Shh信号通路变化对小鼠胚胎腭突间充质细胞自噬的影响

目的：探讨地塞米松（dexamethasone,DEX）及平滑激动剂（smoothened agonist,SAG）干预下小鼠胚胎腭突间充质细胞Shh信号通路改变后,小鼠胚胎腭突间充质细胞自噬状态的变化。方法：体外培养胚胎14.5 d的C57BL/6J小鼠胚胎腭突间充质细胞,根据干预方式分为4组：对照组、地塞米松组（DEX组）、地塞米松+SAG组（DEX+SAG组）、单独SAG干预组（SAG组）。通过透射电镜观察各组细胞自噬体或自噬溶酶体的数量,Western blot检测各组Shh、Ptch1、Smo、Gli3A/R、Cyclin D1及自噬标志物LC3Ⅱ/Ⅰ和P62、Beclin-1的表达情况。结果：电镜结果显示,对照组和DEX组中自噬体/自噬溶酶体的数量无明显差异,而SAG干预后可观察到大量自噬体/自噬溶酶体。Western blot结果显示,与对照组相比,P62、Ptch1、Smo、Gli3A/R、CyclinD1在DEX组中明显降低（P<0.05）,LC3Ⅱ/Ⅰ、Beclin1表达无明显变化（P>0.05）;DEX+SAG组及SAG组中Ptch1、Smo、Gli3...     

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors.     

Shh/Gli1信号参与小鼠脊髓损伤后血-脊髓屏障渗透性改变及运动功能恢复

目的:探讨sonic hedgehog/Gli1(Shh/Gli1)信号在小鼠脊髓损伤后病理变化及运动功能恢复中的作用。方法:成年雄性C57野生型、Gli1lz和Gli1lz/lz小鼠行T8节段脊髓夹伤及假手术。Gli1lz小鼠脊髓损伤及假手术后3 d行X-gal染色;7 d行Shh/PDGFr-α、Shh/GFAP、LacZ/GFAP染色,观察Shh/Gli1信号在小鼠脊髓损伤后的激活。C57野生型和Gli1lz/lz小鼠脊髓夹伤后7 d行GFAP染色和实时定量RT-PCR,观察星形胶质细胞反应;术后14 d尾静脉注射伊文氏兰观察血-脊髓屏障改变;术后1、3、5 d和7 d行BMS评分评价小鼠后肢运动功能。结果:脊髓损伤7 d后,损伤区Shh表达升高;Shh/Gli1信号报告基因LacZ表达升高,主要表达于反应性星形胶质细胞;免疫组织化学及实时定量RT-PCR结果显示Gli1基因敲除不影响脊髓损伤后GFAP表达;伊文氏兰染色及其定量分析显示Gli1lz/lz小鼠脊髓损伤后脊髓组织伊文氏兰外渗增加。BMS评分显示Gli1lz/lz小鼠运动功能恢复显著差于野生型小鼠。结论:Shh/Gli1信号在小鼠脊髓损伤后激活,可能参与脊髓损伤后血-脊髓屏障渗透性的改变,并影响脊髓损伤后的运动功能恢复。     

Shh在雷奈酸锶促进骨髓间充质干细胞成骨分化过程中的作用

 目的：研究Sonic Hedgehog（Shh）在雷奈酸锶（strontium ranelate,Sr）促进大鼠骨髓间充质干细胞（BMSCs）向成骨细胞分化中的作用。方法：采用全骨髓贴壁培养法分离、纯化、培养大鼠BMSCs,取第3~5代BMSCs加入成骨诱导液诱导成骨分化,再加入不同浓度Sr及Shh拮抗剂cyclopamine（Cy）,分别观察它们对BMSCs向成骨细胞分化的影响。酶标法检测成骨细胞分化早期标志物碱性磷酸酶(alkaline phosphatase,ALP)的活性;茜素红染色检测细胞钙化水平;Western blotting法检测Shh和Runx2蛋白的表达情况。结果：Sr（3 mmol/L）可以使细胞ALP活性增高,钙结节形成增加。Sr（0.1～5 mmol/L）作用BMSCs 7 d,可明显促进Shh和Runx2蛋白的表达,且Shh蛋白在1 mmol/L Sr作用时表达最多,而Runx2在3 mmol/L Sr作用时表达最多。1 mmol/L Sr作用BMSCs不同时间（1、3、5、7 d）,呈时间依赖性地上调Shh和Runx2蛋白的表达。Cy（10 μmol/L）不仅拮抗Sr对Shh和Runx2表达的上调作用,还抑制Sr对ALP和钙结节形成的促进作用。结论：Sr可通过上调Shh蛋白及成骨特异性转录因子Runx2的表达促进BMSCs向成骨细胞分化。     

Development of astrocytes in the vertebrate eye

Astrocytes represent the earliest glial population in the embryonic optic nerve, contributing critically to retinal angiogenesis and formation of brain‐retinal‐barrier. Despite of many developmental and clinical implications of astrocytes, answers to some of the most fundamental questions of this unique type of glial cells remain elusive. This review provides an overview of the current knowledge about the origination, proliferation, and differentiation of astrocytes, their journey from the optic nerve toward the neuroretina, and their involvement in physiological and pathological development of the visual system. Developmental Dynamics 243:1501–1510, 2014. © 2014 Wiley Periodicals, Inc.     

蝎毒多肽干预K562/BALB/c-nu小鼠Hedgehog通路传导的机制研究

目的探究蝎毒多肽干预K562/BALB/c-nu白血病荷瘤小鼠Hedgehog通路信号传导的机制,从基因、蛋白水平解析蝎毒多肽抑制慢性粒细胞白血病发展的分子机制和作用靶点。方法将K562/BALB/c-nu白血病荷瘤小鼠分为对照组、模型组、伊马替尼(50 mg/kg)组和蝎毒多肽高、中、低剂量(0.3、0.6、1.2 mg/kg)组,药物干预14 d后,实时荧光定量PCR(q RT-PCR)法检测小鼠瘤组织Hedgehog信号通路上游因子Shh、Ptch、Smo m RNA表达水平,Western blotting法检测Shh、Ptch、Smo蛋白表达水平,ELISA法检测小鼠瘤组织Hedgehog信号通路下游因子Gli1蛋白表达水平。结果与模型组比较,蝎毒多肽干预组小鼠瘤组织Shh m RNA和蛋白表达水平均升高;蝎毒多肽低、中剂量组小鼠瘤组织Ptch、Smo m RNA及蛋白表达水平均降低;伊马替尼组各上游因子与模型组比较差异不显著;蝎毒多肽低、中剂量组小鼠瘤组织Gli1蛋白表达水平降低,蝎毒多肽高剂量组、伊马替尼组Gli1蛋白表达水平无显著差异。结论蝎毒多肽能够抑制Hedgehog信号通路上游因子Ptch、Smo以及下游因子Gli1的表达,而伊马替尼对Hedgehog信号通路无明显抑制作用。