Differential expressions of BMPR1α, ACTN4α and FABP7 in Hirschsprung disease

Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. Recent studies have shown that the bone morphogenetic protein receptor-type IA (BMPR1α), actinin-alpha 4 (ACTN4α) and fatty acid binding protein 7 (FABP7) play important roles in the differentiation and development of neurons. The aganglionic (stenotic) and the ganglionic (normal) colon segment tissues of 60 HSCR patients were collected to investigate the expression pattern of BMPR1α, ACTININ-4α and FABP7 using RT-PCR, quantitative real-time RT-PCR (qRT-PCR) and immunohistochemical staining. The mRNA and protein expressions of BMPR1α and ACTN4α were higher in the stenotic colon segment tissue than those in the normal colon segment tissue. However, the mRNA and protein expressions of FABP7 were lower in the stenotic colon segment tissue than those in the normal colon segment tissue. The study in HSCR patients, findings in mRNA and protein alterations to expecting provide more information to in order to find some clue for the pathomechanism of HSCR disease.     

Expression analysis of BMP2, BMP5, BMP10 in human colon tissues from Hirschsprung disease patients

Objective: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF β) superfamily. BMP2, BMP5 and BMP10 exert their biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family. Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. However, putative Notch function in enteric nervous system (ENS) development and the etiology of HSCR is unknown. Methods: Aganglionic and ganglionic colon segment tissues of 50 HSCR patients were investigated for the expression pattern of BMP2, BMP5 and BMP10 using real-time RT-PCR, Western blot analysis and immunohistochemical staining. Results: The mRNA levels of BMP2, BMP5 and BMP10 in the stenotic colon segment from HSCR patients were significantly higher than those in the normal ones. Similar increased expressions of them in the stenotic colon segments were detected by Western blotting coupled with densitometry analysis. Lastly, immunohistologicl stain showed significant BMP2, 5 and 10 increases in mucous and muscular layers from stenotic colon segments compared to normal segments. Conclusions: BMP2, BMP5 and BMP10 are elevated in the stenotic colon segment of HSCR, and BMPs signaling plays a pivotal role in the development of HSCR.     

Expression of dishevelled gene in Hirschsprung’s disease

Hirschsprung’s disease (HSCR) is a congenital disorder of the enteric nervous system and is characterized by an absence of enteric ganglion cells in terminal regions of the gut during development. Dishevelled (DVL) protein is a cytoplasmic protein which plays pivotal roles in the embryonic development. In this study, we explore the cause of HSCR by studying the expression of DVL-1 and DVL-3 genes and their proteins in the aganglionic segment and the ganglionic segment of colon in HSCR patients. Materials and Methods: Specimen of aganglionic segment and ganglionic segment of colon in 50 cases of HSCR patients. Expression levels of mRNA and proteins of DVL-1 and DVL-3 were confirmed by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry staining between the aganglionic segment and the ganglionic segment of colon in HSCR patients. Results: The mRNA expression of DVL-1 and DVL-3 were 2.06 fold and 3.12 fold in the aganglionic segment colon tissues compared to the ganglionic segment, respectively. Similarly, the proteins expression of DVL-1 and DVL-3 were higher (39.71 ± 4.53 vs and 53.90 ± 6.79 vs) in the aganglionic segment colon tissues than in the ganglionic segment (15.01 ± 2.66 and 20.13 ± 3.63) by western blot. Besides, immunohistochemical staining showed that DVL-1 and DVL-3 have a significant increase in mucous and submucous layers from aganglionic colon segments compared with ganglionic segments. Conclusion: The study showed an association of DVL-1 and DVL-3 with HSCR, it may play an important role in the pathogenesis of HSCR.     

WNT3A gene expression is associated with isolated Hirschsprung disease polymorphism and disease status

WNT3A has been regarded as an activator of the canonical Wnt signaling pathway. It has been found Wnt signaling pathway is closely related with embrionic development and Hirschsprung disease (HSCR). A common haplotype consisting of minor SNPs alleles located in the WNT3A gene has been described as a risk factor for various genetic disorders. However, whether WNT3A contributes to the onset of HSCR has not been identified. The present study aims to detect the interactions of genetic variations in the WNT3A gene and examine the biological expression levels with Hirschsprung disease (HSCR) in the Chinese people. We analyzed WNT3A gene (rs61743220, rs192966556 and rs145882986) variants in the whole blood samples from HSCR patients and normal children (control groups). WNT3A expression was also examined by quantitative real-time PCR (qRT-PCR), western blotting and immunostaining. Consequently, when rs192966556 and rs145882986 alleles of the WNT3A gene lack the SNPs, they are especially associated with a greater risk of HSCR (OR [95% confidence interval] = 1.791, p = 0.001; OR [95% confidence interval] = 1.556, p = 0.003, respectively). The mRNA and protein expressions of WNT3A were higher in the aganglionic colon segment tissues than in the normal ganglionic segments tissues. Immunostaining indicates that the staining of WNT3A was much stronger (brown) in the aganglionic colon segment tissues than that in the normal ganglionic colon segment tissues (colorless or light yellow) in the mucous layer and muscular layer. Although preliminary, these results suggest that WNT3A may play an important role in the pathogenesis of HSCR.     

Detection and identification of peroxiredoxin 3 as a biomarker in hepatocellular carcinoma by a proteomic approach

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Thus, alternative therapeutic strategies need to be established. In order to search for a useful biomarker to improve its efficacy, we conducted a two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis to profile the differentially expressed proteins between HCC tumor tissues with histological evidence and the adjacent non-tumor tissues. Twenty-two out of 43 dysregulated proteins were identified, including 15 upregulated proteins, and 7?downregulated proteins (over 2-fold, P<0.01). The expression of peroxiredoxin?3 (PRDX3) at the mRNA and protein levels was confirmed by RT-PCR and western blotting in HCC cell lines, and HCC samples, and further analysed by immunohistochemistry in HCC samples of different clinical pathological stages. The results indicated that overexpression of PRDX3 was associated with 94.9% HCC, and correlated with poor differentiation (P<0.05), which suggest that PRDX3 has substantial clinical impact on the progression of hepatocarcinoma, and may be a potential therapeutic target for HCC.     

Differential expression of FOXA1, DUSP6, and HA117 in colon segments of Hirschsprung’s disease

Objective: To describe the expression profiles of FOXA1, DUSP6, and HA117 in different portions of the colon of patients diagnosed with Hirschsprung’s disease (HSCR). Methods: Colon specimens were collected from34 HSCR patients and grouped into 3 segments: proximal anastomosis, dilated segment and stenotic segment. Levels of FOXA1, DUSP6, and HA117 RNA were evaluated by real-time PCR. Levels of FOXA1 and DUSP6 protein were analyzed by immunohistochemistry and Western blotting. Results: The levels of FOXA1 and DUSP6 RNA were significantly lower in the stenotic segment compared to proximal anastomosis (P < 0.05). The level of HA117 RNA was significantly higher in the stenotic segment compared to proximal anastomosis (P < 0.05). In proximal anastomosis, FOXA1 and DUSP6 were both expressed at the protein level in ganglion cells and nerve fibers between the circular and longitudinal muscles. In the stenotic segments, positive staining for FOXA1 and DUSP6 was diminished. The levels of FOXA1 and DUSP6 protein were significantly lower in the stenotic segment compared to proximal anastomosis (P < 0.05). Conclusion: Suppression of the FOXA1/DUSP6 signaling pathway may contribute to the development of HSCR. LncRNA HA117 may have an anti-differentiation function, and play a pivotal role in the progression of HSCR.     

Up-regulated FHL1 Expression Maybe Involved in the Prognosis of Hirschsprung's Disease

Background: In a subset of patients with Hirschsprung''s disease (HSCR), gastrointestinal motor dysfunction persisted long after surgical correction. Gastrointestinal motility is achieved through the coordinated activity of the enteric nervous system, interstitial cells of Cajal, and smooth muscle (SMC) cells. Inhibition of four-and-a-half LIM protein-1 (Fhl1) expression by siRNA significantly decreases pulmonary artery SMCs migration and proliferation. Furthermore when up-expressing FHL1 in atrial myocytes, K (+) current density markedly increases, therefore changing myocytes'' response to an electrical stimulus. However whether FHL1 in colon SMCs (the final effector organ) influences intestinal motility in HSCR patients has not been clarified. Methods: FHL1 mRNA and protein expressions were analyzed in 32 HSCR colons and 4 normal colons. Results: Smooth muscle layers were thicken and disorganized in HSCR. FHL1 was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the ganglionic colon. FHL1 mRNA relative expression level in aganglionic colons was 1.06±0.49 (ganglionic colon relative expression level was 1) (P=0.44). FHL1 protein gray level relative to GAPDH in normal colons was 0.83±0.09. FHL1 expression level in ganglionic colon (1.66±0.30) or aganglionic colon (1.81±0.35) was significantly higher than that in normal colons (P=0.045 and P=0.041, respectively). Meanwhile, we found FHL1 expression in aganglionic colon was slightly stronger than that in ganglionic colon (P=0.036). Conclusion: These data suggested that up-regulated FHL1 in smooth muscle in HSCR might be associated with intestinal wall remodeling in HSCR and might be one of the risk factors for gastrointestinal motor dysfunction.     

结肠肿瘤中wisp-1的表达及意义

目的探讨wisp-1在原发性结肠癌组织中的表达及临床意义。方法应用免疫组化EnVision两步法检测wisp-1蛋白在138例结肠癌组织(48例含癌旁组织)、37例结肠腺瘤组织及66例结肠正常黏膜组织中的表达;应用实时荧光定量PCR检测wisp-1基因在37例新鲜原发性结肠肿瘤标本及对照组37例正常结肠中的表达;分析wisp-1蛋白、基因表达与患者临床病理特征的关系。结果 wisp-1蛋白在正常结肠、结肠腺瘤、癌旁组织及结肠癌中阳性率分别为53.0%、67.6%、75.0%、76.1%,其在结肠癌及癌旁组织中表达高于正常结肠(P<0.05);wisp-1蛋白表达与淋巴结转移及浸润深度有关(P<0.05);wisp-1基因在结肠肿瘤中平均表达是正常结肠中的1.349倍(P>0.05)。结论 wisp-1蛋白异常可能在结肠癌的发生过程中起重要作用,而其基因在结肠癌中的作用尚需进一步研究。     

先天性肠无神经节细胞症患儿中血清维生素A及肠组织维甲酸受体α和HA117的表达特点

目的检测先天性肠无神经节细胞症(HD)患儿中血清维生素A及肠组织维甲酸受体α(RARα)、抗分化长链非编码RNA(LncRNA)HA117的表达特点,探讨其与HD发病机制的可能联系。方法收取17例临床诊断为HD的患儿术前外周血及手术结肠组织标本,结肠组织由近端至远端分为3组:吻合口组、扩张段组和痉挛段组。检测血清维生素A浓度,采用Real-time PCR、免疫组织化学染色、Western blotting方法检测RARα及HA117在各组结肠组织的表达水平。结果 17例HD患儿均有不同程度维生素A缺乏,血清维生素A平均浓度为(0.73±0.05)μmol/L。在吻合口段,RARα在肌间神经丛可见棕黄色的阳性染色,而在痉挛段几乎未见阳性神经丛染色;在吻合口组、扩张段组和痉挛段组,RARαmRNA相对表达量依次为3.62±1.12、1.81±0.69、0.65±0.32,与吻合口相比,痉挛段表达量降低(P0.05,n=17);HA117 mRNA相对表达量依次为0.47±0.15、1.25±0.40、2.57±0.70,与吻合口相比,痉挛段表达量增高(P0.001,n=17);RARα蛋白相对表达量依次为1.16±0.05、0.99±0.05、0.88±0.04,与吻合口相比,扩张段及痉挛段表达量降低(分别为P0.05、P0.001,n=17)。结论维生素A、RARα及HA117可能共同参与肠神经系统(ENS)的发育过程;其中,维生素A和RARα可能促进了ENS的正常分化发育,而高表达的HA117可能对抗了前者的促分化作用,阻碍了ENS正常分化发育过程,进而导致HD的发生。     

低氧预适应小鼠脑皮层内与nPKCe相互作用蛋白的鉴定

目的鉴定脑低氧预适(HPC)小鼠脑皮层组织内与新奇型蛋白激酶Cε(nPKCε)相互作用的蛋白。方法利用免疫沉淀(IP)和双向电泳(2-DE)技术分离小鼠脑皮层中与nPKCε相互作用的蛋白;以ImageMaster2D Platinum软件对双向电泳图谱进行差异表达分析;目标蛋白用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)及蛋白印迹(Western blot)进行鉴定分析。结果与对照组比较,HPC小鼠脑皮层内与nPKCε相互作用的胞质成分中有34个蛋白点上调,20个蛋白点下调,膜相关成分中有27个蛋白点上调,28个蛋白点下调;HPC后脑皮层胞质及膜相关成分中与nPKCε相互作用的热休克蛋白(HSP)70和14-3-3γ的蛋白表达量均升高,而HSP60仅在膜相关成分中升高(P<0.05,n=3)。结论nPKCε可能通过调节HSP60、HSP70和14-3-3γ等多种与其相互作用的蛋白参与脑HPC的形成。     

Concurrent alterations of RAGE,RECK, and MMP9 protein expression are relevant to Epstein-Barr virus infection,metastasis, and survival in nasopharyngeal carcinoma

This study aimed to concurrently investigate the expressions of receptor for advanced glycation end products (RAGE), reversion inducing cysteine-rich protein with Kazal motifs (RECK) and matrix metalloproteinase 9 (MMP9) in nasopharyngeal carcinoma (NPC) and their correlations with clinicopathological properties. Using immunohistochemistry, we found that RECK expression was downregulated in NPC tissues compared with chronic nasopharyngitis (CNT) tissues, while RAGE and MMP9 expressions were upregulated. We further found that RECK expression level was inversely correlated with MMP9 expression level in NPC, whereas RAGE expression level was positively correlated with MMP9 expression level. Moreover, aberrant expressions of these proteins had a positive correlation with the titers of EBVCA-IgA, lymphatic metastasis, recurrence and survival. Together, these findings suggest that dysregulations of RECK and RAGE expressions may be collectively involved in tumor progression of NPC by regulating MMP9 expression and that they may be a good prognostic predictors for NPC.     

Sonic hedgehog mRNA及其蛋白在大肠癌的表达及其临床意义

目的 探讨大肠癌中Hedgehog信号通路相关基因Sonic hedgehog(SHH)的表达及其临床意义.方法 应用RT-PCR及免疫组织化学法(Envison二步法)检测本院2008年12月至2009年4月收集的43例大肠癌组织和距离癌10 cm以上的癌旁组织的SHHmRNA及蛋白的表达情况,并与20例非肠癌患者的正常大肠组织对照,分析其与大肠癌患者临床和病理各变量的相关性.结果 凝胶成像分析显示阳性表达的样本SHH mRNA片段大小为280 bp,与理论值相符,未表达的样本则未见相应条带.大肠癌组织SHHmRNA表达阳性率为27.9%(12/43),与癌旁组织的18.6%(8/43)差异无统计学意义(P＞0.05),但均显著高于正常大肠黏膜的0%(0/20)(均P＜0.05).SHH mRNA在大肠癌组织的表达强度显著高于癌旁组织(P＜0.05),在正常大肠黏膜则未见其表达.免疫组织化学显示SHH蛋白在大肠癌组织和癌旁组织均有阳性表达,细胞膜及胞质内出现棕黄褐色颗粒,背景不着色;正常大肠黏膜阴性表达.SHH蛋白在大肠癌组织的表达阳性率显著高于正常大肠黏膜[25.6%(11/43)比0%(0/20),P＜0.05],在大肠癌组织与癌旁组织、癌旁组织与正常大肠黏膜的表达阳性率差异则无统计学意义(均P＞0.05).SHH蛋白的表达强度大肠癌组织＞癌旁组织＞正常大肠黏膜(均P＜0.05).大肠癌组织中的SHH mRNA和蛋白表达强度与患者的年龄、性别、临床分期、肿瘤部位、肿瘤浸润深度、病理分型等变量均无明显关系(均P＞0.05).结论 SHH mRNA和蛋白在大肠癌组织中表达显著增强,但与临床和病理各变量无关联.     

Transthyretin, identified by proteomics, is overabundant in pancreatic juice from pancreatic carcinoma and originates from pancreatic islets

Analyses of pancreatic juice by proteomics have identified many proteins that are overabundant in pancreatic cancer (PC) juice. The mechanism by which secretion of these proteins occur remains unclear. Pancreatic juice was collected from patients with three pancreatic diseases: PC, chronic pancreatitis (CP), and simple choledocholithiasis (CDS), and analyzed by 2-DE, MALDI-TOF/MS, and Western blot. Five PC cell lines, 30 PC tissues and their corresponding adjacent pancreatic tissues were used to validate the expression of genes which code for overabundant proteins in PC juice. The mRNA and protein levels were measured by RT-PCR and immunohistochemistry, respectively. Using proteomics, it was demonstrated that the protein transthyretin (TTR) was upregulated more than 2-fold in PC juice compared with CP and CDS, while apolipoprotein A-I, lithostathine, and regenerating islet-derived 1 beta precursor were downregulated more than 2-fold. Western blots confirmed that TTR was overabundant in the PC juice. However, TTR mRNA was not detected in any of the five PC cell lines, and was only detected in islet cells. By microscopy, it was shown that islet architecture was almost completely destroyed, and the islet's maximum diameter appeared larger in PC tissues than in normal. Some overabundant proteins in PC juice, such as TTR expressed only in islets, leak into the pancreatic ductal system due to hyperplasia and architectural damage in PC tissues. The destruction of organ and tissue architecture by tumor growth may result in novel tumor markers even if the markers are not secreted directly by tumor cells.     

基因沉默PRDX1 表达对人结直肠癌SW480 细胞侵袭迁移的影响

目的:探讨RNA 干扰过氧化还原酶1(Peroxiredoxin 1,PRDX1)表达对人结直肠癌SW480 细胞侵袭转移能力的影响。方法:筛选RNA 干扰PRDX1 的慢病毒质粒,与阴性对照慢病毒质粒分组转染结直肠癌SW480 细胞,转染后的SW480 细胞可分为PRDX1 基因沉默组(si-PRDX1)和阴性对照组(Vector)。实时荧光定量PCR(qRT-PCR)和免疫印迹法(Western blot)分别检测两组细胞中PRDX1 mRNA 和蛋白表达;采用Transwell 侵袭和迁移实验检测基因沉默PRDX1 表达对结直肠癌细胞侵袭及迁移能力的影响;通过Western blot 检测两组细胞中基质金属蛋白酶(MMP)家族部分蛋白表达水平。结果:基因沉默PRDX1 表达可有效抑制结直肠癌SW480 细胞中PRDX1 mRNA 和蛋白水平的表达,与阴性对照组相比(Vector),差异均具有统计学意义(P<0.01),说明基因沉默PRDX1 的SW480 细胞系构建成功;Transwell 侵袭和迁移实验显示si-PRDX1组细胞的侵袭及迁移能力较对照组均明显降低(P<0.01);Western blot 结果显示,与Vector 组相比,si-PRDX1 组细胞中组织基质金属蛋白酶抑制剂2(TIMP-2)的表达明显增加,而MMP-2 及MMP-9 的表达显著下降,且差异均具有统计学意义(P<0.05)。结论:基因沉默人结直肠癌SW480 细胞的PRDX1 表达可有效抑制细胞的侵袭、迁移及转移能力,其机制可能会通过调控TIMP-2、MMP-2 及MMP-9 的表达介导。     

Modulation of LILRB2 protein and mRNA expressions in septic shock patients and after ex vivo lipopolysaccharide stimulation

Septic patients develop immune dysfunctions, the intensities and durations of which are associated with deleterious outcomes. LILRB2 (leukocyte immunoglobulin-like receptors subfamily B, member 2), an inhibitory member of the LILR family of receptors, is known for its immunoregulatory properties.In a microarray study, we identified LILRB2 as an upregulated gene in septic shock patients. On monocytes primed with LPS ex vivo, LILRB2 mRNA and protein expressions were dose-dependently downregulated and subsequently highly upregulated versus non-stimulated cells. This is concordant with clinical data, since both LILRB2 mRNA and protein expressions were significantly increased in septic shock patients at day 3. In a cohort of more than 700 patients, only after septic shock were LILRB2 mRNA levels increased compared with non-infected or less severely infected patients. This was preceded by a phase of downregulated mRNA expression during the first hours after septic shock. Interestingly, the intensity of this decrease was associated with increased risk of death after septic shock.LILRB2 protein and mRNA expressions are deregulated on monocytes after septic shock and this can be reproduced ex vivo after LPS challenge. Considering LILRB2 inhibitory properties, we can hypothesize that LILRB2 may participate in the altered immune response after septic shock.     

压力负荷性左室肥厚大鼠心肌钠钙交换体和肌浆网钙泵的表达

目的 观察压力负荷性左室肥厚大鼠心功能异常及心肌钠钙交换体(NCX)和肌浆网钙泵(SERCA2a)的表达变化.方法 缩窄大鼠腹主动脉制备压力负荷性心肌肥厚模型,测定在体血流动力学及左室重量指数(LVWI),用RT-PCR和Western blot法检测左室组织NCX及SERCA2a的表达.结果 与假手术组相比,模型大鼠左室收缩压(LVSP)及左室舒张末压(LVEDP)均显著升高(P<0.01,P<0.001);左室重量指数显著增加(P<0.001)及左室NCX mRNA表达上调(P相似文献   

Long segment and short segment familial Hirschsprung''s disease: variable clinical expression at the RET locus.

Hirschsprung's disease (aganglionic megacolon, HSCR) is a frequent condition of unknown origin (1/5000 live births) resulting in intestinal obstruction in neonates and severe constipation in infants and adults. In the majority of cases (80%), the aganglionic tract involves the rectum and the sigmoid colon only (short segment HSCR), while in 20% of cases it extends toward the proximal end of the colon (long segment HSCR). In a previous study, we mapped a gene for long segment familial HSCR to the proximal long arm of chromosome 10 (10q11.2). Further linkage analyses in familial HSCR have suggested tight linkage of the disease gene to the RET protoncogene mapped to chromosome 10q11.2. Recently, nonsense and missense mutations of RET have been identified in HSCR patients. However, the question of whether mutations of the RET gene account for both long segment and short segment familial HSCR remained unanswered. We have performed genetic linkage analyses in 11 long segment HSCR families and eight short segment HSCR families using microsatellite DNA markers of chromosome 10q. In both anatomical forms, tight pairwise linkage with no recombinant events was observed between the RET proto-oncogene locus and the disease locus (Zmax = 2.16 and Zmax = 5.38 for short segment and long segment HSCR respectively at 0 = 0%) Multipoint linkage analyses performed in the two groups showed that the maximum likelihood estimate was at the RET locus. Moreover, we show that point mutations of the RET proto-oncogene occur either in long segment or in short segment HSCR families and we provide evidence for incomplete penetrance of the disease causing mutation. These data suggest that the two anatomical forms of familial HSCR, which have been separated on the basis of clinical and genetic criteria, may be regarded as the variable clinical expression of mutations at the RET locus.