kidney tissues were taken. The pathological changes of the sections of pancreas and kidney after HE staining were observed under light microscope and the pathological injury degree of pancreas and kidney were scored respectively. The levels of amylase, creatinine, urea nitrogen urea nitrogenbun in serum were detected by automatic biochemical analyzer. Western Blotting method was used to detect the expression of total signal transduction and transcription activator 3(signal transducer and activator of transcription-3STAT3 and phosphorylated STAT3p-STAT3 in renal tissues. The relative expression of Bcl-2 and Bcl-XL in renal tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). The statistical analysis was carried out by SPSS19.0, the statistical data was analyzed by t test, the statistic was expressed by the mean 卤standard deviation (x 卤s), the death rate was analyzed by fisher exact probability method, and the test standard was taken 伪 -0. 05% as the difference. Results the pathological sections of NS mice stained with HE were observed under optical microscope: there was no necrosis and edema in pancreatic tissue and the arrangement of pancreatic cells, and no obvious pathological changes in renal tissue. Compared with NS group, SAP group and PBS group induced by L-arginine showed necrosis of pancreatic cells, swelling of tissues, infiltration of inflammatory cells, dilation of tubule lumen and widening of interstitial space in renal cortex. All of them had significant statistical significance (P 0.05). Compared with the PBS group, the pancreatic tissue injury in the IL-22 group was significantly alleviated, and there was no obvious pathological injury in the renal tissue section, and the serum AMYP0. 05 bun level was decreased in varying degrees. There was no significant difference in the total expression of STAT3 protein between the two groups. However, the expression of p-STAT3 protein in the kidney of IL-22 group was increased, and the expression of IL-22RA1Bcl 2 and Bcl-XL mRNA was increased in IL-22 group. The mortality rate of P0.05 + IL-22 group was 8.3g / ml. The mortality rate of PBS group was 41.7% (P 0.77), there was no significant difference between them. Conclusion Intraperitoneal injection of high concentration of L-arginine can establish the model of severe acute pancreatitis associated renal injury in mice. Exogenous IL-22 has protective effect on renal injury associated with severe acute pancreatitis, which may be related to activation of STAT3 pathway, up-regulation of downstream anti-apoptotic gene expression, maintenance of pancreatic and renal tissue cell activity and cell membrane stability. To protect the pancreas and kidney function.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R576;R692

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