Physiological pharmacokinetics and pharmacodynamics of (+/-)-verapamil in female rats

Tissue distribution and pharmacodynamics of verapamil were evaluated during steady state intravenous (i.v.) infusion and after single dose intraperitoneal (i.p.) drug administration to female Sprague-Dawley rats. In one group of rats, verapamil was infused to a steady state concentration at which time animals were killed. Verapamil-induced decreases in mean arterial pressure (MAP) were monitored during infusion and correlated with concomitantly obtained plasma verapamil concentrations. Tissue (lung, liver, renal medulla, renal cortex, cardiac muscle, skeletal muscle, perirenal fat, brain stem, cerebral cortex, and cerebellum) and plasma samples were obtained immediately after animals were killed and verapamil and norverapamil concentrations determined. Another group of rats, after receiving i.p. verapamil, were killed at 1, 3, 5, 19, and 24 h. Elimination from each tissue evaluated was described by a first order process. Elimination half-life of verapamil was similar among plasma and tissues evaluated (1.5 to 2.2 h). The per cent verapamil not bound to plasma proteins was concentration-independent and similar between rats receiving i.p. (mean +/- S.D.) (2.28 +/- 0.72 per cent) and i.v. (2.08 +/- 0.03 per cent) verapamil. MAP and verapamil concentration in plasma (r = 0.75; p less than 0.01) and cardiac muscle (r = -0.82; p less than 0.01) were inversely correlated in a highly significant fashion during both i.v. and i.p. drug administrations. The tissue-to-plasma distribution ratio for verapamil and norverapamil was similar among animals receiving i.p. verapamil at all points of sampling, suggesting distribution equilibrium had been achieved. After steady state i.v. infusion, both verapamil and norverapamil tissue: plasma concentration ratios were greater than after i.p. administration. Higher tissue: plasma verapamil concentration ratios after i.v. administration than after i.p. administration suggest either only a pseudoequilibrium is attained after i.p. administration or that determinants of tissue distribution of racemic verapamil differ with different routes of drug administration. In these studies, MAP provided a reasonable pharmacodynamic marker for verapamil tissue and plasma concentrations.     

人血浆作为真皮支架对自体皮肤增殖的影响：组织学和免疫组织化学试验●☆

背景：组织工程是一个多学科研究的交叉学科，其目标是使人体损伤的组织和器官再生，通过这种假设，几乎所有的动物组织都可以在实验室进行培养。一般的方法是从需要移植的患者身上提取干细胞，在一定的支持条件下允许其生长、增殖、生产为可替换的组织。另一方面，寻找细胞能够互相联结并形成分层结构的合适的支持条件，如基质或支架等非常必要。目前用于烧烫伤治疗的材料有很多种，如胶原，透明质酸、纤维蛋白和聚乳酸及其共聚物。 目的：讨论以新型的生物材料自体血浆为支架对自体成纤维细胞和角质化细胞生长、扩张、增殖的影响。 设计：建立一种真皮再生的方法，将自体成纤维细胞浸于人血浆基质中，排除各种影响样本安全性和排斥反应的问题，应用同样的方法在新的真皮上获取角质化细胞。 时间及地点：实验于2008年在意大利曼多瓦C. Poma医院动物工厂完成。 材料：人角质化细胞和成纤维细胞取自1例58岁乳房切除患者的皮肤碎片。实验得到C. Poma医院独立伦理委员会批准，患者知情同意。 方法：从自体活组织皮肤样本中分离人角质化细胞和成纤维细胞，置于培养瓶中增殖，随后将自体血浆作为皮肤移植形成构造的支架，免疫和免疫组织化学特征显示与正常皮肤相似。 主要观察指标：将血浆样本用甲醛固定，埋入石蜡，苏木精-伊红染色，用显微镜进行细胞计数。所有样本均经免疫组织化学评估。 结果：在血浆基质上获得了多层规则形状的角质化细胞和成纤维细胞，基底膜的形成说明自体血浆是角质化细胞和成纤维细胞的生长、分化、扩展的良好支架，真皮和表皮细胞联结重建皮肤移植与正常皮肤无异。 结论：血浆作为角质化细胞和成纤维细胞分化、扩展的支架表现出良好的性能，同时实验还特别发现成纤维细胞浓缩血浆可以暂时替代皮肤，也是角质化细胞生长的强有力的支架。此外血浆还具有廉价，易于制备等优点。自体角质化细胞和成纤维细胞及自体血浆的应用，可以避免血种类型的免疫排斥反应。这种治疗方法给有慢性缺损并且需要连续移植的患者带来了希望。 doi:10.3969/j.issn.1673-8225.2009.47.001     

组织工程血管管状支架的制备与动态培养在血管组织工程中的应用

目的探索体外构建组织工程血管及细胞种植的方法。方法(1)制备管状支架模具,采用模具成形,冷冻干燥等技术,应用胶原和透明质酸复合体外预构管状支架;(2)分动态种植和静态培养两组进行细胞分层种植,每组12个,应用水溶性磺酸四唑(WST-1)测定,扫描电镜观察,5-溴2-脱氧嘧啶核苷(BrdU)细胞标记鉴定比较两种方法的细胞生长情况。结果(1)成功制备了管状支架,在培养液中约3周可保持管腔形态不变形,不塌陷;2.WST-1测定值在培养6、14、20 d分别为:动态组0.842±0.110、1.124±0.102、1.132±0.112,静态组0.386±0.121、0.489±0.245、0.499±0.265,两组比较差异有统计学意义,电镜和BrdU标记表明动态组细胞排列整齐有序。结论应用模具成形,冷冻干燥技术预构管状支架的方法切实可行,动态培养有助于细胞种植,本研究为组织工程血管的构建方法提供了思路。     

力学刺激促进骨髓基质干细胞体外软骨分化

目的探讨离心力刺激对猪骨髓基质干细胞(BMSCs)体外成软骨分化的作用,以及对三维支架上构建组织工程化软骨的影响,明确力学刺激与细胞分化及组织形成的关系,为体外软骨构建提供适当参数。方法抽取8周龄猪髂嵴骨髓,应用贴壁法分选单个核细胞,体外培养扩增后获得第2代BMSCs,以5.0×107/cm3的细胞密度接种到聚羟基乙酸(PGA)制成的圆柱形三维支架上,7d后分成4组,在不同的力学条件及诱导条件下培养。8周后取材,行相关检测。结果力学诱导组形成的组织呈白色,细腻光泽,形状规则,体积无明显改变,有良好的弹性和硬度,并具有典型的软骨陷窝结构和大量软骨特异性细胞外基质,Ⅱ型胶原及丰富的聚合蛋白多糖(GAG)成分。GAG含量为(6.0±1.2)mg/g,抗压强度为(2.2±0.8)kPa,弹性模量为(7.4±1.6)kPa。各项指标均明显优于其他各组。结论力学刺激有利于促进BMSCs成软骨分化,并在三维支架材料上构建组织工程化软骨。     

组织工程化表皮膜片构建及其在供皮区的应用研究

目的构建组织工程化表皮细胞膜片并应用于供皮区创面治疗。方法利用患者少量自体或同种异体正常皮肤分离、培养、扩增的表皮细胞,接种于壳聚糖明胶膜构建成表皮细胞膜片;将膜片移植于治疗组供皮区创面、适度加压,同时设立对照组:空白材料对照以及传统油纱布对照覆盖创面。于术后5～10d、30d、90d行大体观察、组织学检查、广谱角蛋白、外皮蛋白、层粘连蛋白、免疫组织化学检测以及Ⅰ、Ⅲ型胶原比值测定(偏光显微镜、RTPCR)。结果利用自体及异体表皮细胞和壳聚糖明胶膜能够构建出人表皮细胞膜片,应用于临床供皮区创面治疗15例,经过3个月随访,疗效肯定。移植膜片创面愈合时间(8.1±1.3)d,空白材料对照区为(16.2±3.8)d,空白油纱布对照区为(23.0±5.8)d。移植膜片存活良好,结构较完整、术后30d及90d观察:12例无明显增生,3例有轻度增生(20.0%),而空白油纱布对照区11例遗留增生性瘢痕(74.4%,χ2=8.127,P<0.01)。结论表皮细胞-壳聚糖明胶膜片能促进供皮区创面早期愈合并减少后期瘢痕增生,具有良好治疗效果。     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

支架及无支架条件下构建组织工程软骨的研究进展

支架材料的研究是组织工程的研究热点之一,软骨细胞复合培养给组织工程软骨的构建带来希望,单一的材料诸如胶原、透明质酸、壳聚糖、纤维蛋白凝胶等已经证明可以与软骨细胞复合培养,两种或者多种材料复合可以提高材料的性能,更好地用于组织工程软骨的构建,并满足软骨缺损修复的需要;同样,在无支架情况下应用软骨细胞聚集培养、沉淀培养的方法,可以构建组织工程软骨,并给软骨缺损的修复带来新希望,但目前的研究较少。两者是组织工程软骨构建的两个主要方向。     

复方壳多糖组织工程皮肤的组织学特性

目的评价制备的复方壳多糖组织工程皮肤在组织学方面的特性，为其修复皮肤缺损创面提供实验依据。方法通过苏木精-伊红（HE）染色、高碘酸-希夫（PAS）染色、免疫组织化学染色及电镜，观察组织工程皮肤的组织学特征，包括表皮、真皮及两者的连接结构基底膜。结果制备的复方壳多糖组织工程皮肤表皮细胞增殖活跃，分层分化良好，厚约150μm，真皮层细胞生长正常，排列有序。PAS染色示真表皮间有均匀红染的条带出现，免疫组织化学染色结果显示Ⅳ型胶原、Ⅶ型胶原、层黏蛋白反应阳性，在电镜下可见角质形成细胞间大量桥粒，基底层内侧较多半桥粒，与机体皮肤结构相似。结论复方壳多糖组织工程皮肤组织结构良好，符合新型皮肤替代物在治疗皮肤缺损时的组织学要求。     

颜面凹陷畸形的临床分型及整复

目的为规范各类颜面凹陷畸形的临床治疗提供参考性建议。方法根据凹陷部位支撑组织的异常或被覆组织的异常情况。临床上将凹陷畸形分为三型：Ⅰ型为骨性凹陷畸形，包括先天性发育不良。后天性局部骨组织缺损、错位或塌陷；Ⅱ型为软组织凹陷畸形。包括局部软组织萎缩、缺损或黏连；Ⅲ型为复合性凹陷畸形。骨与软组织同时存在萎缩、缺损或较严重的塌陷。并依照此分型分别采用游离皮瓣、肌皮瓣或带蒂肌皮瓣填充法。局部组织瓣填充法。带蒂组织瓣加医用材料混合填充法，游离脂肪颗粒填充法。HA人工骨填充法。硅胶假体填充法。有机玻璃填充法，奥美定填充法。复杂凹陷畸形的综合治疗整复方法。为216例患者修复颜面凹陷畸形。结果随访216例患者6个月至5年，未发现严重并发症，外观效果满意。结论此颜面凹陷畸形的临床分型简单、合理。便于临床对颜面凹陷畸形的整复。具有一定的实用价值。     

Evidence for motility and pinocytosis in ramified microglia in tissue culture

Ramified microglial cells were investigated in primary cultures of dissociated cerebral cortical tissue from rats. The identification of these cells was confirmed through immunohistochemical staining with 7 monoclonal antibodies selective for microglia. While there was significant variation in staining intensity with different antibodies, all stained the identified ramified cells; the antibodies OX-42 and ED1 yielded the most intense immunoreactivity. Based on distinctive morphological features, the microglia could be identified in living cultures where they were monitored using time-lapse video recording. This technique revealed extremely dynamic features of cellular plasticity and motility. Ramified microglia exhibited constant and rapid alterations in the size and shape of their cell body with an associated extension and retraction of processes; concomitantly, the cells moved about in a circumscribed area. These features of plasticity and motility were unique to this cell type, and correlated with OX-42 immunostaining. The microglia also possessed a differentially high level of pinocytotic activity; this too was correlated with OX-42 staining. From the nature of their morphological plasticity and motility, high pinocytosis, and cellular distribution, it is hypothesized that the ramified microglia specifically function as a system of fluid cleansing in normal brain tissue.