不同血清浓度及体外培养时间对小鼠成纤维细胞L929细胞系生长曲线和细胞形态学的影响

目的 探讨不同实验条件下不同血清浓度及体外培养时间对小鼠成纤维细胞L929细胞系生长曲线和细胞形态学变化的影响.方法 采用体外细胞培养技术进行小鼠成纤维细胞L929细胞的培养,并用磺基罗丹明B(sulforhodamine B,SRB)酶标仪法(A490 nm波长)测量各孔的吸光值(OD值)并转化为小鼠成纤维细胞L929细胞系生长曲线.同时,分别采用HE染色法、吖啶橙荧光染色进行小鼠成纤维细胞L929细胞系形态学观察.结果 小鼠成纤维细胞L929细胞系分别在不同浓度的血清中培养2、4、6、8 d,其表现出不同的生长和生存情况及细胞形态学改变.将该细胞系处于相同培养时间和不同血清浓度(0%、20%、40%、60%、80%、100%)时,在20%和40%血清浓度组中,成纤维细胞的生长基本表现为OD值呈现快速增加趋势、且对细胞生长促进作用较强;而血清浓度为0%和100%时对小鼠成纤维细胞L929细胞生长表现出明显抑制作用及OD值呈逐渐减小的趋势.当该细胞系处于不同培养时间、且相同血清浓度时,除0%组OD值呈现减弱趋势,其他浓度组随时间的延长OD值呈增加趋势,在4 d之内OD值增长较快,4~6 d增长趋势减弱,6~8 d增长趋势回升,尤其是100%组.在这种实验条件下,小鼠成纤维细胞L929细胞的形态学也发生了相应改变.结论 不同血清浓度及不同培养时间等体外培养环境对小鼠成纤维细胞L929细胞系生长和生存及细胞学形态均有明显影响,提示利用血清进行体外细胞培养时应充分考虑所添加血清的浓度以及细胞培养的时间.     

血清介质对体外培养L929系小鼠成纤维细胞表达产物Ⅰ、Ⅲ型胶原及其比值的影响

目的 探讨不同浓度血清、不同体外培养时间对L929系小鼠成纤维细胞表达产物Ⅰ、Ⅲ型胶原及其比值的影响.方法 采用双抗体夹心法检测细胞培养液中Ⅰ、Ⅲ型胶原的含量,并同时计算出Ⅰ/Ⅲ型胶原的比值.结果 除0%血清浓度组的Ⅰ、Ⅲ型胶原呈现无明显变化的微弱升高趋势外,其余各血清浓度组的Ⅰ、Ⅲ型胶原均处于增加状态,且在6~8 d时增加最为明显;另外,Ⅰ/Ⅲ型胶原比值随着血清浓度和细胞生长时间不同,表现出不同的变化趋势.其中20%和40%血清浓度组随着培养细胞生长时间延长,Ⅰ/Ⅲ型胶原比值逐渐增加;而其他血清浓度组,随着细胞培养时间的延长,Ⅰ/Ⅲ型胶原比值增加并不明显.结论 L929细胞在不同体外培养时间和不同浓度血清下其表达产物Ⅰ、Ⅲ型胶原含量及其比值有一定变化,为今后优化体外细胞培养条件提供了相关的实验数据.     

太白梅木对成纤维细胞增殖及形态学的影响

[目的]探讨太白橡木对成纤维细胞增殖及形态学的影响及抗肝纤维化机制。[方法]采用体外培养的NIH3T3成纤维细胞作为肝星状细胞（HSC）的替代模型，常规培养，秋水仙碱、齐墩果酸及太白橡木中药血清作用于细胞，在光镜及电镜下观察药物血清对NIH3T3细胞形态学的影响；用放射免疫法测定培养上清透明质酸（HA）的生成。[结果]太白橡木、齐墩果酸、秋水仙碱血清组细胞与对照组细胞在形态、数目上差别显著；3者均可抑制细胞增殖（P〈0．01）；显著抑制HA的合成，降低NIH3T3细胞培养上清中HA水平，与对照组比较差异有统计学意义（P〈0．01）。[结论]太白橡木中药血清可显著抑制NIH3T3细胞增殖和细胞外HA的合成，在体外具有一定的抗肝纤维化作用。     

光动力学疗法诱导卵巢癌细胞系Skov3和宫颈癌细胞系Hela凋亡的体外研究

目的 研究光动力疗法对人宫颈癌细胞系Hela及卵巢癌细胞系Skov3的体外增殖及凋亡的影响,探讨光动力学在老年女性妇科肿瘤治疗的优势。 方法 采用不同能量激光对不同浓度光敏剂（5-ALA）处理过的宫颈癌细胞系(Hela)及卵巢癌细胞系(Skov3)进行照射,48 h后应用光镜、电镜进行形态学观察,并采用四甲基偶氮唑蓝(MTT)比色法检测光动力学治疗对肿瘤细胞体外生长的抑制作用,采用流式细胞仪检测细胞凋亡情况。 结果 实验组细胞经光动力学处理后体积缩小,细胞变形、皱缩,细胞核浓缩、深染,染色质密集成斑块状,细胞间失去彼此连接,透射电镜下可见致密的染色质沿核膜下聚集,有凋亡小体形成。0.1、0.5、1.0、2.5 mmol/L的5-ALA处理过的肿瘤细胞经不同能量0.1、0.5、2.5、12.5 J/cm2激光照射后细胞系Skov3和Hela的抑制率分别为12.9％～90.7％和3.9％～70.0％;光敏剂浓度、光照强度对卵巢癌细胞系Skov3与宫颈癌细胞系Hela吸光度值(A)均有影响(P＜0.01),光敏剂与光照间交互作用显著(P＜0.01)。随光敏剂浓度与光照强度的增加两细胞系A值呈降低趋势,呈光敏剂浓度与光照时间的依赖性(P＜0.01)。 结论光动力学治疗对体外生长的卵巢癌细胞系Skov3及宫颈癌细胞系Hela有抑制作用。     

银杏叶提取物对人胚肺成纤维细胞氧化应激的保护作用

目的探讨银杏提取物（EGb）对体外培养的人类成纤维细胞的抗氧化作用。方法分别于常态氧（氧浓度为21%）及高氧（氧浓度为40%）环境下培养人胚肺成纤维细胞（MRC-5），并给予不同浓度（0，7.5，15，30，60μg/ml）的EGb，以400μmol/L的α-苯基-N-4-丁基硝酮（PBN）为阳性对照，观察不同条件下细胞的生长曲线，并用流式细胞仪测定不同时期、不同条件下MRC-5细胞内活性氧（ROS）的含量。结果常规培养条件下，MRC-5在不同浓度EGb下的生长曲线无明显差别，不同时期测定的ROS含量与阴性或阳性对照比较，总体上无显著差异；而在高氧环境中，使用EGb的MRC-5细胞生长绝对数明显比空白对照多，且呈现EGb浓度越高，细胞绝对数越多、不同时期ROS含量越低、衰老细胞越少的趋势。结论银杏叶提取物对慢性氧化应激下的MRC-5有保护及抗衰老作用。     

血清中肿瘤坏死因子对人的恶性疟原虫有细胞毒作用

选6～12周龄C3H/HeN雌雄性小鼠,静脉注射2×10~6活的牛卡介苗,14天后注射10μg细菌内毒素,收集血清,作为测定细胞毒性的卡介苗-脂多糖血清(BCG-LPS血清)。用部分纯化的肿瘤坏死因子(TNF)免疫家兔,制备TNF抗血清。用微量板培养L929鼠成纤维细胞,每井1.5×10~4L929鼠成纤维细胞,0.1mlRPMI-1640+10%灭活马血清(20μg/ml庆大霉素)。加入不同浓度的BCG-LPS血清,孵     

细粒棘球蚴生发层细胞系的建立

目的：为获得能在体外稳定繁殖的细粒棘球蚴细胞系。方法：用研磨法将绵羊源细粒棘球蚴生发层处理得分散的生发细胞，将其在含10％—20％小牛血清的RPMI1640培养基在24孔培养板和包被胶原的24孔细胞培养板上交替培养至14代，随后在常规24孔培养板上连续传代培养；用倒置显微镜观察细胞形态及生长繁殖情况；将培养细胞接种BALB／c小鼠以观察其形成继发性包囊的能力；用ELISA法研究细胞系抗原的免疫学特征。结果：生发层细胞已在体外连续培养75代以上，生长良好；细胞系细胞呈圆形，具有形成合胞体乃至类组织块 的趋势，在4℃冰箱中至少可保存半个月，在液氮中至少可保存10个月；将细胞接种BALB／c小鼠后3个月至7个月，解剖未见细粒棘球蚴包囊生长; 细胞系抗原可和抗生发层体抗原、原头节可溶性抗原、囊液抗原的小鼠血清及人工感染原头节的阳性小鼠血清起反应。结论: 细粒棘球蚴生发层细胞系已经建立。     

无机砷诱导体外培养人皮肤细胞的增殖作用

目的　观察亚砷酸钠诱导体外培养的人皮肤细胞的作用 ,探讨砷性皮肤损伤的分子机制。方法　原代培养皮肤成纤维细胞和表皮细胞 ,通过直接细胞计数法和噻唑蓝 (MTT)还原法检测亚砷酸钠诱导对 2种细胞系生长的影响。结果　成纤维细胞较表皮细胞易于培养 ,与对照组比较 ,不同剂量的亚砷酸钠诱导后 ,吸光度值均有升高 ,有剂量 -效应关系 (P <0 .0 1)。 0 .5～ 8.0 μmol/ L 浓度的亚砷酸钠对成纤维细胞有明显的增殖作用 ;低浓度 (0 .8,1.6 μm ol/ L)对表皮细胞有明显的增殖作用 ,浓度高于 3.2 μm ol/ L 时 ,表皮细胞生长受抑制。 10 μmol/ L亚砷酸钠对皮肤成纤维细胞有明显的毒性作用。结论　无机砷对人皮肤细胞有双向调节的促增殖作用 ,可能是无机砷引起慢性砷中毒皮肤损伤不同表现的一个重要原因 ,具体作用机制有待进一步研究     

碱性成纤维细胞成长因子、新生小牛血清和肝素对大鼠血管平滑肌细胞迁移的影响

为研究血管平滑肌细胞迁移及其影响因素，建立体外检测血管平滑肌细胞迁移的实验方法，并观察不同浓度的新生小牛血清、碱性成纤维细胞生长因子和肝素作用于血管平滑肌细胞不同时间后对其迁移的影响。结果发现，新生小牛血清和碱性成纤维细胞生长因子均可显著促进血管平滑肌细胞迁移（P＜0.001或P＜0.01）；肝素对新生小牛血清诱导的血管平滑肌细胞迁移产生显著抑制作用（P＜0．001）．三者的作用强度均与浓度呈正相关。提示碱性成纤维细胞生长因子对血管平滑肌细胞具有促增殖作用．而肝素具有抑制血管平滑肌细胞迁移的作用。     

人肛瘘管壁成纤维细胞的体外分离培养和鉴定

通过组织块法分离并原代培养人肛瘘管壁组织，用胰蛋白酶和乙二胺四乙酸（EDTA）消化成纤维细胞、上皮细胞，获得更加纯化的成纤维细胞；以DMEM培养液为基础培养蒸，添加胎牛血清（体积分数10％）、青霉素（100U／L）和硫酸链霉素（100U／L），置37℃、5％CO2培养箱中培养。倒置相差显微镜和细胞爬片HE染色。观察成纤维细胞形态结构，并对培养成纤维细胞行角蛋白、波形蛋白免疫细胞化学鉴定．结果分离后的成纤维细胞在第4～12h于体外快速贴壁，第2～4天进入对数期生长、增殖期。细胞起源鉴定波形蛋白免疫细胞化学染色为阳性，角蛋白免疫细胞化学染色为阴性。提示该方法所获得的肛瘘管壁成纤维细胞可在体外稳定培养，不含有杂质细胞。可为在细胞分子水平上研究肛瘘瘘管愈合的机制提供可靠的细胞模型。     

Establishment,growth properties,and morphological characteristics of permanent human small cell lung cancer cell lines

Summary Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in estabilishing and permanently growing SCLC. Serum-free SIT medium and SIT 2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R10 medium was suprior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.Abbreviations SCLC small cell lung cancer - FBS fetal bovine serum - R 10 Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS - D 10 Dulbecco's modified Eagle's medium supplemented with 10% FBS - SIT RPMI 1640 medium supplemented with selenium, insulin, transferrin - HITES RPMI 1640 medium supplemented with hydrocortisone, insulin, transferrin, estradiol, selenium - SIT 2.5 SIT medium supplemented with 2.5% FBS - PBS phosphate buffered saline - PDT population doubling time This work was supported by the SFB 215 of the German Research Society. The authors Thank Dr. Adi F. Gazdar for reviewing the morphology     

EGF与miRNA-21联合构建雄激素非依赖性前列腺癌LNCaP亚细胞模型

目的构建雄激素非依赖性的人前列腺癌细胞株LNCaP亚型。方法前期常规RPMI 1640培养基培养LNCaP细胞,然后给予无酚红的RPMI 1640培养基,其中加入10%活性碳/葡聚糖处理的胎牛血清,细胞传代后将10μg/L表皮生长因子（EGF）加入培养基,50 nmol/L miRNA-21类似物转染细胞,长期培养。镜下观察细胞形态变化,MTT检测细胞增殖活性,RT-PCR检测雄激素受体基因扩增情况。结果细胞培养约2.5个月后,LNCaP细胞成功转变为非雄激素依赖的前列腺癌细胞LNCaP亚细胞,细胞增殖能力升高,雄激素受体表达上调。结论体内雄激素依赖性前列腺癌可向雄激素非依赖性前列腺癌转变,而且miRNA-21和EGF对该转变起促进作用。     

Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied in an attempt to optimize the maintenance of glucose stimulated insulin biosynthesis and release in the cultured specimens. Islets which had been cultured at a physiological glucose concentration (5.5 mmol/l) in the absence of serum had an impaired glucose-stimulated insulin biosynthesis and release as well as a reduced insulin content. Thus, insulin biosynthesis was three times higher after culture in a serum supplemented medium. Further, the insulin secretion of islets cultured in the presence of serum was markedly enhanced in acute incubations with high concentrations of glucose. This response was most pronounced in islets which had been cultured free-floating. A comparison between different culture media showed that islets cultured in RPMI 1640 had the highest insulin production. The present data suggest that the most favourable conditions for long-term storage of isolated islets in culture may be obtained when the islets are maintained as free-floating explants in a culture medium consisting of RPMI 1640 supplemented with serum.     

In vitro development of Haplorchis taichui (Trematoda: Heterophyidae)

Newly excysted metacercariae of Haplorchis taichui were cultured in a candle jar set at 37 degrees C. Both monophasic culture media [0.85% NaCl, RPMI 1640, RPMI 1640+10% fetal calf serum (FCS)] and diphasic culture media [RPMI 1640 + egg yolk agar, RPMI 1640 + 5%, 10% or 15% blood in blood agar (BA), RMPI 1640 + 5%, 10% and 15% FCS with 5% blood in BA] were used in vitro. Parasites survived for only 1 day in 0.85% NaCl without any development. In RPMI 1640 with egg yolk agar and RMPI 1640 + 5%, 10% FCS, the parasite survived for 3-5 days. In contrast, worms survived for 12-14 days in RPMI 1640 with blood agar without any change in result in a different concentration of blood in BA. The ovary and testes were observed after 3 days incubation in this media. Nevertheless, only 1 parasite in RPMI 1640 with 15% blood in BA had vitellaria and eggs at day 6. RPMI 1640 with blood agar can be used as short-term maintenance for the in vitro culture of H. taichui. However, further studies are needed.     

L-谷氨酰胺对克隆的胰岛β细胞株INS-1E细胞和小鼠胰岛的急性刺激作用

目的探讨L-谷氨酰胺对INS-1E细胞和小鼠胰岛分泌胰岛素的作用。方法INS-1E细胞经传代培养2d后，在Krebs-Ringer缓冲液中37℃培养箱预培养30min，再用含有不同浓度葡萄糖和不同浓度L-谷氨酰胺的改良Krebs-Ringer缓冲液培养60min，然后留取上清液进行胰岛素测定。雌性NMRI小鼠，6～10周龄，苯巴比妥腹腔麻醉，应用胶原酶技术消化胰腺分离胰岛，置于RPMI1640培养皿中在37℃培养箱（5％CO2，95％空气）过夜培养。次日在Krebs-Ringer缓冲液中37℃水浴培养箱预培养30min，然后分别把单个胰岛小心放入100bd含有不同浓度葡萄糖和不同浓度L-谷氨酰胺的改良Krebs-Ringer缓冲液37℃水浴培养箱培养60min，然后留取50μ1上清液进行胰岛素测定。结果L-谷氨酰胺在0．1～5mmol／L范围不增加葡萄糖刺激的INS-1E细胞的胰岛素分泌，仅10～20mmol／L的L-谷氨酰胺促进葡萄糖诱导的胰岛素分泌。L-谷氨酰胺在0．1～10mmol／L范围不促进葡萄糖诱导的小鼠胰岛的胰岛素分泌，仅20mmol／L的L-谷氨酰胺促进葡萄糖诱导的胰岛素分泌。结论大剂量L-谷氨酰胺能增加葡萄糖诱导的INS-1E细胞和小鼠胰岛分泌胰岛素。     

苦参碱对人膀胱癌EJ细胞株凋亡的影响及机制

目的为苦参碱用于膀胱癌治疗提供依据。方法将EJ细胞贴壁培养于10％胎牛血清、10万U／L青霉素、100mg／L链霉素的1640培养液中,37℃、5％CO2饱和湿度培养箱中培养,取对数生长期细胞随机分为两组,苦参碱组加入质量浓度为0．5、1．0,2．0mg／ml的苦参碱;对照组不加苦参碱,每天更换RPM11640培养液。采用MTT法检测各组细胞生长抑制率;荧光显微镜观察EJ细胞形态学变化;流式细胞仪检测细胞凋亡率;并对凋亡相关Bcl-2蛋白表达进行检测。结果0．5—2．0mg／ml苦参碱可有效抑制EJ细胞增殖,抑制作用呈时间-剂量依赖性,作用72h时2．0mg／ml的抑制率为50．65％±2．78％,凋亡率为36．35％±2．06％,与对照组比较,P均〈0．01;荧光显微镜下可观察到细胞内空泡与核碎裂现象;Bcl-2蛋白表达随药物浓度增高和作用时间延长而下降。结论苦参碱能诱导EJ细胞凋亡,其机制可能与下调Bcl-2蛋白表达有关。     

胎肝细胞培养上清液对小鼠急性肝衰竭的保护作用

为探讨胎肝细胞治疗重型肝炎的机制,将84只小鼠随机分为3组(每组28只),经D-氨基半乳糖/内毒素(D-galn/LPS)诱导急性肝衰竭后,分别给予生理盐水(NS)、RPMI1640培养液(RPMI)和人胎肝细胞培养上清液(FHCS)治疗,各组分别取14、8、6只小鼠进行存活率、血清谷丙酶(ALT)及肝脏病理观察。结果显示,NS组和RPMI组24小时存活率分别为14.3％和21.4％;ALT升高达207.2±41.3IU/L和188.4±52.6IU/L。FHCS组小鼠存活率为57.1％,ALT为92.5±28.7IU/L,与对照组相比差异显著(P<0.05,P<0.01)。此外,对照组分别有5(83.3％)只和4(66.7％)只动物肝组织病理改变在Ⅲ级以上,而FHCS组仅有2(33.3％)只。上述结果表明,FHCS对急性肝衰竭有保护作用,同时提示胎肝细胞分泌产物可能与保护肝细胞、治疗重型肝炎及肝衰竭的机制有关。     

Molecular epidemiology of malaria in Cameroon. XXIII. Experimental studies on serum substitutes and alternative culture media for in vitro drug sensitivity assays using clinical isolates of Plasmodium falciparum

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.     

Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience

Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.