小鼠胸腺和脾脏细胞的体外转化培养

建立了小鼠脾脏、胸腺细胞体外转化微量培养系统,确定了最佳实验条件.点蜡法充CO_2,简便易行,效果好.RPMI-1640培养基内补充胎牛血清,其浓度确定为在脾细胞培养为10%,在胸腺细胞培养为20%.脾细胞培养最适细胞密度为2～5×10~6/ml,Con A最适量为2～4μg/培养;胸腺细胞培养细胞最适密度为1×10~7/ml,Con A最适量为2～4μg/培养.两种已知免疫活性的药物在脾细胞培养体系有明确肯定的反应.     

Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria

Summary Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observedin vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of β-glycerophosphate. The technique of dropinoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm²) in 3 weeks of culture.     

PsD007对四株NPC细胞放射敏感性的影响

采用ＭＴＴ方法观察癌光啉结合＾６０Ｃｏγ射线放射对四株人鼻咽癌细胞体外放射增敏作用，结果表明在一定的ＰｓＤ００７浓度范围内，随ＰｓＤ００７浓度的升高，放射对细胞的杀灭作用明显增强，当浓度分别为１．２５，６．２５，１２．５，２５和５０μｇ／ｍｌ时，对四株细胞杀灭增加倍数分别为１．０１－１．０６，１．１５－１．２９，１．２８－１．５９，１．７９－３．１８，２．８６－５．５５；放射量效存活曲线按多靶单击     

动态旋转系统构建组织工程化血管模型中血管内皮细胞分泌功能监测

目的 比较静态和动态旋转系统中构建组织工程化血管模型中血管内皮细胞分泌功能。方法新型可降解材料聚羟基丁酸酯(PHB),用胶原包埋,形成多孔状PHB 胶原管形支架。分离传代、分化人脐静脉内皮细胞,接种于PHB管型支架内腔面,分别在静态、动态旋转系统中培养14 d后,测定血管内皮细胞分泌一氧化氮(NO)、前列环素(PGI2)水平。结果 在动态旋转系统构建组织工程化血管模型中血管内皮细胞分泌NO、PGI2水平显著高于静态系统,在11 d NO分别为(120.52±3.83)μmmol／L、(80.98±5.98)μmmol／L,PGI2分另9为(20.48±1.52)μg／L,(16.59±1.29)μg／L,与静态系统相比,差异有显著性(P<0.05)。结论胶原包埋PHB支架有利于细胞的黏附和生长,可作为组织工程化血管的支架材料。在动态旋转系统构建组织工程化血管模型中血管内皮细胞,具有与正常血管类似的"生理功能"。     

分离肝细胞纯化培养方法的比较研究

目的 评价一种可提高肝细胞纯度和存活率的分离培养方法。方法 以体外两步胶原酶灌流法分离肝细胞,然后将其分成两组,对照组在接种培养前不经进一步处理,试验组则在应用适宜的Percoll梯度液离心纯化之后再行培养。藉台盼蓝拒染法比较两组肝细胞的存活率,采用MTT法动态比较两组肝细胞的增殖状态,在相差显微镜下观察细胞的纯度和形态。结果 未经进一步纯化处理的猪肝细胞存活率为90％±5％,鼠肝细胞存活率为80％±5％,两者纯度均约90％;经Percoll梯度液离心纯化后,其高活力肝细胞比率均提高至98％±2％,纯度可达99％以上。从开始接种到大部分肝细胞贴壁生长,试验组比对照组肝细胞的时间有所缩短。结论 用Percoll梯度液纯化新分离肝细胞,可提高肝实质细胞的活力与纯度。     

大鼠成体心肌细胞的分离和培养

目的: 探索稳定的大鼠成体心肌细胞的分离和培养方法. 方法: 应用主动脉逆行生物酶(5 g/L胰蛋白酶及1 g/L胶原酶)灌流法分离大鼠成体心肌细胞,免疫荧光染色法鉴定分离的心肌细胞,比较有无血清培养对心肌细胞的影响. 结果: 心肌细胞的存活率为95%以上;存活细胞呈杆状,形态完整,长宽比约为4～6∶ 1;无血清培养心肌细胞只能存活1 wk,形态不发生改变;而加血清培养心肌细胞可存活2 wk以上,但超微结构发生了改变,且不同血清浓度对成纤维细胞生长具有不同的作用. 结论: 主动脉逆行生物酶(胶原酶和蛋白酶)灌流法是比较理想的心肌细胞分离方法;血清对成体心肌细胞的培养起着重要作用.     

乳猪肝细胞的分离、培养及功能检测

目的 为构建生物人工肝进行肝细胞的准备。方法 采用胶原酶半原位灌流法分离单个乳猪肝细胞,并对其活力及单层和聚集培养后的白蛋白、尿素合成功能进行检测。结果 采用本方法从每头乳猪中分离到的单个肝细胞数为(3.1±1.5)×10~(10),活性超过95％。在加入激素和生长因子的培养基中单层培养时,肝细胞功能良好,可维持2周左右。而在未加入激素和生长因子的培养中肝细胞虽能存活1周,但功能于24h后即丧失。球形聚集培养可实现肝细胞的大量培养,且生物学活性较单层培养显著提高。结论 采用胶原酶半原位灌注法所得单个乳猪肝细胞基本能满足构建生物人工肝对肝细胞数量的要求。聚集培养接种密度大,细胞生物学活性高,可用于构建生物人工肝。     

3种培养基体外培养人芽囊原虫的效果观察

目的　观察人芽囊原虫在 3种培养基中的生长繁殖情况 ,以探讨最佳的培养方法。　方法　从大学生粪便中分离人芽囊原虫 ,转种至 3种不同培养基 ,定时观察人芽囊原虫的生长繁殖情况 ,根据原虫密度评价培养效果。　结果培养 96h后 ,Locke’s鸡蛋血清 (LES)双相培养基中的人芽囊原虫密度最高 ,改良Jones单相液体培养基次之 ,Locke’s琼脂血清 (LAS)双相培养基原虫密度最低 ,差异有显著性 (P <0 .0 5 ) ;观察 3种培养基中的原虫密度高峰持续时间分别为 3 60h、96h和 72h ,最长存活时间分别为 5 5 2h、3 3 6h和 168h。　结论　Locke’s鸡蛋血清双相培养基较适用于人芽囊原虫体外增殖培养。     

还原型谷胱甘肽和L-NAME对体外培养的脊髓运动神经元的影响

目的 :探讨还原型谷胱甘肽 (GSH)和L NAME对体外培养的脊髓运动神经元的保护作用。方法 :不同浓度的GSH和L NAME作用于脊髓运动神经元 ,3d后计算存活率。并测量存活率高的两组和对照组的免疫细胞化学标本的神经元形态学指标。结果 :GSH 10mmol·L-1和L NAME 1× 10 -3 mol·L-1组存活率最高。两实验组轴突长度、树突总长度、树突分叉点数目和胞体面积高于对照组。结论 :抗氧化剂和NOS抑制剂可以提高脊髓运动神经元的存活率 ,促进神经元生长     

The thymus in seronegative myasthenia gravis patients

Summary In 5–10% of all patients with typical generalised myasthenia gravis (MG), serum antibody to the acetylcholine receptor (AChR) is undetectable. To determine whether these represent a distinct subgroup, we have compared the thymuses of 14 seronegatives, 70 seropositives and 12 non-myasthenic controls. By quantitative immunohistology on coded sections, the 7 seronegative samples were clearly distinguishable from the controls by the pronounced lymph node-type T-cell areas in the medulla. While these closely resembled those in the seropositive cases, germinal centres were significantly sparser, and total in vitro IgG production was disproportionately low (per B cell) in the 12 cases tested. Furthermore, specific anti-AChR production was never detected in any of these cultures. The data support the view that the medullary T-cell areas are the most consistent abnormalitiy in the MG thymus (though it may not be primary), and they strongly imply that seronegative and seropositive MG are distinct entities.