新生C57小鼠耳蜗螺旋神经节细胞的原代培养*****☆

背景：目前在原代细胞培养领域内,对耳蜗螺旋神经节细胞培养条件的报道各有差异,个别方法重复性较差,不利于实际应用。 目的：原代培养并鉴定新生C57小鼠螺旋神经节细胞。 方法：显微解剖分离新生C57小鼠蜗轴组织,经胰酶消化+差速贴壁+化学药物相结合方法培养;倒置相差显微镜及苏木精-伊红染色观察细胞生长状态,免疫组织化学染色鉴别细胞来源。 结果与结论：蜗轴组织细胞纯化后,胞体呈椭圆形或三角形,有细长的突起,Nuen染色胞核呈棕黄色阳性反应,β3-Tubulin染色细胞胞浆与轴突均呈棕黄色阳性反应。提示实验成功培养出小鼠螺旋神经节细胞。     

应用改良差速贴壁法和有限稀释技术分离培养小鼠来源肌源干细胞*☆

背景：研究者们通过多种方法从肌组织中分离得到肌源干细胞，并应用于各类组织工程和再生医学研究。 目的：结合改良的差速贴壁法和有限稀释技术分离小鼠来源肌源干细胞，并培养其单细胞克隆和亚克隆集落。 方法：以新生C57BL/6小鼠四肢作为肌组织取材对象，经三重酶消化和细胞筛过滤，运用改良的差速贴壁法分离出肌源干细胞，予细胞特异标记物以免疫组织化学染色；以有限稀释技术克隆培养的方法，获得稳定的肌源干细胞单克隆和亚克隆集落。 结果与结论：差速贴壁培养过程中，肌性细胞占比逐渐增高，首次贴壁1 h可以获得足够数量的细胞进行第6次贴壁培养；肌源干细胞需72 h左右贴壁生长，经10 d左右可以增殖为300~500细胞数量的集落，细胞形态以小圆形细胞为主，并有少量梭形细胞，肌源干细胞能够维持形态并持续增殖；应用有限稀释技术可获得肌源干细胞单克隆和亚克隆集落，肌源干细胞克隆细胞均呈现Desmin染色阳性，Sca-1染色阳性，阳性率为(92.3±4.1)%。提示应用preplate法和有限稀释技术可以分离得到小鼠来源肌源干细胞及其克隆集落。     

大鼠背根神经节细胞的纯化培养

目的:在NB1培养基中建立体外胚胎大鼠背根神经节(DRG)细胞的纯化培养体系。方法:取胎鼠的背根神经节,用胰蛋白酶消化法分离成单细胞,通过差速贴壁法进行背根神经节神经元的分离纯化,在NB1中培养,用活细胞计数和神经元特异性的烯醇化酶(NSE)免疫组织化学染色相结合判定培养神经元的纯度。结果:纯化培养神经元生长状态良好,培养4天时神经元纯度为91％左右。结论:采用差速贴壁法可获得高纯度的神经元,可作为神经科学研究的重要工具。     

大鼠乳鼠和成鼠雪旺细胞提取纯化的对照研究*

背景：许旺细胞是神经创伤修复的种子细胞,获取大量高纯度、高活性许旺细胞是研究的关键。 目的：比较乳鼠和成年鼠许旺细胞的体外培养、纯化和形态学的差别,探讨简单可行的、可以获得高纯度许旺细胞的培养方法。 方法：出生1~3 d SD大鼠20只和成年大鼠10只(体质量150~200 g),实验按细胞的来源分为新生组和成年组。双酶分步消化,二次接种差速分离纯化细胞;倒置显微镜观察细胞形态、贴壁速度;细胞计数,纯度计算;MTT法检测细胞增殖能力,绘制两组许旺细胞的增殖曲线,判定增殖速度;S-100免疫化学法鉴定细胞。 结果与结论：乳鼠的许旺细胞贴壁速度快于成纤维细胞,成鼠的许旺细胞贴壁速度慢于成纤维细胞,两组许旺细胞纯度均达96%以上;MTT法检测两组许旺细胞增殖均活跃,由增殖曲线显示乳鼠许旺细胞增殖更快(P < 0.05);S-100免疫化学反应均呈阳性。提示双酶分步消化,二次接种差速分离纯化细胞,可以获取纯度高、活性良好的许旺细胞;乳鼠许旺细胞的增殖、贴壁能力更强。     

大鼠成骨细胞的原代培养和鉴定

背景：组织工程需要大量的种子细胞,成骨细胞已成为骨组织工程构建的重要种子细胞之一。但成骨细胞取材困难,获得的成骨细胞的纯度不一。 目的：建立大鼠新生乳鼠颅骨来源成骨细胞的分离培养纯化方法,观察颅骨来源成骨细胞生物学特点。 方法：采用二次酶消化法对SD大鼠乳鼠成骨细胞进行原代培养,扩增。通过差速贴壁法进行成骨细胞纯化。通过形态学、细胞碱性磷酸酶检测、茜素红染色、钙结节Von kossa法染色、超微结构以及细胞增殖曲线,确定其增殖与成骨活性。 结果与结论：二次酶消化法培养颅骨来源成骨细胞可获得原代细胞增殖,传代扩增细胞具有典型成骨细胞形态学和生物学活性。碱性磷酸酶、茜素红染色、钙结节Von kossa法染色均呈阳性结果。超微结构显示为高分化功能活跃成骨细胞,细胞增殖曲线显示细胞生长活跃。提示,新生SD大鼠颅骨来源成骨细胞具有良好的增殖与成骨活性,能连续传代增殖,纯度高,细胞生物学特征稳定,适用于做体外实验研究。     

人胚胎干细胞饲养层的制备及生物学活性

背景：建立一种既可以大量制备,又能保存并保持较高活性的饲养层是胚胎干细胞培养研究不可缺少的环节。 目的：体外分离培养、冻存复苏ICR小鼠胚胎成纤维细胞,观察其生物学特性。 方法：取ICR小鼠13.5 d胚胎,用胰蛋白酶分步消化法分离培养小鼠成纤维细胞,对冻存复苏后的小鼠胚胎成纤维细胞形态、生长曲线、贴壁率、细胞化学染色及支持人胚胎干细胞生长特性等进行观察。 结果与结论：复苏后的小鼠胚胎成纤维细胞在体外传代30 min时80%以上细胞贴壁,生长曲线显示细胞增殖活跃,细胞化学染色AKP、PAS、POX阴性,能长期支持人胚胎干细胞传代生长。提示此方法所获得的小鼠胚胎成纤维细胞复苏后有较高的生物学活性,可为人胚胎干细胞扩增提供稳定、优质的饲养层细胞。     

以差速贴壁与化学药物并胰酶限时消化法纯化新生大鼠嗅鞘细胞：优于单一差速贴壁和化学药物法吗？★

背景：目前国内外对嗅鞘细胞培养条件的报道各不相同，个别方法重复性较差，不利于实际应用。 目的：观察差速贴壁+化学药物+胰酶限时消化法纯化大鼠嗅鞘细胞的效果，并与化学药物法、差速贴壁法培养结果进行比较。 设计、时间及地点：细胞学体外对照观察，于2007-06/2008-06在福建医科大学人体解剖与组织胚胎学系实验室完成。 材料：新生2 d的SD大鼠8只，由福建医科大学试验动物中心提供。 方法：无菌条件下完整取出大鼠双侧嗅球，剥除嗅球表面的软脑膜及毛细血管网，剪成0.5 mm3的小块进行胰蛋白酶消化，过筛后按4.0×108 L-1密度种植于包被多聚左旋赖氨酸的培养瓶中进行原代培养。设立4组：①未经纯化的常规对照组。②化学药物组加入5 μmol/L阿糖胞苷抑制成纤维细胞分裂。③差速贴壁组采用Nash差速贴壁法纯化嗅鞘细胞。④差速贴壁+化学药物+胰酶限时消化组首先采用Nash差速贴壁法去除大部分成纤维细胞，而后对于少量残留的成纤维细胞加入阿糖胞苷处理，培养6 d贴壁细胞大部分融合后，加入1.25 g/L胰蛋白酶消化1 min，显微镜下见突起回缩、细胞变圆、少量细胞浮起时终止消化。 主要观察指标：嗅鞘细胞的形态，NGFR p75免疫细胞荧光染色结果。 结果：体外培养的新生大鼠嗅球嗅鞘细胞主要为双极或三极细胞，其突起细长；未经纯化的常规对照组培养7 d时视野内成纤维细胞已达60%以上，至14 d成纤维细胞完全长满；经过纯化处理的另外3组始终以嗅鞘细胞居多，嗅鞘细胞的形态与原代基本相同。存活的双极和3极嗅鞘细胞呈NGFRp75阳性反应，但化学药物组、差速贴壁组嗅鞘细胞纯度偏低，仅为75%；差速贴壁+化学药物+胰酶限时消化组嗅鞘细胞纯化效率明显增高，达85%以上。 结论：实验结果证实了差速贴壁+化学药物＋胰酶限时消化法的三合一操作技术，是一种高效率的纯化培养嗅球嗅鞘细胞的方法。     

人正常神经组织许旺细胞的体外培养*

背景：周围神经组织工程所需要的许旺细胞数量巨大。在以往的研究中,由于正常人神经组织的缺乏,常选用大鼠、兔等动物神经组织分离许旺细胞进行培养,而作为异种细胞在临床的应用相对受限。 目的：体外培养人正常周围神经的许旺细胞,寻找一种最佳的获取、培养、纯化的方法。 方法：切取脑瘫患者周围神经缩窄术中的正常周围神经,以消化培养法为主,结合差速贴壁法纯化培养许旺细胞。将神经组织剪成碎块,接种于含胎牛血清、胶原酶、Dispase酶的培养液中消化培养,离心,将组织块加入培养基中,吹打成单细胞悬液,再移入有多聚赖氨酸的DMEM培养皿中,加入碱性成纤维细胞生长因子培养,当贴壁细胞覆盖培养皿达85%~90%即可开始传代培养。锥虫蓝染色对培养后的第 2,3,4,5,6,7,8,9,10天不同时间许旺细胞进行计数,并经S-100蛋白免疫组织化学染色鉴定计算细胞纯度。 结果与结论：4 d后镜下可见基本为纯净的许旺细胞,密度在0.5×108 L-1以上。传3代后,许旺细胞计数可达到9×108 L-1以上;许旺细胞纯度达85 %以上。结果表明,以消化培养法结合差速贴壁方法培养许旺细胞所需时间短,可获得较高纯度的人许旺细胞,能够为进一步的神经组织工程提供细胞来源。     

C57胎鼠、乳鼠及成年小鼠心室肌细胞分离、培养及鉴定**☆

背景：培养的心肌细胞被广泛应用于心肌细胞的生理特性、毒性实验、基因工程、疾病模型和药物筛选等方面的研究。获得纯度较高活性良好的品系小鼠心肌细胞是研究的关键前提。 目的：分离和培养C57小鼠胎鼠、乳鼠及成年小鼠心室肌细胞。 方法：应用机械切碎心室肌后,胰蛋白酶消化不同发育阶段的C57小鼠心室肌细胞,差速贴壁1 h纯化心室肌细胞,锥虫蓝染色判定心肌细胞活力,体外分别培养48~72 h后分别行倒置显微镜、扫描及透射电镜观察细胞形态,微电极阵列评价细胞电生理指标,免疫组化鉴定。 结果与结论：经3~6次消化后,心室组织消化完全,即刻细胞存活率大于85%。倒置显微镜下观察,细胞呈梭形、多角形。12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30~90次/min。说明用胰蛋白酶组织消化法可以成功地分离、培养,并获得形态、活力良好的胎鼠、乳鼠及成年C57小鼠心室肌细胞。     

人早孕绒毛滋养层细胞原代培养：差异贴壁法与消化排除法联合应用的可行性

背景：国内外有关人绒毛膜滋养层细胞的体外培养方法,大多步骤繁琐,细胞纯度低而且成本很高,不适于普通实验室推广应用。 目的：拟求建立一种人妊娠5~10周绒毛滋养层细胞简便有效的分离培养及鉴定方法。 方法：采用改进的胰酶消化法分离培养妊娠5~10周人绒毛滋养层细胞,加0.062 5%胰酶,37 ℃消化25~40 min;以差速贴壁法和消化排除法纯化细胞,倒置显微镜观察细胞形态,细胞免疫化学方法鉴定细胞来源和纯度。 结果与结论：倒置显微镜下原代培养滋养层细胞接种后见大量圆形细胞悬浮存在,1 h后可见部分细胞贴壁,24 h后70%~80%细胞贴壁,五六天细胞数量明显增多,细胞呈三角形、多边形平铺片状生长,核大卵圆形居中,部分细胞连接成片,部分细胞呈长梭形。七八天长满瓶壁的80%~90%可传代。9~10 d铺满培养瓶底部。细胞碎屑不贴壁。传代接种后1.5h贴壁,迅速增长,三四天爬满瓶底,各代细胞形态基本一致。可见细胞为上皮样细胞形态,呈片状铺展生长。细胞角蛋白染色阳性,波形蛋白染色阴性细胞达70%~80%。采用低浓度胰酶进行长时间消化分离培养人早孕绒毛滋养层细胞,利用差异贴壁法和消化排除法以及反复换液法进行纯化,可简单、快捷地获得较高纯度的人早孕绒毛滋养层细胞。     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.