Sonic hedgehog

摘要： 目的探讨胰腺肿瘤细胞中Shh(Sonic Hedgehog)的分泌情况及对巨噬细胞极化的影响。方法应用GEPIA 2数据库分析Shh在胰腺癌组织与癌旁正常组织中的表达差异;体外培养正常导管上皮细胞株HPDE6-C7和胰腺癌细胞株PANC-1和CFPAC-1,通过酶联免疫吸附技术(ELISA)检测其上清液中Shh的含量,应用胰腺癌细胞上清液作用于巨噬细胞株RAW264.7,以及单纯性应用重组Shh蛋白作用于巨噬细胞,通过细胞免疫荧光染色检测M2型巨噬细胞标志物精氨酸酶-1(Arg-1)、干扰素调节因子4(IRF4)的表达水平。结果GEPIA 2数据库显示,与癌旁正常组织相比,Shh在胰腺癌组织中的表达明显升高(P<0.05);同时ELISA分析显示,与正常导管上皮细胞HPDE6-C7相比,胰腺癌细胞PANC-1和CFPAC-1中Shh的表达水平明显增高(P<0.05)。应用胰腺癌细胞的上清液作用于巨噬细胞RAW264.7后,其Arg1的表达明显增加(P<0.05)。同样,应用重组Shh作用后可增加巨噬细胞中Arg-1的表达(P<0.05);胰腺癌细胞上清液或Shh处理巨噬细胞后,与M2型极化相关的转录因子IRF4的表达明显增加(P<0.05)。结论胰腺癌细胞通过释放Shh提高巨噬细胞中IRF4的表达,进而诱导巨噬细胞向M2型极化。

摘要： Hedgehog(HH)信号通路在胚胎发育过程中发挥着重要作用.HH主要有三个配体,其中sonic hedgehog(SHH)主要与肾损伤后修复和肾脏发育相关.近期研究发现SHH信号通路的激活与肾纤维化发生发展有紧密联系.在各种慢性肾脏病(chronic kidney disease,CKD)中,SHH在肾小管上皮细胞中高表达后直接作用于肾间质成纤维细胞.其扮演生长因子的角色并调控各级纤维化相关基因,从而导致细胞外基质沉积.本文将阐述SHH信号通路原理以及在肾脏疾病中的调控.并讨论SHH促进肾纤维化的可能机制以及分析阻断信号通路后的影响.SHH信号通路可能成为CKD患者新的治疗靶点.

摘要： 目的:探讨Sonic hedgehog(Shh)信号通路相关的单核苷酸多态性(single-nueleotide polymorphism,SNP)与非综合征型唇腭裂(non-syndromic cleft lip and/or palate,NSCL/P)之间的关联,并对唇腭裂疾病的致病风险因素进行探索.方法:收集197例个体的外周血(NSCL/P患者100例,健康对照97例),基于国际人类基因组单体型图计划中中国北京汉族人口数据,使用Haploview软件进行单倍体型分析和标签SNP选择.针对Shh信号通路中的4个候选基因SHH、PTCHI、SMO和GLI2共选择了27个SNP.使用Sequenom质谱技术检测27个SNP在4个Shh信号通路中候选基因的基因型,并进行分析.结果:所选择的SNP基本涵盖了候选基因的潜在功能性SNP,其最小等位基因频率(minor allele frequency,MAF)＞ 0.05:GLI2 73.5％,PTCH1 91.0％,SMO 100.0％,SHH 75.0％.发现位于SMO基因的SNP(rs12674259)和位于PTCHI基因的SNP(rs2066836)的基因型频率在NSCL/P病例组和对照组之间的差异有统计学意义.同时在4个候选基因所在的3条染色体(第2、7、9号染色体)中均发现了连锁不平衡,但在连锁不平衡单倍体型分析中,病例组和对照组之间的差异无统计学意义.结论:提示Shh信号通路参与NSCL/P的发生,其信号通路中关键基因的某些特殊SNP位点与唇腭裂相关,为NSCL/P的病因研究提供了新的探索方向,可能为NSCL/P的早期筛查与风险预测提供帮助.

摘要： 目的:探讨Shh协同骨髓间充质干细胞在体外非接触共培养中对造血干细胞增殖的影响及其机制.方法:采用全骨髓贴壁培养法体外培养间充质干细胞,采用miniMACS磁珠分选仪分选人骨髓CD34+细胞,流式细胞术鉴定两种细胞纯度;将CD34+细胞与MSC分别种至Transwell上下室进行非接触共培养,并添加外源性shh蛋白进行干预.收集非接触共培养d7的样本检测HSC及MSC细胞数量、RNA总量、HSC的ki67的mRNA表达量,以了解HSC增殖情况;测量HSC细胞Tie-2 mRNA表达量和MSC的VEGF、Ang-1 mRNA表达量,以了解细胞因子的表达情况.结果:非接触共培养d7,共培养组2种细胞数量、RNA总量以及ki67、Tie-2、VEGF、Ang-1相对表达量增加并呈以下趋势:MSC+HSC组和shh+HSC纽均高于HSC组(P＜0.05),MSC+shh+HSC组高于MSC+HSC组(P＜0.05).结论:shh可协同MSC体外非接触共培养促进HSC增殖,其机制可能与血管生成因子相关.

摘要： 目的 初步探讨促分裂原活化的蛋白激酶/胞外信号调节激酶(MAPK/ERK)信号通路在Sonic Hedgehog (Shh)调控RA成纤维样滑膜细胞(RA-FLS)增殖中的作用.方法 收集病情活动(DAS28≥3.2)的RA患者经关节镜下滑膜清理术或关节置换术切除的滑膜组织,组织块培养法培养分离RA-FLS,分别予Shh激动剂purmorphamine、抑制剂cyclopamine及MAPK/ERK信号通路抑制剂U0126处理,采用蛋白质印迹法检测RA-FLS细胞MAPK/ERK信号通路关键蛋白phospho ERK1/2(p-ERK1/2)磷酸化水平,细胞增殖与毒性试剂盒(CCK8)检测细胞增殖活力,以及流式细胞术检测细胞增殖情况,采用单因素方差分析和Kruskal-Wallis H (K)方法对数据进行分析.结果 与对照组相比,浓度为1μmol/L的purmorphmine可短时间内引起p-ERK1/2蛋白表达增加,作用15 min时显著(P＜0.01),经U0126和cyclopamine处理后,p-ERK1/2蛋白表达明显下降(P＜0.01).RA-FLS经purmorphmine(1μmol/L)处理后,细胞增殖活力为(114±5)％,明显高于对照组(100±0)％(P＜0.01),purmorphmine(1μmol/L)组S期细胞占(8.39±0.60)％,较对照组(3.29±0.69)％明显增加(P＜0.01);经cyclopamine(10 μmol/L)处理后,细胞增殖活力为(89±1)％(P＜0.05),S期细胞占(1.53±0.22)％(P＜0.05);经purmorphamine(1μmol/L)和U0126(10 μmol/L)共同处理后,细胞增殖活力为(89±2)％(P＜0.05),S期细胞占(1.07±0.25)％(P＜0.05).结论 Shh可能通过调控MAPK/ERK信号通路,促进RA-FLS细胞增殖,促进滑膜增生,导致RA疾病的进展.%Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

摘要： 目的:探讨Sonic hedgehog(Shh)信号通路对髁突肥大软骨细胞凋亡活性的影响.方法:取正常髁突软骨3例,髁突肥大软骨6例,酶消化法分离培养其软骨细胞后,用Western blotting和Real-time PCR法分别检测两种软骨细胞中凋亡效应因子cleaved-caspase-3和BCL-2的表达;用免疫组化和Western blotting法比较正常髁突软骨和髁突肥大软骨中Shh和Smo的表达;用Shh、Cyclopamine、LY294002和U0126分别刺激两种软骨细胞,观察cleaved-caspase-3和BCL-2表达的变化.结果:与正常髁突软骨相比,髁突肥大软骨细胞中cleaved-caspase-3的表达水平降低,BCL-2 mRNA的表达水平升高(P<0.05);在髁突肥大软骨中Shh、Smo呈高表达;Shh刺激组软骨细胞的凋亡活性降低(P<0.05),Cyclopamine刺激组软骨细胞的凋亡活性增加(P<0.05),LY294002及U0126刺激组软骨细胞的BCL-2 mRNA表达水平均降低(P<0.05).结论:Shh可通过介导PI3K/AKT和MAPK/ERK通路而抑制髁突肥大软骨细胞的凋亡,促进髁突肥大的病理进程.%AIM:To investigate the effect of Shh signaling pathway on the apoptosis of the chondrocytes in the cartilage with condylar hyperplasia(CH).METHODS:Chondrocytes were isolated from 6 CH and 3 normal cartilage specimens.Western blotting and Real-timePCR were used to detect the expression levels of cleaved-caspase-3 and BCL-2.Then the expression levels of Shh and Smo were assessed by immunohistochemistry and Western blotting.Finally,chondrocytes were treated with Shh,cyclopamine,LY294002 or U0126,the expression levels of cleaved-caspase-3 and BCL-2 were examined.RESULTS:As compared with normal chondrocytes,CH chondrocytes showed lower apoptotic activity and expressed higher level of BCL-2 mRNA and lower level of cleaved-caspase-3 (P<0.05).Shh and Smo had higher expression in CH chondrocytes.Shh stimulation significantly inhibited the apoptosis of chondrocytes.On the contrary,cyclopamine stimulation significantly increased the apoptosis of chondrocytes.LY294002 and U0126 treatments reduced BCL-2 mRNA expression in both groups (P<0.05).CONCLUSION:Shh promotes human TMJ cartilage overgrowth in the developing process of CH by inhibiting chondrocyte apoptosis via PI3K/AKT and MAPK/ERK pathway.

摘要： 目的:构建Sonic Hedgehog(Shh)基因慢病毒载体,并进行鉴定.方法:构建携带Shh基因慢病毒表达质粒载体pLV.Ex2d.P/puro-EF1A-mShh,RT-PCR反应和基因测序证实质粒载体构建成功.对载体进行包装,测定病毒滴度.将携带Shh基因慢病毒颗粒以感染复数(MOI=10)转导HT1080细胞,使用RT-PCR法检测转导细胞中Shh基因的表达.结果:成功构建携带Shh基因的慢病毒表达载体(Lenti-Shh),病毒滴度为2.9×107TU/mL.RT-PCR检测结果证明Lenti-Shh转导细胞成功表达Shh基因.结论:成功构建小鼠Shh慢病毒载体系统,为基因增强型骨组织工程提供可靠的基因传递载体.%Objective:Constructing and characterizing a lentiviral vector carrying Sonic Hedgehog( Shh) gene. Meth-ods:Mouse Shh gene was amplified and subcloned into the lentiviral plasmid pLV. Ex2d. P/puro-EF1A-mShh. RT-PCR and gene sequencing were performed to validate the successful construction of the vector. The vector was packed and examined for titer. HT1080 cells were transfected with the multiplicities of infection(MOI=10). Shh expression in the transfected cells was assayed using RT-PCR. Results:Mouse Shh gene was successfully subcloned into a lentiviral vector. The viral titer was 2. 9 × 107 TU/mL. The RT-PCR result confirmed the expression of Shh gene in the transfected cells. Conclusion:A lentiviral vector containing Shh gene was successfully constructed,which can serve as an ideal tool for gene-enhanced bone tissue engineer-ing.

摘要： In order to reveal the hypothalamus Sonic hedgehog (Shh) positive cells in the brain,and track Shh + offspring cell fate,constructed the SBE2-Cre transgenic mice,and combining the Rosa26 mTmG and Rosa26 LacZ reporter genes in mice,SBE2 + cells for the study of lineage tracing,according to the results in mice embryonic period E10.5 SBE2 + cells began to appear in the preoptic area (preoptic area),and then along the hypothalamus area to the hypothalamus back area of the side,covering the ventral diencephalon most area;In embryonic period E12.5,SBE2 + cells mainly appeared in the capsule,the hypothalamus of the side,the ventral epithelial cells of the hypothalamus,papillae lateral nucleus,papillary nucleus,lateral hypothalamus,has strong expression in the ventral hypothalamus.In embryonic period E16.5 and 10th day after birth,SBE2 expressed in much of the hypothalamus.Compare the endogenous Shh expression,SBE2 specificity on behalf of the hypothalamus Shh+ cells,and is one of the main source for cell differentiation to form the hypothalamus nucleus,and SBE2-Cre mice can be used as the important tool of the functioning of genes in the region of the hypothalamus.%为了揭示下丘脑Sonic hedgehog (Shh)阳性细胞及其子代细胞的发育去向,利用特异性的增强子SBE2(Shh brain enhancer-2,SBE2),构建了SBE2-Cre转基因小鼠,并结合Rosa26-mTmG和Rosa26-LacZ报告基因小鼠,对SBE2+细胞进行了谱系追踪研究.结果显示,小鼠胚胎期E10.5,SBE2+细胞开始出现在视前区(preoptic area),然后沿下丘脑前侧区到下丘脑后侧区形成条带,覆盖了腹侧间脑大部分区域;胚胎期E12.5,SBE2+细胞主要出现在视囊,前侧下丘脑,腹侧下丘脑上皮细胞,乳头体外侧核,乳头状核,外侧下丘脑,在腹侧下丘脑有着较强的表达.胚胎期E16.5和出生后第10 d,SBE2在下丘脑大部分区域表达.对比内源性Shh表达,SBE2+细胞特异性的代表下丘脑Shh+细胞,是分化形成下丘脑神经核的主要细胞来源,而SBE2-Cre小鼠可作为研究基因在下丘脑区域功能的重要工具鼠.

摘要： Sonic hedgehog信号通路存在于多种动物体内,在调节胚胎发育、组织损伤后修复、组织再生等中发挥重要作用,其异常激活参与多种肿瘤与癌症的发生过程;除此之外该通路也与神经系统相关,促进神经再生、调控轴突导向,并在多种应激损伤中有重要作用.本文综述了Sonic hedgehog信号转导通路在多种应激损伤,如激素应激损伤、氧化应激损伤、缺血缺氧应激损伤等中的重要作用,其可能是通过调节与这些应激损伤相关的通路或者凋亡相关通路以及发挥其促进神经再生、损伤修复功能而起作用的,因此在治疗多种相关神经系统疾病方面具有研究前景.

摘要： Sonic hedgehog(Shh)是Hedgehog(Hh)家族中的一员,在胚胎发育和器官形成过程中发挥重要作用.法氏囊是鸟类所特有的中枢免疫器官,在机体的免疫防御方面发挥重要作用.利用切片原位杂交的方法探究Shh基因在鸡胚法氏囊发育过程中的表达模式,检测发现Shh基因主要在鸡胚法氏囊的囊下上皮细胞、血管周围上皮细胞以及网状细胞中表达.

摘要： Sonic hedgehog(Shh)是Hedgehog(Hh)家族中的一员,在胚胎发育和器官形成过程中发挥重要作用.法氏囊是鸟类所特有的中枢免疫器官,在机体的免疫防御方面发挥重要作用.利用切片原位杂交的方法探究Shh基因在鸡胚法氏囊发育过程中的表达模式,检测发现Shh基因主要在鸡胚法氏囊的囊下上皮细胞、血管周围上皮细胞以及网状细胞中表达.

摘要： Sonic hedgehog(Shh)是Hedgehog(Hh)家族中的一员,在胚胎发育和器官形成过程中发挥重要作用.法氏囊是鸟类所特有的中枢免疫器官,在机体的免疫防御方面发挥重要作用.利用切片原位杂交的方法探究Shh基因在鸡胚法氏囊发育过程中的表达模式,检测发现Shh基因主要在鸡胚法氏囊的囊下上皮细胞、血管周围上皮细胞以及网状细胞中表达.

摘要： Sonic hedgehog(Shh)是Hedgehog(Hh)家族中的一员,在胚胎发育和器官形成过程中发挥重要作用.法氏囊是鸟类所特有的中枢免疫器官,在机体的免疫防御方面发挥重要作用.利用切片原位杂交的方法探究Shh基因在鸡胚法氏囊发育过程中的表达模式,检测发现Shh基因主要在鸡胚法氏囊的囊下上皮细胞、血管周围上皮细胞以及网状细胞中表达.

摘要： 啮齿目动物的味乳头在其出生前便开始发育,随后大约在出生时味蕾会出现在味乳头中.因此,乳头的形成是哺乳动物味觉系统开始发育的第一步,有关对小鼠和大鼠的研究表明,在味乳头形态形成期间Shh的表达先于味蕾的形成,Shh(SonicHedgehog)在舌上皮细胞分化中可能是一种活性信号分子,它对乳头的发育可能起多重的作用。

摘要： 啮齿目动物的味乳头在其出生前便开始发育,随后大约在出生时味蕾会出现在味乳头中.因此,乳头的形成是哺乳动物味觉系统开始发育的第一步,有关对小鼠和大鼠的研究表明,在味乳头形态形成期间Shh的表达先于味蕾的形成,Shh(SonicHedgehog)在舌上皮细胞分化中可能是一种活性信号分子,它对乳头的发育可能起多重的作用。

摘要： 啮齿目动物的味乳头在其出生前便开始发育,随后大约在出生时味蕾会出现在味乳头中.因此,乳头的形成是哺乳动物味觉系统开始发育的第一步,有关对小鼠和大鼠的研究表明,在味乳头形态形成期间Shh的表达先于味蕾的形成,Shh(SonicHedgehog)在舌上皮细胞分化中可能是一种活性信号分子,它对乳头的发育可能起多重的作用。

摘要： 啮齿目动物的味乳头在其出生前便开始发育,随后大约在出生时味蕾会出现在味乳头中.因此,乳头的形成是哺乳动物味觉系统开始发育的第一步,有关对小鼠和大鼠的研究表明,在味乳头形态形成期间Shh的表达先于味蕾的形成,Shh(SonicHedgehog)在舌上皮细胞分化中可能是一种活性信号分子,它对乳头的发育可能起多重的作用。

摘要： 啮齿目动物的味乳头在其出生前便开始发育,随后大约在出生时味蕾会出现在味乳头中.因此,乳头的形成是哺乳动物味觉系统开始发育的第一步,有关对小鼠和大鼠的研究表明,在味乳头形态形成期间Shh的表达先于味蕾的形成,Shh(SonicHedgehog)在舌上皮细胞分化中可能是一种活性信号分子,它对乳头的发育可能起多重的作用。