小鼠气管平滑肌原代细胞的分离、培养与鉴定

目的建立一种可靠的通过组织块贴壁法分离培养原代小鼠气管平滑肌细胞及免疫组化鉴定的方法。方法体视显微镜下立体分离小鼠气管平滑肌组织,组织块贴壁法培养原代细胞,对分离培养细胞通过免疫组化方法进行鉴定,并用MTT法对其增殖特性进行检测。结果从BALB/c雄性小鼠分离气管平滑肌组织,剪碎为1 mm3,用含1%青-链霉素的PBS及培养液漂洗,使组织块贴于培养皿底,并加入5 m L培养液,放入37℃、5%CO2的细胞培养箱培养,3~5 d后有明显梭状细胞从组织块爬出,5~6 d后,细胞可见明显"峰-谷"结构。经免疫荧光鉴定,在传代、纯化后,可得纯度为99%以上的气管平滑肌细胞。用MTT法测量其生长曲线。结论本方法操作简单、经济,获得的气管平滑肌细胞具有较好增殖能力,细胞数量和纯度能够满足后续细胞生物学实验研究的需要。     

组织贴块法建立人气道平滑肌细胞体外培养模型的研究

背景:人气道平滑肌已被证实参与气道重塑,气道平滑肌的重塑已成为慢性呼吸道疾病的主要病理改变之一。人气道平滑肌细胞的培养对慢性呼吸道疾病的研究有重要意义。组织贴块法培养人气道平滑肌细胞是原代培养人气道平滑肌细胞的基本方法之一。目的:采用组织贴块法建立人气道平滑肌体外培养模型。方法:采集人气道组织,用组织贴块法进行人气道平滑肌细胞的原代培养,获得的细胞经形态学和免疫细胞化学染色鉴定。结果:培养的细胞呈典型的"谷峰"状生长,胞浆内特异性的平滑肌肌动蛋白阳性表达,符合平滑肌细胞的形态学特征和生物学特性。结论:组织贴块法易操作,结果可信,并可培养出高纯度活性好的人气道平滑肌细胞,成功建立了体外人气道平滑肌细胞增殖模型,提供了研究慢性呼吸道疾病的细胞培养模型。     

组织贴块法建立人气道平滑肌细胞体外培养模型的研究

背景：人气道平滑肌已被证实参与气道重塑,气道平滑肌的重塑已成为慢性呼吸道疾病的主要病理改变之一。人气道平滑肌细胞的培养对慢性呼吸道疾病的研究有重要意义。组织贴块法培养人气道平滑肌细胞是原代培养人气道平滑肌细胞的基本方法之一。目的：采用组织贴块法建立人气道平滑肌体外培养模型。方法：采集人气道组织,用组织贴块法进行人气道平滑肌细胞的原代培养,获得的细胞经形态学和免疫细胞化学染色鉴定。结果：培养的细胞呈典型的”谷峰”状生长,胞浆内特异性的平滑肌肌动蛋白阳性表达,符合平滑肌细胞的形态学特征和生物学特性。结论：组织贴块法易操作,结果可信,并可培养出高纯度活性好的人气道平滑肌细胞,成功建立了体外人气道平滑肌细胞增殖模型,提供了研究慢性呼吸道疾病的细胞培养模型。     

改良组织块消化法建立大鼠气道平滑肌细胞模型

目的：建立改进大鼠气道平滑肌细胞（ASMC）的体外培养方法,为相关研究提供实验材料。方法：将组织块连续贴壁进行细胞原代培养,胰酶消化传代培养,差速贴壁进行细胞纯化,形态学及免疫细胞化学染色法进行细胞鉴定。MTT法检测PDGF-BB诱导的ASMC增殖。结果：成功培养大鼠ASMC,以改良组织块消化法最为理想。第四代平滑肌细胞纯度可达95％以上。相差显微镜下培养细胞呈典型“峰谷状”生长。免疫荧光化学染色显示特异性平滑肌肌动蛋白阳性表达。随着PDGF浓度的升高（2-80ng／ml）,MTT比色A490值呈上升趋势。与对照组相比较,80ng／ml、20ng／ml PDGF—BB组有统计学意义（P〈0．01）。结论：改良组织块消化法可缩短培养周期,在充分利用标本的基础上获得大量气道平滑肌细胞。     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

新生大鼠肺成纤维细胞的原代培养改良法及细胞鉴定

探讨新生大鼠肺成纤维细胞原代培养的改良方法及细胞鉴定。用胰酶消化组织块结合的方法提取新生大鼠肺成纤维细胞,并纯化细胞,对肺成纤维细胞进行形态学观察,用HE染色及免疫组化染色法对细胞进行鉴定,并用MTT法测定细胞生长曲线。倒置相差显微镜下观察选用改良法获得的细胞,3 d后可见组织块周边有少许细胞,5 d后组织块周围有大量细胞爬出,生长迅速,10 d接近融合。经改良后的方法纯化细胞,细胞活性状态较好的为3~5代,5代以后的细胞增殖能力下降。对第3代肺成纤维细胞进行HE染色,镜下可见形态典型的成纤维细胞,免疫组化结果显示波形蛋白(Vi-mentin)阳性表达,细胞角蛋白(cytokeratin)阴性表达。MTT法检测第3代细胞于3~5 d处于对数生长期。胰酶消化组织块结合法是一种可靠快速的肺成纤维细胞分离纯化的培养方法,使用这种方法可得到具有典型形态特征且活性较好的肺成纤维细胞,初学者容易掌握。     

小鼠主动脉血管平滑肌原代细胞的分离培养及鉴定

目的:血管平滑肌细胞在人类心血管疾病中具有重要的作用,而作为重要的遗传学研究模式生物的小鼠血管平滑肌材料有限,因此建立一种简单高效的小鼠血管平滑肌原代细胞分离培养方法很重要。方法:分离小鼠主动脉中膜层,胶原酶消化法获得原代平滑肌细胞,免疫荧光方法检测细胞的纯度和分化状态;分离平滑肌细胞特异的报告小鼠的平滑肌细胞,LacZ染色鉴定。结果:用该方法分离的原代平滑肌细胞生长迅速,3d后即可达5×106个。免疫荧光显示,细胞传至第3代后纯度在98%以上,细胞传至8代分化状态没有改变。LacZ染色鉴定报告小鼠分离的3代平滑肌细胞98%以上显示特异的蓝染。两种实验证明,应用此方法分离原代平滑肌细胞可以满足平滑肌体外功能实验的需求。结论:与传统的组织块培养法相比,该方法操作简便、经济,可以获得更多高纯度的血管平滑肌细胞。     

SD大鼠胸腹主动脉平滑肌细胞的原代培养及鉴定

目的建立原代大鼠胸腹主动脉平滑肌细胞培养方法,为研究心脑血管动脉粥样硬化疾病提供重要的载体和工具细胞。方法选取6～8周龄SD大鼠2只,剪开胸腹腔,剥离主动脉,刮除血管内、外膜,经0.2%Ⅱ型胶原酶/弹性蛋白酶消化后,剪碎成块,种瓶进行原代培养。通过细胞形态学观察、α平滑肌肌动蛋白免疫细胞化学染色法鉴定所培养的目的细胞。结果接种于培养瓶中的血管组织块培养48h后开始贴壁;72h后细胞以组织块为中心,向外迁移,"岛屿状"细胞团簇初步形成;96h后原代细胞集落逐渐融合,铺满瓶底,呈现典型的"峰-谷"样生长;第一代传代细胞形态基本保持不变,高倍镜下细胞呈三角形或星形。免疫细胞化学染色显示,细胞α平滑肌肌动蛋白阳性率达99%以上。结论复合酶消化法结合组织块法能够成功高效地分离培养出原代大鼠胸腹主动脉平滑肌细胞。     

大鼠主动脉内皮细胞的分离培养和鉴定

目的:探索大鼠主动脉原代内皮细胞体外培养方法,为体外研究提供细胞模型。方法:分离大鼠主动脉,直接贴壁于培养皿中,荧光倒置显微镜观察细胞形态,免疫组化Ⅷ因子相关抗原染色鉴定细胞。结果:约24小时组织块边缘有游离的新生细胞长出,7天即融合成片。消化传代后细胞呈短梭形或三角形,单层生长,铺路石状,Ⅷ因子表达阳性,呈指数增殖。冻存后复苏细胞活性均超过90%。结论:用贴壁法成功建立了大鼠血管内皮细胞体外培养方法,冻存细胞存活率高,为体外研究提供了稳定的模型。     

人妊娠子宫平滑肌细胞的原代培养Transgelin蛋白的检测

为了建立最佳的人妊娠子宫平滑肌细胞的原代培养方法和初步检测子宫平滑肌细胞中Transgelin蛋白的表达,采用组织块贴壁法和酶消化法进行人妊娠子宫平滑肌原代培养.发现组织贴块培养的细胞呈典型的梭状肌细胞样生长,经过传代纯化,通过免疫细胞化学方法检测平滑肌肌动蛋白(smooth muscle acting,SMA)进行细胞鉴定及检测Trangsgelin(smooth muscle 22 alpha,SM22-α)蛋白,得到SMA、snd2-α荧光免疫细胞化学染色为阳性.结果表明组织贴块法对妊娠子宫平滑肌细胞损伤小,可获得状态良好的纯净的子宫平滑肌细胞,子宫平滑肌细胞中大量表达sm2-α,为进一步研究sm22-α在子宫平滑肌细胞中作用打下基础.     

Photoperiodic influence on the innervation of the ductus epididymidis and ductus deferens of the Djungarian hamster,Phodopus sungorus: electron-microscopic and biochemical results

Summary The influence of long (light: dark 168) and short (light:dark 816) photoperiods on the autonomic innervation of the ductus epididymidis and the ductus deferens of Phodopus sungorus was studied using electron microscopy with morphometric analyses, and biochemical methods. At short photoperiods, only the large smooth muscle cells in the ductus deferens became atrophic, the number of mainly adrenergic varicosities in the smooth muscle layer decreased, and the mean distance between varicosity and smooth muscle cells increased. The content of noradrenaline was determined by high-performance liquid chromatography with electrochemical detection. For the ductus deferens, the noradrenaline content was reduced at LD 816 to less than 10% of the initial value. Short photoperiods are proposed to influence only the adrenergic innervation of the large smooth muscle cells of the ductus deferens. These cells are believed to exert a trophic influence on their nerves.     

Expression of cysteine sulfinate decarboxylase (CSD) in male reproductive organs of mice

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine, but it is still controversial whether the male reproductive organs have the function to synthesize taurine through CSD pathway. The present study was thus undertaken to detect CSD expression in male mouse reproductive organs by RT-PCR, Western blot and immunohistochemistry. The results show that CSD is expressed both at the mRNA and protein levels in the testis, epididymis and ductus deferens. The relative levels of both CSD mRNA and protein increase from the testis to the epididymis and to the ductus deferens. Immunohistochemical results demonstrate that the main cell types containing CSD are Leydig cells of testis, epithelial cells and some stromal cells throughout the efferent ducts, epididymis and ductus deferens. These results suggest that male genital organs have the function to produce taurine through the CSD pathway, although quantifying the relation of CSD expression to taurine synthesis and the exact functions of taurine in male genital organs still need to be elucidated in future studies.     

Cholinesterases and choline acetyltransferase in the ductus deferens of the guinea-pig

Summary Several authors have reported that longitudinal and circular muscle layers of the guinea-pig ductus deferens possess a rich adrenergic innervation. Cholinergic innervation has been doubted by several authors, especially its presence in the longitudinal muscle layer. Acetylcholinesterase and butyrylcholinesterase were demonstrated, by electronmicroscopic examination of both muscle layers of the guinea-pig ductus deferens, to be localized on the axolemma of the nerve endings and in smooth-muscle fibres, on sarcolemma, in the intracellular caveolae and in the intercellular space. Activity of cholinesterases and choline acetyltransferase was measured by the radiometric method and was found in both muscle layers. The activity of butyrylcholinesterase was higher than that of acetylcholinesterases in the homogenates of the whole ductus deferens and in the longitudinal muscle layer. In the circular muscle layer, the activity of acetylcholinesterase was higher than the activity of butyrylcholincsterase. In both muscle layers, we also found choline acetyltransferase, the activity being stronger in the circular layer. The localization of cholinesterases in smooth-muscles in the same places as the calcium and muscarinic acetylcholine receptors is discussed, together with the possibility that the enzyme is in some way involved in the excitation-contraction mechanism of smooth muscle.Part of this work was presented at the 2^nd International Meeting on Cholinesterases, Bled, Yugoslavia, 1983     

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster

Summary The postnatal maturation of regions of the epididymis and intragonadal segment of the deferens duct was studied in the rat by light-and transmission electron microscopy. Maturation of the genital duct starts in the distal cauda epididymidis and ductus deferens after one week of life, and one week later, in the more cranial segments of the epididymis. Epithelial principal cells and peritubular contractile cells are structurally mature 35 days after birth. The synchronous changes of these cells indicate that the same factors control their postnatal maturation. The epithelial principal cells obtain an endocytotic apparatus and long stereocilia, whereas peritubular cells acquire contractile features. These changes are associated with a progressive increase in the immunoreaction for smooth muscle actin in both cell types. Smooth muscle myosin is detected in the apical region of the epithelial cells and the peritubular cell cytoplasm by day one of postnatal development. The differentiation of contractile cells in the wall is accompanied by progressive organization of the pericellular matrix into a continuous basement membrane. Although fibronectin is visible at birth, it is gradually removed from the tubule wall.     

Inhibitory effect of mitragynine, an analgesic alkaloid from Thai herbal medicine, on neurogenic contraction of the vas deferens

The effect of an indole-alkaloid mitragynine isolated from the Thai medicinal herb kratom (Mitragyna speciosa) on neurogenic contraction of smooth muscle was studied in guinea-pig vas deferens. Mitragynine inhibited the contraction of the vas deferens produced by electrical transmural stimulation. On the other hand, mitragynine failed to affect the responses to norepinephrine and ATP. Mitragynine did not reduce KCl-induced contraction in the presence of tetrodotoxin, prazosin and alpha,beta-methylene ATP. Mitragynine inhibited nicotine- or tyramine-induced contraction. By using the patch-clamp technique, mitragynine was found to block T- and L-type Ca2+ channel currents in N1E-115 neuroblastoma cells. In the Ca2+ measurement by a fluorescent dye method, mitragynine reduced KCl-induced Ca2+ influx in neuroblastoma cells. The present results suggest that mitragynine inhibits the vas deferens contraction elicited by nerve stimulation, probably through its blockade of neuronal Ca2+ channels.     

Occurrence of ampulla in the ductus deferens of the Indian garden lizard Calotes versicolor daudin

During the breeding season, the terminal end of the ductus deferens of Calotes versicolor appears swollen and is comparable to the ampulla of the mammalian ductus deferens. Its anatomy was studied from paraffin sections. It differentiates along its length into five zones. The first has thick smooth muscle and pesudostratified epithelium; the second has luminal trabeculae with an epithelium showing evidence of secretory activity; the third has the epithelial mucosa abutting against the smooth muscle in the form of pocketlike indentations; the fourth has crypts between epithelial folds; and the fifth zone is a sphincter. The anatomy of this ampullary region is indicative of secretory as well as spermatophagous roles. It undergoes seasonal change and appears to be androgen-dependent. © 1995 Wiley-Liss, Inc.     

Immunohistochemical localization of taurine in the male reproductive organs of the rat.

The amino acid taurine has been implicated in several aspects of reproductive system physiology. However, its localization in these organs has not been previously analyzed. The aim of this study was to characterize its distribution in male rat reproductive organs by immunohistochemical methods. Taurine was localized in the smooth muscle cells of the tissues studied and in the skeletal fibers of the cremaster muscle. In the testis, taurine was found in Leydig cells, vascular endothelial cells, and other interstitial cells. No immunoreactivity was observed in the cells of the seminiferous tubules, either in germ cells at all spermatogenic stages or in Sertoli cells. However, peritubular myoid cells were immunostained. Most epithelial cells of the efferent ducts were immunolabeled, whereas the epithelial cells of the rete testis (extratesticular segments), epididymis (caput, corpus, and cauda regions), and ductus deferens were unstained. However, most epithelial cells from the intratesticular segments of the rete were immunopositive. Some cells identified as intraepithelial macrophages and lymphocytes, apical cells, and narrow cells were intensely immunolabeled. Regional differences in the distribution of these cell types along the ducts studied were also noted. The possible functional roles for taurine in these cells are discussed.     

Microanatomy of the testes and testicular ducts of the butterfly lizard,Leiolepis ocellata Peters, 1971 (Reptilia: Squamata: Agamidae) during the active reproductive period

The microanatomy of the testes and testicular ducts (rete testis, ductuli efferentes, ductus epididymis and ductus deferens) of Leiolepis ocellata (Agamidae) was investigated using light microscopy including histochemistry. Each testis contains seminiferous tubules and interstitial tissues. The former house spermatogenic cells (spermatogonia A & B, preleptotene, primary and secondary spermatocytes, spermatids (steps 1–8) and spermatozoa) and Sertoli cells, while the latter comprise peritubular and intersitial tissues. The rete testis is an anastomosing duct, having intratesticular and extratesticular portions. The proximal region of ductuli efferentes has wider outer ductal and luminal diameters than those of the distal region. The convoluted ductus epididymis is subdivided into four regions (initial segment, caput, corpus and cauda), based on the ductal diameter, epithelium characteristics and cell components. The ductus deferens has the greatest diameter and is divided into the ductal and ampulla ductus deferens. The ductal portion is subdivided into the proximal and distal regions, based on the epithelium types and ductal diameters. The ampulla ductus deferens is a fibromuscular tube, having numerous mucosal folds projecting into the lumen. Spermiophagy is detectable in the ductus epididymis and ductus deferens. The present results contribute to improved fundamental knowledge on the microanatomy of the reptilian reproductive system.     

The long-term influence of decentralisation or preganglionic hypogastric nerve stimulation in vivo on the reinnervation of minced vas deferens in the guinea-pig

Previous studies have shown that minced regenerating smooth muscle of the guinea-pig vas deferens becomes reinnervated by nerves growing in from the surrounding intact vas deferens. Using electron microscopy, we have examined the effect of altering activity in the preganglionic nerves, either by decentralisation, or by chronic stimulation of the hypogastric nerve, in vivo, on the reinnervation of regenerating smooth muscle cells. Chronic stimulation induced earlier reinnervation than that seen in unstimulated (sham-operated) or decentralised preparations; the number of nerve profiles present in four preparations stimulated for up to 7 days was approximately 10-20 times that seen in unstimulated or decentralised preparations. However, electron micrographs revealed that "empty" nerve terminals were a feature following stimulation for longer periods. Decentralised preparations showed little change of reinnervation, at least up to 7 weeks. Compensatory changes in the density of innervation were found in the unstimulated contralateral vas deferens.