慢病毒介导绿色荧光蛋白转染人胚胎干细胞及其培养

目的 稳定培养人胚胎干细胞,并通过慢病毒载体对其进行绿色荧光蛋白标记.方法 利用小鼠胚胎成纤维细胞作为饲养层或Matrigel作为基质培养人胚胎干细胞,包装带有GFP序列的慢病毒转染人胚胎干细胞.对转染前后的人胚胎干细胞进行了碱性磷酸酶和SSEA-3免疫组化鉴定.结果 在MEF饲养层和Matrigel上均可培养出呈克隆样生长,表达标志抗原的人胚胎干细胞,经慢病毒转染及抗生素筛选后仍可稳定表达GFP.结论 成功地培养了人胚胎干细胞系,并进行了GFP标记.     

CREG基因沉默诱导小鼠ESCs来源的EBs自发性凋亡

目的: 探讨E1A激活基因阻遏子(CREG)基因沉默诱导鼠源性胚胎干细胞(ESCs)来源的胚胎小体(EBs)自发性凋亡的作用。方法: 应用pDS-shRNA-CREG逆转录病毒真核表达载体和绿色荧光蛋白(GFP)的对照载体,感染鼠源性胚胎干细胞R1,筛选获得稳定的细胞克隆(R1-shCREG和R1-GFP);用小鼠胚胎成纤维细胞株STO作为饲养细胞,进行R1、R1-shCREG和R1-GFP细胞培养。倒置显微镜观察3种ESCs的生长情况。碱性磷酸酶(AKP)染色和小鼠心肌接种畸胎瘤形成实验检测转染后ESCs分化情况。当ESCs生长至亚融合状态时进行传代,去除白血病抑制因子(LIF)并悬浮培养制备EBs。用RT-PCR技术和Western blotting方法分别检测3组EBs培养7 d时CREG和cleaved caspase-3蛋白和mRNA的表达, Annexin V/PI流式细胞术检测EBs细胞凋亡情况。结果: 经pDS-shRNA-CREG逆转录病毒真核表达载体及 GFP的质粒载体转染获得稳定转染的细胞克隆。AKP染色证实R1、R1-GFP和R1-shCREG 3组ESCs均保持未分化状态。同时,体内和体外分化研究证实,R1和R1-GFP组细胞具有三胚层分化能力,小鼠心肌内注射的ESCs形成畸胎瘤组织;而R1-shCREG则不能够形成畸胎瘤类似物,细胞分化能力降低,且细胞死亡现象增加。培养7 d的R1-shCREG/EB组 CREG蛋白表达量较R1/EB组和R1-GFP/EB组均降低; CREG的mRNA表达下降;而cleaved caspase-3 mRNA和蛋白表达均显著升高;同时, R1-shCREG/EB细胞凋亡率明显升高。结论: 下调CREG表达可抑制ESCs分化,促进ESCs来源的EBs细胞凋亡的发生。     

绿色荧光蛋白、菲立磁体外双标记神经干细胞及其活性的研究

目的探索用绿色荧光蛋白(green fluorescent protein,GFP)重组逆转录病毒和菲立磁(超顺磁性氧化铁,,Superparamagnetic Iron Oxide,SPIO)体外双标记神经干细胞的可行性及其对细胞活力的影响。方法用病毒介导的GFP基因转导大鼠胚胎神经干细胞。用SPIO和Lipofectamine体外磁化标记经GFP标记的胚胎神经干细胞。用荧光显微镜观察双标记神经干细胞,并行普鲁士蓝染色,了解转染成功率。MTT(四甲基偶氮唑蓝)法检测标记神经干细胞的细胞增殖活力。结果双标记神经干细胞分化的细胞荧光显微镜下观察见发出绿色荧光,普鲁士蓝染色呈阳性。而未标记神经干细胞荧光显微镜下观察未见发出绿色荧光,普鲁士蓝染色呈阴性。MTT法检测发现GFP和SPIO双标记神经干细胞的增殖活力与未标记神经干细胞相比无改变。结论绿色荧光蛋白重组逆转录病毒和菲立磁可以有效地标记体外分离培养的大鼠胚胎神经干细胞。双标记神经干细胞的增殖、分化活力与未标记神经干细胞相比无改变。     

绿色荧光蛋白小鼠骨髓间充质干细胞的体外培养及成骨诱导

目的分离培养绿色荧光蛋白(GFP)小鼠骨髓间充质干细胞(MSCs)并成骨诱导分化。方法密度梯度离心法分离GFP小鼠MSCs，体外传代扩增，条件培养基成骨方向诱导，利用荧光显微镜、HE染色、细胞化学和免疫组织化学方法进行观察。结果MSCs在传代和诱导分化过程中稳定表达GFP，经成骨方向诱导10d后，出现大量碱性磷酸酶和骨钙素染色阳性细胞。结论成功分离了GFP小鼠MSCs，并体外向成骨方向诱导分化。     

人包皮成纤维细胞来源的多能性干细胞的建立

目的 探讨人包皮成纤维细胞重编程为诱导多能性干细胞(iPSCs)的方法.方法 利用反转录病毒携带绿色荧光蛋白(GFP)作为指示,转导到人包皮成纤维细胞中,确定最佳的病毒感染剂量,应用经典的Yamanaka方法,联合小分子物质组蛋白去乙酰化酶抑制剂丙戊酸盐( VPA)诱导人包皮成纤维细胞为多能性干细胞.通过碱性磷酸酶(AKP)染色及免疫荧光细胞化学方法,检测人胚胎干细胞多能性标记物在所建立的诱导多能性干细胞(iPSCs)中的表达,RT-PCR检测拟胚体(EB)三胚层特异基因的表达.结果 以GFP作为指示,建立了高效的反转录病毒感染体系,将人包皮成纤维细胞重编程为iPSCs;所建立的iPSCsAKP染色阳性,表达人胚胎干细胞多能性标记物OCT4、TRA-1-60和TRA-1-81,体外诱导分化后具有三胚层特异基因的表达;小分子物质VPA的添加并未显著提高重编程效率.结论 在GFP所指示的最佳病毒感染剂量下,联合小分子物质VPA成功建立了人包皮成纤维细胞来源的iPSCs.     

可视化鼻咽癌细胞模型CNE2-EGFP的建立

目的利用pEGFP-N1质粒转染鼻咽癌细胞株,建立稳定表达绿色荧光蛋白的鼻咽癌细胞株CNE2-EGFP．为整体鼻咽癌可视化研究提供良好的试验材料。方法利用脂质体转染法,将pEGFP—N1质粒转染鼻咽癌细胞株．通过G418抗性筛选、亚克隆扩增获得GFP稳定表达的人鼻咽癌细胞株CNE2-EGFP。结果成功获得多个稳定表达GFP的人鼻咽癌细胞CNE2-EGFP细胞株,比较CNE2和CNE2-EGFP发现,两者生长曲线、细胞周期分布、裸鼠成瘤能力等均无显著性差异。结论建立了非G418依赖、稳定表达GFP且保持母株细胞特性的人鼻咽癌细胞株CNE2-EGFP,为鼻咽癌整体可视化研究打下了坚实的基础。     

Sox-2基因表达的变化在小鼠EST模型药物胚胎毒性评价中的应用

目的: 建立小鼠胚胎干细胞实验 (EST) 模型,验证该模型检测胚胎毒性的有效性,探讨未分化基因 Sox-2 表达水平的变化对药物胚胎毒性评价的作用。方法: 体外培养小鼠胚胎干细胞(ES),通过形态学观察、核型分析和碱性磷酸酶(AKP)染色等方法鉴定ES;MTT法检测5-氟尿嘧啶(5-FU)、苯妥英钠(DPH)和青霉素G(penicillin G)对ES的毒性作用,RT-PCR半定量分析法检测3种药物对未分化基因 Sox-2 表达的影响。结果: 成功建立EST 模型。5-FU、DPH和penicillin G细胞毒性检测结果表明,其胚胎毒性依次为强、弱和无,与临床药物毒性相一致。 Sox-2 基因表达结果显示,随药物浓度增高及毒性增加, Sox-2 基因表达量逐渐高于阴性对照组,但均低于未分化ES细胞。结论: 研究结果显示, Sox-2 基因的表达水平与药物毒性密切相关,可用于初步评价EST模型中药物的胚胎毒性。     

20S蛋白酶体β5亚单位基因沉默对人骨髓间充质干细胞增殖能力的影响

目的:应用基因沉默技术,观察下调20S蛋白酶体β5亚单位(PSMB5)对人骨髓间充质干细胞(hBMSCs)增殖能力的影响及其潜在机制,寻求调节干细胞活力的关键靶点。方法:构建PSMB5-shRNA慢病毒载体感染早期hBMSCs,根据感染条件不同将细胞分为绿色荧光蛋白(GFP)对照组和shRNA组。倒置荧光显微镜观察转染效率,RT-PCR和免疫印迹检测PSMB5沉默效率,荧光分光光度法检测蛋白酶体活性;BrdU掺入实验观察PSMB5基因沉默对早期hBMSCs增殖潜能的影响,流式细胞术检测细胞周期分布,免疫印迹检测细胞周期相关蛋白1(cyclin D1)及其激酶CDK4的表达变化。结果:慢病毒感染早期hBMSCs 72 h可见约95%以上细胞表达绿色荧光蛋白。shRNA组PSMB5 mRNA和蛋白表达水平较GFP对照组分别降低93.31%±0.59%和56.83%±13.31%,且蛋白酶体活性较对照组降低37.47%±0.41%。此外,shRNA组BrdU阳性率26.14%±8.13%较对照组49.53%±11.18%显著降低,G1期细胞较GFP对照组增多,而S期和G2/M期细胞却较对照组减少,Cyclin D1和CDK4的表达水平分别下降54.55%±7.76%和63.26%±15.76%。结论:PSMB5基因沉默能够下调Cyclin D1和CDK4的表达,导致细胞G1/S转换停滞,影响hBMSCs增殖潜能。     

鸡胚精原干细胞转染EGFP获得转基因精子的研究

目的:通过精原干细胞体外转染外源基因获得转基因精子.方法:孵化16 d的鸡胚睾丸,酶消化获取精原干细胞(SSCs),纯化后体外培养和传代扩增,碱性磷酸酶(AKP)活性和胚胎阶段特异性抗原(SSEA-1)免疫荧光鉴定.电穿孔介导EGFP转染SSCs,G418筛选8 d后将阳性SSCs移植入经白消安处理的实验鸡体内.结果:移植后25 d采集实验鸡精液检测,精子密度为3.87(1010/L),表达EGFP的成熟精子比率为4.25%;移植后85 d,精子密度为3.27(1011/L),表达EGFP的成熟精子比率为16.25%.睾丸组织冰冻切片显示,曲精细管中转染后的SSCs定居在曲精细管的基底部,有表达EGFP蛋白生精细胞.收集实验鸡精液,提取DNA进行PCR扩增EGFP基因,经凝胶电泳检测,精液DNA的条带清晰,与目的片段大小一致.结论:鸡胚精原干细胞体外转染EGFP可获得转基因精子,为进一步生产转基因模型动物拓宽了途径.     

慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞

背景:对牙髓干细胞进行稳定高效安全的体外标记是示踪技术中首先需要解决的问题,也是牙齿再生体内研究的基础。目的:探讨慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞的理想条件及方法,并确定其转染后是否保持干细胞特性。方法:通过改良酶消化法获得大鼠牙髓干细胞,对其免疫表型及分化潜能进行鉴定,以感染复数为5,10,25,50和100的慢病毒载体介导绿色荧光蛋白作用24 h和48 h,倒置显微镜下检测转染率和荧光强度,并对大鼠牙髓干细胞感染前后的克隆增殖能力、细胞周期及牙向分化能力进行比较,评价感染对其生物学特性的影响。结果与结论:流式细胞仪检测结果显示大鼠牙髓干细胞STRO-1和CD146表达阳性,CD34和CD45表达阴性,经相应诱导培养后可向成骨和成脂分化。当感染复数为50,作用时间为48 h时,转染效率最高,荧光表达最强;感染前后细胞增殖、克隆形成率及细胞周期等方面差异无显著性意义(P0.05),碱性磷酸酶阳性表达。说明感染复数为50作用48 h是慢病毒载体介导绿色荧光蛋白转染大鼠牙髓干细胞的理想条件,且不影响牙髓干细胞生物学特性,为大鼠牙髓干细胞的体内研究提供了可靠的示踪方法。     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

The Beauty of the Network in the Brain and the Origin of the Mind in the Control of Behavior

Behavior is not adequately described as a stimulus-response process. It is initiated by the animal and is generated because of its expected outcome in the future. The outcome can be good or bad for the animal. The brain is in charge of the selection process. This is the basic function of the brain. Taking Drosophila as a study case, this paper discusses initiating activity, several examples of outcome expectations, trying out (the internal search for a suitable behavior), chaining of actions, and the functional roles of chance in action selection. It takes mental processes and states such as goals, intentions, feelings, memories, cognition, and attention as higher levels of behavioral control that have their origin in biological evolution.     

Adequate function of the cell: Interactions between the needs of the cell and the needs of the organism

The continuous maintenance of cell adequate function (cellular stress) is considered to be the main feature which characterizes the activity of a live cell. In accordance with the concept of cellular stress, the transition of the cell to the state of adequate funcitoning represents a dual commitment. The cell must function adequately to respond to the demands of the organism and to provide for its own needs. Thus, it is proposed that two systems exist for the regulation of the adequate function of the cell: one enabling the cell to respond to the demands of the organism, and the other providing for the needs of the cell itself. The hormonal system of the organism represents the first of these mechanisms, and I believe that another system exists for regulation of intracellular needs. Adequate function of the cell is determined by the regulative, controlling and defense mechanisms. The balance between the needs of the cell and of the organism should be a concern of physician, since it provides for the optimal function and health of the organism as a whole.     

On the development of the sympathetic chain and the adrenal medulla in the mouse

Summary The first visible primordia of the sympathetic chain appear in the anterior thoracic region and run caudally through 8 segments in an embryo of 11 gestation days. At 12 gestation days the trunk reaches from the base of the skull into the sacral region. During the following days the trunk develops into ganglia with interganglionic fibers.At 16 gestation days there is a well developed trunk with a cranial ganglion cervicale superior and a ganglion stellatum in the anterior part of the thoracic region. In the remaining sympathetic chain there are segmentally arranged ganglia but in this common pattern large differences are noticed.At the 13th gestation day the first signs of the adrenal medulla and the splanchnic plexus appear in the form of sympathoblasts ventral to the sympathetic chain. The migration of sympathoblasts into the primordia of the adrenal cortex goes on for 3 days while the migration to the splancnic plexus in the mesenchym ventral and lateral to the aorta goes on at least 2 more days.     

Anatomical relationship between the morphology of the styloid process of the ulna and the attachment of the radioulnar ligaments

The radioulnar ligaments are the major stabilizers of the distal radioulnar joint under dynamic loading; however, anatomical detail regarding their attachment on the middle and distal thirds of the styloid process of the ulna remains unclear. Because previous anatomical studies included only old cadavers, their anatomical findings might not reflect the morphological features of younger and healthy specimens. This study investigated the anatomical features of the distal ulna, particularly the styloid process, to determine the attachment of the radioulnar ligaments to the styloid process and verified their direction and attachment to the styloid process in younger and healthy donors using magnetic resonance imaging (MRI). We investigated the morphological features of the distal ulna of 12 cadaveric wrists using micro-computed tomography (micro-CT). We also visualized and measured the distribution of the cortical bone thickness. We histologically analyzed three specimens in the axial plane and macroscopically analyzed seven specimens to examine the attachment of the radioulnar ligaments to the styloid process. In addition, we evaluated five wrists from living volunteers using 3.0 Tesla MRI. The distal ulna has a ridge on the dorsoradial aspect of the styloid process that corresponds to the attachment of the radioulnar ligaments. Micro-CT images after data processing revealed that the cortical thickness of the dorsoradial quadrant was thicker than that of the other quadrant at the proximal slice of the styloid process (p < 0.01), and that of the dorsoulnar (p = 0.021) and ulnopalmar (p < 0.01) quadrants at the middle slice. Histological analyses showed that the radioulnar ligaments were attached to the middle and distal thirds of the styloid process via chondral-apophyseal entheses. The direction of the fiber was dorsal in the middle third of the styloid process and changed to palmar in the distal third of the styloid process. The direction and attachment of the radioulnar ligaments on the styloid process were confirmed using MRI for younger and healthy participants. The radioulnar ligaments were attached to the dorsoradial ridge of the styloid process, which was confirmed by cortical bone thickening, histology at the attachment sites, and in vivo MR imaging. The directions of the radioulnar ligaments sterically intersected, which would satisfy both slipping stability and rotational mobility. These anatomical findings may provide the basis for biomechanical consideration of distal radioulnar joint stabilization.     

指背腱膜滑动与近侧指间关节屈曲关系及临床意义

目的:探讨指背腱膜滑动距离与近侧指间关节(PIP)屈曲关系,为临床修复提供解剖学基础。方法:男性成人新鲜尸体标本10侧30指(示、中、环指各10指),切除手指皮肤,不破坏腱鞘、屈肌支持带、伸肌支持带、内在肌及外在肌,使肌腱保持正常的生理状态,分别测量各指中央束(CS)、侧束(LB)在PIP屈曲45°和90°时的滑动距离。结果:当PIP屈曲45°时,CS滑动距离为(2.7±0.4)mm,LB滑动距离为(2.8±0.6)mm;当PIP屈曲90°时,CS滑动距离为(4.3±0.7)mm,LB滑动距离为(4.8±0.6)mm。结论:指背腱膜滑动距离减少,严重影响手指的屈曲功能。对于指背腱膜的新鲜性损伤应予以精确修复;对于陈旧性损伤的修复应确保指背腱膜的正常滑动范围。     

On the musculature of the gastro-intestinal tract of the guinea-pig

Summary The small intestine, caecum, colon and part of the stomach of guinea-pigs were studied by light microscopy, using semithin sections of plastic embedded specimens. The muscular coat is thicker in the duodenum than in the rest of the small intestine. The ratio between longitudinal and circular muscle is 1:4.6 in the duodenum and it increases regularly along the small intestine, to reach 1:2 in the terminal ileum. In the caecum, shape and sectional area of the muscle tissue were analyzed along the full length of the taeniae. In the caecal circular muscle there is a characteristic change in the arrangement of the muscle bundles from the regions in the centre of the haustrations to the regions of the grooves between haustrations or to those lying beneath a taenia. The functional significance of the taeniae is discussed in terms of an arrangement allowing reduction of the lumen of the organ (which at the level of the grooves between haustrations acquires a triangular outline) more efficiently than if the longitudinal musculature were spread over the entire surface of the organ. Haustrations are present also on one side of the wall of the ascending colon where there is no longitudinal muscle layer. In the descending colon the structure of the wall is examined in different functional states, namely in the regions between fecal pellets (constricted regions) and in the regions around a fecal pellet (moderately distended regions). The musculature increases considerably in thickness in the constricted regions (both muscle layers being actively contracted), while the mucosa and submucosa are thrown into prominent longitudinal folds. These folds produce occlusion of the lumen when the circular muscle has shortened by about 50%.     

On the origin of the corticotectal projections in the cat

Summary Stereotaxic injection of horseradish peroxidase into the superior colliculus produced retrograde labelling of layer V pyramides in the Clare Bishop area and the lateral bank of the suprasylvian sulcus, in area 17,18 and 19. Single labelled cells were also found scattered in the splenial, the suprasplenial, the lateral and the suprasylvian gyri. In the cruciate sulcus no labelled cells were observed. Autoradiographically, the lateral bank of the suprasylvian sulcus was also shown to give rise to fibres to the superior colliculus.