激活素受体相互作用蛋白1在小鼠组织中的表达与分布

激活素具有调节激素分泌以及神经保护等多种作用,最近在小鼠脑内发现的激活素受体相互作用蛋白1(ARIP1)具有介导激活素信号传导作用,但有关ARIP1的分布情况仍然不清楚。本研究采用RT-PCR及免疫组织化学染色分析ARIP1在脑及脑外的表达与分布情况。RT-PCR检测发现ARIP1 mRNA不仅在大脑、小脑表达,在垂体、肾上腺以及睾丸也有明显表达。免疫组化染色显示大脑、小脑、垂体、肾上腺和睾丸均有不同程度的ARIP1免疫染色反应,小脑中浦肯野细胞着色明显,大脑主要是海马和下丘脑,在神经垂体、腺垂体的嗜碱细胞以及肾上腺网状带、球状带、束状带中均有表达,睾丸间质细胞也可见ARIP1成熟蛋白表达。结果提示,ARIP1不仅参与脑神经细胞的信号传导调节,也可能参与神经内分泌腺的功能调节。     

Expression and distribution of trihydrophobin 1 in postnatal developing mouse testis

The human trihydrophobin 1 (TH1) is a highly conserved and widely expressed protein. It is clear that TH1 serves as a new specific negative regulator of A-Raf kinase. In this study, we found that TH1 associated with A-Raf in mouse testis by using coimmunoprecipitation analysis. Then we characterized the gene expression of TH1 in mouse testis and analyzed the changes of TH1 protein during postnatal development. The protein expression of TH1 in mouse testis was further analyzed by immunohistochemistry staining. Strong signals were detected in the seminiferous tubules and the distribution patterns varied with the different ages of postnatal mouse testis. TH1 was distributed in spermatocytes and Sertoli cells at 2 weeks postnatal, and was abundant in spermatogonia at 8 weeks postnatal. Leydig cells were positive to TH1 throughout testicular development. A high expression of TH1 in both Leydig cells and mouse Leydig tumor cells (mLTC-1cells) was found to be concentrated in the cytoplasm. The colocalization of TH1 and A-Raf in mLTC-1 cells or in adult testis was also observable.     

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.     

PDCD4和DNMT1在卵巢上皮性癌中的表达及其临床意义

目的:检测PDCD4和DNMT1在正常卵巢组织、卵巢良性肿瘤组织、卵巢上皮性癌组织中的表达,并探讨其临床意义。方法:采用免疫组化法检测20例正常卵巢、25例卵巢良性肿瘤、40例卵巢上皮性癌组织中PDCD4和DNMT1的表达情况,分析其与卵巢上皮性癌临床病理参数之间的关系。结果:正常及良性卵巢组织中PDCD4的阳性表达率明显高于卵巢癌组(P0.05),卵巢癌的FIGO分期越高,PDCD4的表达越低,卵巢癌的病理分化程度越低,PDCD4的表达也越低,PDCD4的表达与卵巢癌的组织类型、腹水、年龄、是否绝经无关。正常及良性卵巢组织中DNMT1的阳性表达率明显低于卵巢癌组(P0.05),卵巢癌的病理分化程度越低,DNMT1的表达越高,但其与卵巢癌的FIGO分期、组织类型、腹水、年龄、是否绝经均无关。卵巢癌中DNMT1的与PDCD4的表达呈显著负相关(P0.05)。结论:PDCD4和DNMT1在卵巢癌中的表达呈负相关,PDCD4的表达下调和DNMT1的表达上调可能在卵巢癌的发生及发展中起重要作用。     

Gfat1基因在2型糖尿病大鼠肺组织中的表达

观察2型糖尿病大鼠不同时期肺组织谷氨酰胺果糖转移酶1(Gfat1)的表达情况.方法 SD大鼠随机分为正常对照组和模型组,对照组18只,模型组28只.模型组高脂饲料喂养2个月后,腹腔注射链脲佐菌素(STZ 15mg/kg)复制糖尿病模型,统计体重变化及空腹血糖值.RT-PCR方法检测造模成功后2周、4周和6周肺组织Gfat1 mRNA表达.结果 模型组大鼠体重增长较快,造模开始第28天,第42天,第56天和第70天高脂模型组与对照组体重差异有显著性(P＜0.05).注射STZ的高脂模型组空腹血糖值较高(FBG≥10.0),和对照组比较,FBG差异有极显著性(P＜0.01).糖尿病大鼠造模成功后2周,模型组肺组织Gfat1的表达低于对照组,与对照组比较差异无显著性(P＞0.05),4周模型组Gfat1的表达高于对照组,模型组与对照组比较差异有显著性(P＜0.05).6周模型组Gfat1的表达高于对照组,但模型组与对照组比较差异无显著性(P＞0.05).结论 大鼠饲喂高脂饲料结合腹腔注射STZ可成功建立2型糖尿病大鼠模型;在不同时期2型糖尿病大鼠肺组织中,Gfat1表达水平发生改变.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中,构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1,转化大肠杆菌BL21（DE3）获得表达菌株,经IPTG诱导发酵的菌体超声破碎后,裂解液用Ni离子亲和层析纯化得到融合蛋白,经肠激酶裂解,再次Ni离子亲和层析,得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测,样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa,肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断,均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。     

Expression of Chx10 and Chx10-1 in the developing chicken retina

We have isolated full-length cDNAs of chick Chx10 and Chx10-1, two members of the paired type homeobox/CVC gene family. A comparison of sequences suggests that Chx10 is closely related to Alx/Vsx-2 and Vsx-2 of zebrafish and goldfish, respectively; while Chx10-1 is closely related to Vsx-1 of zebrafish and goldfish. Chx10 and Chx10-1 are expressed in the early retinal neuroepithelium, but not in the pigment epithelium and lens. The expression of Chx10 is present in most retinal neuroblasts, while Chx10-1 exhibits a novel pattern along the nasotemporal border. In the differentiating retina, both Chx10 and Chx10-1 are restricted to bipolar cells and are maintained at a low level in bipolar cells of the mature retina.     

大鼠咬合创伤牙周膜中MCP-1、ICAM-1的表达变化

目的:研究咬合创伤大鼠牙周组织中MCP-1、ICAM-1的表达情况。方法:12周龄雄性SD大鼠24只,随机分为4组(1个正常对照组和3个实验组),每组6只。正常对照组不作任何处理,实验组通过在大鼠左上颌第一磨牙颌面粘接树脂并内置不锈钢丝形成高出颌面0.6-0.8 mm的树脂层以建立同侧下颌咬合创伤实验动物模型,分别于建模后3、5、7 d处死各组大鼠,分离大鼠下颌组织,运用HE、Masson染色观察咬合创伤牙周组织形态变化,同时用免疫组织化学染色法检测MCP-1和ICAM-1的表达变化。结果:HE染色显示,正常组牙周膜纤维排列整齐,牙骨质表面较为平整,牙槽骨结构致密。实验组牙周膜纤维排列紊乱,牙周膜血管水肿充血、间隙改变,牙槽骨和牙骨质表面不平整,出现骨吸收。Masson染色显示,正常组牙周组织未见异常表现;实验组牙周膜纤维排列紊乱,可见水解断裂,局部有血流障碍和血管破裂。免疫组织化学显示,各实验组MCP-1和ICAM-1的表达变化均较正常对照组增多,差异有显著性(P0.05)。其中7 d组表达水平最高,与其他2组相比有统计学意义(P0.05)。结论:咬合创伤可引起大鼠牙周组织形态变化,MCP-1、ICAM-1的表达随时间呈现递增的趋势。     

hAng-1真核表达载体的构建及其在MSCs中的表达

目的:通过基因重组技术,构建人血管生成素-1(human angiopoietin 1,hAng-1)真核表达载体体系,并将其转染至大鼠的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)内进行培养,进而验证hAng-1的表达.方法:将hAng-1编码序列(互补脱氧核糖核酸)通过酶切,插入至pcDNA3.1(+)质粒的多克隆位点,构建质粒pcDNA 3.1 (+)/hAng-1真核表达质粒;重组质粒经脂质体介导转染鼠MSCs.应用逆转录聚合酶联反应(RT-PCR)、蛋白免疫印迹法(Western blot)等方法检测hAng-1的表达情况.结果:pcDNA 3.1 (+)/hAng-1真核表达质粒转染鼠MSCs后,应用流式细胞仪检测,转染效率约为15％.同时应用RT-PCR能够检测出目的基因mRNA,Western blot能够检出hAng-1的蛋白表达.结论:本实验通过基因重组技术,构建的pcDNA3.1 (+)/hAng-1真核表达载体能够在转染的鼠MSCs中表达,且表达较为持续,为hAng-1基因应用于基因治疗的研究奠定了基础.     

Twist1调控FoxM1 在上皮性卵巢癌中的表达

目的:探讨Twist1调控转录因子Fox M1在上皮性卵巢癌中的表达。方法:采用免疫组织化学SP法检测Fox M1、Twist1在上皮性卵巢癌组织中表达情况,斯皮尔曼秩相关分析其在上皮性卵巢癌组织中的表达相关性;双荧光素酶报告系统检测Twist1对Fox M1的调控作用;Real-time quantitative RT-PCR和蛋白免疫印迹技术验证Twist1对Fox M1的调控作用。结果:Twist1、Fox M1在上皮性卵巢癌组织中的阳性表达率分别是71.4%(40/56)、78.6%(44/56),明显高于正常卵巢组织,差异有统计学意义(P<0.05);且其在上皮性卵巢癌中的表达显著相关(r=0.896,P<0.01);Twist1可以结合并激活Fox M1启动子(P<0.05);上调Twist1表达可以激活Fox M1在卵巢癌细胞中的表达(P<0.05),而干扰Twist1后可以下调Fox M1的表达(P<0.05)。结论:Twist1参与调控增殖相关转录因子Fox M1在卵巢癌中的表达,可能是一个潜在的治疗靶点。     

Expression of Musashi-1 in the rat stomach and changes during mucosal injury and restitution

Musashi-1 is involved in maintenance of neural stem cells. In the rat stomach, we found some cells in the luminal compartment of the mucosa that stained positively for Musashi-1. These cells were distinct from other cells of epithelial lineage, except for parietal cells, and coexpressed HES5. The Musashi-1-positive cells exfoliated after damage, while Musashi-1 expression in neck cells were upregulated, and proliferating cells diminished before reappearing and increasing in number thereafter. We conclude that a subpopulation of parietal cells acts as a source of Musashi-1, which contributes to rapid re-epithelization by restoration of stem cells and regulation of cell differentiation.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.     

Expression of Nerve Growth Factor and Nerve Growth Factor Receptor Genes in Human Tissues and in Prostatic Adenocarcinoma Cell Lines

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.     

p27kip1蛋白在胶质瘤细胞中的表达及定位研究

目的：探讨胶质瘤细胞中p27酬蛋白的表达、定位,为进一步研究p27kip1在胶质瘤发生、发展过程中的功能奠定理论基础。方法：用免疫荧光方法检测U87、LN308细胞p27kiP1蛋白的定位情况;进一步分离两种细胞的细胞质与细胞核,在显微镜下观察细胞核形态并用DAPI染色分析细胞核完整性,提取蛋白用Westernblotting。方法检测分离的细胞质与细胞核蛋白的纯度,并检测p27kip1,在细胞中的表达情况。结果：成功分离了细胞的细胞浆与细胞核,并得到纯度较好的细胞浆蛋白与细胞核蛋白。确定了p27kip1蛋白主要表达于U87和LN308细胞的细胞质中。结论：p27kip1蛋白在恶性胶质瘤中可能主要表达在细胞质中,并且其亚细胞定位可能与胶质瘤的恶性程度相关。     

Expression of Lhx6 in the adult and developing mouse retina

PurposeTo investigate the expression patterns of LIM Homeobox 6 (Lhx6) in the adult and developing mouse retina.MethodsThe Lhx6-GFP knock-in allele was used to activate constitutive expression of a GFP reporter in Lhx6 expressing cells. Double labeling with GFP and retinal markers in the mouse retina at postnatal day 56 (P56) was performed to identify the cell types expressing Lhx6. To determine the neuronal cell types that express Lhx6, double labeling with GFP and various retinal markers was employed in the differentiating retina at P7 and P15.ResultsGFP + Lhx6 lineage cells were determined in Brn3a + retinal ganglion cells (RGCs), ChAT + amacrine cells (ACs), and Islet-class LIM-homeodomain 1 (Isl1+) ACs in the mouse retina at P56. In the ganglion cell layer (GCL), Lhx6 was expressed in Brn3a + RGCs but not Brn3b + RGCs at P15. Moreover, in the inner nuclear layer (INL), Lhx6 was not expressed in Bhlhb5+ ACs at P15. However, Lhx6 was weakly expressed in Glyt1+ ACs and Pax6+ ACs, and strongly expressed in Isl1+ and ChAT + ACs at P15.ConclusionLhx6 was expressed in RGCs and ACs in both the adult and developing mouse retina.     

DDR1在人垂体腺瘤组织中的表达及其临床意义

目的:检测盘状结构域受体1(DDR1)在垂体腺瘤中的蛋白表达水平并探讨其临床意义。方法:收集81例手术切除的垂体腺瘤和5例正常垂体前叶组织标本及其临床资料,应用免疫组化SP法检测和比较其DDR1蛋白的表达情况,并分析垂体腺瘤组织中DDR1蛋白的表达与患者临床及病理特征的相关性。结果:垂体腺瘤组织中DDR1的阳性表达率为64.2%(52/81例);侵袭性垂体腺瘤组织中DDR1蛋白的阳性表达率显著高于非侵袭性垂体腺瘤(x2=16.40,P=0.000);功能性垂体腺瘤组织中DDR1蛋白的阳性表达率显著高于无功能性垂体腺瘤(x2=18.06,P=0.000);直径≥10 mm垂体腺瘤组织中DDR1蛋白的阳性表达率显著高于直径10 mm的垂体腺瘤(x2=11.68,P=0.003)。结论:DDR1的表达与垂体腺瘤的侵袭性、内分泌类型和肿瘤大小有关,可能在垂体腺瘤发病机制中起重要作用。     

半滑舌鳎FTZ-F1cDNA克隆及表达分析

为研究性别相关基因FTZ-F1在半滑舌鳎鱼中的表达特征,采用同源克隆策略,从其精巢分离了3143bp长的半滑舌鳎FTZ-F1(hsFTZ-F1)的全长cDNA,该序列包含1458bp开放阅读框,66bp长的5'末端非编码区(UTR),1619bp长的3'末端UTR。mRNA的组织分布、氨基酸序列和系统发生分析表明:hsFTZ-F1属于SF-1/Ad4BP类群。RT-PCR分析表明:hsFTZ-F1mRNA的分布广泛,几乎在所有组织都有表达,但在性腺、肾脏、脑和头肾组织中表达最强,其他组织表达较弱,雌鱼脑和头肾中的表达量明显高于雄性。胚胎发育过程中表达量都高于孵化后仔鱼的表达量,表明hsFTZ-F1可能参与了半滑舌鳎的器官形成过程。