SHH通路对小鼠内毒素性肺损伤及早期脏器发育作用机制研究

发布时间：2018-05-04 05:02

本文选题：Sonic + hedgehog信号通路　；参考：《山东大学》2016年博士论文

【摘要】：Sonic Hedgehog(SHH)信号通路在胚胎发育和成年组织稳态中起关键作用。经典Hh信号通路是SHH配体与跨膜蛋白受体PTCH(Patched)相结合,进而激活第二个跨膜蛋白SMO(Smoothened),活化的SMO导致GLI蛋白稳定和核内积累。GLI 家族包括 GLI1(GLI family zinc finger 1),GLI2(GLI family zinc finger 2)和GLI3(GLI family zinc finger 3)。有充分的证据表明,SHH信号通路是胚胎期肺脏形成和发育过程中重要的信号传导通路,但在出生后的一段时间和成年动物体内该信号通路表达大为减少。近年来不少研究表明SHH信号通路在一些肺部疾病中异常激活,如在肺发育不良中存在SHH信号通路的表达上调,间质性肺疾病中发现有SHH信号通路的异常激活,在哮喘和慢性阻塞性肺部疾病中观察到SHH信号通路的表达上调,在肺小细胞癌和肺非小细胞癌中也发现SHH信号通路表达的上调。但该信号通路在急性肺损伤的表达及意义尚不清楚。最新研究表明SHH信号通路在早期脏器形成和发育过程中发挥重要作用。SHH信号通路的突变导致脊柱、肛门、气管、食管、肾和肢体联合综合征(VACTERL syndrome)。本研究选取肾脏作为代表器官,深入研究SHH信号在早期脏器发育中的作用。目前研究表明SHH错义突变和无效突变与"肾发育不全"和"泌尿生殖系统畸形"相关联。SHH基因敲除鼠肾发育不全,表现为输尿管缩短、肾皮质及髓质体积减少及肾单位减少。然而SHH调节肾脏发育的具体机制及其与调节肾脏发育的其他因子之间的相互作用尚不可知。在本研究中,我们分别探讨了 SHH信号通路在脂多糖(LPS)致急性肺损伤(acute lung injury,ALI)疾病进展过程中的表达变化及意义;以及SHH信号通路在肾脏发育过程中的表达及调控。第一部分Sonic hedgehog信号通路:其对内毒素性肺损伤小鼠模型的保护作用目的Sonic hedgehog(SHH)信号通路在脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)模型中的表达变化及意义。方法雄性Balb/c小鼠随机分为以下4组:对照组、LPS组、LPS-环巴胺组和环巴胺组。急性肺损伤模型是通过腹腔注射LPS(5mg/kg)建立,LPS-环巴胺组是在LPS注入30min后给予SHH信号通路的抑制剂环巴胺(50mg/kg),通过HE染色观察肺组织病理改变,半定量肺组织损伤评分,测定肺湿干质量比值(W/D),PCR检测肺组织肿瘤坏死因子α(TNF-α)及SHH、Patched(PTC)及下游转录因子GLI1的mRNA表达,同时Western blot检测肺组织SHH和GLI1的蛋白表达情况。结果LPS组小鼠的肺组织病理损伤评分、肺泡隔厚度、W/D、TNF-α mRNA表达水平明显高于正常对照组(P0.05)。LPS组小鼠SHH、PTC、GLI1的mRNA表达水平在LPS注入后的12h、24h明显升高,但在6h时与对照组无明显差异。与对照组相比,LPS组在6、12、24h的SHH、GLI1蛋白表达水平均明显升高,且与时间呈正相关。单独环巴胺组对正常小鼠肺组织的病理改变无明显影响。LPS-环巴胺组出现SHH、PTC、GLI1的mRNA表达及SHH、GLI1的蛋白表达水平降低,同时,肺组织病理损伤评分、肺泡隔厚度、W/D及TNF-α mRNA表达均高于LPS组。结论在LPS所致的ALI模型中存在SHH信号通路的激活,且SHH信号通路表达上调可减轻肺损伤,参与肺组织损伤后修复。第二部分小鼠体外培养胚肾组织中SHH蛋白表达及其与Fgf8关系的研究目的探讨Sonic hedgehog(SHH)蛋白在小鼠体外原代培养胚肾组织中的表达及与成纤维生长因子8(Fibroblast growth factor,Fgf8)的关系。方法检测胚龄E11.5至E14.5小鼠肾脏组织中SHH及Fgf8表达水平,体外培养小鼠胚肾组织,分别加入外源性SHH蛋白以及SHH信号通路抑制剂环巴胺,分为正常对照组、SHH组和SHH+环巴胺组。通过组织染色和免疫荧光检测小鼠原代胚肾组织中输尿管芽发育及肾单位数目以评估肾脏发育水平。Real-time PCR法检测SHH及FGF8基因在不同阶段胚肾组织中的表达差异。Real-time PCR及Western blot方法分析三个不同处理组胚肾组织中Fgf8的分布和表达。结果在不同的时间段,小鼠原代胚肾组织中SHH及Fgf8mRNA表达水平均呈时间依赖性增高。外源性SHH蛋白通过促进输尿管芽发育及增加肾单位数目促进肾脏发育。与正常对照组比较,SHH组中Fgf8mRNA及蛋白水平显著降低。抑制SHH信号通路后,Fgf8表达恢复至正常水平。结论SHH通过Fgf8途径促进输尿管芽发育及增加肾单位数目,参与肾脏发育过程,为进一步探讨SHH和Fgf8在肾脏发育及先天性肾脏疾病中的作用提供了实验依据。
[Abstract]:The Sonic Hedgehog (SHH) signaling pathway plays a key role in the development of embryo and the homeostasis of adult tissues. The classical Hh signaling pathway is the combination of the SHH ligand with the transmembrane protein receptor PTCH (Patched) and the activation of the second transmembrane protein SMO (Smoothened). The activated SMO leads to the stabilization of the GLI protein and the accumulation of the nuclear.GLI family. Inger 1), GLI2 (GLI family zinc finger 2) and GLI3 (GLI family zinc finger 3). There is sufficient evidence that the SHH signal pathway is an important signal transduction pathway in the formation and development of the embryonic lung, but the expression of the signal pathway is greatly reduced in a period of time after birth and in adult animals. Signal pathways are abnormal activation in some pulmonary diseases, such as the up regulation of SHH signaling pathway in lung dysplasia, abnormal activation of SHH signaling pathway in interstitial lung disease, and the expression of SHH signaling in asthma and chronic obstructive pulmonary diseases, and in small and small cell lung cancer and lung cancer. The expression and significance of the SHH signaling pathway in acute lung injury is not clear. The latest research shows that the SHH signaling pathway plays an important role in the early organ formation and development of.SHH signaling pathways leading to the spinal, anus, trachea, feeding tube, kidney and limb joint syndrome (VACTERL syndrome). In this study, the role of the kidneys as the representative organ is to study the role of SHH signal in the early organ development. The present study shows that the SHH missense mutation and the invalid mutation are associated with the "renal dysplasia" and the "genitourinary system malformation" in the.SHH knockout rats, showing the ureter shortening, the decrease of the renal cortex and medulla volume and the kidney. However, the specific mechanism of SHH regulation of kidney development and its interaction with other factors regulating renal development are unknown. In this study, we explored the changes and significance of SHH signaling pathway in the progression of acute lung injury (acute lung injury, ALI) disease induced by lipopolysaccharide (LPS); and SHH Expression and regulation of signal pathway in the process of renal development. Part 1: Sonic hedgehog signaling pathway: its protective effect on mice with endotoxic lung injury; the expression and significance of Sonic hedgehog (SHH) signaling pathway in the model of acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. Methods male Balb/c mice The machine was divided into 4 groups: the control group, the LPS group, the LPS- cyclic amine group and the cyclic amine group. The acute lung injury model was established by intraperitoneal injection of LPS (5mg/kg), and the LPS- cyclic amine group was the inhibitor of the SHH signaling pathway after LPS injection 30min (50mg/kg). The pathological changes of the lung tissue were observed through HE staining, and the semiquantitative lung tissue injury score was measured. Lung wet dry mass ratio (W/D), PCR detection of lung tissue tumor necrosis factor alpha (TNF- alpha), SHH, Patched (PTC) and downstream transcription factor GLI1 mRNA expression, and Western blot detection of lung tissue SHH and GLI1 protein expression. The mRNA expression level of SHH, PTC, GLI1 in the normal control group (P0.05) group.LPS was significantly higher than that of the control group after LPS injection, but there was no significant difference between the control group and the control group. Pathological changes did not significantly affect the expression of SHH, PTC, GLI1, mRNA and SHH, and the level of protein expression in GLI1 was decreased in the.LPS- cyclic amine group. At the same time, the pathological damage score of the lung tissue, the thickness of the alveolar septum, the expression of W/D and TNF- alpha mRNA were higher than that in the LPS group. The expression of SHH protein and the relationship between the SHH protein and Fgf8 in the embryo and kidney tissue of the mice in vitro were studied to investigate the expression of Sonic hedgehog (SHH) protein in the primary cultured embryonic kidney tissue in vitro and the relationship with the fibroblast growth factor 8 (Fibroblast growth factor, Fgf8). The expression of SHH and Fgf8 in the kidney tissues of E11.5 to E14.5 mice was detected by the method. The mouse embryo and kidney tissue was cultured in vitro, and the exogenous SHH protein and the SHH signaling pathway inhibitor, cyclic diamine, were divided into normal control group, SHH group and SHH+ cyclic amine group. The ureteral buds in the renal tissue of mice were detected by tissue staining and immunofluorescence. Development and renal unit number to evaluate the expression of SHH and FGF8 gene in different stages of embryo and kidney tissue by.Real-time PCR method. The distribution and expression of Fgf8 in three different treated groups of embryo and kidney were analyzed by.Real-time PCR and Western blot method. Results in different time periods, SHH and Fgf in mouse primary embryo kidney tissue The expression level of 8mRNA increased in time dependent. Exogenous SHH protein promoted the development of kidney by promoting the development of ureter buds and increasing the number of renal units. Compared with the normal control group, the level of Fgf8mRNA and protein in the SHH group decreased significantly. After the inhibition of the SHH signaling pathway, the expression of Fgf8 was restored to the normal level. Conclusion SHH promotes the transfusion through Fgf8 pathway. The development of urethral buds and the increase of the number of renal units involved in the process of renal development, which provide experimental basis for further study of the role of SHH and Fgf8 in renal development and congenital renal diseases.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R726.9;R725.6

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