大鼠睾丸和附睾中钙调素免疫组织化学定位的研究

用免疫组织化学ＡＢＣ法，观察了钙调素在大鼠睾丸和附睾的定位与分布。结果表明，钙调素免疫反应产物分布在精母细胞，精子细胞和精子中，而精原细胞，Ｓｅｒｔｏｌｉ细胞和Ｌｅｙｄｉｇ细胞呈阴性反应。钙调素免疫反应既见于胞质中，也见于胞核中。相邻曲细精管钙调素免疫反庆强度呈明显的不均一性。不同发生阶段的生精细胞间免疫反应强度也存在差异。大附睾，钙调素免疫反应见于附睾管尾段主细胞顶端胞质内，偶见于主细胞的基底部     

骨骼生长发育中生长激素释放肽的重要作用

文题释义：生长激素释放肽(Ghrelin)：也被称为饥饿素及内源性脑肠肽,20世纪80年代人们通过从大鼠胃组织中分离提取出一种多肽类激素,发现其具有促进生长激素分泌而被命名为生长激素释放肽。骨代谢：骨的功能是为肌肉收缩提供附着处及保护内脏等重要的生命器官。一般认为骨在细胞水平上是不活跃的,事实上骨的细胞在不停地进行着细胞代谢,不仅骨的细胞之间会相互作用,还存在骨髓中的红细胞生成细胞、基质细胞相互作用,以进行骨的改建和重建。背景：生长激素释放肽具有促进生长激素分泌、调节机体能量代谢平衡及一些尚在探索的药理学特性,这些都与机体代谢状态密切相关。目的：就生长激素释放肽对于骨骼细胞功能的调节及其在骨代谢和能量代谢中的整合作用做一综述,以期阐明生长激素释放肽在骨骼发育过程中发挥的重要作用。方法：检索 PubMed数据库1993年1月至2017年1月相关文献,检索词为“ghrelin,Bone metabolism,leptin,osteoblast,osteoclast,castric resection,cartilage cells”。排除重复研究及与综述内容关系不密切的文献,对最终纳入的74篇文献进行分析。结果与结论：机体骨骼系统的重塑是一个高度耗能的过程,与之相应的是,骨骼的生长发育涉及到外周和中枢的能量代谢,包括交感神经系统和瘦素等激素的作用。国外研究表明,生长激素释放肽能够调节成骨细胞的分化和功能,诱导骨髓干细胞的增殖及分化,通过不同的方式调节生长激素-胰岛素轴的状态,并能够通过与瘦素相作用来调配骨骼代谢与机体能量代谢从而影响骨骼的生长状态。ORCID: 0000-0003-1053-8093(叶楠)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

子宫颈癌中miR-153和Survivin的表达及其临床意义

目的探讨miR-153和Survivin在子宫颈癌组织中的表达及其对子宫颈癌的临床意义。方法收集50例子宫颈癌和40例正常子宫颈组织,采用RT-PCR法和免疫组化SP法检测miR-153和Survivin在子宫颈癌和正常子宫颈组织中的表达,分析两者表达与子宫颈癌临床病理特征的关系及对患者预后的意义。结果miR-153在子宫颈癌组织中的相对表达量低于正常子宫颈组织,Survivin在子宫颈癌组织中的相对表达量高于正常子宫颈组织,差异均有统计学意义(t=16.333,t=6.407,P<0.01)。通过散点图及相关性分析,在子宫颈癌组织中miR-153和Survivin相对表达量呈负相关,差异有统计学意义(P<0.01);miR-153、Survivin表达与患者年龄、肿瘤大小、TNM分期均无关(P>0.05),与淋巴结转移明显相关(χ^2=6.455,χ^2=5.937,P<0.05)。通过散点图及相关性分析,淋巴结转移患者的淋巴结受侵数目与miR-153表达量呈负相关,与Survivin表达量呈正相关,差异均有统计学意义(P<0.01)。预后生存分析发现,miR-153高表达患者生存率高于低表达患者,Survivin高表达患者生存率低于低表达患者,差异均有统计学意义(χ^2=19.390,χ^2=27.921,P<0.01)。结论miR-153在子宫颈癌组织中低表达,与Survivin在子宫颈癌组织中表达水平呈负相关,可能与子宫颈癌的增殖、侵袭转移有关。     

大鼠延髓后角神经降压肽(NT)的亚细胞定位和胞吐释放

神经降压肽(NT)广泛分布于哺乳动物的中枢神经系统,具有明显的镇痛作用,为了探索其镇痛机理的形态学基础,本文应用电镜免疫组化技术,对大鼠延髓后角NT的超微结构和胞吐释放进行了研究。超微结构显示延髓后角浅层NT轴突终末形态多样,大小不一,含有圆形或多形性清亮小泡及数量不等的大颗粒小泡,它们主要与未标记的树突形成轴-树突触,其突触后成分有的还含有少量清亮小泡。NT免疫反应阳性树突可分为两类:一类主要含微管;另一类主要含大颗粒小泡,有的尚可见少量清亮小泡。这两类NT树突可成为未标记的含圆形小泡终末、多形性小泡终末以及突触小球中央轴突终末的突触后成分,提示后角浅层NT神经元可接受不同种类轴突终末(包括一级伤害性传入纤维)的传入(?)动,然后可能再通过一个抑制性中间神经元,抑制痛觉的传递。本文还观察到有少量NT终末内的大颗粒小泡靠近突触活性区处,而更多见它们沿非突触部位轴膜分布,并与其融合,形成胞吐。本文认为NT既可在突触活性区处又可能在非突触部位释放。     

应激猪睾丸葡萄糖转运蛋白1和葡萄糖转运蛋白2的表达与定位

目的 探讨正常和热应激条件下,葡萄糖转运蛋白1（GLUT1）和葡萄糖转运蛋白2（GLUT2）在成年猪睾丸的表达和定位。 方法 性成熟长白公猪9头,随机分为3组。局部阴囊热刺激组（n=3）,用自制电热毯置阴囊42 ℃加热1 h;环境热应激组（n=3）,每天置于37～40 ℃猪舍环境3 h,连续7 d,每天于热处理结束后,将实验猪驱赶回21～25 ℃猪舍环境;对照组（n=3）,饲养在21～25 ℃猪舍环境。局部热刺激6 h后和环境热应激处理结束24 h后,手术摘除双侧睾丸。用Real-time PCR、Western blotting和免疫组织化学技术检测猪睾丸组织内GLUT1和GLUT2的表达。 结果 Real-time PCR和Western blotting结果显示,与对照组相比,环境热应激组GLUT1蛋白和mRNA的表达差异不显著,局部阴囊热刺激组GLUT1蛋白和mRNA表达显著升高;环境热应激组和局部阴囊热刺激组,GLUT2蛋白和mRNA表达均显著升高。免疫组织化学结果发现,热处理前后,GLUT1蛋白在曲精小管内定位于精母细胞和圆形精子细胞;环境热应激组GLUT1蛋白染色与对照组相比,无明显差异,局部阴囊热刺激后,GLUT1染色变深,表达升高。热处理前后,GLUT2蛋白在曲精小管定位于生精细胞和支持细胞,环境热应激和局部阴囊热刺激导致GLUT2染色变深,表达升高。 结论 葡萄糖转运蛋白GLUT1和GLUT2表达于猪睾丸曲精小管,环境高温和阴囊局部热刺激导致GLUT1和GLUT2在猪睾丸的表达水平改变,提示这两种葡萄糖转运蛋白在猪精子发生过程中发挥重要作用。     

电针上调脊髓损伤后大鼠c-fos mRNA及其蛋白的表达

目的:探讨电针对大鼠脊髓损伤后c-fos mRNA及蛋白表达的影响.方法:将SD雄性大鼠随机分为假手术组、模型组和电针组,采用改良Allen氏重物坠落法制成脊髓损伤模型.分别在1、2、3、5和7d处死动物,使用实时荧光定量PCR技术检测c-fos mRNA表达;使用免疫组织化学显色结合图像定量分析方法检测c-fos蛋白表达.结果:实时荧光定量PCR结果显示,模型组c-fos mRNA表达呈现升高趋势,电针能够促进这种趋势,在术后1d和3d与模型组比较,差异具有统计学意义.免疫组织化学结合图像分析结果显示,模型组c-fos蛋白表达升高趋势较为明显,电针促进这种趋势,且在术后2d与模型组比较,差异具有统计学意义.结论:电针能够促进脊髓损伤后大鼠脊髓损伤部cfos mRNA及蛋白的表达.     

子宫内膜异位症TGF-β_1及其基因的表达及意义

目的研究子宫内膜异位症中转化生长因子β1（TGF-β1）及其基因表达,探讨TGF-β1在子宫内膜异位症中的作用机制。方法 54例子宫内膜异位症异位内膜、在位内膜,50例对照组正常子宫内膜中,采用免疫组化方法检测TGF-β1的表达,SYBR Green实时定量聚合酶链反应（RQ-PCR）检测TGF-β1 mRNA的表达量。结果 TGF-β1在子宫内膜异位症异位内膜组中的蛋白表达显著高于在位内膜组及对照组（P〈0.05、P〈0.05）;TGF-β1在子宫内膜异位症在位内膜组中的表达与对照组中的表达无显著性差异（P〉0.05）;TGF-β1mRNA在子宫内膜异位症异位内膜中的表达量显著高于在位内膜组和对照组（P〈0.05、P〈0.05）,TGF-β1mRNA在位内膜组的表达量与对照组中的表达量无显著性差异（P〉0.05）。结论 TGF-β1可能参与子宫内膜异位症的发病机制,影响其发生发展。     

丝胶对2型糖尿病大鼠睾丸生长激素/胰岛素样生长因子-1轴的作用

目的 探讨丝胶对2型糖尿病大鼠睾丸生长激素（GH）/胰岛素样生长因子-1（IGF-1）轴的作用。 方法 40只雄性SD大鼠随机分为正常对照组、糖尿病模型组、丝胶治疗组和阳性对照组,每组均为10只。链脲佐菌素连续腹腔注射制作2型糖尿病大鼠模型,1周后以血糖≥16.7mmol/L作为成模标准。待模型成功建立后,模型组大鼠不做任何处理,丝胶治疗组大鼠给予丝胶灌胃［2.4g/(kg&#8226;d)］,阳性对照组大鼠给予二甲双胍［55.33mg/(kg&#8226;d)］灌胃,均为35d。采用酶联免疫吸附测定（ELISA）方法检测大鼠血清睾酮、GH和IGF-1水平;分别采用免疫组织化学染色、免疫印迹法和RT-PCR法检测睾丸GH、GH受体（GHR）和IGF-1的表达。 结果 丝胶可明显降低糖尿病大鼠血GH水平、下调睾丸GH的表达,升高血IGF-1和睾酮水平,上调睾丸IGF-1和GHR的表达（P＜0.05,P＜0.01）。并且丝胶治疗组各项指标与阳性对照组比较无明显差别（P＞0.05）。 结论 丝胶可通过调节糖尿病时GH/IGF-1轴紊乱改善生精功能,发挥对糖尿病生殖功能损害的保护作用,其作用与二甲双胍相当。     

生长激素释放肽对心力衰竭大鼠血清和心肌组织中脂联素的影响

目的研究生长素释放肽(GHRP)对脂联素(APN)的影响,探讨GHRP在慢性充血性心力衰竭(CHF)发生、发展中的作用,以期为临床更好地防治CHF提供理论依据。方法采用腹腔注射阿霉素(Adriamycin,ADR)方法建立大鼠CHF模型,采用酶联免疫吸附剂测定(ELISA)检测大鼠血清和心肌组织匀浆中APN含量的变化。结果各组间血清和心肌组织匀浆中APN的含量比较结果显示:模型组APN明显低于对照组和治疗组(P<0.05);治疗组APN比对照组轻度降低,但无明显差异(P>0.05)。结论APN在CHF发生过程中有着重要作用,GHRP具有保护和改善CHF大鼠心功能的作用。     

人类腺体激肽释放酶2(hK2)的研究进展

前列腺癌是男性常见疾病,前列腺特异抗原(PSA)是当今泌尿外科中最重要的肿瘤标志物,它在前列腺癌的诊断和治疗中得到了广泛的应用[1],但PSA在诊断灰区4ng/mL～10ng/mL的特异性不高,不能完全区别前列腺癌与良性前列腺增生.近年来大量研究证实,人类腺体激肽释放酶2(hK2)能改进前列腺癌早期诊断的特异性.由于测定方法的不断改进,对hK2的认识也逐渐清晰.现将近年来对hK2的研究综述如下:     

Aberrant SATB1 expression is associated with Epstein-Barr virus infection,metastasis and survival in human nasopharyngeal cells and endemic nasopharyngeal carcinoma

Special AT-rich sequence-binding protein 1 (SATB1) has been identified as a key factor in the progression of some cancers, functioning as a global genome organizer and chromatin regulator. We examined the levels of SATB1 mRNA expression in NPC cell lines 5-8F (high metastasis) and 6-10B (low metastasis) and immortalized human nasopharyngeal epithelial cells NP69-SV40T by quantitative real-time PCR. We also examined the protein expression levels of SATB1 in 72 cases of nasopharyngeal carcinoma (NPC) tissues and 30 cases of normal nasopharyngeal (NNP) tissues by immunohistochemistry, and then assessed the correlations between SATB1 expression and clinicopathological factors. The expression level of SATB1 mRNA in 5-8F was much higher than those in 6-10B and NP69-SV40T (P < 0.05). The expression level of SATB1 mRNA in 6-10B was higher than in NP69-SV40T, but the difference was not statistically significant (P > 0.05). The positive expression rates of SATB1 protein in NPC (38/72, 52.8%) were significantly higher than in NNP (4/30, 13.3%) (P < 0.05). SATB1 protein levels in NPC were not associated with gender, age, and T stage (P > 0.05), but positively correlated with the titers of EBVCA-IgA, metastasis (N and M stage), recurrence, and survival (P < 0.05). Multivariate analysis showed that the overexpression of SATB1 protein is an independent prognostic factor for NPC. The expression levels of SATB1 were obviously upregulated in primary NPC tissues and human NPC cell lines. Therefore, SATB1 may be a valuable predictor in assessing the metastasis, recurrence, and prognosis of NPC.     

生长素在成年皖西白鹅肝和胰中的免疫组织化学定位

目的:研究皖西白鹅肝和胰内是否有生长素细胞分布。方法:应用免疫组织化学SABC法检测皖西白鹅肝和胰生长素表达。结果:肝细胞有生长素表达,肝中的淋巴组织也有生长素免疫阳性细胞;在胰的外分泌部和内分泌部(胰岛)均可观察到生长素的表达。结论:生长素在成年皖西白鹅肝和胰中分布广泛,生长素可能以自分泌/旁分泌的形式调节消化腺的功能。     

实时荧光定量PCR检测结直肠癌UbcH10基因的表达

目的：通过检测UbcH10 mRNA在结直肠癌中的表达,探讨UbcH10作为结直肠癌诊断标志物的可能性。方法：采用Trizol提取组织总RNA,RT-PCR获得UbcH10 cDNA,实时荧光定量PCR检测45例结直肠癌患者肿瘤组织与相应癌旁正常组织中UbcH10 mRNA的丰度。结果：在所有肿瘤组织样本中UbcH10 mRNA的相对表达量与其自身的癌旁正常组织样本中的相对表达量相比明显升高,平均表达水平是正常组织的10倍以上（0.0802±0.0907vs0.0079±0.0088,P〈0.01）,其中超过20倍的占37.78%（17/45）。结论：UbcH10基因在结直肠癌患者肿瘤组织中过度表达,可作为结直肠癌诊断的潜在肿瘤标志物及结直肠癌基因治疗的新靶点。     

原发性肝癌患者血清AFP及外周血AFP mRNA结果分析

目的:探讨血清AFP及外周血AFP mRNA联检对原发性肝癌(PLC)患者的早期诊断和微小转移的临床意义。方法:采用电化学发光法和实时荧光定量PCR分别检测40例PLC患者血清AFP和外周血AFPmRNA。结果:PLC患者血清AFP和外周血AFP mRNA阳性率分别为72.50%(29/40)和57.50%(23/40),显著高于肝硬化患者(8.33%和4.17%,P<0.05)。其中血清AFP阳性和阴性的PLC患者分别有21例(72.41%)和2例(18.18%)检出AFP mRNA。结论:外周血AFP mRNA可作为血循环中存在肝癌细胞的一个早期标志物,与血清AFP联检有助于提高PLC的确诊率,更好地预测肿瘤转移和复发。同时为开展PLC靶向治疗提供依据。     

Studies on the association of BF1/BF2 gene expression patterns with traits of genetic resistance to Marek's disease in chickens

The aim was try to clarify the association of BF1 and BF2 gene expression patterns with the traits of genetic resistance to Marek's disease (MD) in chickens. An MD-resistant line and a common line of Xiayan chickens were used in the experiment. The productions of mRNA of BF1 and BF2 and Marek's disease virus (MDV) gene, meq, were quantified by quantitative real-time PCR on different days post-infection (dpi) with virulent MDV in the peripheral blood leukocytes (PBLs). Results indicated that the MDV loads were lower in the MD-resistant line compared to the common line. The mRNA levels of BF1 were significantly lower and levels of BF2 higher in the MD-resistant line whereas levels of BF1 were significantly higher and levels of BF2 lower in the common line. The results demonstrated that the expression modes of BF1 and BF2 genes might be related to traits of genetic resistance to MD in the chicken.     

Suprathreshold manipulations of testosterone and reproductive functioning in gonadally intact sexually experienced and inexperienced male rats

Circulating titers of testicular and gonadotrophic hormones differ widely among conspecific mammalain males. The behavioral and physiological consequences of these differences were examined by injecting gonadally intact male rats with extra testosterone or an antiandrogen. Serum testosterone values increased and decreased, respectively, relative to control animals, although all values remained within normal physiological ranges. Results suggested that hormone-sensitive behavior and physiology were related to suprathreshold androgen differences in a dose-response fashion among virgin males. The relationship was more complex among sexually experienced males. Additional testosterone to the experienced males increased the frequency of both aggressive and sexual responding as well as the activity of sex accessory glands. Reductions of circulating testosterone, however, had minimal effects on behavior despite substantial atrophy of hormone-sensitive tissues. Indeed, fecundity was independent of hormone therapy as all experienced males successfully fathered pups. Suprathreshold hormone differences in natural populations of males may reflect sexual selection that is maintained by female choice of a mate.     

Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understoood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co‐localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co‐localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co‐localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post‐translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON‐1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down‐regulated by post‐translational modulation and shows potential antiproliferative properties in vitro. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Development of a real-time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture

Mycoplasma hominis is an opportunistic human mycoplasma species that can be either commensal or pathogenic. Its detection by culture is considered to comprise the reference technique. Previously reported PCR techniques target the 16S rRNA or the gap gene, although sequence variations among clinical isolates may lead to variations in clinical sensitivity. The present study aimed to develop a specific TaqMan quantitative real-time PCR assay, targeting a gene conserved in all M. hominis isolates, and to compare it with quantitative culture. With the knowledge of the M. hominis PG21 genome sequence, the yidC gene, encoding a membrane protein translocase, was chosen as target. Its intraspecies heterogeneity was checked at the nucleotide level using 31 reference or clinical strains. The limit of detection, the analytical specificity and the reproducibility of the assay were assessed. Moreover, PCR and culture results were compared using 153 urogenital specimens. The limit of detection was seven copies/μL. The analytical specificity was 100%, with good inter- and intra-assay reproducibility. Among the 153 urogenital specimens, the yidC PCR and culture allowed detection of 55 and 45 M. hominis-positive samples, respectively. Comparison of the bacterial load among the 45 specimens found to be M. hominis-positive by both techniques revealed a statistically significant association between the quantitative results obtained. In conclusion, we developed a specific, sensitive and reproducible real-time PCR to detect all M. hominis clinical isolates. This PCR was shown to have higher sensitivity than culture, although both methods were correlated for quantification of M. hominis loads in urogenital specimens.     

rpoB作为蜡样芽胞杆菌群快速检测的标志基因的实验研究

目的探讨常规PCR和实时荧光定量PCR（Q-PCR）方法检测蜡样芽胞杆菌群rpoB基因的特异性和敏感性。方法提取蜡样芽胞杆菌群和其他各种对照细菌的基因组DNA,合成蜡样芽胞杆菌群rpoB基因扩增引物,采用常规PCR和SYBRgreen实时定量PCR两种方法扩增rpoB基因片段,并将PCR产物克隆到pMD18-T载体后进行DNA测序。结果常规PCR和Q-PCR均能扩增出蜡样芽胞杆菌群rpoB基因的174bpDNA片段,而各种对照菌株均未见扩增。序列比对发现蜡样芽胞杆菌群细菌在该片段中存在5处核苷酸的不同,差异率为2.88%。以炭疽芽胞杆菌基因组DNA系列稀释作为扩增模板显示常规PCR最小检出量为3.42pg,Q-PCR的敏感性达到171fg,3次重复实验显示Q-PCR检测rpoB基因的灵敏度为（3.32×101±7.45×100）拷贝。结论以rpoB基因为检测靶基因的Q-PCR方法具有高度的特异性和良好的敏感性,能实现对蜡样芽胞杆菌群快速而准确的检测。