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。     

Hedgehog信号通路在脑胶质瘤的表达及其预后意义

目的 观测Hedgehog信号通路在脑胶质瘤的表达,探讨其表达在脑胶质瘤的预后意义.方法 选取118例原发性脑胶质瘤患者的手术切除标本,运用免疫组织化学方法检测Sonic hedgehog(Shh)、受体Patched(Ptch)及下游转录因子Gli1的表达,采用Kaplan-Meier生存分析和Cox比例风险回归模型评价脑胶质瘤患者的预后.结果 免疫染色结果显示Shh、Ptch和Gli1的阳性表达率随胶质瘤病理等级升高呈增强趋势(P＜0.01);随KPS评分的下降而成增强趋势(P＜0.01).生存分析表明,阳性表达Shh、Ptch和Gli1的胶质瘤患者总体存活率低于三者不表达的患者(P＜0.01).多因素Cox分析显示KPS(P＜0.05)、WHO grade(P＜0.01)、Shh(P＜0.05)、Ptch (P＜0.05)和Gli1(P＜0.05)是影响脑胶质瘤预后的独立因素.结论 脑胶质瘤的Shh-Ptch1-Gli1 信号通路处于激活状态,与脑胶质瘤的临床病理特征及预后参数密切相关,提示Hedgehog信号通路的活化在脑胶质瘤的恶性潜能和患者的生存时间起重要的预示作用.     

Ptch和Smo在舌鳞状细胞癌Tca8113细胞中的表达及其意义

目的:研究Hedgehog(Hh)信号通路相关因子Ptch和Smo在舌鳞状细胞癌Tca8113细胞株中的表达及其意义。方法:Hh信号通路抑制剂Cyclopamine处理Tca8113细胞后,采用MTT实验检测细胞增殖抑制率,再利用RT-PCR法和Western-Blot方法检测 Ptch和Smo的mRNA和蛋白表达情况。结果:Cyclopamine对Tca8113细胞增殖具有抑制作用。加药后的不同时间段中,显示Ptch、Smo的mRNA和蛋白在Cyclopamine药物组中的表达低于对照组。Smo的mRNA和蛋白分别在加药后24 h、72 h中的表达随着药物浓度增高而降低。结论:Cyclopamine可以抑制Tca8113细胞的生长,降低Smo mRNA和蛋白的表达,Hh信号通路可能在舌癌的发生、发展中起重要作用。[关键词] Hh信号通路 Tca8113细胞 Cyclopamine Ptch Smo     

MicroRNAs regulate distal region of mandibular development through Hh signaling

Mandibular anomalies are often seen in various congenital diseases, indicating that mandibular development is under strict molecular control. Therefore, it is crucial to understand the molecular mechanisms involved in mandibular development. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating the level of gene expression. We found that the mesenchymal conditional deletion of miRNAs arising from a lack of Dicer (an essential molecule for miRNA processing, Dicer^fl/fl;Wnt1Cre), led to an abnormal groove formation at the distal end of developing mandibles. At E10.5, when the region forms, inhibitors of Hh signaling, Ptch1 and Hhip1 showed increased expression at the region in Dicer mutant mandibles, while Gli1 (a major mediator of Hh signaling) was significantly downregulated in mutant mandibles. These suggest that Hh signaling was downregulated at the distal end of Dicer mutant mandibles by increased inhibitors. To understand whether the abnormal groove formation inDicer mutant mandibles was caused by the downregulation of Hh signaling, mice with a mesenchymal deletion of Hh signaling activity arising from a lack of Smo (an essential molecule for Hh signaling activation, Smo^fl/fl;Wnt1Cre) were examined. Smo^fl/fl;Wnt1Cre mice showed a similar phenotype in the distal region of their mandibles to those in Dicer^fl/fl;Wnt1Cre mice. We also found that approximately 400 miRNAs were expressed in wild-type mandibular mesenchymes at E10.5, and six microRNAs were identified as miRNAs with binding potential against both Ptch1 and Hhip1. Their expressions at the distal end of the mandible were confirmed by in situ hybridization. This indicates that microRNAs regulate the distal part of mandibular formation at an early stage of development by involving Hh signaling activity through controlling its inhibitor expression level.     

Sonic hedgehog signaling during adrenal development

It has been speculated for a number of years that Sonic hedgehog (Shh) signaling plays an important role in adrenal development. Over the past two years several reports have described the expression and function of Shh pathway genes in the adrenal cortex, using primarily mouse models. The key findings are that Shh signals produced by a population of partially differentiated cortical cells located in the outer cortex/zona glomerulosa are received by non-cortical mesenchymal cells located predominantly in the overlying capsule. This signal is required for growth of both the capsule and the cortex, but not for cortical zonation or steroidogenic cell differentiation. Using molecular genetic tools to define the adrenocortical cell lineages that are descended from both Shh signaling and receiving cells, both capsule and cortical cells were found to have properties of adrenocortical stem and/or progenitor cells. Here we place these observations within the context of prior studies on adrenal development, postnatal adrenal maintenance and adrenocortical stem/progenitor cell lineages.     

鼻咽癌中Ptch1和Ptch2基因的表达及关系

目的 探讨Sonic Hedgehog信号通路Ptch1和Ptch2(Ptch1/2)成员在鼻咽癌组织中的表达及其关系.方法 应用免疫组化EnVision两步法检测60例鼻咽癌标本及15例正常鼻咽黏膜中Ptch1/2蛋白的表达,比较Ptch1与Ptch2表达的差异,分析Ptch1和(或)Ptch2与鼻咽癌患者临床病理特征之间的关系.结果 鼻咽癌组织中Ptch1/2蛋白表达的综合评分分别为4.70±3.59、3.58±3.47,明显高于正常鼻咽黏膜上皮组织的2.93±1.98、2.07±1.10(均P＜0.05);肿瘤或正常鼻咽黏膜上皮组织中Ptch1的表达水平均稍高于Ptch2;不同Ptch1及(或)Ptch2蛋白表达水平的鼻咽癌临床病理特征的比较均差异无统计学意义(P＞0.05);Ptch1与Ptch2蛋白表达在正常组织中呈显著相关(r =0.592,P＜0.05),而在肿瘤组织中则无相关性(r=0.114,P＞0.05).结论 鼻咽癌组织中Ptch1/2表达上调可能共同参与鼻咽癌的发生及发展,其中尤以Ptch1占主导地位.     

Shh和Ptch在肝细胞癌中的表达研究

目的探讨肝细胞癌（HCC）中Shh和Ptch的表达及其意义。方法应用组织芯片技术、免疫组化和原位杂交方法检测100例HCC、25例癌旁组织和5例正常肝组织中Shh和Ptch的表达情况。结果免疫组化检测Hcc中Shh和Ptch的阳性表达率分别为19．28％（16／83）、24．68％（19／77）;原位杂交检测结果分别为44．29％（31／70）、15．85％（13／82）;两种基因在HCC中的表达相关,均与非肿瘤组织的表达有显著性差异,并分别与肿瘤的大小及分化程度相关。结论Shh和Ptch的表达参与了HCC的发生,它们可能是HCC治疗的理想靶标。