大鼠血管平滑肌细胞的贴块法培养

目的:探讨大鼠血管平滑肌细胞的原代培养方法与生长特性,为中药研究提供实验材料。方法:采用组织贴块法培养,倒置显微镜观察形态,电镜鉴定,台盼蓝染色活力检测。结果:倒置显微镜和电镜观察培养的血管平滑肌细胞生长状态良好,呈现平滑肌细胞特征性的“峰—谷”状结构,台盼蓝染色约有95%以上的细胞为活细胞。结论:本培养方法简单、经济,经过一定时间的操作训练,即可成功培养出平滑肌细胞。     

膀胱Cajal样间质细胞的分布和结构与功能

西班牙神经解剖学家Cajal于1893年首先在胃肠道发现一种特殊的间质细胞,学术界后将此类细胞命名为Cajal间质细胞( ICC)[1-2].这类细胞分布在消化道自主神经末梢和平滑肌之间,表达c-Kit、波形蛋白和缝隙连接蛋白( Cx43)[3-5].ICC有自发的电节律活动,是胃肠道运动的起搏细胞,作为神经信号向肌肉传递的中转站,在调控消化道运动中起重要作用[6].1996年,Smet等[7]在豚鼠和人逼尿肌上发现了类似胃肠ICC的间质细胞(膀胱ICC样间质细胞).其后,一系列研究也证实膀胱间质具有与胃肠间质细胞类似的功能.我们对膀胱ICC的分布、结构与功能做一综述.     

半簿切片技术

半薄切片(Semithin section)又称厚切片,名称是相对于电镜的超薄切片而讲的。半薄切片厚度介于0.5～2μ,介于光镜石蜡切片(7μ)和电镜超薄切片(0.07μ)之间。半薄切片的制法是按常规电镜制备的树脂包埋组织块,在超薄切片机或经改制的石蜡切片机上用玻璃刀制取的,经一定方法处理后进行染色,光学显微镜下观察。主要用于电镜观察前的定位,但由于半薄切片的制备是按电镜制样程序进行固定、脱水、包埋,组织结构保存较好,又兼切片较薄,故在光镜下分辨率较常规光镜切片为优,若把照相底片加以适当     

家兔动脉血管平滑肌细胞培养方法的改进

目的提高家兔血管平滑肌细胞(vascular smooth mus-cle cell,VSMC)体外培养的成功率。方法以传统的主动脉平滑肌细胞培养方法中的组织块贴壁法为基础,从动物选择、培养基配制、血管取材、组织块的大小和接种间距、培养液的量及换液量、细胞传代的时机等多个重要环节进行改进;用倒置显微镜、电镜对培养细胞进行形态学观察,并用平滑肌细胞特异性α-肌动蛋白(α-actin)单克隆抗体免疫组织化学方法进行鉴定。结果台盼蓝检查传代细胞的存活率大于97%;光镜下见培养细胞平行排列呈长梭形及"峰、谷"样结构特征;免疫组织化学染色鉴定培养细胞中VSMC的纯度为97%;电镜技术观察到VSMC完整的超微结构。结论此培养方法能提高动脉平滑肌细胞原代培养的成功率,传代后血管平滑肌细胞生物学特征的稳定性好,可作为研究血管平滑肌细胞生物学行为的有效模型。     

豚鼠气道平滑肌的急性分离及K_(ATP)通道单通道电流的记录

目的:建立急性分离豚鼠气道平滑肌的方法,并初步分析ATP敏感钾通道(KATP通道)单通道电流的性质。方法:急性分离出豚鼠3~4级支气管,并用链霉蛋白酶E分散气道平滑肌细胞,应用膜片钳技术的内面向外式记录方法,研究气道平滑肌KATP通道单通道电学性质。结果:成功记录到电导为112.4±5.14 pS的、可被优降糖所阻断的KATP通道单通道电流。钳制电压在0~-60 mV之间,通道电流无整流现象,且未见时间依赖性失活。结论:建立了急性分离豚鼠气道平滑肌的方法,并成功记录KATP通道单通道电流,为进一步研究气道平滑肌KATP通道在呼吸道疾病中的作用提供了基础。     

经尿道灌注慢病毒转染豚鼠膀胱的研究

目的 观察慢病毒介导绿色荧光蛋白（GFP）转染膀胱的效果, 并确定取得较好转染效果的病毒量。方法 豚鼠经尿道灌注及静脉注射不同量的携带 GFP 基因的慢病毒, 饲养 7 d 后取膀胱、 肝、 肺、 肾等组织冰冻切片,激光共聚焦显微镜下观察各组织 GFP 分布。结果 滴度为 4×108的慢病毒, 经尿道膀胱灌注 30 μL 时可见组织黏膜下有绿色荧光, 40 μL 时组织肌层有广泛绿色荧光分布, 50 μL 时组织肌层有更强绿色荧光分布, 静脉注射 25 μL 时组织肌层可见有广泛绿色荧光分布; 经尿道灌注各病毒量下未在膀胱外组织见有明显绿色荧光分布, 静脉注射下在肝、 肺等组织见有较膀胱更强绿色荧光。结论 慢病毒可以成功介导 GFP 转染豚鼠膀胱, 并且经尿道灌注可以避免静脉注射带来的病毒在膀胱外组织广泛分布, 能够为膀胱疾病的临床治疗带来新的研究方向和手段。     

肾血管平滑肌脂肪瘤31例病理分析

目的研究肾血管平滑肌脂肪瘤(Renalangiomyolipoma,RAML)的临床病理学特点和鉴别诊断要点。方法收集31例RAML的临床病理资料,常规切片,HE染色及免疫组化染色,2例电镜观察。结果RAML患者的征象及影像学均有其特征,病理形态学上由畸形血管、上皮样或梭形平滑肌细胞及脂肪三种成分构成,部分细胞可有异型性。免疫组化染色Actin、HMB45阳性。电镜下可见类似黑色素前体样的小体。结论RAML是一种好发于中青年女性的良性肿瘤,因其特殊形态、免疫组化表达Actin、HMB45,应归入血管周上皮样细胞相关肿瘤。     

实验性糖尿病大鼠心迷走神经及其心神经节结构变化的研究

目的对糖尿病心脏自主神经的病理生理变化进行深人研究,阐明糖尿病心脏自主神经病变的病理基础。方法采用神经示踪技术顺行标记实验性糖尿病大鼠模型的心迷走传出神经纤维及其支配的心脏神经节的神经纤维末端：同时采用荧光金染料标记心神经节神经元细胞,通过荧光显微镜和激光共聚焦显微镜观察其结构的改变。结果（1）糖尿病大鼠心脏神经元细胞胞体变小,细胞间隙模糊;（2）Dil标记的迷走神经传出纤维直径变细,伴局部肿胀：（3）被心脏迷走神经传出纤维所支配的心脏神经元细胞明显减少。结论糖尿病可导致心脏神经节细胞结构的改变,并减少迷走神经传卅纤维对神经节细胞的支配活动,这种改变可能与糖尿病患者心血管乐力反射敏感性的下降有关。     

膀胱ICCs细胞SCF/c-kit信号通路研究现状

膀胱ICCs细胞是Smet和Jonavicius等于1996年首先在豚鼠和人逼尿肌发现的类似于胃肠道Cajal间质细胞(Interstitial cells of Cajal,ICC)的一种特殊间质细胞.它是泌尿系平滑肌蠕动的起搏者,在膀胱逼尿肌兴奋收缩过程中起着重要作用.最新研究表明SCF/c-kit通路与ICCs细胞形态和功能的维持密不可分,信号通路的异常会导致ICCs表型和功能的转变,这一发现为之后的尿动力方面研究提供了新的平台.     

芥子气软膏治疗豚鼠银屑病样皮损的作用机制

［摘要］目的探讨芥子气软膏对豚鼠银屑病样皮损的治疗机制。方法豚鼠耳廓均匀涂抹 5%普萘洛尔乳膏,bid,持续28 d。从第21天起,再给予芥子气软膏。测量豚鼠耳廓厚度,苏木精 伊红（HE）染色、组织学评分以及免疫组织化学法检测用药前后肿瘤坏死因子相关凋亡诱导配体（TRAIL）的表达变化。结果芥子气软膏可降低豚鼠银屑病样皮损耳廓肿胀度（P＜0.05）,病理组织切片可见皮肤组织逐渐恢复正常,与对照组相比,经芥子气软膏治疗后,皮肤基底层和棘层TRAIL蛋白强阳性表达。结论芥子气软膏治疗银屑病的作用机制可能与诱导TRAIL蛋白的过度表达及促进表皮变异细胞的凋亡有关。     

高糖中膀胱ICCs的变化

目的观察高糖环境下豚鼠膀胱Cajal间质细胞(interstitial cells of Cajal,ICCs)内钙离子荧光及其超微结构的改变,探讨糖尿病膀胱(DCP)的发病机制。方法采用原代培养,激光共聚焦、透射电镜观察5、10、15mmol/L葡萄糖浓度培养24、72h的ICCs,扫描记录胞内荧光强度值,观察其超微结构。结果糖浓度增高培养时间延长,ICCs内钙离子荧光值升高,胞内线粒体大小不等、数量减少、空泡样变甚至溶解,胞质广泛溶解,突起消失。结论高糖环境对豚鼠膀胱ICCs电生理学、超微结构造成显著影响,这种改变可能是造成DCP发病的重要原因之一。     

高糖中膀胱ICCs超微结构的变化

目的观察高糖环境下豚鼠膀胱Cajal间质细胞超微结构的改变,探讨糖尿病膀胱(DCP)的发病机制。方法采用酶消化法原代培养,透射电镜观察不同葡萄糖浓度下培养的ICCs超微结构。结果随糖浓度增高和培养时间延长,ICCs内细胞器明显减少,线粒体大小不等、数量减少、肿胀、空泡样变甚至溶解,内质网扩张、空泡样变,胞质广泛溶解,突起消失。结论高糖环境对豚鼠膀胱ICCs超微结构造成显著影响,这种结构破坏可能是造成DCP发病的重要原因之一。     

高糖对豚鼠膀胱ICCs细胞电生理的影响

目的观察高糖环境下豚鼠膀胱Cajal间质细胞（interstitial cells of Cajal,ICCs）内钙离子荧光的改变,探讨糖尿病膀胱（diabetic cystopathy,DCP）的发病机制。方法采用酶消化法原代培养,激光共聚焦技术观察5、10、15mmol/L葡萄糖浓度下培养24、72h的ICCs,扫描记录每个时间点胞内的钙离子荧光强度值。结果糖浓度增高培养时间延长,ICCs内钙离子荧光值升高（P=0.00）,只有15mmol/L的24h组降低（P=0.00）。结论高糖环境对豚鼠膀胱ICCs电生理学造成显著影响,这种改变可能是造成DCP发病的重要原因之一。     

Characterization of human urinary bladder KATP channels containing SUR2B splice variants expressed in L-cells

The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K(ATP) channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon 17- mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk(-)cells (L-cells) with K(IR) 6.2 were determined by measuring changes in membrane potential responses evoked by K(+) channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) fluorescence. The rank order potency of a variety of structurally distinct K(+) channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17- cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of [125I]A-312110 binding with the same K(+) channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K(ATP) channels containing SUR2B exon 17- represent a major splice variant expressed in urinary bladder smooth muscle.     

3H-muscimol binding sites within guinea pig ovary: a histoautoradiographic study

The distribution of 3H-muscimol within guinea pig ovary was studied using combined radioreceptor binding studies and histoautoradiographic technique performed on frozen sections of ovary mounted on microscope slides. 3H-muscimol was bound by sections of ovary in a manner consistent with the existence of specific GABA receptors. The binding was reversible, saturable, of high affinity (KD was 38 +/- 6 nmol/l and Bmax was about 378 +/- 61 fmol/mg protein, and inhibited by GABA receptor interfering drugs. 3H-muscimol binding sites were found in the blood vessel wall, in the follicle and in the oocytes. The number of oocyte and follicular receptors gradually decreases during the development of ovarian follicles.     

Pharmacological action of cattle prostate extracts (PE). V. Effect on the bladder]

As the extract of cattle prostate (PE) is clinically effective in treating prostatic hypertrophy, a study was carried out on urinary bladders of rat, guinea pig, rabbit and dog as well as on guinea pig ileum. Muscle strip of rat and/or dog bladder contracted with PE with increasing spontaneous movement, and was unaffected by atropine. The isolated ileum of guinea pig also contracted with PE, and the contraction was inhibited by papaverine, but not by atropine. A rise in intravesical pressure was observed with increasing spontaneous movement in guinea pig bladder treated with PE, as well as in rabbit bladder in vitro or in situ. The sphincter vesica of guinea pig was dilated by PE as well as by ACh and methacholine.     

Inhibitory Effects of Urothelium‐related Factors

The urothelium of the bladder has long been recognized as a protective barrier between detrusor and urine. In recent years, it has become more evident that the urothelium plays a role as an active source of mediators. The urothelium can release neurotransmitters and modulators such as acetylcholine, ATP, nitric oxide, prostaglandins and neuropeptides. They exert both excitatory and inhibitory effects in modulating urinary tract motility. In addition, several studies have reported the existence of an urothelium‐derived unknown inhibitory factor in the urinary bladder. By the use of a new serial cascade superfusion bioassay on guinea pig ureter, recent studies confirm that the guinea pig bladder urothelium releases a substance with inhibitory bioactivity, which was resistant to treatment with nitric oxide synthase inhibitor and cyclooxygenase inhibitor and to adenosine A1/A2 receptor blockade. Lately, a marked and quickly inactivated novel release of PGD₂ from the bladder urothelium was discovered, together with localization of prostaglandin D synthase therein. PGD₂ was found to have an inhibitory influence on nerve‐induced contractions in guinea pig urinary bladder and on spontaneous contractions in the out‐flow region. An altered release of excitatory and inhibitory factors is likely to play an important part in bladder motility disturbances, of which the prostanoids are a notable group. Due to the fact that the bladder is relaxed 99% of the time, not only excitatory mechanisms in the bladder are necessary to study, but also inhibitory mechanisms need considerable attention, which will contribute to the discovery of new targets to treat bladder motility disorders.     

Characterization of a new muscarinic receptor antagonist PNU-171990 in guinea pig,cat and human smooth muscle

The present study was done to characterize a new compound, PNU-171990, 2-diisopropyl aminoethyl 1-phenylcyclopentane carboxylate hydrochloride, with functional smooth muscle selectivity at least as high as tolterodine. In vitro homogenates of guinea pig cerebral cortex, parotid gland, heart, urinary bladder, and Chinese hamster ovary (CHO) cells expressing human muscarinic m(1)-m(5) receptors PNU-171990 did not show selectivity for any subtype (pK(i), 7.72-8.64). PNU-171990 caused a parallel shift in the concentration-response curve for carbachol-induced contraction of smooth muscle from guinea pig bladder (pK(B), 7.65), guinea pig ileum (pK(B), 8.48), and human ileum (pK(B), 7.10). In vivo PNU-171990 inhibited urinary bladder contraction with a significantly lower ID(50) than on the salivary secretion (206 and 706 nmol/kg, respectively, P<0.05). In conclusion, PNU-171990 is a competitive and potent muscarinic receptor antagonist in vitro with a numerically better selectivity ratio for the bladder contraction over salivation in vivo than tolterodine.     

阳和平喘颗粒对哮喘模型豚鼠肺功能和形态学变化的实验研究

目的 观察阳和平喘颗粒对哮喘豚鼠肺功能与镜下组织的影响,探讨其治疗哮喘的机制.方法 杂色豚鼠120只雌雄各半随机分作6组(正常组,模型组,地塞米松组,桂龙咳喘宁组,阳和平喘颗粒中剂量组,阳和平喘高剂量组).用卵蛋白成功诱发豚鼠哮喘后,给予阳和平喘颗粒、地塞米松、桂龙咳喘宁胶囊进行治疗,观察肺功能与肺组织光镜和电镜下的变化.结果 与正常对照组相比,模型组肺功能指标吸气阻力(Ri)与呼气阻力(Re)均显著增加,肺顺应性(Cldyn)下降,肺组织电镜下显示:模型组动物的肺组织可见嗜酸性粒细胞片状浸润,II型细胞部分线粒体脊突消失、膨胀,板层小体减少,有较多排空现象.血管腔狭窄,毛细血管内皮肿胀,核浓缩,胞浆内可见到髓样小体(细胞器变性),基底膜结构不清.结论 阳和平喘颗粒可在一定程度上改善损害的肺功能,达到治疗哮喘的目的,其机制尚须进一步探讨.