临床血清循环miRNAs定量技术的建立

目的建立可靠的临床血清（浆）循环miRNAs定量技术。方法常规收集血清（浆）标本,mirVana PARIS试剂盒法抽提血清（浆）总RNA,采用DNaseI消化总RNA提取液,以miRNAs特异性茎-环引物引导反转录,通过TaqMan实时荧光定量PCR对U6及靶miRNAs进行检测。结果10份新鲜血浆总RNA浓度介于3.5～35.4ng/μl之间。对常规收集的400μl临床血清标本中U6、miR-16、miR-224均能实现特异扩增及定量,相应的平均Ct值约为30、25及32。6份不同留置时间血清标本总RNA浓度分别10.24和4.46ng/μl,定量PCR结果显示其中相应miR-16和miR-224的丰度却相对稳定。结论血清（浆）总RNA抽提及循环miRNAs定量切实可行。     

Expression of neurotensin and its receptors in pituitary adenomas

The neurotensin (NT) produced in the hypothalamus and in pituitary gonadotrophs and thyrotrophs participates in neuroendocrine regulation. Recently, the involvement of this peptide in normal and neoplastic cell proliferation has been postulated. In the present study, we evaluated the expression of NT and its receptors (NTR1, 2 and 3) in a series of 50 pituitary adenomas [11 growth hormone (GH)-, eight prolactin (PRL)-, four adrenocorticotrophic hormone (ACTH)- and 27 nonfunctioning adenomas]. NT mRNA expression was significantly higher in functioning compared to nonfunctioning adenomas and with normal pituitary. Nonfunctioning pituitary adenomas showed lower expression of NT mRNA than normal pituitary. In the immunohistochemical study of functioning adenomas, NT was colocalised with GH, PRL and ACTH secreting cells. In nonfunctioning adenomas, the NT immunoreactivity intensity was variable among the samples. NTR3 mRNA expression was observed in all examined samples and was higher in the adenomas, both functioning and nonfunctioning, compared to normal pituitary. By contrast, NTR1 and NTR2 mRNA were not detected in either pituitary adenomas or normal tissue. The higher expression of NTR3, as well as the expression of NT by tumoural corticotrophs, lactotrophs and somatotrophs, which are cells types that do not express this peptide in the normal pituitary, suggests that NT autocrine and/or paracrine stimulation mediated by NTR3 may be a mechanism associated with the tumourigenesis of functioning adenomas.     

胰岛素抵抗小鼠肠道菌群与FGF21的相关性研究

目的研究糖耐量正常人粪菌液移植(fecal bacteria transplantation,FMT)对胰岛素抵抗(insulin resistance,IR)小鼠肠道菌群的影响及其与成纤维细胞生长因子21(fibroblast growth factor 21,FGF21)的相关性,探究肠道菌群影响IR的可能机制。方法采用高脂饮食建立IR模型,筛选30只建模成功的小鼠随机分成3组:IR组、IR+二甲双胍(Met)组、IR+粪菌液移植(FMT)组,及空白对照(Control)组,每组10只。给药8周,记录第8周小鼠的体质量、空腹血糖;采用RT-qPCR检测粪样中靶标菌的数量,肝脏、结肠、回肠中FGF21及其受体mRNA表达水平。结果①与Control组比较,IR组小鼠体质量和空腹血糖升高,肝脏、结肠和回肠中FGF21/β-Klotho/FGFR1/FGFR4 mRNA表达降低,粪样中Bacteroides、B.sartorii降低,P.distasonis、M.schaedleri、R.gnavus升高,Met、FMT干预后上述指标均逆转。②FGF21的表达与FBG、P.distasonis、M.schaedleri、R.gnavus呈负相关,与Bacteroides、B.sartorii呈正相关。结论FMT可增加FGF21的表达量和调节肠道菌群,且二者密切相关,可能是FMT改善胰岛素抵抗的重要机制之一。     

The selection and identification of compound housekeeping genes for quantitative real-time polymerase chain reaction analysis in rat fetal kidney

During quantitative real-time polymerase chain reaction (RT-qPCR) data analysis, the selection of optimal housekeeping gene is necessary to ensure the accuracy of results. It is noteworthy that housekeeping genes commonly used in adult studies may not be applicable for fetus. However, the stability analysis of housekeeping gene in fetal kidney has not been reported. This study intends to screen the applicable compound housekeeping genes in rat fetal kidney. In this study, eight housekeeping genes used in kidney studies based on literature reports (GAPDH, ACTB, 18S, HPRT, YWHAZ, HMBS, PPIA, and TBP) were selected as the research object. Their expression levels in the rat fetal kidney in physiological condition and the intrauterine growth retardation (IUGR) model induced by prenatal dexamethasone exposure (PDE) (0.2 mg/kg·day from gestation Days 9 to 20) was measured. Furthermore, these eight housekeeping genes were used to conduct relative quantitative analysis of nephrin expression in the fetal kidney in PDE-induced IUGR model, to compare the influence of choosing different housekeeping gene on data analysis of nephrin expression and to verify the reliability of selected compound housekeeping genes. In this study, stable housekeeping genes of fetal kidney tissues in PDE-induced IUGR model were identified: ACTB, GAPDH, TBP, and HMBS for males; ACTB, YWHAZ, and GAPDH for females. Besides, our results suggest that ACTB + GAPDH were the best compound housekeeping genes for normalization analysis in male fetal kidney studies, and ACTB + YWHAZ in females. This study will provide an experimental evidence basis for the selection of housekeeping genes in the RT-qPCR experiment in renal development toxicology-related models.     

Bridging integrator 1在胃腺癌中的表达及其预后价值

目的研究Bridging integrator 1（BIN1）在胃腺癌中的表达及其预后价值。方法通过免疫组化研究BIN1在234个胃腺癌组织样本中的表达,之后运用荧光定量PCR（RT-qPCR）和蛋白质印迹（Western blotting）技术对免疫组化结果进行进一步验证。结果通过免疫组化数据表明,在234个胃腺癌病例中,有128例的BIN1表达水平下降（54.7%）。此外,BIN1的表达水平与组织学分级、肿瘤侵袭、淋巴结转移及TNM分期显著相关（P〈0.05）。通过RT-qPCR和Western blotting分析,发现与非胃腺癌组织相比较,大多数肿瘤组织中的BIN1的转录和转录后水平下调（P=0.0015和P=0.012）。Kaplan-Meier存活曲线证实BIN1表达水平的下降与胃腺癌患者的预后不良有关。进一步的多因素分析结果提示BIN1是胃腺癌患者的总存活率的独立预后因子（HR=0.552,95%CI：0.354～0.862,P=0.009）。结论在胃腺癌中BIN1表达的下降可导致疾病预后不良,BIN1作为潜在的肿瘤抑制基因可能在防止肿瘤进展方面起重要作用。     

Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy₀ quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy₀ method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~ 1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.