癌旁组织CD44vmRNA的表达对原发性肝癌术后复发的影响

研究原发性肝癌癌旁组织ＣＤ４４ｖｍＲＮＡ表达的意义。应用ＲＴ－ＰＣＲ检测原发性肝癌癌旁组织ＣＤ４４ｖｍＲＮＡ的表达并结合术后近２年的随访结果进行综合分析。癌旁组织ＣＤ４４ｖｍＲＮＡ表达高于癌组织者的肿瘤包膜缺如、门静脉癌栓和瘤周卫星结节等与转移相 关的病理指标高于癌旁组织ＣＤ４４ｖｍＲＮＡ表达低于癌组织的病例，前者复发率亦高于后者。结论：癌旁组织ＣＤ４４ｖｍＲＮＡ的表达肝癌侵袭潜能以及预测其复发等     

P物质对肉芽组织成纤维细胞表皮生长因子的表达调控及其意义

目的 探讨感觉神经肽P物质(SP)对离体培养的肉芽组织成纤维细胞表皮生长因子(EGF)表达的调控作用及特点。方法 采用甲醛注射的方法造成Wistar大鼠局部无菌性炎性反应，提取肉芽组织进行成纤维细胞原代培养；采用逆转录—聚合酶链反应(RT—PCR)方法检测SP对肉芽组织成纤维细胞EGF基因表达的调控作用，观察时间及剂量—效应关系；采用Western-blotting方法检测EGF蛋白表达情况，观察时间及剂量—效应关系。结果 10^-7mol/L SP可诱导成纤维细胞EGF mRNA的表达，在作用后6h与对照组比，差异有显著性(P＜0．01)；SP在10^-8-10^-6mol/L范围内可以显著促进成纤维细胞EGF mRNA表达，在10^-7mol/L达到峰值，10^-5mol/L时与对照组差异无显著性(P＞0．05)。10^-7mol/L SP作用12h后可检测到EGF蛋白明显表达增强(P＜0．01)，24h达高峰，48h后逐渐有所回落。SP在在10^-8--10^-5mol/L范围可诱导EGF蛋白的表达，均在10^-7mol/L剂量点达到峰值(P＜0．01)。结论 SP可诱导肉芽组织成纤维细胞EGF基因和蛋白的表达，且呈现出一定的时间和剂量特点，在SP调控创伤愈合的作用中具有重要意义。     

Analysis of differentially expressed genes in fetal skin of scarless and scar-forming periods of gestational rats

Objective: To study the differences of gene expression between earlier gestational skin and later gestational skin of rats with the aids of single primer amplification (SPA) and high-density oligonucleotide DNA array to understand the molecular mechanism of scarless healing. Methods: Total RNAs were isolated from fetal rat skin of the scarless (E15) and scar-forming (E18) periods of gestation (term =21.5 days). The RNAs from earlier gestational skin (EGS) and later gestational skin (LGS) were both reversely transcribed to cDNAs, then labeled with the incorporation of fluorescent dCTP for preparing the hybridization probes by SPA method. The mixed probes were then hybridized to the oligonucleotide DNA arrays which contained 5 705 probes representing 5 705 rat genes. After highly stringent washing, these DNA arrays were scanned for fluorescent signals to display the differentially expressed genes between the 2 groups of skin. Results: Among 5 705 rat genes, there were 53 genes (0.93%) with differentially expressed levels between EGS and LGS groups, 27 genes, including fibroblast growth factor 2 ( FGF2 ) and follistatin were up-regulated (0.47%) and 26 genes were down-regulated (0.46%) in fetal skin during scarless period versus scar-forming period. Higher expressions of FGF2 and follistatin in EGS than those in LGS were also revealed by RT-PCR method. Conclusions: High-density oligonucleotide DNA array provided a powerful tool for investigating differential gene expression in earlier and later gestational fetal skins. This technology validates that the mechanism of fetal scarless healing is very complicate and the change of many gene expressions is associated with fetal scarless healing.     

Corticofugal modulation of the information processing in the auditory thalamus of the cat

Summary Single unit activity of 355 cells was recorded in the auditory thalamus of anesthetized cats before, during, and after the inactivation by cooling of the ipsilateral primary auditory cortex (AI). Most of the units (n = 288) showed similar functional characteristics of firing before and after the cryogenic blockade of AI. The spontaneous firing rate remained unchanged by cooling in 20% of the units and decreased in the majority of them (60%). In some regions, i.e. dorsal division of the medial geniculate body (MGB), lateral part of the posterior group of the thalamus, and auditory sector of the reticular nucleus of the thalamus, the maximum firing rate evoked by white noise bursts was generally affected by cooling in the same direction and to the same extent as the spontaneous activity. Units in the ventral division of MGB showed a characteristic increase of signal-to-noise ratio during cortical cooling. The corticofugal modulation led to the appearance or disappearance of the best frequency of tuning in 51 units and changed it by more than 0.5 octave in 34 units. The bandwidths of different response patterns to pure tones stimulation were used to define a set of functional properties. During cryogenic blockade of AI, two cortically modulated sub-populations of units were usually distinguished that exhibited changes for a given functional property. The complexity and diversity of the effects of cortical inactivation suggest that the corticothalamic projection may be the support for selective operations such as an adaptive filtering of the incoming acoustic signal at the thalamic level adjusted as a function of cortical activity.     

Novel multi-probe RNase protection assay set for detection of endotoxin associated receptors gene expression

Objective: To construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells. Methods : The designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR)using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7/ promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method. Results: The proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation. Conclusions: These new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.     

hMSH2、hMLHl在胃癌中的表达及其临床意义

目的:探讨错配修复基因hMSH2、hMLH1蛋白的表达与胃癌临床病理学特性之间的关系。方法:采用免疫组织化学方法检测40例胃癌及50例胃炎组织中hMSH2、hMLH1蛋白的表达情况,并研究其与胃癌临床病理之间的关系。结果:40例胃癌标本中,hMSH2减低表达率为35%,hMLH1蛋白降低表达率为72.5%;淋巴结转移阳性的胃癌中,hMSH2、hMLH1蛋白表达明显减低。结论:hMSH2、hMLH1蛋白可能与早期胃癌和胃癌淋巴结转移有关。     

冠脉内导入重组β-半乳糖苷酶腺病毒的实验研究

目的 :研究经冠状动脉内导入重组 β-gal腺病毒后心肌的转染效率 ,证明冠脉内导入途径是否为重组基因导入心脏的有效的可行的途径。方法 :用定向克隆的方法构建重组 β -gal腺病毒载体 ,将 1 .53× 1 0 1 2 pfu重组 β-gal腺病毒经冠状动脉造影导管由左冠状动脉分别导入到 7只雄性Yorkshire猪心脏中 ,于术后 1周、2周、4周及 8周处死动物 ,取左右冠状动脉、3～ 5mm厚的不同部位的心肌及各实质脏器标本 ,经 1 .2 5 %戊二醛固定 2 0min ,用PBS洗涤 3次 ,在室温下用X -gal染液染色 1 6～ 2 4h ,脱水、石蜡包埋、切片及HE复染 ,光镜下计算出表达 β -gal的心肌细胞百分数即为转染效率。结果 :经冠状动脉内导入重组基因后 ,于术后 1周即有 β-gal表达 ,2周达高峰 ,心肌细胞的转染效率高达 45 .57% ,全层左冠状动脉壁、心肌间质中均有 β-gal表达 ,4周 β-gal表达下降 ,8周 β -gal恢复至对照组的基线水平。结论 :经冠状动脉内导入重组腺病毒 ,心肌细胞及冠脉壁重组基因表达效率高 ,说明冠状动脉内导入重组目的基因将是冠心病基因治疗中有效的可行的方法     

霍乱毒素B亚单位基因的克隆和表达

用DNA重组技术构建CT-B表达性质粒pXB1及进一步修饰的pXB2,使CT-B基因在大肠杆菌中得到较高水平的表达(280～350ng/ml),且有71.5％分泌率。表达动力学研究阐明,克隆子培养24h产物收率较高。一定浓度的Mg~(2+)、L-组氨酸和天冬酰胺能刺激CT-B的表达。GM1-ELISA、CHO细胞测毒结合间接免疫荧光检测和家兔肠段结扎试验证实,表达的CT-B能与游离的或活细胞膜上的GM1受体结合,但无细胞和肠段毒性。这种细胞测毒结合免疫荧光序贯试验,为客观证实CT-B的生物学活性开拓了新途径。     

一种用于基因表达谱分析的基因芯片方法

目的：通过研究SLE患者外周血基因表达谱的改变，建立一种新型的基因芯片技术用于分子发病机理的研究。方法：提取总RNA，逆转录合成单链cDNA、双链cDNA，体外转录合成生物标记的cRNA与基因芯片进行杂交，通过抗体的检测标记荧光染料Cy3，基因芯片扫描仪进行图像扫描。结果：该方法有较高的重复性和稳定性，SLE患者与正常对照组相比较，鉴定出94个基因存在表达差异。结论：该方法能在较少起始标本量的情况下，有效地进行SLE致病基因的筛选，更好的理解SLE发生的分子机理，并且可在其它疾病研究中推广应用。     

基于人工嗅觉的呈香物质识别方法

研究了人工嗅觉在物质识别中的应用，概述了使用模式识别辨别物质气味的基本原理，详细介绍了人工嗅觉系统的硬件设计，算法和软件实现方法，最后使用本系统对不同香气物质进行了识别，取得了较好的结果。