Gli2介导炎性细胞因子调控小鼠急性胰腺炎的机制研究

发布时间：2018-12-06 20:23

【摘要】：目的:Hedgehog信号通路在急性胰腺炎中发挥重要作用,但其下游的细胞因子及具体机制尚不十分清楚。本实验主要是通过雨蛙素诱导小鼠急性胰腺炎并激活Hedgehog信号通路,从细胞因子芯片中筛选出变化比较明显的细胞因子,并从体内和体外实验两个方面进行验证。方法:取30只C57小鼠,随机分为三组:对照组、急性胰腺炎组、抑制组。(1)取胰腺组织进行病理切片和血清淀粉酶检测来验证模型建立成功。用RT-q PCR、Western blot、免疫组化等方法对Hedgehog信号通路中的Shh、Gli2信号表达进行检测。(2)体外培养小鼠胰腺腺泡细胞266-6,过表达Gli2之后用细胞因子芯片筛选出变化明显的细胞因子并用RT-q PCR在体外进行验证。(3)用Gant61(Gli家族特异性抑制剂)抑制Gli2后,取小鼠胰腺组织作病理切片,取小鼠血清行Elisa检测对筛选出的细胞因子进行体内验证。结果:(1)急性胰腺炎组和抑制剂组与对照组相比较,淀粉酶水平明显升高(P0.05),HE染色急性胰腺炎组较对照组胰腺腺泡细胞明显水肿,细胞体积增大;(2)用RT-q PCR、Western blot、免疫组化等方法来检测Shh、Gli2表达,结果与对照组相比较,急性胰腺炎组各组织(胰腺、肺、肝、小肠、肾)中Shh、Gli2表达明显升高,差异有统计学意义(P0.05),且Gli2主要表达于胰腺腺泡细胞、肺泡上皮细胞、肝实质细胞、小肠黏膜细胞、肾小管上皮细胞等细胞的细胞质内;(3)体外培养小鼠胰腺腺泡细胞266-6,过表达Gli2之后用细胞因子芯片筛选出变化比较明显的细胞因子,如IFN-γ、Fasl、IL-6、G-csf、Cxcl9和Tnfr1a,然后用RT-q PCR进行验证,过表达Gli2组中IFN-γ、Fasl、IL-6表达较Vector组明显下降,过表达Gli2组中G-csf、Cxcl9、Tnfr1a表达较Vector组升高,其差异有统计学意义(P0.05);(4)再从体内进行验证,用Gant61抑制Gli2之后,作胰腺Gli2蛋白的western blot,结果发现抑制组中Gli2蛋白表达较急性胰腺炎组和对照组降低,表明Gant61抑制有效,然后作胰腺组织病理损伤评分,结果发现抑制组中胰腺病理损伤评分高于急性胰腺炎组和对照组,随后对上述细胞因子进行Elisa检测,发现抑制剂组中IFN-γ、Fasl、IL-6表达较对照组、急性胰腺炎组均升高(P0.05),G-csf、Cxcl9、Tnfr1a在急性胰腺炎组中与对照组比较明显升高(P0.05),而抑制剂组中G-csf、Cxcl9、Tnfr1a表达较急性胰腺炎组和对照组明显降低(P0.05)。结论:1.在雨蛙素诱导的小鼠急性胰腺炎中,Shh-Gli2信号轴参与了胰腺的损伤及肺、肝、小肠、肾等组织的炎症过程。2.Gli2介导细胞因子IFN-γ、Fasl、IL-6、G-csf、Cxcl9和Tnfr1a参与了对急性胰腺炎的调控过程。
[Abstract]:Objective: Hedgehog signaling pathway plays an important role in acute pancreatitis, but its downstream cytokines and specific mechanisms are not well understood. This experiment was mainly conducted to induce acute pancreatitis and activate Hedgehog signaling pathway in mice. Cytokines with obvious changes were screened from cytokine microarray and verified by in vivo and in vitro experiments. Methods: thirty C57 mice were randomly divided into three groups: control group, acute pancreatitis group and inhibitory group. (1) the pancreatic tissue was taken for pathological section and serum amylase detection to verify the establishment of the model. RT-q PCR,Western blot, immunohistochemical method was used to detect the expression of Shh,Gli2 signal in Hedgehog signaling pathway. (2) cultured mouse pancreatic acinar cells 266-6 in vitro. After overexpression of Gli2, cytokines were screened by cytokine chip and verified by RT-q PCR in vitro. (3) after inhibiting Gli2 with Gant61 (Gli family specific inhibitor), the pancreatic tissues of mice were taken as pathological sections. The selected cytokines were tested by Elisa in serum of mice. Results: (1) the levels of amylase in acute pancreatitis group and inhibitor group were significantly higher than those in control group (P0.05), HE staining showed that the acinar cells in the acute pancreatitis group were significantly edematous and the cell volume was larger than that in the control group; (2) the expression of Shh,Gli2 was detected by RT-q PCR,Western blot, immunohistochemical method. Compared with the control group, the expression of Shh,Gli2 in the tissues (pancreas, lung, liver, small intestine and kidney) of acute pancreatitis group was significantly increased. The difference was statistically significant (P0.05), and Gli2 was mainly expressed in the cytoplasm of acinar cells, alveolar epithelial cells, hepatic parenchyma cells, intestinal mucosal cells and renal tubular epithelial cells. (3) in vitro cultured mouse pancreatic acinar cells 266-6. After overexpression of Gli2, cytokines were screened by cytokine microarray, such as IFN- 纬, Fasl,IL-6,G-csf,Cxcl9 and Tnfr1a,. Then RT-q PCR was used to verify that the expression of IFN- 纬 and Fasl,IL-6 in Gli2 group was significantly lower than that in Vector group, and that in Gli2 group was higher than that in Vector group (P0.05). (4) after Gli2 was inhibited by Gant61 in vivo, the expression of Gli2 protein in the inhibition group was lower than that in the acute pancreatitis group and the control group, which indicated that Gant61 was effective. The results showed that the pancreatic pathological injury score in the inhibition group was higher than that in the acute pancreatitis group and the control group. Then the cytokines mentioned above were detected by Elisa, and IFN- 纬 and Fasl, were found in the inhibitor group. The expression of IL-6 in acute pancreatitis group was higher than that in control group (P0.05), and G-csffCxcl9Tnfr1a in acute pancreatitis group was significantly higher than that in control group (P0.05), while G-csffCxcl9 in inhibitor group was significantly higher than that in control group (P0.05). The expression of Tnfr1a was significantly lower than that in acute pancreatitis group and control group (P0.05). Conclusion: 1. Shh-Gli2 signal axis is involved in pancreatic injury and inflammation in lung, liver, small intestine, kidney, etc. 2.Gli2 mediates cytokines IFN- 纬, Fasl,IL-6,G-csf,. Cxcl9 and Tnfr1a are involved in the regulation of acute pancreatitis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R576

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