生物材料Sapeptide的体外塑型及在体毒性实验

目的：观察生物材料Sapeptide的大体形状及其表面结构特性，并进行在体毒性实验，评测其生物相容性。方法：实验于2004-08／2005-09在解放军第三军医大学外科研究所完成。①采用等渗盐水作为Sapeptide塑型剂，塑型后分别进行大体、光镜和电镜观察材料表面结构。②对成年Wistar大鼠分组进行腿部肌肉注射毒性实验。取成年Wistar大鼠4只，标记后分为2组。A组：左前肢为RAD16-Ⅱ，右前肢为RAD16-Ⅰ，左后肢为RAD16-Ⅱ和RAD16-Ⅰ的均等合剂，右后肢为生理盐水对照。B组：左前肢为RAD16-Ⅱ，左后肢为RAD16-Ⅰ，右前肢和右后肢为1，25g／L胰酶作对照。其中RAD16-Ⅱ和RAD16-Ⅰ水溶液浓度为2g／L，每个肢体注射液体量均为20μL。每组分为两个时相点（9d和35d）用剪刀按照标记部分取出被注射的整条肌肉组织，切片后做苏木精一伊红染色和刚果红染色，观察肌肉组织结构的改变及有无炎症反应。结果：（1）Sapeptide的大体形状与表面结构：Sapeptide材料表面呈网状，网孔直径约54μm，纤维的直径约为9μm。②毒性实验结果：大体观察，各组标本材料植入部位未见出血、积液和组织坏死，注射部位肌肉组织质地光泽正常；刚果红染色，可见所有注射Sapeptide材料的肢体切片中均有片状的红色物质，9d时面积大于35d所见：生理盐水对照组和胰蛋白酶组未见红染物质；苏木精-伊红染色，A组，第9天和第35天四肢肌肉组织取材标本见肌肉纤维形态正常，未见炎性细胞浸润；B组．注射胰蛋白酶侧肢体在第9天可见局部组织有炎症反应，见少量淋巴细胞浸润，但第35天，组织炎症已基本消退。Sapeptide注射处肌肉组织未观察到炎症反应和免疫排斥反应。结论：Sapeptide材料具有生物相容性好，可同种子细胞和宿主组织很好的整合；合适的生物可降解性，降解产物对人体无不良反应，有良好的可塑性；并且应用简便，在神经组织工程中是一种具有良好特性的组织工程材料。     

几种生物材料表面黏附特性的实验

目的：应用微吸管吸吮技术测量大鼠骨髓基质细胞在聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的黏附力,以筛选生物相容性良好的生物材料.方法：实验于2003-11/2004-05在华中科技大学附属协和医院骨科组织工程实验室完成.选取4周龄健康SD大白鼠4只.大鼠麻醉后抽取双侧胫骨和股骨的骨髓基质细胞体外培养,第3代细胞用于实验.制备以玻璃为底、直径约25 mm的特制圆形小腔室,分别将聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物溶液0.3 mL滴入小腔室内,300 r/min离心5 min,在玻璃表面形成聚合物薄膜.用微吸管拉制器拉制微吸管,加工成前端内径为2～4 μm的微管,并用微量进样器向微管内灌满培养液,微管后端接于水压控制的微操作手上,并与自动压力控制系统连接.将第3代骨髓基质细胞接种在表面为不同材料的小腔室内,在每种材料表面随机测量20～30个细胞,以没有涂覆材料的玻璃为对照,测量骨髓细胞于不同时间在三种生物材料表面的细胞黏附力.结果：实验纳入大鼠4只,全部进入结果分析.不同生物材料上骨髓基质细胞黏附力的比较：细胞种植到生物材料表面4,12,24 h,骨髓基质细胞在聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的黏附力均明显高于聚乳酸、聚丙交酯/乙交酯[（172.78&;#177;15.23）,（155.609&;#177;17.29）,（144.84&;#177;21.14）;（324.84&;#177;102.73）,（199.39&;#177;37.20）,（234.09&;#177;62.09）;（180.77&;#177;29.22）,（170.32&;#177;21.14）,（174.38&;#177;44.29）&;#215;10^-10N,P均＜0.05].结论：微细管吸吮法能够精确测量细胞在支架材料上的黏附力.在聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物这三种生物材料中,聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的细胞黏附力最好,是较理想的生物材料.     

三嵌段高分子骨组织工程支架材料的体内生物学性能评价

目的：对三嵌段高分子骨组织工程支架材料-聚（丙交酯-乙交酯）[天冬氨酸-聚乙二醇1的生物相容性进行评价，探讨用于骨组织工程的可行性。 方法：实验于2003-08／12在华中科技大学同济医学院附属协和医院骨科实验室完成。①过敏试验：20只豚鼠随机分为试验组、对照组，每只豚鼠脊柱两侧皮内注射等体积聚（丙交酯-乙交酯）[天冬氨酸-聚乙二醇]浸提液、生理盐水及体积分数为0．05的甲醛溶液，记录激发部位红斑水肿。②急性全身毒性试验：12只小白鼠随机分为试验组和对照组，该两组腹腔分别注射浸提液及生理盐水，4，24，48及72h观察动物的一般状态。③植入试验：聚（丙交酯-乙交酯）[天冬氨酸-聚乙二醇]植入6只新西兰大白兔肌肉内，1，4及8周切取材料周围0．5cm处肌肉组织，苏木精-伊红染色，光镜下观察。 结果：①过敏试验结果：皮内注射浸提液及生理盐水动物皮肤无红斑水肿，注射体积分数为0．05的甲醛溶液出现中度以上红斑水肿。②急性全身毒性试验结果：阴性对照组未见毒性症状，试验组4h后仅1只有轻微的运动减少，24h后恢复正常。③植入试验结果：所有试验动物伤口均一期愈合。 结论：聚（丙交酯-乙交酯）[天冬氨酸-聚乙二醇]具有良好生物相容性，是一种新的骨组织工程支架材料。     

β-磷酸三钙／聚磷酸钙纤维／聚左旋乳酸复合材料的物理及其降解性能

目的：研制一种可任意调控降解速率且具有良好力学性能、生物相容性能的高孔隙率海绵状软骨组织工程支架复合材料。 方法：实验于2002-05／12在兰州交通大学工程材料研究所完成。以自制聚磷酸钙纤维、β-磷酸三钙为增强材料，聚左旋乳酸为基体材料，采用溶媒浇铸／粒子滤取技术与气体发泡相结合的方法制备了β-磷酸三钙／聚磷酸钙纤维／聚左旋乳酸软骨组织工程支架复合材料，测试了该复合材料的物理力学性能和降解性能。结果：①支架复合材料物理力学性能：随着β-磷酸三钙重量分数的增加，支架材料的密度也随之增加。②微观结构观察：β-磷酸三钙在横截面及纵截面上分布较为均匀，且支架材料为三维、连通、网状结构。③降解性能：随着β-磷酸三钙含量的增加，支架复合材料的降解率逐渐减小；随着聚磷酸钙纤维含量的增加，支架复合材料的降解率逐渐增大；该组支架材料在0-3周时压缩模量迅速衰减，3周后压缩模量衰减变缓；随着β-磷酸三钙含量的增加，降解液的pH值稍稍增加，呈微碱性环境。 结论：β-磷酸三钙／聚磷酸钙纤维／聚左旋乳酸支架复合材料的力学性能和生物降解特性基本满足软骨组织工程的要求，特别是β-磷酸三钙的加入使降解液pH值保持在6-7之间，避免了由于降解液呈酸性而引起的无菌性炎症反应。故可用作软骨组织工程支架材料。     

组织工程骨支架的材料学：短棒状纳米羟基磷灰石的细胞毒性检测

目的：体外研究短棒状纳米羟基磷灰石(Na-HA)的细胞毒性．探讨将其应用于组织工程骨支架的可能性。方法：实验于2004—01／05在第四军医大学口腔颌面外科实验室完成。采用MTT检测法，将不同浓度的短棒状Na-HA浸提液与L929成纤维细胞接触1，3，5d，对细胞增殖活性进行检测，计算细胞相对增殖率，用6级毒性分类法评级，并进行形态学观察。用碱性磷酸酶(ALP)测定试剂盒检测浸提液对兔成骨细胞功能表达的影响。结果：不同时间点用不同浓度浸提液培养的细胞均正常增殖．毒性0—1级。浸提液不影响兔成骨细胞的功能表达。结论：短棒状Na-HA无细胞毒性，不影响成骨细胞的成骨活性，可能是一种组织工程骨支架的良好材料。     

几种生物材料表面黏附特性的实验

目的:应用微吸管吸吮技术测量大鼠骨髓基质细胞在聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的黏附力,以筛选生物相容性良好的生物材料。方法:实验于2003-11/2004-05在华中科技大学附属协和医院骨科组织工程实验室完成。选取4周龄健康SD大白鼠4只。大鼠麻醉后抽取双侧胫骨和股骨的骨髓基质细胞体外培养,第3代细胞用于实验。制备以玻璃为底、直径约25mm的特制圆形小腔室,分别将聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物溶液0.3mL滴入小腔室内,300r/min离心5m in,在玻璃表面形成聚合物薄膜。用微吸管拉制器拉制微吸管,加工成前端内径为2～4μm的微管,并用微量进样器向微管内灌满培养液,微管后端接于水压控制的微操作手上,并与自动压力控制系统连接。将第3代骨髓基质细胞接种在表面为不同材料的小腔室内,在每种材料表面随机测量20～30个细胞,以没有涂覆材料的玻璃为对照,测量骨髓细胞于不同时间在三种生物材料表面的细胞黏附力。结果:实验纳入大鼠4只,全部进入结果分析。不同生物材料上骨髓基质细胞黏附力的比较:细胞种植到生物材料表面4,12,24h,骨髓基质细胞在聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的黏附力均明显高于聚乳酸、聚丙交酯/乙交酯[(172.78±15.23),(155.609±17.29),(144.84±21.14);(324.84±102.73),(199.39±37.20),(234.09±62.09);(180.77±29.22),(170.32±21.14),(174.38±44.29)×10-10N,P均<0.05]。结论:微细管吸吮法能够精确测量细胞在支架材料上的黏附力。在聚乳酸、聚丙交酯/乙交酯、聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物这三种生物材料中,聚丙交酯/乙交酯/天冬氨酸-聚乙二醇三嵌段共聚物表面的细胞黏附力最好,是较理想的生物材料。     

新型三维编织型生物支架的研制及体外生物相容性

背景:脊髓损伤后,由于有胶质瘢痕的阻隔,使再生的轴突难以穿越损伤区域,从而影响脊髓功能的恢复.随着组织工程技术的发展,研究人员尝试利用在三维支架的空间诱导作用下,让再生的轴突和支架内部携带的细胞能够三维有序的生长,穿越瘢痕屏障,连接脊髓损伤断端.目的:采用编织工艺研制新型以聚乙交酯-丙交酯为原料的三维支架,检测新型支架与许旺细胞的生物相容性.设计、时间及地点:体外对比观察实验,于2007-06/2008-03在长海医院中心实验室完成.材料:选择聚乳酸:聚羟基乙酸为9:1的聚合材料聚乙交酯-丙交酯,通过熔融纺丝、拉伸、编织等步骤制作聚乙交酯-丙交酯三维支架.取新生3 d SD乳鼠的坐骨神经,分离、纯化许旺细胞.方法:实验分3组:三维支架组将许旺细胞接种在新型三维支架内培养;明胶海绵组将许旺细胞接种在胶原海绵上培养;细胞培养皿组直接接种在预涂左旋多聚赖氨酸的24孔培养板上培养.主要观察指标:扫描电镜观察内部微管道的排列规律,测量其孔径大小、孔隙率等指标.通过倒置相差显微镜和扫描电镜观察许旺细胞在支架上生长情况,包括许旺细胞在支架上的黏附、增殖和凋亡情况等.结果:支架外径为3 mm,微管道内径为100 μm,呈均匀平行排列,支架的孔隙率为68%.三维支架组与细胞培养皿组中的许旺细胞黏附、增殖差异无显著性意义,与明胶海绵组差异有显著性意义.三维支架组与明胶海绵组中细胞凋亡差异无显著性意义,它们均低于细胞培养皿组,差异有显著性意义.结论:新型三维编织型支架具有良好的生物相容性.     

新型组织工程化人工骨的体外初步构建

背景：L-聚乳酸可以用于制备骨组织工程支架材料，但存在某些缺陷，能否复合聚磷酸钙纤维、Ⅰ型胶原来改善材料性能，从而找到新的组织工程化骨体外构建方法?目的：应用组织工程学技术，体外初步构建有活性的新型组织工程化人工骨。设计：体外构建细胞-材料复合体，自身对照前瞻性研究。地点和对象：本研究在解放军第三军医大学西南医院骨科和兰州铁道医学院复合材料研究所完成，对象为5例重庆市健康志愿者的髂骨骨髓。干预：培养、诱导人骨髓间充质干细胞向成骨细胞表型分化，制备性能优越的新型支架材料，再按一定的方法体外构建细胞-材料复合体。主要观察指标：细胞碱性磷酸酶染色的阳性率、扫描电镜观察组织工程化骨的微观结构。结果：由L-聚乳酸、聚磷酸钙纤维、Ⅰ型胶原制备出新型的骨组织工程复合支架材料，孔径300～450μm，孔晾率90．1％；细胞接种前碱性磷酸酶阳性率为(55&;#177;8)％，接种后细胞可在材料内良好贴附生长，碱性磷酸酶阳性率为(52&;#177;6)％，两者差异无显著性。结论：经成骨诱导的间充质干细胞，可以与由L-聚乳酸、聚磷酸钙纤维、Ⅰ型胶原制备的支架材料复合，在体外构建出组织工程化人工骨。     

神经组织工程生物材料的生物相容性及安全性评价

神经组织工程生物材料的出现尽管只有20年左右的时间，但发展迅速，已成为神经系统疾病的一种新的治疗手段和发展趋势。多数组织工程生物材料在动物实验已体现出良好的生物相容性，但是由于神经系统独特性，实际应用于临床还需要大量深入研究。总之，具有良好生物相性和安全性的神经组织工程生物材料仍是当前和今后研究方向。文章回顾神经组织工程生物材料的应用历史及现状，从生物相容性及安全性方面对现有神经组织工程生物材料进行评价，为提高神经组织工程生物材料水平提供依据。     

贻贝粘蛋白创面修复敷料体外细胞毒性的检测方法

背景：在贻贝粘蛋白创面修复敷料的体外细胞毒性检测中,由于蛋白分子表面带正电荷,1∶9的浸提比会使细胞聚团而导致测定产生误差,影响测定结果。 目的：在已有标准的基础上,根据贻贝粘蛋白特殊的性质及使用状态,改进贻贝粘蛋白创面修复敷料的浸提比例或前处理方法。 方法：①浸提液法：将贻贝粘蛋白创面修复敷料与细胞培养液分别以1∶9、1∶131浸提比例制备浸提液,分别以贻贝粘蛋白创面修复敷料浸提液、天然乳胶浸提液、高密度聚乙烯浸提液及细胞培养液培养L929小鼠成纤维细胞。②直接接触法：分别以蒸馏水、贻贝粘蛋白创面修复敷料溶液、二甲基亚砜及细胞培养液培养L929小鼠成纤维细胞。 结果与结论：采用浸提比1∶9测定样品体外细胞毒性时,细胞产生聚团作用,不适用于样品毒性的检测;调整浸提比为1∶131后,絮凝作用和细胞聚团现象明显降低,提高了检测结果的可信度,显示样品无细胞毒性。直接接触法显示样品无细胞毒性。采用经调整过的浸提液法或直接接触法均可适用于贻贝粘蛋白创面修复敷料体外细胞毒性的检测。     

Applications of self-assembling ultrashort peptides in bionanotechnology

Peptides are intriguing building blocks for a variety of applications in bionanotechnology. Peptides can self-assemble into well-ordered nanostructures. Among the various nanomorphology forms, peptide nanofibers and nanotubes are relevant in biomedical applications. In this review, their applications as tissue engineering scaffolds, drug delivery vehicles, three-dimensional printing bioinks and bioimaging nanoprobes will be illustrated. This review article describes di-, tri-, tetra-peptides because they are cost-effective, simple to prepare, and amenable to production on a large scale.

Ultrashort peptide applications in bionanotechnology.     

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.     

乳化异氟烷的体内和体外溶血性实验研究(英文)

Objective Emulsified isoflurane (8%,vol/vol) is a kind of lipid based formation for intravenous administration.The purpose of this study is to evaluate whether emulsified isoflurane induces hemolysis or not in vitro and in vivo.Methods In hemolysis test in vitro,a male rabbit was used to prepare 2% (vol/vol) erythrocyte suspensions for measuring degree of hemolysis of emulsified isoflurane at the doses from 12 to 0.3 g/L. In hemolysis test in vivo,4 male Beagle dogs were intravenously adminstrated emulsified isoflurane 225.6 mg/kg in 3-5 min. 5 ml samples of venous blood were collected from each dog at 0 min (start of injection),5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after the adminstration of emulsified isoflurance for measuring erythrocyte morphology,reticulocyte counts,the concentrations of free hemoglobin,haptoglobin,and bilirubin. At the same time,urinary blood,urinary bilirubin and urobilinogen were also measured before and after adminstration.Results In vitro experiment,emulsified isoflurane led to hemolysis at the concentrations from 12 to 1.2 g/L. However,no hemolysis was found at the concentrations from 0.6 to 0.3 g/L. In vivo experiment,with the exception of a slight reduction in indirect bilirubin and a mild increase in direct bilirubin at 5 min(P<0.05),others such as total bilirubin,retculocyte counts,haptoglobin,free hemoglobin,urinary bilirubin,urinary blood,and urinary urobinogen were not significantly different compared with their corresponding values before injection. There was also no significant difference in erythrocyte fragmentations at 0 min,5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after injection of emusified isoflurane,and none of macrocytes and nucleated red cells was noted on all blood films. Conclusions Emulsified isoflurane at concentrations recommended for clinical trials did not cause hemolysis in vitro and in vivo.     

Efficacy of synthetic peptides RP-1 and AA-RP-1 against Leishmania species in vitro and in vivo

Host defense peptides are naturally occurring molecules that play essential roles in innate immunity to infection. Based on prior structure-function knowledge, we tested two synthetic peptides (RP-1 and AA-RP-1) modeled on the conserved, microbicidal α-helical domain of mammalian CXCL4 platelet kinocidins. These peptides were evaluated for efficacy against Leishmania species, the causative agents of the group of diseases known as leishmaniasis. In vitro antileishmanial activity was assessed against three distinct Leishmania strains by measuring proliferation, metabolic activity and parasite viability after exposure to various concentrations of peptides. We demonstrate that micromolar concentrations of RP-1 and AA-RP-1 caused dose-dependent growth inhibition of Leishmania promastigotes. This antileishmanial activity correlated with rapid membrane disruption, as well as with a loss of mitochondrial transmembrane potential. In addition, RP-1 and AA-RP-1 demonstrated distinct and significant in vivo antileishmanial activities in a mouse model of experimental visceral leishmaniasis after intravenous administration. These results establish efficacy of RP-1 lineage synthetic peptides against Leishmania species in vitro and after intravenous administration in vivo and provide further validation of proof of concept for the development of these and related systemic anti-infective peptides targeting pathogens that are resistant to conventional antibiotics.     

Effect of antimicrobial peptides HNP-1 and hBD-1 on Staphylococcus aureus strains in vitro and in vivo

The aims of this study were: (i) To investigate the activity of recombinant AMPs HNP-1 and hBD-1 in combination with cefotaxime against Staphylococcus aureus strains (MSSA and MRSA) in vitro using checkerboard method; (ii) To investigate the activity of HNP-1 and hBD-1 encapsulated in silicon nanoparticles (niosomes) in the treatment of MRSA-infected wound in rats. For this S. aureus strains (MSSA and MRSA) were isolated from patients with diabetic foot infection. Cefotaxime, recombinant HNP-1 and hBD-1 (in all possible combinations with each other) were used for testing by the checkerboard method. Two niosomal topical gels with HNP-1/hBD-1 were prepared to treat MRSA-infected wounds in rats. Gels were administered once a day, the control group–without treatment. Wound healing rate was calculated on the 4th, 9th and 16th days of the experiment and compared using one-way ANOVA with Bonferroni correction. MIC of HNP-1 for MSSA and MRSA was the same–1 mg/L. MIC of hBD-1 for MSSA and MRSA was also the same–0.5 mg/L. Topical gels with niosomal HNP-1 (or hBD-1) showed a significantly faster wound healing in comparison with the control. The data obtained open up prospects for use of AMPs encapsulated in silica nanoparticles for the development of new antibiotics.     

Interleukin 7 promotes long-term in vitro growth of antitumor cytotoxic T lymphocytes with immunotherapeutic efficacy in vivo

A major obstacle to the effective use of adoptive immunotherapeutic treatment of cancer is the difficulty of obtaining tumor-reactive lymphocytes in either sufficient numbers or with appropriate in vivo function to make such an approach feasible. Previous studies have shown that antitumor cytotoxic T lymphocytes (CTL) with in vivo efficacy can be generated in vitro from lymphoid cells obtained from lymph nodes that drain the anatomical site of a tumor. Results presented here demonstrate that inclusion of interleukin 7 (IL-7) into the medium in which such CTL are cultured can support their growth in vitro for prolonged periods of time in the absence of repeated stimulation with either tumor stimulator cells or tumor antigen. More importantly, antitumor CTL propagated in medium containing IL-7 have retained both their antigenic specificity and their ability to reject tumors in vivo subsequent to intravenous injection. Parallel cultures of antitumor CTL similarly cultured in medium containing only IL-2 could only be maintained for 5-6 wk, after which the number and proportion of viable cells that were recoverable from such cultures progressively decreased. Phenotypic analysis of CTL maintained after extended culture (i.e., 22 mo) in medium containing IL-7 demonstrated them to be CD3+4-8+ T cells. These cells were also found to express lymphocyte function associated 1, intercellular adhesion molecule 1, and Mel-14 cell interaction molecules. The data also demonstrate that these CTL do not require the presence of antigen-presenting cell populations to mount a proliferative response to tumor stimulator cells. Cells in these cultures were also demonstrated to produce IL-2 after stimulation with irradiated tumor cells, thereby indicating that these CTL have become independent of the requirement for CD4+ helper cells to survive and function either in vitro or in vivo. Collectively, the findings that IL- 7 can beneficially augment the generation, and propagate the long-term growth, of antitumor CTL from lymph nodes draining a tumor site may have profound implications for promoting the immunotherapeutic treatment of cancer in humans.     

Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo.

Cationic lipids can deliver genes efficiently in vitro, but are generally inhibited by the presence of serum, and their efficiency in vivo is much lower than in vitro. An attractive strategy is to induce strong DNA compaction by its association with proteins, before addition of lipids. However the use of whole proteins might present both production and immunological limitations. We have devised a system in which DNA is associated with short peptides derived from human histone or protamine, before the addition of a cationic lipid or polymer. Peptides strongly associating with DNA confer to such peptide-DNA-lipid particles an enhanced in vitro transfection efficiency over that observed with classical DNA/lipid lipoplexes, and particularly confer the capacity to transfect in the presence of serum. This acquisition of serum resistance is cell type-independent, and observed with all four lipopolyamines tested and polyethylenimine. Precompacting DNA with a histone H1-derived peptide enhances cationic lipid RPR 115335-mediated gene transfer in an in vivo model of Lewis lung carcinoma. Apart from their use in peptide-DNA-lipid association, such peptides could be useful as part of chimeric gene delivery vectors presenting a DNA-binding moiety that can be easily associated with other functional domains.     

肽刺激培养法体外扩增巨细胞病毒特异细胞毒性T淋巴细胞

目的研制一种扩增特异细胞毒性T淋巴细胞频数的方法。方法用不同浓度的巨细胞病毒（CMV）特异的表位肽pp65刺激人外周血单个核细胞（PBMCs）使CMV特异CTLs在PBMCs中的百分比增高。用四聚体一PE和CD。一FITC双标记PBMCs中的CMV特异CTLs，然后用流式细胞仪分析。结果这个方法能有效扩增CMV特异CTLs，使在外周血中占淋巴细胞百分比不到l％的CMV特异CTLs迅速增加到接近20％，占CDsT淋巴细胞的40％以上，并能从单份血样中获得大量特异CTLs用于流式分析。结论肽刺激培养法简便、易操作，能有效扩增CMV特异CTLs，使不同人的数据更容易对比和连续的评价。该方法也为过继性免疫疗法治疗巨细胞病毒疾病打下了基础。