聚醚砜绒毛膜培养L02细胞的初步研究

目的初步观察一种聚醚砜(polyethersulfone,PES)绒毛膜对人肝细胞系L02细胞培养的支持作用,以探讨该种PES膜材料用于生物人工肝的可行性。方法 PES绒毛膜材料利用非对称性冷却成型技术制备而成。将L02细胞以7.0×104的密度接种于该膜上,分别用悬浮细胞计数法、MTT法、激光共聚焦显微镜、扫描电镜和RT-PCR法来观察L02细胞在该材料上的贴壁率、细胞活性、生长特性及AFP和CYP1A1的基因表达,并以无绒毛型平板膜材料和普通培养板做为对照。结果①PES绒毛膜上L02细胞生长良好,在10h内贴壁率由57.7%增高到91.7%,优于平板膜,与培养板相似;细胞活性8d内增长4至5倍,优于平板膜和培养板。②激光共聚焦显微镜下观察L02细胞在绒毛膜上粘附呈多细胞聚集生长,保持活性;扫描电镜证实了该结果,且逐渐融合形成紧密连接,并见部分细胞呈三维立体生长。③培养于PES绒毛膜上的L02细胞AFP和CYP1A1的表达优于平板膜,与培养板一致。结论 PES绒毛膜能够有效的支持L02细胞在其表面粘附、生长、增殖和AFP及CYP1A1基因的表达,可成为生物人工肝支持系统的膜材料。     

构建与人肝细胞系L02相容的聚丙烯生物杂化界面

学术背景：具有良好生物相容性的肝细胞/聚合物界面是生物反应器设计和构建的关键因素,然而,目前临床实践中使用的生物反应器并不理想。目的：构建与人肝细胞相容的聚丙烯生物杂化界面,为用聚丙烯中空纤维管构建生物人工肝反应器奠定基础。设计、时间及地点：对比观察,细胞相容性实验,于2003-02/10在上海交通大学完成。材料：聚丙烯利用光化学接枝聚合改性技术,在微孔聚丙烯超滤膜表面通过化学键的形式接枝亲水性丙烯酰胺基团形成接枝改性微孔聚丙烯超滤膜。方法：将人肝细胞系L02接种于聚丙烯膜、接枝改性聚丙烯膜表面,以聚苯乙烯作为正常对照。主要观察指标：聚丙烯膜接枝改性前后的静态水相接触角度;人肝细胞系L02在不同材料表面形态、贴壁率及增殖活性。结果：聚丙烯膜接枝改性后的静态水相接触角小于接枝前（P〈0.05）。人肝细胞L02在改性后聚丙烯膜上的贴壁率为0,细胞成球形聚集体生长,其增殖活性明显高于聚苯乙烯和改性前聚丙烯膜。结论：在聚丙烯表面接枝聚丙烯酰胺可建立良好的人肝细胞系L02与聚丙烯生物杂化界面,并可通过简单的静止培养形成肝细胞球形聚集体。     

单皮层聚醚砜膜培养HepG2细胞的初步研究

目的评价单皮层聚醚砜（PES）膜对肝细胞培养的支持作用，为其用于生物人工肝支持系统生物反应器的制备奠定基础。方法通过显微镜观察制备的平板膜结构，在PES膜上培养HepG2细胞，检测培养细胞的形态、生长曲线、白蛋白合成、戊醚试卤灵转化等功能。结果制备的PES膜为单皮层非对称性多孔结构；PES膜上HepG2细胞的生长曲线典型，在8天的培养时间能增殖6至7倍：细胞在PES膜上粘附呈多细胞聚集生长，部分细胞延伸入表面微孔内；PES膜上培养细胞LDH漏出量与对照无显著差异。PES膜上培养HepG2细胞具有较强的蛋白合成和生物转化功能。结论PEs平板膜能较好的支持肝细胞的粘附、生长、增殖和功能的表达，可用于中空纤维的制备。     

生物人工肝系统的生物反应器

目的:介绍生物反应器的基本作用、功能、条件和要求,并对主要生物反应器的研究进展予以探讨.方法:由第一作者应用计算机检索PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed,英文)及CNKI数据库(www.cnki.net/index.htm,中文).检索时限1994-01/2009-08.中文检索关键词:生物人工肝,生物反应器,膜材料.英文检索关键词:bioartificial liver,bioreactor,membrane material.文献筛选、纳入标准:①选取针对性强,相关度高的文献.②对同一领域的文献选择近期发表或权威杂志的文献.③排除较陈旧的理论观点以及一些重复性研究.结果:计算机初检到150篇文献,阅读标题和摘要进行初筛,排除研究目的与本文无关的文献88篇,内容重复性研究32篇,共30篇文献符合标准.结果显示生物人工肝系统正在成为肝衰竭患者体外有效的肝支持治疗手段,生物反应器为肝细胞提供良好的生长代谢环境、物质交换及免疫隔离的平台,是人工肝最重要的组成部分.以纤维素半透膜为基础的生物反应器主要有平板式反应器、中空纤维型反应器,半透膜材料主要有混合纤维素酯膜、醋酸纤维素膜、铜仿膜、聚偏二氟乙烯膜、聚丙烯膜等.结论:生物反应器作为一个动态体系,伴随着生物反应器本身控制系统的优化,能够更好地控制反应器内部的传质,建立仿生物理化学梯度,实现肝细胞最小结构单元的模拟构建,膜材料的选择是构建人工肝支持系统生物反应器的首要和基础环节之一.     

聚羟基丁酸／戊酸酯共聚物膜与人骨髓间充质干细胞的体外生物相容性

背景：聚羟基丁酸/戊酸酯共聚物是近年来受到重视的聚羟基脂肪酸族组织工程支架材料,具有免疫排斥反应低、生物相容性好和降解产物无毒副作用的优点。目的：观察聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞的体外生物相容性。方法：将第3代人骨髓间充质干细胞种植于聚羟基丁酸/戊酸酯共聚物膜上作为实验组,培养板单纯培养细胞作为对照组,计算1,2,4 h两组贴壁细胞的数量,得出细胞贴壁率。MTT比色法观察2,4,6,8 d两组细胞的增殖情况。采用Hoechst33258荧光法,检测3,6,9,12 d两组细胞内的DNA含量。将人骨髓间充质干细胞接种聚羟基丁酸/戊酸酯共聚物膜材料上5d后,电镜扫描观察细胞在材料上的生长情况。结果与结论：共培养1h时,实验组的细胞贴壁率低于对照组;其他时间段两组之间细胞贴壁率差异无显著意义。两组各时点间的细胞增殖差异无显著意义。两组各时间点细胞内DNA含量差异无显著意义。扫描电镜观察人骨髓间充质干细胞在聚羟基丁酸/戊酸酯共聚物膜上生长良好,形态呈梭形,细胞间连接紧密,分泌较多细胞基质。证明聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞有良好的生物相容性。     

有关生物人工肝膜材料的一些问题

1 人工肝膜材料的基本要求? 出于安全考虑,大多数生物反应器采用膜材料将肝细胞与血液相隔离.无论是平板型还是中空纤维型膜材料血液透析器,都已被证实具有运送氧气和血液中溶质成分的能力,并且具有良好的血液、血浆生物相容性.     

鼠尾草酸逆转K562/A02细胞多药耐药的机制研究

目的 探讨鼠尾草酸(Canosic acid,CA)对人类白血病多药耐药(MDR)细胞系K562/A02细胞的逆转作用及机制.方法 MTT法测定CA作用前后K562/A02细胞对阿霉素(ADM)的敏感性.流式细胞术(FCM)和激光扫描共聚焦显微镜(LSCM)测定细胞内ADM的平均荧光强度,计算细胞内ADM浓度.半定量RT-PCR检测细胞mdr1 mRNA表达水平.采用流式细胞术和Western blot 检测细胞膜P糖蛋白(P-gp)表达.结果 CA可将ADM对K562/A02细胞的IC50值由16.31μg/ml降至1.35μg/ml,逆转倍数为12.08倍.流式细胞术检测结果表明CA可将K562/A02细胞内ADM的荧光强度由17.05提高到60.53(P<0.01).LSCM结果显示CA可恢复ADM在K562/A02细胞的细胞核和胞质中的弥散分布,并使细胞内ADM的浓度由4.9 Oμg/ml提高至15.4μg/ml.RT-PCR结果显示K562/A02细胞mdr1 mRNA水平明显高于K562细胞,CA处理后K562/A02细胞mdr1 mRNA水平明显降低(P<0.01).流式细胞术检测K562/A02细胞膜上P-gp的荧光强度在经CA处理后由44.40降至22.80(P<0.05).Western blot结果显示CA处理后的K562/A02细胞膜上P-gp的表达明显降低.结论 在体外,CA可有效逆转人白血病细胞K562/A02的MDR,其逆转耐药的机制可能与P-gp蛋白表达下调并抑制其功能有关.     

聚丙烯酰胺接枝改性聚丙烯膜的血液相容性(英文)

背景:绝大多数高分子材料在与血液接触时都导致不同程度凝血,使其应用受到限制.因此研制有优良抗凝血性能的高分子材料成为生物人工肝材料临床研究中的关键问题.目的:体外检测新型人工肝反应器材料--聚丙烯酰胺接枝改性聚丙烯膜(PP-g-AAm)的血液相容性.方法:对改性前、后的聚丙烯膜行溶血试验、凝血酶原时间和活化部分凝血激酶时间试验,用流式细胞术检测血小板CD62P和CD63的表达率,扫描电镜观察两种膜上血小板的黏附情况.结果与结论:聚丙烯膜和PP-g-AAm膜的溶血率分别为1.32%和1.46%;聚丙烯膜的凝血酶原时间和活化部分凝血激酶时间较PP-g-AAm 膜明显缩短(P<0.05);PP-g-AAm 膜激活血小板表达CD62P、CD63的百分率都明显少于聚丙烯膜(P<0.05);扫描电镜观察两种材料表面黏附的血小板都有明显变形,但PP-g-AAm膜表面黏附的血小板明显少于聚丙烯膜.提示PP-g-AAm膜具有良好的血液相容性.     

聚砜膜生物反应器实验系统构建及对重型肝炎患者血浆的影响

背景：人工肝反应器性能的评价通常需要构建一个密闭实验装置模拟生物人工肝支持系统来对反应器内肝细胞的生物功能以及生物反应效率等进行综合评价，对聚砜膜中空纤维反应器的性能进行初步评价对优化生物人工肝支持治疗系统具有一定意义。目的：构建聚砜膜生物反应器实验系统，了解该系统对重型肝炎患者血浆的影响，观察聚砜膜中空纤维反应器作为生物人工肝反应器的可行性。设计：重复测量实验。单位：第三军医大学西南医院全军感染病研究所。材料：选用7只新生1-7d中国实验小型猪，雌雄不拘，由第三军医大学实验动物中心提供，许可证号码：F99017实验过程中对动物的处置符合动物伦理学标准。肿瘤坏死因子α、转化生长因子β1试剂盒购自中国晶美生物工程公司；聚砜膜中空纤维反应器订制于中国上海德宏生物材料研究所；Cellco培养循环式人工毛细管培养系统购于美国Spectrum公司；7份重型肝炎患者血浆样本均取自住院慢性重型肝炎患者做血浆置换时分离的血浆。患者均对实验项目知情同意，实验经过医院伦理委员会批准。方法：实验于2004—01／2005-07在第三军医大学西南医院全军感染病研究所实验室完成。①实验过程：实验前12h对实验猪进行断乳，清洁皮毛后分离肝细胞。将聚砜膜中空纤维反应器的内腔通过氧和二氧化碳弥散管与培养液贮池和Cellco培养液循环式人工毛细管培养系统连接成为密闭系统，外腔接口用洁净橡胶塞封闭。用磁力搅动法将分离的肝细胞进行球形体培养，接种到本系统的聚砜膜中空纤维反应器外腔，然后加重型肝炎患者血浆样本200mL于贮液池中，以80mL/min的速度在反应器内腔及贮液池中循环。②实验评估：在循环的0，2，4，6h从反应器内腔各取出2mL循环液，用谷氨酸脱氢酶．紫外法检测上清中氨的水平，全自动生化分析仪上检测标本中总胆红素的含量，在全自动血凝仪上检测标本的凝血酶原时间，肿瘤坏死因子α、转化生长因子β1的检测参照试剂盒说明书进行。主要观察指标：聚砜膜生物反应器系统对重型肝炎患者血浆的血氨、胆红素、凝血酶原时间、肿瘤坏死因子α及转化生长因子β1的影响。结果：①血氨、总胆红素、凝血酶原时间检测结果：重型肝炎患者血氨水平持续降低，0-2h下降最明显，其后血氨水平下降趋势减缓；总胆红素水平持续降低，6h的总胆红素水平明显低于0h，差异有显著性意义（P〈0．05）：凝血酶原时间持续降低，6h的凝血酶原时间较0h降低，差异具显著性意义（P〈0．05）。②肿瘤坏死因子α、转化生长因子β1检测结果：重型肝炎患者血浆的肿瘤坏死因子α及转化生长因子β1含量持续降低，其中6h均明显低于0h，差异具显著性意义（P〈0．05）。结论：砜膜生物反应器实验系统能够清除重型肝炎患者血浆中的小分子有毒物质，补充有益成分，降低细胞因子水平，可作为生物人工肝反应器。     

黄芩苷衍生物02-036对Burkitt淋巴瘤CA46细胞株的作用及相关机制

目的:探讨黄芩苷(Baicalin)衍生物02-036对人Burkitt淋巴瘤CA46细胞株的增殖、凋亡的影响及其相关机制。方法:应用MTT法检测和绘制细胞生长曲线;细胞集落形成实验检测黄芩苷衍生物02-036对CA46细胞增殖的影响;AnnexinV-FITC/PI双染流式细胞术检测02-036对细胞凋亡的影响;DNA倍体分析(DNA ploidy analysis)检测02-036对细胞周期的影响;Western blot检测增殖及凋亡相关蛋白BCL-2、Procaspase-9、Procaspase-3、PARP和C-MYC表达水平。结果:细胞生长曲线结果显示黄芩苷衍生物02-036对CA46细胞株有明显的抑制增殖作用,并且呈量效和时效关系(r=0.963,r=0.992),48 h半数抑制浓度(IC50)为(6.04±0.11)μmol/L。细胞集落形成实验显示,02-036作用CA46细胞后,细胞集落形成能力明显降低,并在一定范围内呈量效关系(r=-0.967)。Annexin V-FITC/PI双染流式细胞术分析结果表明,02-036能有效诱导CA46细胞凋亡,并且在一定范围内,细胞凋亡率随着药物作用浓度的增高而增加(r=0.959)。DNA倍体分析检测显示02-036能将CA46的细胞周期阻滞于S期。不同浓度的02-036作用于CA46细胞后,BCL-2、Pro-caspase-9、Pro-caspase-3、PARP和C-MYC的表达逐渐下调,而Cleaved-PARP表达逐渐上调,并呈现量效关系(r值分别为-0.990、-0.939、-0.971、-0.967、-0.936和0.920)。结论:黄芩苷衍生物02-036能有效抑制CA46细胞增殖,诱导其凋亡,在一定范围内呈时效和量效关系;其作用机制可能与BCL-2、Pro-caspase-9、Pro-caspase-3、PARP及C-MYC的表达下调有关。     

Disposition of eslicarbazepine acetate in the mouse after oral administration

Eslicarbazepine acetate is a promising antiepileptic drug structurally related to carbamazepine and oxcarbazepine, which is in the final phase of clinical development. The metabolism of eslicarbazepine acetate is clearly species dependent and, in this case, among small laboratory animals, the mouse seems to be the most relevant species to humans. Hence, the aim of this study was to investigate the plasma, brain and liver disposition of eslicarbazepine acetate in mice to better understand its disposition in humans. Adult male CD-1 mice were treated orally with a single dose of eslicarbazepine acetate 350 mg/kg. Blood samples, brain and liver tissues were taken at 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, 16 and 24 h post-dose. Plasma and tissue levels of eslicarbazepine acetate and its metabolites (S-licarbazepine, R-licarbazepine and oxcarbazepine) were assessed by using high-performance liquid chromatography-ultraviolet detection. Both eslicarbazepine acetate and R-licarbazepine concentrations were below the limit of quantification of the assay in all matrices. Eslicarbazepine acetate was rapidly and extensively metabolized to S-licarbazepine (major metabolite), which was oxidized to oxcarbazepine to a small extent. The brain/plasma ratios suggest that the brain exposure to S-licarbazepine and oxcarbazepine was approximately 30% of their total systemic exposure. However, S-licarbazepine crossed the blood-brain barrier (BBB) less efficiently than oxcarbazepine. On the other hand, the liver/plasma ratios support the notion that S-licarbazepine undergoes hepatic accumulation, whereas oxcarbazepine appears to leave this compartment twice as fast as S-licarbazepine. Thus, the diffusion through the BBB is favourable to oxcarbazepine and the liver acts like a deposit of the pharmacologically active metabolite of eslicarbazepine acetate (S-licarbazepine).     

生物人工肝的研究与进展

目的:分析近些年来生物人工肝相关的研究,对生物人工肝的发展作一总结.资料来源:以bioartificial liver, liver cell, hepatocyte culture, bioreactor为检索词,检索Ovid、SpringerLink数据库(1990-09/2008-09);以生物人工肝、肝细胞、细胞培养、生物反应器为检索词,检索维普数据库、中国期刊网及万方数据库(1990-09/2008-09).文献检索语种限制为英文和中文.资料选择:纳入有关生物人工肝肝细胞、反应器及辅助装置的研究,排除生物人工肝免疫和动物传染的研究.结局评价指标:①生物人工肝肝细胞的来源、数量及培养方式.②生物反应器的类型及主要结构膜的性质及类别.③生物人工肝供氧及温控装置的构成.结果:计算机初检得到3 898篇文献,根据纳入排除标准,对29篇进行分析.生物人工肝是在体外生物反应器中培养肝细胞,当患者的血液流经生物反应器时,通过半透膜或直接接触的方式与培养的肝细胞进行物质交换,使其中的肝细胞发挥解毒、合成、生物转化等功能,以达到支持和治疗目的.它不但具有解毒功能,而且还能参与3大营养物质代谢,分泌促进肝细胞生长的物质等,可有效替代肝脏的解毒功能和合成功能,并可较好地预防肝性脑病、肝昏迷和脑水肿,临床上可作为肝衰竭患者肝移植前的过渡辅助措施.结论:生物人工肝研究虽然取得了重大进展,但仍然面临寻找理想肝细胞来源,长期维持肝细胞的活性和功能,进一步优化反应器设计等问题.     

醋酸棉酚诱导淋巴细胞白血病细胞凋亡的作用及联合地塞米松的效应

本研究旨在探讨醋酸棉酚诱导人急、慢性淋巴细胞白血病细胞凋亡的作用及联合地塞米松的效应。用流式细胞仪检测醋酸棉酚对人急、慢性淋巴细胞白血病原代细胞凋亡的影响。用MTT比色法测定醋酸棉酚对人Burkitt淋巴瘤细胞株Raji细胞及正常骨髓单个核细胞存活率的影响。用流式细胞仪检测醋酸棉酚与地塞米松联合诱导Raji细胞的凋亡率。结果表明,≥5μmol/L醋酸棉酚能诱导原代培养的人急性淋巴细胞白血病细胞凋亡,呈浓度及时间依赖性。醋酸棉酚对原代培养的慢性淋巴细胞白血病细胞诱导凋亡的浓度更高,50μmol/L醋酸棉酚作用48 h,急、慢性淋巴细胞白血病细胞的凋亡率分别为(90.4±6.2)%和(51.7±10.3)%。≤10μmol/L醋酸棉酚对正常骨髓单个核细胞的存活率没有明显影响,醋酸棉酚作用48和72 h,对正常骨髓单个核细胞的IC50值分别为Raji细胞的7.1和9.1倍。≥10μmol/L地塞米松能诱导Raji细胞凋亡,不同浓度地塞米松与10μmol/L醋酸棉酚联用,Raji细胞凋亡率明显增加(P<0.05)。结论:醋酸棉酚能诱导原代培养的人急、慢性淋巴细胞白血病细胞凋亡;对正常骨髓单个核细胞的抑制作用明显低于Raji细胞;与地塞米松联用可增强后者诱导Raji细胞凋亡的效应。     

An application of the high-iron diamine staining for detection of sulfated glycoproteins (glycopeptides) in electrophoresis on cellulose acetate membrane

The high-iron diamine staining (HID), which has been used in histochemistry to stain sulfated glycoconjugates (SGC), was tested for detectability of authentic acidic substances (chondroitin sulfates A plus C, dermatan sulfate, heparan sulfate, chondroitin, hyaluronic acid, alpha 1-acid glycoprotein and ribonucleic acid) in electrophoresis on cellulose acetate membrane (Separax). The results showed that only SGC were detectable by the HID, although all the above substances were stained with alcian blue. The glycoconjugate preparations obtained from the liver, kidney, lung, small intestine, colon, stomach, brain and spleen of rats were examined by two-dimensional electrophoresis on Separax. The new spots (or bands), besides those of sulfated glycosaminoglycans, were detected by the HID on the electrophoretograms of all the samples except for the kidney one. The substances giving the new spots (or bands) were indicated to be sulfated glycopeptides (SGP) by crude heparinase digestion of a representative sample. The present results revealed that the HID was applicable for detection of SGP in electrophoresis on cellulose acetate membrane. Also, it is a novel finding that the liver and spleen contain SGP.     

Starch acetate microparticles for drug delivery into retinal pigment epithelium-in vitro study.

Starch acetates are novel biodegradable polymers which undergo slower degradation and swelling than native starch. Retinal pigment epithelium (RPE) is an important target tissue in ocular treatment. The cellular uptake of starch acetate microparticles and degradation of starch acetate by cultured human RPE-cell line (D407) was examined. Calcein-containing starch acetate microparticles were prepared by a modified water-in-oil-in-water double-emulsion technique. The cellular uptake of the starch acetate microparticles was analysed using flow cytometry and confocal microscopy. Degradation of starch acetate films by the homogenate of lysed RPE cells was determined by gel permeation chromatography. The effect of the microparticles on RPE cell viability was determined by the MTT colorimetric assay. The mean diameter (D50%) of microparticles was 11 microm. During 3-h incubation in RPE-cell culture, 8.1 +/- 0.8% of D407 cells took up starch acetate microparticles. Confocal microscopy confirmed the internalisation of microparticles. Incubation of the starch acetate film in the RPE-cell homogenate considerably decreased the molecular weight of starch acetate in the film during 24 h. The viability of cultured RPE cells was at least 82% after 24-h incubation with the microparticles. The present results show that the starch acetate microparticles are taken up by the RPE cells and the polymer can be degraded by the enzymes in these cells. Starch acetate microparticles may be suitable for drug delivery to the RPE.     

Long-term cultures of murine fetal liver retain very early B lymphoid phenotype

Long-term cultures of murine fetal liver have been successfully established using a modification of our in vitro bone marrow culture system (14, 15). Fetal liver cells from midgestation BALB/c embryos were plated onto BAB-14 bone marrow stromal cell-adherent layers. After a 3-5 wk period, cell growth began to increase and these cells were expanded in number on fresh feeder layers. The cultured fetal liver cells were lymphoid in morphology, 5-20% cytoplasmic Ig-positive, but less than 1% surface Ig-positive. Southern blot analysis of the cultured fetal liver cells, as well as cultured bone marrow-derived B cells, demonstrated a population with germline Ig heavy chain loci, possibly representing very early B cell precursors. Abelson murine leukemia virus (A-MuLV) clonal transformants of such cultured fetal liver cells had a phenotypic distribution similar to that seen with fresh fetal liver transformants but distinct from those obtained with the transformation of either cultured or fresh bone marrow. All A-MuLV transformants isolated had rearrangements at the mu heavy chain locus of both chromosomes, irrespective of Ig production. In addition, most mu heavy chain producers had at least one rearranged kappa gene locus. These long-term fetal liver cultures provide large numbers of cells for studying events early in the B lymphocyte lineage. The cultured fetal liver cells retained phenotypic traits similar to fresh fetal liver B cells and distinctive from bone marrow cells cultured under similar conditions.     

Lactate dehydrogenase and alkaline phosphatase isoenzymes and protein-bound sialic acid in regenerating rat liver

Lactate dehydrogenase (LDH) and alkaline phosphatase (AP) isoenzyme patterns and protein-bound sialic acid content were compared between normal, regenerating rat liver 10 days after partial hepatectomy and fetal rat liver. For this purpose, liver from ten adult rats and two pools of ten fetal livers each were examined. Isoenzymes were separated by electrophoresis on cellulose acetate and their percent distribution calculated after quantitation by densitometry of the bands. LDH-5 and LDH-4 combined represented in all the tissues examined 90%-94% of the total activity. LDH-5/LDH-4 ratios were nearly equivalent in the normal and regenerated liver (7.14, 6.41), but substantially lower in fetal liver (2.50). Two bands of AP were visualized in electropherograms. AP-1/AP-2 ratio was lower in regenerated liver (1.57) as compared to normal liver (2.27) and still lower in fetal liver (1.06). Protein-bound sialic acid was, on protein basis, slightly but not significantly higher in regenerated liver (1.71 microgram/mg protein) than in normal liver (1.43), and significantly higher in fetal liver (1.87). The relatively small differences in isoenzyme patterns and in protein-bound sialic acid between regenerated and normal liver as compared to those between fetal and normal tissue add support to the view that the cells in regenerated liver are not of embryonic origin.     

Effects of octreotide acetate and Saccharomyces boulardii on bacterial translocation in an experimental intestinal loop obstruction model of rats

Intestinal obstruction (IO) induces bacterial translocation (BT) due to mucosal disruption, motility dysfunction, and increased intestinal volume, leading to bacterial overgrowth. This study was conducted to investigate the effects of octreotide acetate (OA) and Saccharomyces boulardii (SB) on the BT and intestinal integrity in an animal model of intestinal loop obstruction (LO). Forty adult male Sprague-Dawley rats (250-300 g) were randomized into 4 groups containing 10 rats each. Complete IO was created in the distal ileum of rats by a single 3-0 silk suture (LO). Group Sham: Sham (Laparotomy only was performed in this group); group LO: LO; group OA: LO plus OA (100 microg/kg, at 0, 12 hours of obstruction); group (SB): LO plus SB (800 mg/kg/day, via orogastric and preoperative for 3 days). After 24 hours, samples of mesenteric lymph nodes (MLN), liver, spleen and blood were obtained and cultured. The terminal ileum specimens were examined histopathologically. There were no BT in group Sham, but BT was noticed totally in 31 (77.5%) cultures in group LO. This rate was reduced to 30% (n = 12), 10% (n = 4) in the groups OA and SB respectively. Bacterial translocations of MLN and the liver in group LO were significantly higher than those of groups OA and SB. Bacterial translocations of the both spleen and blood in group LO were significantly higher than those of groups OA and SB. The mean bacterial counts, colony-forming units per gram tissue (cfu/g), in the MLN, liver and spleen of group LO were found significantly higher than those of groups OA and SB. The mean villus height in group OA was significantly higher than that of group LO and it in the group SB significantly higher than those of groups LO and OA. The present experimental study has demonstrated that OA and SB may have protective effects against BT in mechanical bowel obstruction and additionally SB preserves intestinal mucosal integrity.     

Sanfilippo A Syndrome SULFAMIDASE DEFICIENCY IN CULTURED SKIN FIBROBLASTS AND LIVER

The Sanfilippo A syndrome is an autosomal recessive mucopolysaccharidosis characterized clinically by severe mental retardation and biochemically by storage in tissue and excretion in urine of excessive amounts of heparan sulfate. Since sulfamide groups are present in heparan sulfate, a sulfamidase deficiency could explain the impaired degradation of this polysaccharide. To investigate the enzymic basis of this disease, assays for sulfamidase were performed. Extracts of cultured fibroblasts and post-mortem liver were prepared by suspension of tissues in acetate: NaCl buffer, pH 4.5, containing Triton X-100 (Rohm and Haas Co., Philadelphia, Pa.), sonication, and centrifugation at 10,000 g. The supernatant fluid was incubated with [(35)S]-N-sulfated heparin. The release of inorganic sulfate after 18 h of incubation was determined by chromatography on Sephadex G-25. The liver and fibroblast extracts of patients with the Sanfilippo A syndrome showed a deficiency of sulfamidase.The quantity of heparan sulfate in fibroblasts derived from patients with Sanfilippo A, Hurler's and Hunter's diseases was found to be 7-10%, while it was about 1.25% of the total glycosaminoglycans in fibroblasts of normal controls.