异种去细胞真皮基质在体内转归的初步研究

目的 初步研究异种（猪）去细胞真皮基质在体内的转归。方法 经曲通（Triton）X-100、胰蛋白酶处理得到异种（猪）去细胞真皮基质后，将其包埋于SD大鼠皮下，于术后1、2、4、6、8、12、16、20和30周观察异种（猪）去细胞真皮基质的大体形态及组织学变化。结果 制备的异种（猪）去细胞真皮基质去细胞成分，胶原纤维排列有序，基底膜结构完整。大体观察异种（猪）去细胞真皮基质原始形态随时间延长而逐渐模糊．组织学观察异种（猪）去细胞真皮基质1周以炎性细胞浸润为主，以后成纤维细胞、毛细血管逐渐增多，真皮胶原排列逐渐规则、致密。结论 异种（猪）去细胞真皮基质可在体内作为支架长期存在，并可诱导自身成纤维细胞及毛细血管有序长入。     

人去表皮真皮细胞活性及组织结构特征(英文)

背景:研究证实,去表皮的真皮可以作为真皮替代物,在其上接种角质形成细胞后形成表皮结构.但有关真皮替代物细胞生物活性、组织结构特点及基底膜成分分析的研究报道较少.目的:观察人去表皮真皮细胞活性及组织结构特征.方法:将健康成人皮瓣用56℃PBS溶液处理以去除表皮,用液氮连续冻融处理去除真皮中细胞成分,获得去表皮真皮.以组织块培养法观察去表皮真皮细胞活性.以苏木精染色检测去表皮真皮细胞核,以波形蛋白免疫组化检测去表皮真皮成纤维细胞成分.以PAS染色及Ⅳ型胶原免疫组化检测基底膜及其成分.以VG染色检测去表皮真皮胶原纤维,Weigert染色检测弹力纤维,VG与Weigert双染色检测胶原纤维及弹力纤维,透射及扫描电镜观察去表皮真皮超微结构.结果与结论;用组织块培养方法培养的去表皮真皮2周无细胞生长.苏木精-伊红染色显示去表皮真皮中无细胞核、波形蛋白免疫组化显示去表皮真皮中无波形蛋白表达.VG染色显示去表皮真皮胶原纤维染成玫瑰红色,Weigert染色显示去表皮真皮弹力纤维染成紫黑色,双染色进一步显示胶原纤维与弹力纤维均匀排列.去表皮真皮表面及附属器残留部位PAS反应强阳性,Ⅳ型胶原表达明显.透射及扫描电镜下观察到去表皮真皮中胶原,弹力纤维交错排列,间有孔隙,相互交织成网.去表皮真皮无活细胞成分,真皮基质表面及附属器管腔壁仍保留糖原、Ⅳ型胶原等基底膜成分,真皮基质中富含胶原及弹力纤维,是一种类似在体真皮的三维胶原基质.     

复合壳多糖人工皮肤生物学功能初步研究

目的 研制一种新型的胶原凝胶类人工皮肤。方法 制备壳多糖－胶原－糖胺聚糖（GAGs)－成纤维2细胞真皮替代物（DE），观察成纤维细胞（FB）在凝胶中的生长情况。研究不同含量壳多糖对FB和角质形成细胞（KC）生长的影响，不同含量壳多糖DE的抗感染能力以及DE裸鼠全层皮肤缺失移植试验，组织学研究其重建情况。随后在DE表面接种KC，先浸没培养，再气液界面培养，构建完善的人工皮肤。对DE和人工皮肤行组织学和电镜分析。结果 FB在凝胶中2－9d呈指数增生。DE基质配方对FB的生长无抑制作用，但可促进KC的生长，对金黄色葡萄球菌的抑制作用随壳多糖含量增大而增强。DE移植后支持早期真皮重建和血管化。扫描电镜示DE有丰富的微孔结构。结论 复合壳多糖人工皮肤是生物相容性好，有一定抗感染能力的新型胶原凝胶类活人工皮肤。     

利用牛胶原构建人工真皮

目的制备成纤维细胞胶原凝胶(即真皮替代物),以研究细胞的生长、分化、增生及真表皮间的相互作用,为研制大面积创面覆盖物-复合皮奠定基础。方法(1)酶消化法培养胎儿成纤维细胞;(2)制备细胞胶原凝胶,分为Ca-gel和Cf-gel组,接种后于不同时间观察两组细胞的形态变化和胶原凝胶的收缩情况;(3)细胞计数;(4)SEM观测胶原凝胶的表面是否有成纤维细胞附着;(5)HE染色。结果(1)成纤维细胞形态及增殖情况:成纤维细胞胶原凝胶形成后呈半透明状,可出现较明显的细胞增殖,并可见凝胶收缩现象,但Ca-gel组较小;人工真皮质地与颜色近似真皮组织,有一定的弹性和抗拉性,不易脆。(2)细胞计数法所得两组生长曲线基本相似。Ca-gel和Cf-gel组细胞均具有典型的细胞增殖现象,Cf-gel组细胞在后期的增殖略快于Ca-gel组。(3)扫描显微镜下可见到凝胶表面有梭形的成纤维细胞出现,且成纤维细胞紧密贴附于凝胶上。(4)HE染色可见胶原呈较均一的淡红色网状分布,成纤维细胞分布于胶原凝胶内部和表面。结论(1)培养好的细胞凝胶称之为真皮替代物,是进行皮肤器官型培养和制作复合皮的关键与基础,并可与培养的人工表皮膜片混合移植,起到促进表皮细胞的生长粘附和加速创面愈合的作用。(2)成纤维细胞在胶原凝胶中有着活跃的生物学特性,因此在研究成     

表皮细胞、成纤维细胞复合脱细胞真皮基质构建组织工程皮肤

目的；观察体外培养的人皮肤角朊细胞和成纤维细胞的生物学特性，复合脱细胞真皮基质构建组织工程皮肤，为进一步临床应用奠定基础。方法：分别取人皮肤角朊细胞和成纤维细胞体外培养、扩增，测定细胞生长曲线、克隆形成率和染色体倍性；将培养的角朊细胞、成纤维细胞分别接种于脱细胞真皮基质表面，体外构建组织工程皮肤，观察细胞生长增殖情况。结果：在本培养体系下人皮肤角朊细胞和成纤维细胞增殖良好，细胞染色体倍性检测结果均为二倍体细胞。脱细胞真皮基质对角朊细胞、成纤维细胞无明显毒性作用，体外复合培养细胞可生长增殖。结论：此实验方法可以获得生长状态良好的组织工程皮肤种子细胞，复合脱细胞真皮基质可成功构建组织工程皮肤     

简便快捷获取角朊细胞和真皮成纤维细胞：组织工程皮肤种子细胞培养的3种方法比较

目的：探讨如何以简便快捷的方法获得大量最佳生长状态的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞。方法：用2．5g／L胰酶、35g／L热溶素和9．O&;#215;10^3U／L Dispase分别在A，B，C不同条件下处理皮肤标本，观察表皮与真皮的分离情况，并分别培养角朊细胞和真皮成纤维细胞，观察比较细胞的贴壁率和克隆形成率，通过MTT和BrdU检测观察细胞的生长状态，并分别用不同方法获得的细胞构建组织工程皮肤。结果：A，B，C3种方法角朊细胞的贴壁率(％)分别为：8．7&;#177;0．2，12．1&;#177;0．2，15．9&;#177;0．4；成纤维细胞的贴壁率(％)分别为：20．2&;#177;2．4，30．5&;#177;1．8，38．3&;#177;1．9。组间比较差异有统计学意义(t=3．3506，t0.05/2.18=2．101，P&;lt;0．05)。角朊细胞克隆形成率(％)分别为：0．110&;#177;0．025，0．260&;#177;0．026，0．370&;#177;0．021。组间比较差异有统计学意义(t=2．2875，t0.05/2.18=2．101，P&;lt;0．05)。在各种条件下，Dispase处理组获得的细胞数量、细胞贴壁率和克隆形成率均优于其他消化液处理组，细胞纯度高，生长状态良好；用其他方法获得的细胞生长状态相似，都可成功构建组织工程皮肤。结论：使用9．0&;#215;10^3U／L Dispase可以简便快捷的获得大量生长状态良好的角朊细胞和真皮成纤维细胞以作为皮肤组织工程的种子细胞，合适的消化方法、时间和温度可以最大限度保持原代细胞的活力。     

异体脱细胞真皮基质作为组织工程皮肤真皮支架的可行性

背景:组织工程皮肤是目前研究皮肤损伤修复重建的重要手段之一,异体脱细胞真皮基质不存在免疫原性,在异体移植时不会发生排斥反应,是比较理想的真皮替代物。目的:观察异体脱细胞真皮基质的组织相容性。方法:以正常人体真皮组织作为对照,通过体外、体内细胞毒性实验检测异体脱细胞真皮基质的组织相容性,以膨胀度、饱和含水量及生物力学分析检测异体脱细胞真皮基质的亲水性及机械性能。结果与结论:真皮基质中未见任何细胞成分,其网孔直径介于100～180μm之间。脱细胞真皮基质组饱和含水量为(69.6±3.97)%,膨胀度2.30±0.42,最大断裂力为(3.082±0.046)N,与对照组相比,差异无显著性意义(P>0.05)。体内外细胞毒性检测,未见明显细胞生长抑制及免疫排斥反应。提示异体脱细胞真皮基质机械性能接近正常皮肤,组织相容性好,免疫排斥反应小,是构建组织工程皮肤理想的真皮材料。     

脱细胞真皮基质的研究进展

1　研究背景皮肤缺损的修复一直是外科领域不断深入研究的热门课题。过去曾采用异体皮、异种皮覆盖皮肤缺损创面。起到暂时性的覆盖创面 ,防止体液丢失及防止感染的作用 ,但不能完全修复创面。随着皮肤组织工程的诞生 ,人们开始应用分子生物学、细胞生物学、组织工程学及医学科     

异种无细胞真皮活性复合皮的制备

目的：体外重建异种无细胞真皮活性复合皮,方法：将表皮细胞、成纤维细胞种植于异种无细胞真皮基质表面,体外培养约１周,定时取材行真皮表面ＨＥ染色、常规组织切片ＨＥ染色及扫描电镜观察。结果：表皮细胞,成纤维细胞种植生２４ｈ即贴附于真皮表面,电镜观察可见有伪足形成,培养５～７ｄ可融合为致密单层细胞膜,结论：以无细胞真皮为支架,采用细胞培养技术可于体外重建复合皮。     

复合壳多糖人工皮肤生物学功能初步研究

目的研制一种新型的胶原凝胶类人工皮肤。方法制备壳多糖-胶原-糖胺聚糖(GAGs)-成纤维细胞真皮替代物(DE),观察成纤维细胞(FB)在凝胶中的生长情况。研究不同含量壳多糖对FB和角质形成细胞(KC)生长的影响,不同含量壳多糖DE的抗感染能力以及DE裸鼠全层皮肤缺失移植试验,组织学研究其重建情况。随后在DE表面接种KC,先浸没培养,再气液界面培养,构建完整的人工皮肤。对DE和人工皮肤行组织学和电镜分析。结果FB在凝胶中2～9d呈指数增生。DE基质配方对FB的生长无抑制作用,但可促进KC的生长,对金黄色葡萄球菌的抑制作用随壳多糖含量增大而增强。DE移植后支持早期真皮重建和血管化。扫描电镜示DE有丰富的微孔结构。结论复合壳多糖人工皮肤是生物相容性好、有一定抗感染能力的新型胶原凝胶类活人工皮肤。     

High intensity ultrasound clamp for bloodless partial nephrectomy: In vitro and in vivo experiments

In some patients at risk of disease recurrence of renal cancers, maximum conservation of the kidney is possible through partial nephrectomy. However, bloodless surgery is difficult to achieve. The article describes an ultrasonic clamp, which optimises energy deposition and monitors lesion development with an echo-based technique. Using this novel apparatus, coagulation necroses have been obtained in vitro on substantial thicknesses (23 to 38 mm) over exposure durations ranging from 10 s to 130 s, and with acoustic intensities of less than 15 W/cm(2) per transducer. When used for coagulation purposes, two transducers situated on opposite arms of the clamp are driven, while for monitoring, only one is used. Lesions are monitored in real time by analysing the echo signal returned by the opposite arm of the clamp. The presence of a lesion is evaluated on the basis of energy changes and echo phase as a function of time. Both kidneys of two pigs (30 to 36 mm thick) were treated in vivo with the clamp, and the partial nephrectomies performed proved to be bloodless.     

Complement activation by artificial blood substitute Fluosol: in vitro and in vivo studies

A perfluorocarbon blood substitute, Fluosol, is undergoing clinical trials as an adjunct to chemotherapy. The adverse effects associated with its administration have been postulated to result from complement activation. When gel electrophoresis and Western blotting of Fluosol are used after its incubation with serum, activated C3 and factors Bb and H are bound to the Fluosol particles in a time-dependent fashion, which suggests that complement activation with Fluosol, as does that with zymosan, occurs on the surface of the particles. Paradoxically, it is found, both by the measurement of Fluosol-bound C3d and by fluid-phase C5a, that lower concentrations of Fluosol cause greater amounts of complement activation, which suggests a complex interaction of activators and inhibitors that changes as the available surface area is decreased. Studies performed with bystander red cell-bound C3d demonstrated in vivo complement activation occurring in six patients receiving Fluosol as an adjunct to chemotherapy for colon cancer. In two patients, there was a marked increase in red cell-bound C3d after Fluosol infusion; these two patients also developed adverse reactions during Fluosol infusion. These studies suggest that the Fluosol surface plays a major role in the initiation and regulation of complement activation that is seen during Fluosol infusion.     

Protective behavior of captopril on Hg(++)-induced toxicity on kidney mitochondria. In vivo and in vitro experiments

Mercurials are known to induce morphological and functional modifications in kidney mitochondria. In this work we studied in vitro and in vivo the protective effect of captopril on the deleterious effect of Hg(++)-induced nonspecific membrane permeability changes to Ca++ and membrane de-energization. In vivo the administration of captopril prevented the toxic effects of mercury poisoning on membrane permeability, oxidative phosphorylation and Ca++ homeostasis. Moreover, captopril preserves kidney tissue morphology from Hg(++)-induced damage. The protective effect of captopril is most likely related to the existence of a sulfhydryl group in the drug.     

In vitro and in vivo antibacterial activities of telithromycin

BACKGROUND: Telithromycin is one of the ketolides, characterised by a 3-keto group instead of L-cladinose and a C(11)-C(12) carbamate link by an alkyl chain to a pyridinum and imidazolium ring side chain. We evaluated in vitro and in vivo antibacterial activities of telithromycin against gynaecological pathogens. METHODS: In the vitro study, the antibacterial activity of telithromycin against 180 isolates (isolated in the year 2000) of Streptococcus agalactiae (n = 33), Enterococcus faecalis (n = 22), Neisseria gonorrhoeae (n = 30), Peptostreptococcus anaerobius (n = 20), Finegoldia magna (n = 20), Bacteroides fragilis (n = 25) and Prevotella bivia (n = 30) was compared with that of erythromycin A, clarithromycin, azithromycin, ampicillin and levofloxacin. In the in vivo study, the efficacy of telithromycin was evaluated using experimental intra-abdominal abscesses in mice caused by B. fragilis (minimum inhibitory concentration of telithromycin 0.5 mg/l). RESULTS: In the in vitro study, telithromycin inhibited more than 50% of clinical isolates of S. agalactiae, E. faecalis, N. gonorrhoeae, P. anaerobius, F. magna, B. fragilis and P. bivia at concentrations of 0.016, 0.063, 0.063, 0.032, 0.032, 0.5 and 0.25 mg/l, respectively. Telithromycin inhibited more than 90% of these clinical isolates at concentrations of 0.016, 4, 0.125, 0.063, 0.063, 4 and 1 mg/l, respectively. In the in vivo study, telithromycin inhibited abscess formation and significantly decreased viable cell counts in abscesses in comparison with the untreated group. CONCLUSIONS: These in vitro and in vivo antibacterial activities suggest that telithromycin could be a potential candidate for the treatment of bacterial infections complicated by chlamydial infection.     

Effect of ergotamine on platelet prostaglandin synthesis--in vitro and in vivo experiments

We studied the effect of ergotamine on the formation of metabolites of [1 - 14C] arachidonic acid by human blood platelets incubated with ergotamine (10(-5) or 5.10(-5)M) in vitro or after 0.5 mg i.m. administration in vivo. The in vitro experiments confirmed our earlier observation that ergotamine inhibits prostaglandin synthesis. This effect, however, seemed to be restricted to the toxic dose range of the drug as in vivo it occurred only in those subjects who became nauseated or vomited after administration of the drug.     

Microbubble formation: In vitro and in vivo observation

Injection of liquid through a catheter into the circulation is known to produce clouds of signals detected by sonography. Blood forced through a stenotic conduit produced sonographic clouding, and bubbles of 10–100 μm were observed by light microscopy. The microbubbles persisted up to three and a half minutes. Microbubbles were observed in the microcirculation of the rat by placing the catheter tip into the descending aorta of 15 animals, viewing the mesentery at 400X magnification, and recording the results on videotape. Following injection of the rats' own blood, numerous microbubbles lodged promptly at the arteriolar level and obstructed blood flow for up to 200 sec before shrinking sufficiently to pass downstream and allow restitution of flow.     

In vitro and in vivo P-glycoprotein transport characteristics of rivaroxaban

Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated. Furthermore, the inhibitory effect of rivaroxaban toward P-gp was determined. Rivaroxaban exhibited high permeability and polarized transport across Caco-2 cells. Rivaroxaban was shown to be a substrate for, but not an inhibitor of, P-gp. Of a set of potential P-gp inhibitors, ketoconazole and ritonavir, but not clarithromycin or erythromycin, inhibited P-gp-mediated transport of rivaroxaban, with half-maximal inhibitory concentration values in the range of therapeutic plasma concentrations. These findings are in line with observed area under the plasma concentration-time curve increases in clinical drug-drug interaction studies indicating a possible involvement of P-gp in the distribution and excretion of rivaroxaban. In vivo studies in wild-type and P-gp double-knockout mice demonstrated that the impact of P-gp alone on the pharmacokinetics of rivaroxaban is minor. However, in P-gp double-knockout mice, a slight increase in brain concentrations and decreased excretion into the gastrointestinal tract were observed compared with wild-type mice. These studies also demonstrated that brain penetration of rivaroxaban is fairly low. In addition to P-gp, a further transport protein might be involved in the secretion of rivaroxaban.