【摘要】：目的:Hedgehog信號通路在急性胰腺炎中發(fā)揮重要作用,但其下游的細(xì)胞因子及具體機(jī)制尚不十分清楚。本實(shí)驗(yàn)主要是通過雨蛙素誘導(dǎo)小鼠急性胰腺炎并激活Hedgehog信號通路,從細(xì)胞因子芯片中篩選出變化比較明顯的細(xì)胞因子,并從體內(nèi)和體外實(shí)驗(yàn)兩個(gè)方面進(jìn)行驗(yàn)證。方法:取30只C57小鼠,隨機(jī)分為三組:對照組、急性胰腺炎組、抑制組。(1)取胰腺組織進(jìn)行病理切片和血清淀粉酶檢測來驗(yàn)證模型建立成功。用RT-q PCR、Western blot、免疫組化等方法對Hedgehog信號通路中的Shh、Gli2信號表達(dá)進(jìn)行檢測。(2)體外培養(yǎng)小鼠胰腺腺泡細(xì)胞266-6,過表達(dá)Gli2之后用細(xì)胞因子芯片篩選出變化明顯的細(xì)胞因子并用RT-q PCR在體外進(jìn)行驗(yàn)證。(3)用Gant61(Gli家族特異性抑制劑)抑制Gli2后,取小鼠胰腺組織作病理切片,取小鼠血清行Elisa檢測對篩選出的細(xì)胞因子進(jìn)行體內(nèi)驗(yàn)證。結(jié)果:(1)急性胰腺炎組和抑制劑組與對照組相比較,淀粉酶水平明顯升高(P0.05),HE染色急性胰腺炎組較對照組胰腺腺泡細(xì)胞明顯水腫,細(xì)胞體積增大;(2)用RT-q PCR、Western blot、免疫組化等方法來檢測Shh、Gli2表達(dá),結(jié)果與對照組相比較,急性胰腺炎組各組織(胰腺、肺、肝、小腸、腎)中Shh、Gli2表達(dá)明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05),且Gli2主要表達(dá)于胰腺腺泡細(xì)胞、肺泡上皮細(xì)胞、肝實(shí)質(zhì)細(xì)胞、小腸黏膜細(xì)胞、腎小管上皮細(xì)胞等細(xì)胞的細(xì)胞質(zhì)內(nèi);(3)體外培養(yǎng)小鼠胰腺腺泡細(xì)胞266-6,過表達(dá)Gli2之后用細(xì)胞因子芯片篩選出變化比較明顯的細(xì)胞因子,如IFN-γ、Fasl、IL-6、G-csf、Cxcl9和Tnfr1a,然后用RT-q PCR進(jìn)行驗(yàn)證,過表達(dá)Gli2組中IFN-γ、Fasl、IL-6表達(dá)較Vector組明顯下降,過表達(dá)Gli2組中G-csf、Cxcl9、Tnfr1a表達(dá)較Vector組升高,其差異有統(tǒng)計(jì)學(xué)意義(P0.05);(4)再從體內(nèi)進(jìn)行驗(yàn)證,用Gant61抑制Gli2之后,作胰腺Gli2蛋白的western blot,結(jié)果發(fā)現(xiàn)抑制組中Gli2蛋白表達(dá)較急性胰腺炎組和對照組降低,表明Gant61抑制有效,然后作胰腺組織病理損傷評分,結(jié)果發(fā)現(xiàn)抑制組中胰腺病理損傷評分高于急性胰腺炎組和對照組,隨后對上述細(xì)胞因子進(jìn)行Elisa檢測,發(fā)現(xiàn)抑制劑組中IFN-γ、Fasl、IL-6表達(dá)較對照組、急性胰腺炎組均升高(P0.05),G-csf、Cxcl9、Tnfr1a在急性胰腺炎組中與對照組比較明顯升高(P0.05),而抑制劑組中G-csf、Cxcl9、Tnfr1a表達(dá)較急性胰腺炎組和對照組明顯降低(P0.05)。結(jié)論:1.在雨蛙素誘導(dǎo)的小鼠急性胰腺炎中,Shh-Gli2信號軸參與了胰腺的損傷及肺、肝、小腸、腎等組織的炎癥過程。2.Gli2介導(dǎo)細(xì)胞因子IFN-γ、Fasl、IL-6、G-csf、Cxcl9和Tnfr1a參與了對急性胰腺炎的調(diào)控過程。
[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- 緯, Fasl,IL-6,G-csf,Cxcl9 and Tnfr1a,. Then RT-q PCR was used to verify that the expression of IFN- 緯 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- 緯 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- 緯, Fasl,IL-6,G-csf,. Cxcl9 and Tnfr1a are involved in the regulation of acute pancreatitis.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R576

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