应用引物原位标记技术快速检测SOX2基因

目的 应用引物原位标记(primed in situ labeling,PRINS)代替荧光原位杂交技术对SOX2基因进行快速检测.方法 常规制备人外周血染色体并制片,利用SOX2基因特异性引物在染色体上原位扩增包含生物素标记的探针.用酪胺信号放大(tyramide signal amplification,TSA)生物素系统处理经标记的探针.最后用链霉亲和素-德克萨斯红(streptavidin-Texas red)对生物素信号进行荧光染色,用DAPI染料对染色体进行复染.作为对照,MCF-10F细胞用慢病毒载体转染导人SOX2基因,使用相同的方法检测结合位点.结果 VideoTesT-FISH软件分析提示,实验组3号染色体长臂端有特异红色荧光信号表达,空白组与未经TSA信号处理的对照组则无特异性的红色荧光信号.经过转染的MCF-10F细胞则表现出不同的SOX2基因整合效果.结论 PRINS技术利用高敏感度的原位PCR技术(in situ PCR)结合荧光标记的脱氧核苷酸可在细胞内原位合成形成探针,可大大减少探针的费用与检测的时间,提高检测效率.在诱导多能干细胞的鉴定与分类中具有较为广泛的应用前景.     

检测副溶血弧菌TDH斑点免疫胶体金渗滤法的研究

目的:制备特异性检测副溶血弧菌TDH的斑点免疫胶体金渗滤测试盒(Dot immunogold filtration assay,DIG-FA)。方法:T6D4和T9H4两株抗体分别标记于金颗粒和点样于硝酸纤维素膜,通过双抗体夹心法来开发斑点免疫胶体金渗滤测试盒,并对测试盒的特异性、重复性、稳定性做了分析。结果:研制的斑点免疫胶体金渗滤测试盒能够特异性检测副溶血弧菌TDH,其最低检测限达到250 ng/ml TDH,该测试盒在4℃恒温条件下保存12周后仍表现出准确和稳定的检测结果。结论:成功制备了检测副溶血弧菌TDH的斑点免疫胶体金渗滤测试盒,对现场快速检测TDH提供了可靠的测试工具。     

Morphological alterations and distribution of occludin in rat testes after bilateral vasectomy

The aim of study was to investigate the fate and the morphology of the cells which constitute the spermatogenic line, and to determine the distribution of occludin in the testis in adult vasectomized Wistar rats. The rats were divided into two groups: control group (sham-operated) and vasectomized group. One, 3 and 6 months after sham and vasectomy operations, testis samples were examined. The weight of the testes was found to be reduced 3 and 6 months after vasectomy. There was vacuolization in the seminiferous tubules one month after vasectomy. The tubules showed severe atrophy 3 and 6 months after vasectomy. The occludin immunolabeling in the 3- and 6-month groups was weak and diffuse, and the density of the protein was found to be decreased. The increase in the number of apoptotic cells was accompanied by a time-dependent decrease in the number of haploid, diploid and tetraploid cells. This study demonstrated that vasectomy causes degeneration in the seminiferous tubules with alterations in occludin distribution with a decrease in the number of spermatogenic cells. Moreover, these alterations increase in a time-dependent manner.     

双极金属钩电极体表刺激大鼠坐骨神经电生理特性检测

目的:探讨双极金属钩电极体表刺激检测大鼠坐骨神经电生理特性的可行性。方法:麻醉后依次用体表刺激坐骨神经和暴露坐骨神经直接刺激2种方式,记录腓肠肌复合肌动作电位(CMAP),测量CMAP平均振幅和运动神经传导速度(MCV)并行配对t检验分析。结果:经体表刺激可记录到稳定腓肠肌CMAP,其波形与直接刺激神经干所记录CMAP波形基本一致,2种方式记录的CMAP平均振幅分别为13.8±3.0mV和13.3±1.8 mV,MCV分别为53.2±5.1m/s和54.7±2.6 m/s,2种刺激方式之间各指标差异均无统计学意义。结论:直接经双极金属钩电极体表刺激坐骨神经记录CMAP是可行的,这为采用电生理方法动态监测周围神经再生提供参考依据。     

经颅磁刺激与经颅直流电刺激的比较

对经颅磁刺激和经颅直流电刺激技术在基本原理、安全性、脑功能检测、临床应用治疗方面进行比较,以便于合理地选择和有效利用这两种方法。     

直视下额叶造瘘治疗脑室出血铸型16例临床分析

目的探讨脑室出血铸型的有效治疗方法,以降低脑室出血的病死率,缩短住院时间。方法采用直视下额叶造瘘血肿清除加外引流术,早期改善脑脊液循环,防治并发症等综合治疗。结果术前GCS:3~11分的脑室出血患者16例,其中治愈5例,显著进步6例,进步3例,死亡2例。结论直视下血肿清除彻底,止血可靠,迅速降低颅内压,改善脑脊液循环,防治并发症是治疗成功的关健,直视下额叶造瘘是治疗脑室出血铸型的一种安全、可行、有效的方法。     

糖原染色在急性淋巴细胞白血病亚型诊断中的应用

目的分析急性淋巴细胞白血病(ALL)中糖原染色(PAS)的不同阳性率及分布形态及与ALL亚型(B型及T型)的关系,探索PAS在ALL亚型区分中的应用价值。方法 (1)对2009年以来每例急性白血病患者送检的骨髓涂片均加做过氧化物酶(POX)染色,将根据法国、美国、英国(FAB)协作组提出的急性白血病分型诊断标准诊断的ALL患者骨髓涂片与流式细胞术(FCM,流式)分型结果作回顾性比较,计算吻合率;(2)对上述ALL患者骨髓涂片加做PAS,计算PAS阳性率并描述其分布形态,比较PAS不同阳性率及分布形态与ALL亚型(B型及T型)的关系。结果 (1)根据FAB分型标准诊断ALL 57例,其中FCM证实ALL 56例(1例微分化型急性髓细胞白血病),与FCM吻合率为98.2%。(2)44例B-ALL中,PAS阳性38例(占86.4%),其中PAS阳性率大于或等于51.0%的32例(占72.7%)。12例T-ALL中,PAS阳性5例(占41.7%),其中PAS阳性率大于或等于51.0%的1例(占8.3%)。(3)38例PAS阳性B-ALL中,34例PAS颗粒(++～++++);5例PAS阳性T-ALL中,PAS颗粒均为(+)。结论 PAS阳性率大于或等于51%并且PAS呈粗颗粒状时,多数为B-ALL,PAS可作为ALL免疫亚型诊断的辅助手段。     

微粒蛋白质组学研究进展

目的：比较高效价抗人球（效价≥1：512）蛋白、抗人球蛋白试剂（效价≥1：4）（低效价）与微柱凝胶法在自身免疫性溶血性贫血（AIHA）实验室检测中的优势。方法：首先以微柱凝胶法直抗试验筛选阳性血液标本,然后对上述标本分别用高效价抗人球蛋白（效价≥1：512）、抗人球蛋白试剂（效价≥1：4）做直抗半定量试验,γ中和试验。结果：微柱凝胶法易出现假阳性,抗人球蛋白试剂（效价≥1：4）易出现假阴性。高效价抗人球（效价≥1：512）未发现假阳性,而且凝集梯度明显,易于结果判断分析。结论：高效价抗人球（效价≥1：512）在AIHA直抗试验中,较抗人球蛋白试剂（效价≥1：4）与微柱凝胶法具有结果更加准确的明显优势。     

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.     

Galvanic vestibular stimulation reduces the pathological rightward line bisection error in neglect-a sham stimulation-controlled study

Patients with right hemisphere lesions often show left spatial neglect and the typical rightward deviation in horizontal line bisection. Previous studies have shown that sensory stimulation modulates line bisection. A less well-known but promising sensory stimulation method is galvanic vestibular stimulation (GVS). This non-invasive technique leads to activation of the vestibular cortices and adjacent cortical areas in the temporo-parietal cortex via polarization effects of the vestibular nerves. This is accomplished by application of weak direct currents, delivered by two electrodes attached to the mastoids. Despite the relative benefits of GVS its effects on line bisection have not yet been studied in neglect patients. Thus, the present study investigated the impact of GVS on performance in a modified line bisection task in right-brain damaged patients with versus without leftsided visual neglect. In neglect patients, but not in control patients, left-cathodal and right-cathodal GVS significantly reduced the rightward line bisection error as compared to Baseline (without GVS) and sham stimulation. A larger decrease of the rightward line bisection error was observed during right-cathodal GVS. Sham stimulation showed no specific effects on line bisection. The beneficial effects of GVS might be due to activation of preserved structures of the lesioned right posterior parietal cortex which is known to be involved in line bisection.