切割海马伞大鼠海马中65kD和83kD差异蛋白诱导神经干细胞迁移和向神经元分化的作用

目的：探讨切割海马伞大鼠海马中65 kD、83 kD差异蛋白是否具有诱导神经干细胞迁移和向神经元分化的作用。方法：切割海马伞后的海马进行非变性聚丙烯酰胺凝胶电泳,将65 kD、83 kD差异蛋白胶条与大鼠神经干细胞球共培养,计数神经球朝胶条方向1/4扇区内迁移出的细胞数。10 d后行微管相关蛋白2(MAP-2)免疫荧光,计数其中MAP-2阳性神经元数,图像处理其胞体面积和细胞周长。结果：83 kD切割组朝胶条方向迁移的细胞最多,迁移速度最快。其分化的神经元数量多,胞体大、周长长,明显地优于83 kD正常组、65 kD切割组、65 kD正常组和空白对照组。结论：大鼠海马组织中83 kD蛋白具有诱导神经干细胞迁移和向神经元分化的作用。     

牙龈卟啉单胞菌菌毛fimA基因的克隆及表达纯化

目的克隆牙龈卟啉单胞菌菌毛fimA基因并使其在大肠杆菌中正确表达。方法利用PCR方法克隆牙龈卟啉单胞菌fimA基因,构建其原核表达质粒pT-BAD/fimA,获得其在大肠杆菌中的表达,基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF-MS）鉴定重组蛋白。以不同质量浓度咪唑缓冲液（250、200、150、100、50 μmol·L-1）洗脱结合在His-tag纯化柱的目的蛋白,选择最佳洗脱质量浓度。结果克隆基因测序结果与GeneBank数据库中的序列呈现99.9%同源性;诱导表达后可观察到相对分子量3.8×104的融合蛋白,经MALDI-TOF-MS检测进一步证实为FimA蛋白。100 μmol·L-1咪唑缓冲液洗脱得到的蛋白质纯度最高,其纯度为95%。结论本实验成功克隆了fimA基因,并获得在大肠杆菌中的正确表达,同时得到纯度较高的FimA蛋白,为后续研制增强免疫应答的高效牙周炎真核表达质粒奠定了基础。     

切割穹窿海马伞后大鼠与正常大鼠海马蛋白质组双向电泳图谱比较

目的：比较切割穹窿海马伞后大鼠海马与正常大鼠海马蛋白质组双向电泳图谱的差异,为研究切割穹窿海马伞后大鼠海马的神经再生内环境提供实验依据。方法：制作切割双侧穹窿海马伞大鼠动物模型,术后7 d分别取切割穹窿海马伞后大鼠和正常大鼠海马组织,提取总蛋白,进行双向电泳,比较切割穹窿海马伞后大鼠与正常大鼠海马组织的蛋白质表达差异。结果：通过双向电泳软件分析,切割穹窿海马伞后大鼠海马和正常大鼠海马组织双向电泳图谱经匹配比对后发现有7个蛋白点仅在切割穹窿海马伞海马蛋白双向电泳图谱中表达,35个蛋白在两组大鼠海马组织中含量发生了2倍以上的变化,其中切割穹窿海马伞大鼠组上调31个,下调4个。结论：初步建立了切割海马伞大鼠海马比较蛋白质组学的技术方法;切割穹窿海马伞后7 d大鼠海马与正常海马蛋白表达存在明显差异,差异点的发现为今后进一步深入研究切割穹窿海马伞后大鼠海马的神经再生机制提供了实验依据。     

针刺督脉穴对血管性痴呆模型大鼠行为及细胞凋亡影响的实验研究

目的:评价针刺督脉穴对血管性痴呆(Vascular dementia,VD)大鼠模型的学习记忆改善情况及可能机制,为针刺治疗血管性痴呆提供理论依据。方法:采用两侧颈总动脉血管阻断加硝普钠降压法复制VD大鼠模型;对VD大鼠模型进行西药(喜得镇)、针刺和电针干预;采用morris水迷宫法观察学习记忆情况;应用免疫组化法测定海马CA1区Bcl-2、Bax蛋白表达及原位末端标记法检测细胞凋亡。结果:(1)morris水迷宫学习记忆能力检测,经过6次训练,与对照组比较,模型组跳台时间延长;与模型组比较,各治疗组跳台时间明显缩短,差异均有统计学意义(P<0.05)。(2)凋亡相关蛋白检测结果显示:与对照组比较,模型组海马组织bcl-2、bax及细胞凋亡明显增高,差异均有统计学意义(P<0.01);与模型组比较,各治疗组海马组织bcl-2水平增高,bax及细胞凋亡水平明显降低,差异均有统计学意义(P<0.01)。结论:(1)各治疗组均可改善VD模型大鼠的认知能力,提高学习记忆成绩。(2)各治疗组均可抑制VD大鼠海马组织细胞凋亡。     

TLR4, ATF-3 and IL8 inflammation mediator expression correlates with seizure frequency in human epileptic brain tissue

PurposeData from animal models has nicely shown that inflammatory processes in the central nervous system (CNS) can modulate seizure frequency. However, a potential relationship between the modulation of seizure frequency and gene expression of key inflammatory factors in human epileptic tissue is still unresolved. Brain tissue from pharmacoresistant patients with mesial temporal lobe epilepsy (mTLE) provides a unique prerequisite for clinico-neuropathological correlations. Here, we have concentrated on gene expression of the human key inflammatory mediators, TLR4, ATF-3 and IL8, in correlation to seizure frequency and additional clinical parameters in human epileptic brain tissue of pharmacoresistant mTLE patients. Furthermore, we characterized the cell types expressing the respective proteins in epileptic hippocampi.MethodsTotal RNAs were isolated from n = 26 hippocampi of pharmacoresistant mTLE patients using AllPrep DNA/RNA Mini Kit. cRNA was used for hybridization on Human HT-12 v3 Expression BeadChips with Illumina Direct Hybridization Assay Kit and resulting gene expression data was normalized based on the Illumina BeadStudio software suite by means of quantile normalization with background subtraction. Corresponding human hippocampal sections for immunohistochemistry were probed with antibodies against TLR4, ATF-3, IL8 and glial fibrillary acidic protein (GFAP), neuronal nuclear protein (NeuN) and the microglial marker HLA-DR.ResultsWe observed abundant TLR4 gene expression to relate to seizure frequency per month. For ATF-3, we found an inverse correlation of expression to seizure frequency. Lower expression of IL8 was significantly associated with high seizure frequency. Further, we detected TLR4 expression in neurons and GFAP-positive astrocytes of pharmacoresistant mTLE patients. Only neurons of human epileptic hippocampi express ATF-3. IL8 was expressed in microglia and reactive astrocytes.ConclusionOur results suggest a differential correlation of key inflammatory factor expression in epileptic hippocampi and seizure frequency in patients. The modulation of such processes may open new therapeutic perspectives for treating seizures.     

间歇性缺氧对大鼠认知功能和海马CA1区超微结构的影响

目的观察间歇性缺氧对大鼠认知功能及海马CA1区神经元超微结构的影响。方法SD雄性大鼠16只,随机分为正常组(UC,n=8)和间歇性缺氧组(IH,n=8)。UC组正常饲养,IH组每日间歇缺氧7 h建立间歇性缺氧大鼠模型,通过Morris水迷宫测试检测其学习和记忆能力,并于灌注固定后应用透射电镜观察海马CA1区超微结构。结果(1)Morris水迷宫学习成绩(定位航行实验):第5 d训练结束时IH组大鼠逃避潜伏期明显长于UC组,两组之间差异有统计学意义(P<0.05)。(2)Morris水迷宫记忆成绩:IH组穿越平台次数[(1.38±0.92)次]较UC组减少[(3.75±1.04)次](P<0.01);IH组跨越目标象限时间占整个游泳时间的百分率[(20.52±3.41)%]也较UC组减少[(39.89±5.63)%](P<0.01)。(3)电镜下可见IH组神经元细胞质的电子密度增加,核染色质浓缩并聚集核膜附近,粗面内质网排列紊乱或扩张并出现脱颗粒现象,线粒体主要表现为肿胀、空泡变性及嵴消失,突触结构分界不清,突触小泡稀疏。UC组无明显病变。结论IH可致大鼠学习记忆障碍和海马神经元超微结构改变,IH所引起的大鼠学习记忆障碍与海马神经元超微结构的改变密切相关。     

银杏叶提取物对切割海马伞大鼠隔区胆碱能神经元的保护作用

目的：研究银杏叶提取物对切割海马伞大鼠隔区胆碱能元的保护作用。方法：实验组和对照组动物在切割海马伞前后分别用银杏叶提取物和蒸馏水灌胃，术后第4周灌注固定，取隔区切片进行Nissl染色及胆碱乙酰转移酶（ChAT)免疫组化染色，显微镜下记数切割海马伞侧内侧隔核和斜角带垂直支ChAT阳性神经元数目。用CMM－301图像分析系统对切割海马伞侧隔区ChAT阳性神经元的面积、周长等指标进行分析处理，用t检验作统计学分析。结果：实验组切割海马伞侧隔区的ChAT阳性神经元数明显多于对照组（P＜0．01），其神经元的胞体面积及周长指标实验组均好于对照组（P均＜0．01）。结论：银杏叶提取物对切割海马伞大鼠的隔区胆碱能神经元具有保护作用。     

A hemagglutinating variant of Prevotella melaninogenica isolated from the oral cavity

Strains resembling Prevotella melaninogenica were isolated from healthy subjects and patients with periodontal disease and were identified using: a 5-test phenotypic screen; commercial identification kits; and a 16S rRNA-based polymerase chain reaction (PCR) method. Eleven clinical isolates closely resembling P. melaninogenica, and all from patients with periodontitis, were able to agglutinate erythrocytes. In the electron microscope, hemagglutinating isolates showed fimbrialike structures, that were not seen on non-hemagglutinating isolates. Some strains were further classified with PCR-restriction fragment-length polymorphism (RFLP) of 16S rRNA genes. Amplified 16S rDNA was digested using five different endonucleases, separated with agarose gel electrophoresis, stained and photographed. Photographs were then scanned, digitized and a distance matrix calculated using Dice coefficient, where the presence or absence of a band was used as a character. The distance matrix was plotted as a phenogram. At 70% similarity six clusters were seen. Type strains of separate Prevotella species did not fall into any cluster. Hemagglutinating isolates fell into three clusters: four clustered with the type strains of P. melaninogenica and Prevotella veroralis; four with other P. melaninogenica isolates and two hemagglutinating isolates clustered together Prevotella loescheii. The PCR-RFLP results showed that the hemagglutinating strains did not form a homogenous group inside the Prevotella genus.     

载有活性物质的载体对新生大鼠海马细胞的影响

目的采用自制的载有神经细胞营养因子的载体与新生大鼠海马神经细胞共同培养，观察其生长特点。方法取新生零天大白鼠海马组织，进行原代分离培养，观察、记录培养过程中活细胞的生长情况，定期取出培养皿中长有细胞的盖玻片进行固定及染色。结果大鼠海马神经细胞在含有神经细胞营养因子的载体表面和周围生长旺盛。结论所选的载体对细胞无毒性，海马细胞在含有神经细胞营养因子的载体表面和周围长势旺盛，可望为组织工程学的研究提供参考。