Expression of 5-HT2A receptor mRNA in rat spinal dorsal horn and some nuclei of brainstem after peripheral inflammation

The expression of 5-hydroxytryptamine 5-HT2A receptor mRNA was studied in the lumbar spinal dorsal horn, nucleus of raphe magnus (NRM), ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) following carrageenan inflammation using in situ hybridization technique. The findings of this study demonstrated that 5-HT2A receptor mRNA was expressed with low to moderate levels in lumbar spinal dorsal horn, NRM, vlPAG and DRN. Following carrageenan inflammation, the expression of 5-HT2A receptor mRNA in ipsilateral dorsal horn, bilateral NRM, vlPAG and DRN was significantly increased. The peak occurred at 3 h and then there was a clear decrease but still a substantial number of labeled cells at 24 h after injection of carrageenan. This result suggested that the synthesis of 5-HT2A receptor is enhanced in spinal dorsal horn, NRM, vlPAG and DRN during inflammatory pain.     

Phosphorothioate oligonucleotide inhibits tissue factor expression in endothelial cells induced by blood flow shear stress in rats

Objective： To determine the effect of antiparallel phosphorothioate triplex-forming oligonucleotide （apsTFO）, which was designed according to shear stress response element （SSRE） in tissue factor （TF） gene promoter region, on the expression of endothelial TF in carotid artery stenosis rats. Methods： Rat model of severe carotid artery stenosis were inflicted by silica gel tube ligation. Half an hour before the model infliction, GT20-apsTFO, GT20-psTFO and GT21-apsTFO labeled with green fluorescence （FITC） were injected into the vena caudalis of rat at a dose of 0.5 mg/kg. Half an hour, 4 or 9 h after the ligation, the distribution of TFO in the common carotid artery, the liver and the kidney was detected with aid of fluorescence microscopy. And the mRNA and protein expressions of TF, Egr-1 and Spl in the above-mentioned organs were determined with in situ hybridization and immunohistochemical assay respectively in 6 h after the model establishment, and the results were analyzed with an image analysis system. Results： Only in 1 h after TFO injection, fluorescent granules appeared in the liver, the kidney and the vascular wall and lumen of carotid artery, and then in 4.5 h, they still deposited in above sites except the vascular lumen. GT20-apsTFO and GT21-apsTFO significant down-regulated the mRNA and protein expressions of TF compared to the rats without treatment （P〈0.05）, and the former apsTFO had a more stronger effect than the later （P〈0.05）. GT20-psTFO had no such effect （P〉0.05）. The 3 TFOs had no inhibition on the mRNA and protein expressions of Egr-1 and Spl. Conclusion： Pretreated apsTFO can partly come into the vascular endothelial cells, and inhibit TF expression induced by shear stress, but had no effect on Egr-1 and Spl gene expressions.     

C-myc反义寡核苷酸对人胆管癌细胞增殖和侵袭力的影响

目的 探讨C-myc反义寡核苷酸(ASODN)对人胆管癌QBC939细胞增殖和侵袭力的影响.方法 合成C-mycASODN,并将其转染QBC939细胞;四甲基噻唑蓝(MTT)实验检测C-myc ASODN抑制QBC939细胞增殖的影响;Transwell法检测C-myc ASODN抑制QBC939细胞侵袭力的影响.结果 ASODN组细胞的存活率低于空白对照组(P<0.05);ASODN组较空白对照组的细胞穿透数目下降(P<0.01);空载体组细胞的存活率及细胞穿透数目较空白对照组差异无统计学意义(P>0.05).结论 C-myc ASODN能抑制QBC939细胞的增殖和侵袭力.     

Selection and their antitumor activity of antisense oligonucleotides targeting messenger RNA of vascular endothelial growth factor receptor 2

Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.     

供、受者细胞因子及其受体单核苷酸多态性对肾移植近期急性排斥反应的影响

目的研究肾移植供受者的细胞因子和细胞因子受体单核苷酸多态性对术后早期移植物急性排斥反应的影响。方法（1）根据有无急性排斥发生,129例肾移植受者分成急性排斥组和无排斥组（6个月内）,根据HIJA—A—B—DR错配、神经钙蛋白抑制剂、肾功能延迟恢复、ATG或MAb诱导治疗、冷缺血时间等情况,对两组患者进行比较,并比较两组5种细胞岗子及5种受体的21个位点的基因型分布情况。（2）根据HLA—DR配型情况分成0～1个HLADR位点错配组（0—1MM）、HLA—DR完全错配组（2MM）,比较两组基因多态性的分布情况。结果（1）129例肾移植受者中,39例（30．2％）发生急性排斥反应。（2）急性排斥组HLA—DR错配数和无排斥组中差异有统计学意义（P〈0．05）。（3）细胞因子和细胞因子受体位点的多态性在急性排斥组和无排斥组中的分布有明显不同,差异有统计学意义。（4）HIA—DR完全错配组（2MM组）和0～1个位点错配组（0～1MM组）进行比较,两者多态性分布亦有不同。结论肾移植供受者不同基因多态性对预测排斥的发生有指导意义。     

新生儿常见感染病原菌的16SrDNA寡核苷酸阵列快速诊断

目的探讨PCR结合寡核苷酸阵列杂交快速诊断常见新生儿感染病原细菌方法。方法通过对40多种常见病原菌的16SrDNA序列比较分析,在中间找到一段两端高度保守中间相对变异的序列。依据其保守序列设计一对通用引物,用于扩增细菌16SrDNA目的基因。同时依据相对变异区域的序列设计常见的8种病原细菌的探针,阵列于带正电的尼龙膜上,与通用引物的PCR产物杂交,在一个反应体系中1次可检测出标本中可能存在的常见致病菌。结果设计的通用引物能有效扩增出常见的细菌目的序列。实验结果表明寡核苷酸阵列均能特异地与其检测细菌的通用引物PCR产物杂交,而不与其它所试细菌通用引物PCR产物杂交,说明探针具有很强的特异性。进一步采用寡核苷酸阵列对临床分离的菌株进行鉴定,以细菌自动鉴定仪做对比,结果表明寡核苷酸阵列能检出常见的8种病原菌。结论PCR结合寡核苷酸阵列杂交是快速诊断常见新生儿感染病原菌有效方法。     

应用基因芯片技术筛查卵巢癌相关基因

目的 探讨卵巢癌组织中相关基因的差异表达。方法 应用含有512条人类基因的cDNA芯片,检测卵巢癌组织和正常组织中相关差异表达的基因。结果 共筛查与卵巢癌相关的差异表达的基因37条,其中14条基因表达增加(上调),23条基因表达减少(下调)。结论 应用基因芯片技术,可筛查出新的卵巢癌相关基因。     

鲑鱼鱼白DNA对小鼠胸腺作用的差异表达基因筛选

目的　探讨鲑鱼鱼白DNA(salmonmiltDNA ,SMD)对小鼠胸腺增龄性萎缩的作用及作用机制。方法　 10月龄雌性BALB/C小鼠按体重随机分为高剂量组 (333 33mg·kg-1·d-1)、低剂量组 (16 6 6 7mg·kg-1·d-1)和对照组 (0mg·kg-1·d-1)。喂饲 5周后 ,测定胸腺脏器指数 ;组织切片后以Image ProPlus专业图像分析系统 (4 0版 )进行胸腺细胞计数和皮质厚度测量 ,所得数据经SAS统计软件分析。应用基因芯片技术在对照组和高剂量组胸腺组织中筛选差异表达的基因片段、RT PCR鉴定部分片段。结果　SMD对小鼠的体重、胸腺重量和胸腺指数均无影响 ;高剂量组小鼠的胸腺皮、髓质细胞数量与对照组经统计学分析相比均显著增多 ;低剂量组的胸腺皮、髓质细胞数量与对照组相比经统计学分析差异均不显著 ;高、低剂量组的胸腺皮质平均厚度经统计学分析均显著高于对照组 ;经基因芯片技术初筛出 112条差异表达的基因片段 ;Genebank登录号为Aw2 0 910 2、U2 3789、X80 2 32的 3条上调表达的基因片段 ,经RT PCR鉴定 ,在高剂量组确实存在上调表达。结论SMD有通过促进细胞增殖相关基因的表达并同时促进发育、分化相关基因表达而逆转胸腺增龄性萎缩的可能。     

Distribution and biotransformation of therapeutic antisense oligonucleotides and conjugates

Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients.     

Localization of the mRNA for the 5-HT2 receptor by in situ hybridization histochemistry. Correlation with the distribution of receptor sites

³²P-labelled oligonucleotides complementary to rat 5-HT₂ receptor mRNA were used as probes to study the distribution of cells in rat brain containing the mRNA coding for this receptor by in situ hybridization histochemistry. 5-HT₂ receptor binding sites were visualized by autoradiography using [¹²⁵I]DOI as ligand. Both distributions were comparable, demonstrating that 5-HT₂ receptors are expressed by cells intrinsic to the neocortex (lamina Va), claustrum, olfactory bulb and several nuclei of the brainstem.