氯甲基苯甲酰氨荧光染料标记大鼠骨髓间质干细胞的体内示踪

背景：目前广泛应用于骨髓间质干细胞的标记方法主要为绿色荧光蛋白标记法，但标记与固定程序复杂，操作繁琐，易出现偏差。氯甲基苯甲酰氨(Chloromethyl-benzamidodialkylcarbocyanine, CM-Dil)是亲脂性膜荧光染料，能够与含有肽和蛋白质的硫氢基结合进而标记整个细胞。 目的：探讨CM-Dil对大鼠骨髓间质干细胞体内示踪的可行性。 设计、时间及地点：体内外细胞学观察，于2007-05/12在中山大学干细胞与组织工程研究中心、中山大学动物实验中心完成。 材料：SPF级Wistar大鼠40只，由中山大学实验动物中心提供。CM-Dil为美国Sigma公司产品。 方法：在体外，以标记CM-Dil的骨髓间质干细胞作为实验组，以未标记CM-Dil的骨髓间质干细胞作为对照组，比较两组细胞生长与增殖情况，MTT法检测细胞生长增殖情况。在体内，分别将标记CM-Dil的骨髓间质干细胞经门静脉移植及肝内培养，于移植后第7，15，21，30天制备肝脏切片。 主要观察指标：骨髓间质干细胞CM-Dil标记率，标记细胞在肝内定植、生长与分布。 结果：体外培养结果显示，两组骨髓间质干细胞在细胞生长、增殖、分裂以及形态学上均基本相似，在绿色光激发下，CM-Dil发出红色荧光，24 h后细胞标记率为100%，21 d后CM-Dil标记的骨髓间质干细胞荧光开始淬灭。体内实验中，经门静脉移植的骨髓间质干细胞在肝脏内主要位于间质，呈椭圆形或不规则形的分化细胞；肝内培养的骨髓间质干细胞在培养孔内呈“贴壁生长”，为椭圆形分化细胞，未发现骨髓间质干细胞进入并定植于肝组织内；CM-Dil标记的骨髓间质干细胞荧光开始淬灭的时间亦为21 d。 结论：CM-Dil染色简单、方便、稳定，荧光开始淬灭时间较长，可望成为大鼠骨髓间质干细胞体内示踪的新方法。     

荧光显微镜划痕标记染料示踪技术的改进

划痕标记染料示踪(scrape-loading and dye transfer,SLDT)技术是一种简单快速研究培养细胞间缝隙连接通讯(gap junction intercellular communication,GJIC)的方法。适用于各种培养细胞,具有方法简单、实验周期短、成本低廉的特点,自问世以来即在研究细胞通讯方面得到了广泛应     

PKH26荧光标记大鼠内皮祖细胞在慢性损伤肝内迁移示踪

背景：课题组早期研究发现门静脉回输内皮祖细胞可以改善肝纤维化，但是将其应用于临床尚有很多问题需要解决，如回输路径、细胞的分布情况等。 目的：探讨PKH26荧光标记的大鼠内皮祖细胞在慢性肝损伤肝内迁移过程中的示踪。 设计、时间及地点：细胞学体内实验，于2008-08/2009-02在北京大学人民医院肝病研究所完成。 材料：SPF级健康成年SD大鼠100只，由解放军军事医学科学院动物中心提供。红色荧光染料PKH26为SIGMA公司产品。 方法：取16只大鼠，贴壁法分离培养内皮祖细胞，并行PKH26体外标记。实验设立3组：正常对照组4只大鼠，不进行任何干预；二甲基亚硝胺组40只大鼠，通过腹腔注射二甲基亚硝胺 10 mg/kg建立肝纤维化模型；四氯化碳组40只大鼠，通过四氯化碳/橄榄油灌胃建立肝纤维化模型。造模后，二甲基亚硝胺组、四氯化碳组大鼠经尾静脉注入1 mL PKH26标记的内皮祖细胞悬液(1×106个细胞)。 主要观察指标：PKH26标记率和细胞存活率，通过荧光共聚焦显微镜观察大鼠损伤肝内内皮祖细胞的迁移及CD31的表达。 结果：流式细胞仪检测结果显示PKH26标记的内皮祖细胞阳性率为99%，荧光显微观察发现锥虫蓝染色后PKH26标记细胞存活率均>90%。输注1 d后，两种慢性肝损伤模型中的肝内均可见PKH26标记阳性的细胞出现在肝小叶内，并且随时间延长阳性细胞数增加。PKH26标记阳性的细胞主要存在于沿纤维分布的血管内皮和肝小叶内的窦内皮，且血管内皮可见CD31/PK26双阳性细胞。 结论：PKH26可以在体外成功标记大鼠内皮祖细胞，并在损伤肝内示踪。     

大鼠胚胎后肾间充质细胞体外标记的方法

背景：干细胞移植为慢性肾脏病的治疗提供了新的视角，特异性标记干细胞并体内示踪是这一领域研究的基础，目前尚无一种公认的适用于所有细胞的标记方法。 目的：观察DAPI及绿色荧光蛋白标记细胞在阿霉素肾病大鼠体内的定位及分化，探讨便捷的大鼠胚胎后肾间充质细胞的体外标记方法。 设计、时间及地点：分组对比观察，于2007-04/12在中南大学湘雅二医院完成。 材料：清洁级雌性Sprague-Dawley(SD)大鼠60只，体质量180~220 g，用于制备阿霉素肾病大鼠模型。 方法：分别将DAPI、DAPI体外标记后肾间充质细胞、绿色荧光蛋白体外标记后肾间充质细胞，通过尾静脉注入阿霉素肾病大鼠体内；于细胞移植后1，3，5周行肾组织冰冻切片，观察DAPI及绿色荧光蛋白荧光分布，并ABC免疫酶染色法检测肾组织中绿色荧光蛋白表达。 主要观察指标：比较DAPI及绿色荧光蛋白两种不同方法标记后肾间充质细胞在阿霉素慢性肾病大鼠肾内的定植情况，以及绿色荧光蛋白标记细胞不同时间点移植在肾内的定植改变。 结果：①DAPI标记细胞及单纯DAPI尾静脉注射后1周，均可在肾小管上皮细胞中观察到DAPI阳性细胞，至移植后5周仍可观察到，但随时间延长荧光有减弱趋势。②绿色荧光蛋白标记细胞移植后1周，可在肾小管上皮细胞中观察到绿色荧光蛋白阳性细胞，至移植后5周仍可观察到，荧光无明显减弱。③ABC免疫酶染色法检测，显示绿色荧光蛋白阳性细胞主要表达于近端肾小管中，肾小球系膜区亦可观察到少量表达；绿色荧光蛋白阳性产物主要表达于胞浆。绿色荧光蛋白表达半定量评分显示，标记细胞的早期应用阳性率大于晚期应用者，且随观察时间延长，阳性率有升高趋势。 结论：后肾间充质细胞的脂质体感染法绿色荧光蛋白标记是一便捷、有效的体外标记、示踪方法，适于移植细胞的短期内示踪、观察。     

改良小鼠骨髓间充质干细胞培养方法及长效荧光标记的可行性

背景：小鼠骨髓间充质干细胞培养不同于人及大鼠,培养扩展难度高,传代后干细胞活性保持时间短;又鉴于干细胞本身特点,使现有干细胞示踪方法作用时间短,这些因素均影响小鼠骨髓间充质干细胞的实验研究。 目的：观察改良小鼠骨髓间充质干细胞分离培养方法及长效荧光标记干细胞的可行性。 设计、时间及地点：观察性实验,于2008-06/12在解放军军事医学科学院完成。 材料：C57BL/6小鼠4~6周龄,体质量20 g,雌雄不拘。 方法：通过贴壁筛选和Percoll分离法,优化干细胞培养液血清和换液方式,培养扩增小鼠骨髓间充质干细胞。参照以往人和猕猴间充质干细胞培养经验,对小鼠间充质干细胞培养血清选择Hyclone的顶级胎牛血清,控制血清为培养液的10%。用LG-DMEM培养液冲出骨髓细胞后,滤去骨渣和小肌肉碎块。然后加在相对密度1.082的percoll分离液上,以1.5×106/cm2浓度接种75 cm2培养瓶。对第2代骨髓间充质干细胞进行长效CM-DiI标记。 主要观察指标：原代及传代小鼠骨髓间充质干细胞形态变化;对第2代小鼠骨髓间充质干细胞表面抗原,CD105,CD44,CD25,CD34进行流式细胞仪检测,评价改良方法获取干细胞纯度;通过成脂成骨分化,鉴定小鼠骨髓间充质干细胞的活性;观察多次传代后荧光细胞强度。 结果：①小鼠骨髓间充质干细胞原代培养在接种48 h后可见贴壁细胞,培养第7天细胞多数伸展呈梭形,也有三角形、多角形和扁平形。3代后形态趋于统一,融合状态时细胞排列呈束状、漩涡状或放射状。②骨髓间充质干细胞特异性抗原高表达CD105,CD44;造血系抗原低表达CD34和CD45。③骨髓间充质干细胞向成骨分化过程中,纺锤形的突起逐渐消失,胞体增大,培养10 d后,部分细胞呈聚集生长,随细胞生长密度的增长形成多层的结节结构。在脂肪诱导体系中,可见细胞由成纤维样逐渐缩短,9 d后胞浆中出现脂肪滴,油红O染色可见红色的脂肪颗粒。④首次标记,荧光显微镜观察可见长效CM-DiI标记在细胞膜发橙色光,标记率在80%以上。流式细胞仪检测CM-DiI细胞标记率可到47%以上,第4代培养细胞仍可见较多标记细胞。 结论：改良方法早期第2代就可获得高浓度干细胞,同时长效CM-DiI标记方法稳定,标记率高,可作体内细胞示踪。     

Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro

Summary The effect of several benzodiazepines (clonazepam, diazepam, Ro 5-4864, Ro 15-1788) and two pineal gland indoleamines (N-acetylserotonin, melatonin) on the spontaneous proliferation of mouse spleen lymphocytes was estimated in vitro by the 3 H-thymidine uptake assay. It was found that diazepam and Ro 5-4864 (a selective peripheral-type benzodiazepine receptor ligand) produced the concentration-dependent inhibition of 3 H-thymidine incorporation into the DNA of these cells. Ro 15-1788, a specific central-type receptor ligand, evoked a slight inhibitory effect in a high concentration (10^–4M), whereas clonazepam did not produce any significant inhibition. When Ro 5-4864 was tested in combination with diazepam, the inhibition of lymphocyte proliferation did not exceed the effect of diazepam given alone. Ro 15-1788 was unable to reverse the inhibitory action of diazepam in the same experimental conditions. Melatonin and its precursor N-actetylserotonin tested in the concentration range of 10^–4–10^–8 M had no significant influence on the spleen lymphocyte DNA replication in our assay system.These data suggest that diazepam inhibition of lymphocyte proliferation is mediated by peripheral-type sites. Additionally, the fact that melatonin and N-acetylserotonin were unable to affect 3 h-thymidine incorporation argues against any benzodiazepine receptor mediated effect of pineal indoleamines on a cellular proliferation.     

T lymphocytes affect the development of sympathetic innervation of mouse spleen

We investigated whether the development of sympathetic innervation of the spleen is affected by lymphoid cells. Splenic noradrenaline (NA) levels of athymic nude mice (nu/nu) and normal thymus-bearing littermates (nu/+) were determined at different times during ontogeny. While no differences were detected at birth, higher splenic NA levels were found in 7-, 11-, and 21-day-old athymic mice. Thymus transplantation or thymocyte injection to newborn nude mice resulted in splenic NA levels comparable to those of normal nu/+ mice. Histochemical studies fully confirmed such differences. Taken together with previous studies, these results suggest that T lymphocytes or their products exert an inhibitory influence on sympathetic nerve fibers, thus leading to decreased NA content in the spleen. The data also illustrate the capacity of a nonneuronal cell in a peripheral organ to affect the process of autonomic innervation of this organ.     

Histological methods for ex vivo axon tracing: A systematic review

Objectives: Axon tracers provide crucial insight into the development, connectivity, and function of neural pathways. A tracer can be characterized as a substance that allows for the visualization of a neuronal pathway. Axon tracers have previously been used exclusively with in vivo studies; however, newer methods of axon tracing can be applied to ex vivo studies. Ex vivo studies involve the examination of cells or tissues retrieved from an organism. These post mortem methods of axon tracing offer several advantages, such as reaching inaccessible tissues and avoiding survival surgeries.

Methods: In order to evaluate the quality of the ex vivo tracing methods, we performed a systematic review of various experimental and comparison studies to discern the optimal method of axon tracing.

Results: The most prominent methods for ex vivo tracing involve enzymatic techniques or various dyes. It appears that there are a variety of techniques and conditions that tend to give better fluorescent character, clarity, and distance traveled in the neuronal pathway. We found direct comparison studies that looked at variables such as the type of tracer, time required, effect of temperature, and presence of calcium, however, there are other variables that have not been compared directly.

Discussion: We conclude there are a variety of promising tracing methods available depending on the experimental goals of the researcher, however, more direct comparison studies are needed to affirm the optimal method.     

Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract

The mouse is an extremely valuable model for studying vagal development in relation to strain differences, genetic variation, gene manipulations or pharmacological manipulations. Therefore, a method using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was developed for labeling vagal innervation of the gastrointestinal (GI) tract in embryonic and postnatal mice. DiI labeling was adapted and optimized for this purpose by varying several facets of the method. For example, insertion and crushing of DiI crystals into the nerve led to faster DiI diffusion along vagal axons and diffusion over longer distances as compared with piercing the nerve with a micropipette tip coated with dried DiI oil. Moreover, inclusion of EDTA in the fixative reduced leakage of DiI out of nerve fibers that occurred with long incubations. Also, mounting labeled tissue in PBS was superior to glycerol with n-propyl gallate, which resulted in reduced clarity of DiI labeling that may have been due to DiI leaking out of fibers. Optical sectioning of flattened wholemounts permitted examination of individual tissue layers of the GI tract wall. This procedure aided identification of nerve ending types because in most instances each type innervates a different tissue layer. Between embryonic day 12.5 and postnatal day 8, growth of axons into the GI tract, formation and patterning of fiber bundles in the myenteric plexus and early formation of putative afferent and efferent nerve terminals were observed. Thus, the DiI tracing method developed here has opened up a window for investigation during an important phase of vagal development.     

Characterization of the central nervous system innervation of the rat spleen using viral transneuronal tracing

Splenic immune function is modulated by sympathetic innervation, which in turn is controlled by inputs from supraspinal regions. In the present study, the characterization of central circuits involved in the control of splenic function was accomplished by injecting pseudorabies virus (PRV), a retrograde transynaptic tracer, into the spleen and conducting a temporal analysis of the progression of the infection from 60 hours to 110 hours postinoculation. In addition, central noradrenergic cell groups involved in splenic innervation were characterized by dual immunohistochemical detection of dopamine-beta-hydroxylase and PRV. Infection in the CNS first appeared in the spinal cord. Splenic sympathetic preganglionic neurons, identified in rats injected with Fluoro-Gold i.p. prior to PRV inoculation of the spleen, were located in T(3)-T(12) bilaterally; numerous infected interneurons were also found in the thoracic spinal cord (T(1)-T(13)). Infected neurons in the brain were first observed in the A5 region, ventromedial medulla, rostral ventrolateral medulla, paraventricular hypothalamic nucleus, Barrington's nucleus, and caudal raphe. At intermediate survival times, the number of infected cells increased in previously infected areas, and infected neurons also appeared in lateral hypothalamus, A7 region, locus coeruleus, subcoeruleus region, nucleus of the solitary tract, and C3 cell group. At longer postinoculation intervals, infected neurons were found in additional hypothalamic areas, Edinger-Westphal nucleus, periaqueductal gray, pedunculopontine tegmental nucleus, caudal ventrolateral medulla, and area postrema. These results demonstrate that the sympathetic outflow to the spleen is controlled by a complex multisynaptic pathway that involves several brainstem and forebrain nuclei.     

In vivo application of HRP crystals: a new and efficient method for tracing neural connections in insects

Solid HRP pellets prepared with a 2.5% Triton X-100 aqueous solution were implanted either into corpora allata or applied onto neurohemal organs of a cricket. The method presents two advantages: it allows one to perform "in vivo" instead of "in vitro" experiments, and detergent HRP pellets are easy to manipulate. Thus, this method combines simplicity with accuracy and appears to be very useful in tracing neural connections in the insect nervous system.     

In vitro biocytin injection into perinatal mouse brain: a method for tract tracing in developing tissue

Injection of biocytin provides an effective method for labeling axonal projections. Several difficulties arise when this technique is employed in fetal or early postnatal animals in vivo, including limited access to injection sites and extended post-injection survival periods. To circumvent these problems, we adapted the technique of extracellular biocytin injection for use in explanted brain hemispheres of developing mice. Briefly, entire brain hemispheres from perinatal mice (E16-P9) were removed and placed in oxygenated aCSF in a brain slice recording chamber. Following visually guided injection of biocytin (2%) into the prelimbic cortex, the brains were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) for varying periods of time and then immersion-fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. The next day, the brains were sectioned and processed for biocytin histochemistry using the avidin-biotin-complex method. We examined the method of injection, electrode type, time of injection, and post-injection incubation period. We found that in E16-P9 animals iontophoresis of biocytin using 8- to 12-megaohm patch clamp electrodes for a duration of 10 min provides optimal axonal labeling. Post-injection incubation times of four or more hours are sufficient for labeling fine caliber collaterals as well as axon bundles that reach distances over 3 mm. In vitro injection of biocytin into explanted brain hemispheres provides a quick and easy method for tract tracing in developing brains.     

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.     

A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques

A simple method for the simultaneous localization of an antigen and a retrogradely transported fluorescent dye within single neurons is described. The method is based upon: (1) the efficiency of retrograde neuronal labeling with the fluorescent marker ‘true blue’; (2) the near-quantitative persistence of retrogradely transported true blue localizations after subsequent processing of the tissue for immunohistochemistry; and (3) the ability to distinguish clearly between true blue and immunohistochemically-stained cells by simply using appropriate excitation wavelengths for each.

First we examined the characteristics of two fluorescent tracers which are effectively transported over long distances in the rat. The results confirm that true blue and bisbenzimide are transported from terminal fields to parent cell bodies and that both tracers are taken up and transported by damaged fibers and by undamaged fibers-of-passage. No evidence for transneuronal transport of either dye in the anterograde or in the retrograde direction was found. Next, using the projection of the paraventricular nucleus of the hypothalamus (PVH) to the spinal cord as a test system, it was found by direct cell counts that a considerably greater percentage of cells in a specific subdivision of the nucleus was labeled following true blue injections into the spinal cord (88%) than was labeled after comparable injections of bisbenzimide (58%), or horseradish peroxidase (HRP) (24%), or after HRP-polyacrylamide gel implants (39%). A comparison of cell counts of true blue-labeled cells in normal material and in series of adjacent sections that were processed for immunohistochemistry suggested that only about 5% of the true blue-labeled cells are no longer detectable following the immunohistochemical procedures employed. Finally, by coupling the fluorescent retrograde tracing method with an indirect immunofluorescence technique, we have been able to localize reproducibly both retrogradely transported true blue and an antigen in individual neurons. The perfusion and staining method employed provided adequate staining of cell bodies that cross-reacted with antisera to one or another of the 9 peptides and enzymes tested.

The results indicate that true blue is a versatile and highly sensitive marker for retrograde tracing studies that can be used alone, or in conjunction with immunohistochemical methods to establish the identity of cell groups with respect to their biochemistry as well as their efferent connections.