4,6-联脒-2-苯基吲哚标记超薄表皮片移植效果体内示踪评价

目的:评价4,6-联脒-2-苯基吲哚作为荧光示踪剂在标记超薄表皮片移植中的效果,为研究表皮片移植后细胞增殖和分化现象奠定基础。方法:实验于2005-01/05在解放军总医院第一附属医院全军创伤修复实验室完成。表皮片用中性蛋白酶分离自人环切术的包皮。①分离出的表皮片分为两组,空白对照组和实验组,实验组移植前用激发蓝色荧光的4,6-联脒-2-苯基吲哚标记皮片,空白对照组以磷酸盐缓冲液代替。②将表皮片随机移植于8只裸鼠32个创面,分别于移植4d和7d后取材。③用冰冻技术将皮片及其下组织切成厚度约为5μm的冰冻切片,选用罗丹明标记的广谱抗角蛋白抗体作为参照荧光,常规处理后在荧光显微镜观察皮片的染色效果。结果:8只裸鼠均进入结果分析。①皮片移植后的生长情况:空白对照组16个创面13个成活良好;4,6-联脒-2-苯基吲哚组16个创面14个成活良好。两组皮片成活率差异无显著性意义(χ2=1.34,P>0.05)。②皮片移植后的荧光显色结果:荧光显微镜下,4,6-联脒-2-苯基吲哚组可见圆点状的蓝色激发荧光;皮片激发的蓝色荧光与鼠源性组织的自发荧光有明显的对比度,在红色波长激发下可见皮片呈现均匀不一的红色荧光,合成荧光显色表明蓝色荧光多位于中央细胞核区域,红色荧光位于胞浆区域,荧光的对比度明显,且在皮片标记的8d之内荧光未见明显的衰退现象。结论:用4,6-联脒-2-苯基吲哚示踪移植表皮片,效果良好且标记稳定,故4,6-联脒-2-苯基吲哚可以在表皮片移植的细胞增殖分化研究中加以运用。     

移植超薄表皮片中成熟细胞逆向分化为表皮干细胞的现象

目的:观察移植超薄表皮片中成熟表皮细胞的分化及分布状态,以及其向表皮干细胞逆分化现象在创面修复过程中的存在。方法:采用荧光标记和免疫组织化学方法,检测解放军空军总医院2004-11/2005-05经环切术切取的成人的包皮12例,用中性蛋白酶消化获得表皮片,反复冲洗使基底层干细胞脱落。在一部分行苏木精-伊红、免疫组织化学染色的同时,另一部分皮片用DAPI标记后移植于由北京协和动物中心提供的12只裸鼠全层皮肤缺损的创面,于第4和7天取材制作冰冻切片,行免疫组化染色,荧光显微镜观察创面修复过程中表皮细胞角蛋白19、角蛋白14、角蛋白10的分布情况与表达特征。结果:①苏木精-伊红染色可见未标记移植的表皮片示有正常的层次结构,但标记移植的皮片组织学观察显示细胞层次有松弛、紊乱的迹象。②免疫组化染色及荧光显微镜示未标记移植前的皮片颗粒层以下几乎未见角蛋白19阳性细胞而有少量角蛋白14阳性细胞和大量角蛋白10阳性细胞,而标记移植的皮片有孤立成团同时发蓝-红双荧光的角蛋白19阳性细胞,且角蛋白14阳性细胞明显增多。③这些现象在移植后的第4天最为显著。结论:在超薄表皮片移植中显示有成熟表皮细胞向表皮干细胞方向的逆分化,起到了维持皮片活力和参与创面修复的作用。     

应用4,6-联脒-2-苯基吲哚大体染色法快速观察生物支架材料体外内皮化程度

目的:建立一种新的形态学方法,以简便快捷地观察生物支架材料体外内皮化的程度。方法:实验于2005-07/2006-02在复旦大学上海医学院分子生物学实验室完成。①新鲜的猪心瓣膜经过十二烷基磺酸钠和脱氧胆酸处理,获得去细胞生物支架。②采用消化法获得脐静脉内皮细胞。③将脐静脉内皮细胞(2×105/cm2)种植于支架上,静态培养10d。用0.5mg/L的4,6-联脒-2-苯基吲哚溶液对再内皮化支架进行直接的大体染色及染色后冰冻切片,同时用苏木精-伊红染色,免疫荧光染色和扫描电镜对支架的内皮化情况进行平行观察。结果:①猪心瓣膜的细胞成分完全脱去,胞外基质保存完好。②原代分离的脐静脉内皮细胞,培养5d后融合成扁平单层,梭形或多角形,具有典型的铺路石样形态;CD31免疫荧光染色细胞表面呈现黄绿色荧光。③脐静脉内皮细胞种植10d后,支架经4,6-联脒-2-苯基吲哚大体染色显示内皮化程度达90%以上,观察结果与电镜观察相同;冰冻切片观察显示支架表面有一融合的内皮细胞单层,与免疫荧光染色及常规苏木精-伊红染色结果相吻合。结论:4,6-联脒-2-苯基吲哚大体染色法可直接观察生物支架再内皮化程度,简便、准确直观,是体外构建组织的一种有效的形态学检测方法。     

应用4，6-联脒-2-苯基吲哚大体染色法快速观察生物支架材料体外内皮化程度料

目的：建立一种新的形态学方法，以简便快捷地观察生物支架材料体外内皮化的程度。方法：实验于2005-07／2006-02在复旦大学上海医学院分手生物学实验室完成。①新鲜的猪心瓣膜经过十二烷基磺酸钠和脱氧胆酸处理。获得去细胞生物支架。②采用消化法获得脐静脉内皮细胞。③将脐静脉内皮细胞（2&;#215;10^5/cm^2）种植于支架上，静态培养10d。用0．5mg／L的4，6-联脒-2-苯基吲哚溶液对再内皮化支架进行直接的大体染色及染色后冰冻切片，同时用苏木精一伊红染色，免疫荧光染色和扫描电镜对支架的内皮化情况进行平行观察。结果：（1）猪心瓣膜的细胞成分完全脱去，胞外基质保存完好。②原代分离的脐静脉内皮细胞，培养5d后融合成扁平单层，梭形或多角形，具有典型的铺路石样形态；CD31免疫荧光染色细胞表面呈现黄绿色荧光。③脐静脉内皮细胞种植10d后，支架经4，6-联脒-2-苯基吲哚大体染色显示内皮化程度达90％以上，观察结果与电镜观察相同；冰冻切片观察显示支架表面有一融合的内皮细胞单层，与免疫荧光染色及常规苏木精-伊红染色结果相吻合。结论：4，6-联脒-2-苯基吲哚大体染色法可直接观察生物支架再内皮化程度，简便、准确直观，是体外构建组织的一种有效的形态学检测方法。     

超顺磁性氧化铁和DAPI双标记骨髓间充质干细胞

背景:骨髓间充质干细胞目前已经成为重要的组织工程种子细胞,而若想深入研究其在体内外增殖、分化的规律,首先需要解决如何能高效、安全地标记骨髓间充质干细胞。目的:观察超顺磁性氧化铁及DAPI对大鼠骨髓间充质干细胞的双标记效果及其对细胞存活、增殖以及凋亡的影响。方法:分离培养大鼠骨髓间充质干细胞,用超顺磁性氧化铁及DAPI双标记后分别用普鲁士蓝染色法和激光共聚焦显微镜观察其铁标记率及荧光标记率。用锥虫蓝检测细胞活力;MTT法检测标记干细胞增殖力;Calcein-AM/PI以及AO/PI双染细胞,检测细胞存活率以及凋亡率。结果与结论:超顺磁性氧化铁及DAPI在体外双标记大鼠骨髓间充质干细胞效率高,可达100%;锥虫蓝染色显示标记细胞的存活率为97%;MTT法检测发现双标记干细胞组的活力以及增殖力与未标记干细胞组相比差异均无显著性意义(P>0.05);Calcein-AM/PI染色,未标记细胞及双标记细胞的存活率分别为96%和95%;AO/PI染色,未标记及双标记细胞的凋亡率均为1%。提示超顺磁性氧化铁和DAPI双标记大鼠骨髓间充质干细胞后,对于干细胞的存活、增殖以及凋亡无影响。     

In vivo and in vitro evaluation of three controlled release principles of 6-N-cyclohexyl-2'-O-methyladenosine.

6-N-Cyclohexyl-2'-O-methyladenosine was formulated into controlled release formulations exhibiting comparable in vitro release profiles using different formulation principles, i.e. osmotic pump tablets, membrane-coated pellets and hydrophilic matrix tablet. Dissolution behaviour of these formulations was evaluated in vitro under various testing conditions to assess the effect of pH and hydrodynamic conditions. It was found that osmotic tablets were not sensitive to dissolution media pH and hydrodynamics change, while drug release from monolithic hydrophilic matrix tablets were pH-dependent. When tested in vivo in dogs, it was found that metabolism of 6-N-Cyclohexyl-2'-O-methyladenosine was extensive and appeared to be saturable based on a pharmacokinetic study. Cumulative percent input in vivo (%dose) was obtained by numerical deconvolution, and compared to in vitro release profiles. A linear correlation between fraction absorbed (FRA) in vivo and fraction dissolved (FRD) in vitro was established for osmotic tablets--a true zero-order release formula, whereas only a nonlinear correlation was obtained for membrane-coated pellets. The difference in the in vivo behaviour of these formulations, despite their similar in vitro release characteristics, demonstrated the effect of different controlled release principles on their in vivo bioavailability. The curvature of fraction absorbed in vivo vs. fraction dissolved in vitro for membrane-coated pellets indicated that there was a time-scale difference between in vivo and in vitro testing. In conclusion, drug release from the osmotic system was independent of in vitro and in vivo conditions, where best sustained release effect was achieved, whereas the in vitro dissolution test employed for membrane-coated pellets and hydrophilic matrix tablets needed to be optimized to be biorelevant.     

In vivo study of schistosomicidal action of 1-benzyl-4-[(4-fluoro-phenyl)-hydrazono]-5-thioxo-imidazolidin-2-one

Praziquantel has been the drug most widely used therapy against different forms of schistosomiasis around the world. However, this treatment has shown ineffective in humans and in experimental models of Schistosoma mansoni. New therapeutic alternatives have been tested, including the imidazolidine derivative LPSF/PT-09, which has shown high therapeutic potential in vitro. In this work, we tested the schistosomal activity of this derivative in doses of 250 mg/kg and 200 mg/kg in mice experimentally infected with a high parasite load of S. mansoni. Parasitological evaluations related to the number of S. mansoni worms and their oviposition were performed during the acute phase of the disease and have demonstrated moderate effectiveness of 30–54,4%. However, LPSF/PT-09 did not influence oviposition of the parasites or the embryonic development of the eggs. The results obtained in this model showed that the imidazolidine derivative LPSF/PT-09 presented significant antischistosomal activity in vivo, posing as a potential candidate for this class of drugs. However, a better understanding of the pharmacokinetics and pharmacodynamics of the imidazolidine derivative LPSF/PT-09 is needed.     

Micropuncture evaluation of the site of action of 2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride (MK447) in the rat kidney

Free-flow micropuncture and in situ microperfusion techniques were used to define the site of action and relative effect of MK447 [2-aminomethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride] vs. furosemide in the rat kidney. MK447 was administered i.v. at 5 mg/kg/hr. Infusion of this drug had little effect on proximal tubule reabsorption of water, Na+ and K+. In contrast, reabsorption of these constituents by the loop of Henle was significantly reduced. There was a tendency for water and Na+ reabsorption to rise and for K+ secretion to fall along the distal tubule. These latter effects can be explained by the contributions of an increased distal flow rate and increased tubule fluid K+ concentration. Net addition of K+ beyond the distal tubule was observed. This may be due to effect of the drug on the collecting duct system or juxtamedullary nephrons. The effects of MK447 and furosemide on loop of Henle reabsorption were compared in microperfusion experiments. Furosemide reduced Na+, K+ and water reabsorption by the loop, whereas MK447 had no effect. A 6-bromophenol sulfate ester of MK447 significantly reduced loop reabsorption. From these observations, we conclude that MK447 affects water and electrolyte reabsorption by the loop of Henle and beyond the superficial late distal tubule. The fact that a potential metabolite, but not MK447, significantly reduced reabsorption by the in situ, perfused loop of Henle supports the hypothesis that the p.o. and i.v. effects of MK447 are dependent on metabolism.     

The glutathione-activated thiopurine prodrugs trans-6-(2-acetylvinylthio)guanine and cis-6-(2-acetylvinylthio)purine cause less in vivo toxicity than 6-thioguanine after single- and multiple-dose regimens

trans-6-(2-Acetylvinylthio)guanine (trans-AVTG) and cis-6-(2-acetylvinylthio)purine (cis-AVTP) are glutathione-activated prodrugs of 6-thioguanine (6-TG) and 6-mercaptopurine, respectively. In tumor cell lines, these prodrugs exhibit similar IC50 values that are comparable to or lower than those of 6-TG and 6-mercaptopurine, respectively. In this study, the in vivo toxicity and metabolism of the prodrugs were assessed. Mice given multiple treatments of 6-TG and, to a lesser extent, trans-AVTG exhibited decreased peripheral WBC and RBC counts and increased myeloid:erythroid ratios in bone marrow; no change was observed in mice given cis-AVTP. Similarly, intestinal epithelial crypt cell apoptosis was more extensive in mice treated with 6-TG than in those treated with trans-AVTG, whereas mice given cis-AVTP had little apoptosis. Epithelial crypt cell apoptosis was more extensive in the small intestine than in the large intestine in all treatment groups. Histopathological examination detected no kidney or liver toxicity, whereas mild increases in the activities of hepatocellular leakage enzymes were observed in mice treated with trans-AVTG. Only metabolites of trans-AVTG and cis-AVTP were recovered in urine. A higher fraction of the dose was recovered in urine as the parent thiopurine and the metabolites thiopurine riboside, thioxanthine, and thiouric acid after 6-TG treatment than after trans-AVTG treatment; cis-AVTP recovery was slightly less than that of 6-TG. Thioxanthine and thiouric acid comprised a higher fraction of the recovered dose after cis-AVTP treatment than after trans-AVTG or 6-TG treatment. Overall, the results suggest that the prodrugs exhibit less in vivo toxicity than 6-TG. Thus, investigations into their antitumor efficacy are warranted.     

mGluR5 antagonists 2-methyl-6-(phenylethynyl)-pyridine and (E)-2-methyl-6-(2-phenylethenyl)-pyridine reduce traumatic neuronal injury in vitro and in vivo by antagonizing N-methyl-D-aspartate receptors

The effect of selective group I metabotropic glutamate receptor subtype 5 (mGluR5) antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB-1893) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. Treatment with MPEP and SIB-1893 showed significant neuroprotective effects in rat cortical neuronal cultures subjected to mechanical injury. Application of the antagonists also attenuated glutamate- and N-methyl-D-aspartate (NMDA)-induced neuronal cell death in vitro. Intracerebroventricular administration of MPEP to rats markedly improved motor recovery and reduced deficits of spatial learning after lateral fluid percussion-induced traumatic brain injury. Lesion volumes as assessed by magnetic resonance imaging were also substantially reduced by MPEP treatment. Although we show that MPEP acts as a potent mGluR5 antagonist in our culture system, where it completely blocks agonist-induced phosphoinositide hydrolysis, electrophysiological and pharmacological studies indicate that MPEP and SIB-1893 also inhibit NMDA receptor activity at higher concentrations that are neuroprotective. Taken together, these data suggest that MPEP and SIB-1893 may have therapeutic potential in brain injury, although the mechanisms of neuroprotective action for these drugs may reflect their ability to modulate NMDA receptor activity.     

Pronounced in vitro and in vivo antiretroviral activity of 5-substituted 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy] pyrimidines

OBJECTIVES: To discover new potent and selective anti-human immunodeficiency virus (HIV) acyclic nucleoside phosphonate (ANP) drugs with in vivo antiretroviral activity. METHODS: New acyclic pyrimidine nucleoside phosphonate derivatives that mimic the structure of the anti-HIV purine nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA, tenofovir) were designed by linking the acyclic side chain of the ANPs through an ether bond to the C-6 position instead of the N-1 position of the pyrimidine ring. The compounds were evaluated against HIV and Moloney murine sarcoma virus (MSV) in cell culture, including a broad variety of HIV-1 clade clinical isolates and relevant mutant (drug-resistant) HIV-1 isolates. Their antiviral activities were correlated and investigated in an in vivo model consisting of MSV-infected newborn mice. MSV-induced tumour formation and associated death were recorded in drug-treated animals. RESULTS: Several 5-substituted 6-[2-(phosphonomethoxy)ethoxy]-2,4-diaminopyrimidine (PMEO-DAPy) analogues were found to inhibit a broad variety of HIV-1 clinical isolates. They showed a more favourable cross-resistance profile to mutant virus isolates than adefovir and tenofovir. There was a close correlation between inhibition of MSV in C3H/3T3 cells and inhibition of HIV-1 in CEM cells. The PMEO-DAPy derivatives potently inhibited MSV-induced tumour cell formation in newborn mice. The 5-methyl analogue PMEO-5-Me-DAPy proved markedly more inhibitory to MSV-induced tumour cell formation and associated animal death than its unsubstituted parent PMEO-DAPy derivative. When compared with adefovir, PMEO-5-Me-DAPy was less toxic and more antivirally active in MSV-infected mice. CONCLUSIONS: PMEO-5-Me-DAPy deserves further (pre)clinical investigations as a candidate anti-HIV drug.