阴道毛滴虫在两种培养基中的生长与形态观察

目的 通过对改良肝浸汤培养液及RPMI1640培养基内阴道毛滴虫生长情况的观察,选择适合教学和科研的培养基。 方法 用临床分离的阴道毛滴虫标本,分别接种至改良肝浸液及RPMI1640两种培养基内进行培养,pH值为5.6~5.8,通过计数了解生长情况并观察阴道毛滴虫的形态。 结果 肝浸汤培养液中培养48 h,阴道毛滴虫达到生长高峰,虫体呈现出多分裂相,虫体变大、呈圆形,内含4~12个细胞核,细胞膜外缘可见数丛鞭毛。RPMI1640培养基中阴道毛滴虫72 h达到生长高峰,虫体密度约为肝浸液高峰期的2／3,未见多分裂现象,虫体与生理盐水涂片中阴道毛滴虫形态、大小相近。 结论 改良的肝浸液培养基适于阴道毛滴虫的药物试验及其他实验项目的研究,RPMI1640培养基更适用于教学、标本的制作及实验室保种工作。     

人羊膜可作为研究滴虫病中宿主与寄生虫相互关系的模型

本实验将人羊膜作为新的实验模型来研究阴道毛滴虫与宿主上皮细胞的相互关系。将阴道毛滴虫置于含5%马血清的Feinberg-Whittington 培养基(无琼脂)中,于37℃洗2次后,1 2009离心,悬浮于RPMI一1640与reinberg     

低频电磁场对兔动脉平滑肌细胞增殖的抑制作用研究

目的:探讨不同场强、不同作用时间的低频电磁场对兔主动脉平滑肌细胞增殖的调控作用,以观察磁场是否能用于 PTCA支架后冠脉再狭窄的防治.方法:用含10%小牛血清的RPMI-1640培养液体外培养新西兰白兔的主动脉平滑肌细胞至3～7代,将含5％小牛血清的RPMI-1640培养液与动脉平滑肌细胞的混悬液100 靗加入96孔培养板中培养.按照实验分组,每日磁场作用一次,共二日.用MTT法检测动脉平滑肌细胞的增殖数量.结果:磁场作用时间大于10分钟时,20 mT、40 mT和60 mT磁场对动脉平滑肌细胞的增殖均有显著的抑制作用(P<0.05),其中作用时间为30分钟的40 mT磁场对动脉平滑肌细胞增殖的抑制作用最显著(P<0.001).结论:磁场对动脉平滑肌细胞增殖有显著的抑制作用,磁场的这种抑制作用具有强度敏感性、时间依赖性和空间距离性;磁场对PTCA支架后的冠脉再狭窄可能具有防治作用.     

当归促进大肠癌HCT-8细胞增殖及抑制5-氟尿嘧啶致凋亡的作用

1 材料和方法 1.1 材料 大肠癌HCT-8细胞为中国医学科学院药物研究所提供;5-氟尿嘧啶(5-Fu)注射液系天津氨基酸公司人民制药厂出品;当归注射液系北京第四制药厂出品;RPMI-1640培养基为Gibco BRL产品;小牛血清购自天津生化制品厂;胰蛋白酶购自天象人生物制品有限公司;DMSO系天津化学试剂六厂出品;PI(碘化丙啶)、RNAse(RNA酶)、MTT(四甲基偶氮唑盐)均为Sigma产品。     

铁离子对体外培养的阴道毛滴虫生长的影响

目的研究铁离子对体外培养的阴道毛滴虫生长的影响。方法在TYM(trypticase-yeast extract-maltose)培养基(pH6.0)中,分别加入100、200、300和400μmol/L铁离子,并设不加铁离子组为对照组,阴道毛滴虫初始浓度为1×105/ml,于37℃定量纯培养。采用台盼蓝染色法镜下观察并计数活滴虫数和死滴虫数,绘制生长曲线和存活率曲线,并计算对数生长期内世代时间。通过连续稀释的方法测定在加入200μmol/L铁离子培养基和对照组培养基中甲硝唑对阴道毛滴虫的最小致死浓度(minimal lethal concentration,MLC)。结果在加入100、200、300和400μmol/L铁离子的培养基中,阴道毛滴虫均于40h达最高密度,分别为2.9×106、3.2×106、3.1×106和2.8×106/ml,对照组阴道毛滴虫于54h达最高密度,为2.5×106/ml。400μmol/L铁离子组阴道毛滴虫的世代时间为(6.8±0.7)h,较100～300μmol/L铁离子组的世代时间[(4.8±0.3)、(4.8±0.2)和(5.0±0.4)h]延长(均P﹤0.05),而100～400μmol/L铁离子组的世代时间均短于对照组[(10.2±3.1)h](均P﹤0.05)。在加入200μmol/L铁离子的培养基中阴道毛滴虫的甲硝唑最小致死浓度为(23.44±11.56)μg/ml,显著低于对照组[(31.25±15.44)μg/ml](均P﹤0.05)。结论阴道毛滴虫在加入100～400μmol/L铁离子的TYM培养基中生长较快,且甲硝唑对阴道毛滴虫的最小致死浓度较小。     

旋毛虫肌幼虫在不同培养基中发育情况的观察

目的观察旋毛虫肌幼虫在不同培养基中的发育情况。方法将旋毛虫肌幼虫接种在不同培养基中于37℃5%CO2条件下培养18 d,观察幼虫开始蜕皮的时间和蜕皮率,并测量幼虫长度。结果肌幼虫在RPMI-1640、含5%胎牛血清(FBS)的RPMI-1640培养液、PBS和生理盐水中开始蜕皮的时间分别为15、23、19和37 h;肌幼虫在RP-MI-1640与5%FBS+RPMI-1640中分别蜕皮1~4层和1~2层,在PBS与生理盐水中仅蜕皮1层。培养5 d后,RP-MI-1640中培养幼虫的蜕皮率(77.94%)显著高于在其他3种培养液培养的幼虫蜕皮率(39.83%、44.64%和8.84%)(Z1640+5%FBS=-2.268,ZPBS=-2.641,Zsaline=-6.826,P0.05);培养18 d后,在RPMI-1640中蜕2~4层皮的幼虫蜕皮率(25.6%、1.32%及0.26%)显著高于在5%FBS+RPMI-1640中培养的幼虫蜕皮率(0.45%、0和0)(Z2=-16.111,Z3=-7.991,Z4=-3.885,P0.05);在2种培养液中培养1~18 d的幼虫长度差异无统计学意义(t=0.138,P0.05),幼虫长度均随培养时间的延长而减小(F1640=54.639,P0.05;F1640+5%FBS=26.928,P0.05)。结论绝大多数旋毛虫肌幼虫在RPMI-1640与5%FBS+RPMI-1640培养液中不能蜕下完整表皮,不能发育到成虫阶段。     

钙对恶性疟原虫入侵人红细胞的作用

作者用几种增加或减少红细胞内、外游离钙技术,观察了钙在恶性疟原虫入侵人红细胞过程中的作用。实验用泰国恶性疟原虫T_9分离株的克隆T_(9/960)用Percoll进行等密度离心分离裂殖体,加入含10%人血清和A型人红细胞的RPMI-1640培养基中连续培养,经4h虫体再侵入新的红细胞后,残存的裂殖体用山梨醇溶解,如此反复3次使虫体培养同步化而获得纯化裂殖体。此外,以PBS洗涤红细胞     

彭亨丝虫感染期幼虫和在哺乳动物体内早期虫体的体外培养

按 Chen 和 Howells(1979) 的方法,收集彭亨丝虫感染期幼虫及在哺乳动物体内早期虫体进行体外培养。从埃及伊蚊口部分离获得第3期幼虫移至盛有5毫升0. 05%“Chloro”溶液的生长培养基(GM199) 的消毒离心管内进行虫体表面消毒。GM199为组织培养基加10%新生小牛血清和400微克/毫升晶霉素。幼虫培养在组织培养基199、 Puck培养基、RPMI-1640和 Eagle 基础培养基中,亦使用含有犬肉瘤细胞系、幼仓鼠肾细胞系,仓鼠腹膜细胞系和 HeLA 细胞系。在Falcon25厘米~2的组织培养瓶内含上述各种培养基及细胞系,加50～100条消毒的感染期幼虫作培养实验。     

3种不同培养基体外培养阴道毛滴虫效果的比较

目的　探索阴道毛滴虫体外培养的适宜条件。　方法　用临床分离的阴道毛滴虫 ,按 9.0× 10 4 / ml的接种量转种至 3种不同培养基进行培养 ,p H值为 5 .6。　结果　经 3种培养基培养 ,96 h后阴道毛滴虫数量存在差异 ,其中以半胱氨酸 -肝 -胨 -麦芽糖培养基 培养基 )虫数较多 ,肝 -胨 -麦芽糖培养基 培养基 )次之 ,大豆 -肝 -胨 -麦芽糖培养基 培养基 )较少。培养基 与培养基 及培养基 相比较 ,滴虫存活率差异具有显著性意义 P<0 .0 1,P<0 .0 5 ) ,滴虫生长密度差异也具有显著性意义 P<0 .0 1,P<0 .0 5 ) ,生长密度高峰持续时间分别为 192 h、14 4 h和 96h,最长存活时间分别为 2 88h、2 16 h和 192 h。　结论　半胱氨酸 -肝 -胨 -麦芽糖培养基较适于阴道毛滴虫体外增殖     

中性粒细胞杀灭阴道毛滴虫作用的研究

目的 研究中性粒细胞对阴道毛滴虫的杀灭作用。 方法 将滴虫性阴道炎患者的阴道分泌物接种于肝浸汤培养基，获阴道毛滴虫。取患者静脉血分离血清，取其1 ml 于56 ℃ 30 min获补体失活血清。另取1 ml患者血清于0 ℃以阴道毛滴虫吸附3次，获去除抗体血清。用密度梯度离心法及聚合物加速沉降法分离、纯化患者静脉血中性粒细胞。用氮蓝四唑（NBT）和沙黄O（safranin O）染色，显微镜观察中性粒细胞与阴道毛滴虫相互作用及甲臢（NBT还原产物)颗粒（formazan）沉积。取300个阴道毛滴虫和3×104个中性粒细胞，分别在有氧或厌氧、有或无超氧化物岐化酶（SOD）及过氧化氢酶（CAT）、有或无补体等不同条件下，培养10、20、30、40、50及60 min，再接种于固态琼脂培养基，在37 ℃厌氧条件下继续培养5 d。观察计数阴道毛滴虫存活率。 结果 显微镜下可见几个中性粒细胞同时围攻杀灭1个阴道毛滴虫。含有中性粒细胞时培养的虫体存活率，厌氧条件下为85%，有氧条件为3%（P﹤0.01）。SOD及CAT可明显降低其杀虫作用，培养60 min虫体存活率分别为98%及94%，而无SOD及CAT时虫体存活率为2%（P值均<0.05）。加入去除抗体血清，可将虫体全部杀灭。加入补体失活血清则无杀虫作用。 结论 中性粒细胞杀灭阴道毛滴虫作用依赖于氧及患者血清中补体的存在。     

Establishment and characterization of a carcinoembryonic antigen producing cell line derived from human pancreatic exocrine cancer

A human pancreatic carcinoma cell line derived from well differentiated tubular adenocarcinoma of the pancreas head has been established and maintained for nearly 4 years. This established cell line produces and releases carcinoembryonic antigen into the culture medium. The cell line grows as a monolayer in RPMI-1640 medium containing 10 percent fetal calf serum. Xenotransplantation in athymic nude mice after subcutaneous injection of EDTA-trypsin treated cells did not succeed.     

Possible mechanism for the regulation of glucose on proliferation, inhibition and apoptosis of colon cancer cells induced by sodium butyrate

AIM： To study the effect of glucose on sodium butyrate- induced proliferation inhibition and apoptosis in HT-29 cell line, and explored its possible mechanisms.
METHODS： HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, and were allowed to adhere for 24 h, and then replaced with experimental medium. Cell survival rates were detected by MTr assay. Apoptosis was detected by TUNEL assay. Glucose transport protein 1 （GLUT1） and monocarboxylate transporter 1 （MCT1） mRNA expression was detected by RT-PCR. RESULTS： Low concentration of glucose induced apoptosis and regulated proliferation in HT-29 cell line, and glucose can obviously inhibit the effect of proliferation inhibition and apoptosis induced by sodium butyrate. Glucose also down-regulated the expression of MCT1mRNA （0.28 ± 0.07 vs 0.19± 0.10, P 〈 0.05）, and decreased the expression of GLUTlmRNA slightly （0.18 ± 0.04 vs 0.13 ± 0.03, P 〈 0.05）.
CONCLUSION： Glucose can regulate the effect of proliferation inhibition and apoptosis induced by sodium butyrate and this influence may be associated with the intracellular concentration of glucose and sodium butyrate.     

班氏丝虫和马来丝虫感染期幼虫体外培养的进一步研究

在4种培养系统中,马来丝虫Ⅲ期幼虫在改良RPMI1640、20%小牛血清和人胚肾细胞系为饲养层的培养系统中生长发育良好,可以从Ⅲ期幼虫蜕皮2次发育到童虫,最长存活时间为54d。在单纯改良RPMI1640、TC199和20％小牛血清中培养,从Ⅲ期幼虫发育到童虫的最长存活时间为42d。班氏丝虫Ⅲ期幼虫在上述2种培养系统中,分别存活57和36d,从Ⅲ期幼虫蜕皮进入Ⅳ期幼虫和童虫。对马来丝虫幼虫体外培养蜕皮时间、虫体大小与沙鼠体内发育情况进行了比较。     

塞克硝唑苯甲酸酯体外抗阴道毛滴虫作用的研究

目的 研究塞克硝唑苯甲酸酯体外抗阴道毛滴虫的效果。 方法　 以肝浸汤培养基培养阴道毛滴虫,将滴虫混悬液接种于96孔培养板中,分塞克硝唑苯甲酸酯组、塞克硝唑组、甲硝唑组、不加药物的对照组及空白组,通过四甲基偶氮唑盐微量酶反应比色法（MTT法）观察塞克硝唑苯甲酸酯的体外抗滴虫效果。另将滴虫混悬液接种于试管中,分塞克硝唑苯甲酸酯组、塞克硝唑组、甲硝唑组和不加药物的对照组,通过计数法观察塞克硝唑苯甲酸酯的体外抗滴虫效果。 结果　 培养24 h后,塞克硝唑苯甲酸酯浓度为0.15、0.31、0.63、1.25、2.50、5.00、10.0及20.0 μg/ml时呈浓度依赖性地抑制滴虫增殖（t=9.02, P<0.01）,MTT法相对抑制率分别为14.6%、28.7%、31.3%、60.4%、89.0%、89.2%、95.6%和100%;计数法相对抑制率分别为18.2%、31.1%、39.7%、68.8%、84.6%、90.1%、94.6%和100%。培养6～24 h,呈时间依赖性地抑制滴虫增殖。体外抗阴道毛滴虫的最低杀灭浓度为20 μg/ml,最低抑制浓度为0.15 μg/ml。 结论　 塞克硝唑苯甲酸酯具有较强的体外抗滴虫效果。     

去氢骆驼蓬碱通过下调COX-2表达抑制胃癌细胞迁移和侵袭

目的探讨去氢骆驼蓬碱对人胃癌MKN-45细胞环氧化酶-2(cyclooxygenase-2,COX-2)表达、迁移和侵袭的影响。方法 MKN-45细胞接种于含10%胎牛血清的RPMI-1640培养液中,常规培养24h后加去氢骆驼蓬碱(2、4、8、16、32μg/ml),同时设置对照组不加药物,空白组只加培养液不含细胞,分别培养24h、48h、72h,MTT法检测细胞增殖率;western blot法检测COX-2表达;划痕损伤愈合实验及Transwell小室基质侵袭实验检测胃癌细胞体外迁移和侵袭。结果去氢骆驼蓬碱剂量依赖性抑制MKN-45细胞COX-2表达(P0.01);与对照组相比,去氢骆驼蓬碱组MKN-45细胞迁移和侵袭能力明显下降(P0.01)。结论去氢骆驼蓬碱可能通过下调COX-2表达抑制胃癌细胞迁移和侵袭。     

MTT法观察三七总甙对NIH/3T3成纤维细胞毒性作用与增殖的影响

目的：ＭＴＴ法观察三七总甙的体外细胞毒性与抗肝纤维化活性。方法：传代培养ＮＩＨ／３Ｔ３ 细胞,不同浓度接种细胞、不同血清浓度培养、不同ＭＴＴ用量等观察ＭＴＴ的转化Ａ５７０值,建立合适的试验条件。分别以无血清或含５％ 小牛血清的１６４０ 培养液稀释不同浓度的三七总甙或三七浸膏冻干粉,温育细胞１８ ｈ,测定ＭＴＴ转化Ａ５７０ 值,观察三七总甙及三七对ＮＩＨ／３Ｔ３ 细胞的毒性作用与对细胞增殖的影响。结果：细胞数量与ＭＴＴ Ａ５７０值明显正相关,在一定范围内血清温育浓度及ＭＴＴ用量与ＭＴＴ Ａ５７０值也呈明显正相关。三七总甙浓度＞ １．６ ｍ ｇ／ｍ ｌ时,对细胞有明显毒性作用,其ＩＤ５０ 约为０．４ ｍ ｇ／ｍ ｌ,对小牛血清刺激的细胞增殖有明显的抑制作用。与三七冻干粉相比,三七总甙对细胞的毒性作用与增殖抑制影响均较明显。结论：三七总甙能抑制成纤维细胞增殖,具有体外抗肝纤维化作用,可能是三七的抗肝纤维化的主要药效成分。     

Methotrexate cytotoxicity determination using the MTT assay following enzymatic depletion of thymidine and hypoxanthine

Methotrexate, an important agent in the treatment of childhood acute lymphoblastic leukaemia, has generally failed to induce dose-dependent cytotoxicity of patient-derived leukaemic blasts when tested in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This effect is apparently due to salvage from the medium, by surviving leukaemic cells, of metabolites such as hypoxanthine and thymidine. In an attempt to address this problem, we have examined the effect, on leukaemic cell populations, of enzymatically depleting these metabolites from the culture medium employed during the MTT assay, using xanthine oxidase and thymidine phosphorylase. Specifically we have assessed methotrexate cytotoxicity in the paediatric acute lymphoblastic T cell leukaemia, GKTL, which is maintained as a xenograft, and like primary leukaemias, has poor viability in vitro. Although little cytotoxicity of GKTL cells was observed when the MTT assay was performed in supplemented RPMI-1640 medium, dose-dependent cytotoxicity of these cells was clearly apparent when the same medium was enzymatically depleted. In contrast, the ID₅₀ for methotrexate of control CCRF-CEM cells was unaltered in enzymatically depleted medium. In the absence of methotrexate, enzymatic depletion of the medium did not affect leukaemic cell survival. We are currently investigating the general applicability of this approach for assaying the response to methotrexate of primary leukaemia samples.Abbreviations ALL acute lymphoblastic leukaemia - MTX methotrexate - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Supported by a grant from the National Health and Medical Research Council (Australia) and also by the Children's Leukaemia and Cancer Foundation Inc. (Australia)     

Stable Down-Regulation of HLA Class-I by Serum Starvation in Human PBMCs

Background: The human leukocyte antigen (HLA) matching between organ donor and recipient is an acceptable strategy in clinical transplantation since 1964. However, in bone marrow transplantation, finding matched donors is often problematic. Thus new method for down regulation of HLA can be an alternative strategy to solve this problem. Objective: To examine the effect of serum starvation on HLA class I expression in human peripheral blood mononuclear cells (PBMCs). Methods: PBMCs were cultured in RPMI-1640 supplemented with 10% FBS (non-starved cells) as well as in medium only (starved cells) for 16, 24, 48, 72, 96h under standard cell culture conditions. The pattern of cell death and HLA class I expression was determined by flowcytometry. Antigenicity of the starved PBMCs was evaluated in a one-way mixed lymphocyte culture by MTT assay. Results: Mean fluorescence intensity (MFI) of different indicated starved PBMCs gradually decreased and this reduction was stable after 96h of re-feeding with medium containing FBS. Under serum starvation condition, PBMCs showed apoptotic cell death pattern. There was a linear correlation between percentages of cells, which exhibited the late apoptosis death pattern and serum starvation period (r=0.88, p<0.01). Surprisingly, the starved PBMCs lost their stimulatory property in mixed culture with allo-reactive lymphocyte. Conclusions: Membrane HLA class I expression could be stably reduced in 96h starved human PBMCs culture condition, decreasing their allo-reactivity while their viability rate is enough for possible clinical application.     

Response of nasal ciliated cells of the guinea-pig during allergic reactions in human blood

Nasal mucosal samples from thirty-five guinea-pigs were placed in a chamber, containing the medium RPMI 1640. Ciliary activity (beats.s-1) of the most active cell in each mucosal culture was measured using a photoelectric method. The RPMI 1640 was then replaced by 2 ml of RPMI 1640 and 1 ml of heparinized human blood from a non-allergic patient, with a ragweed-sensitive nasal allergy, or patients with D. farinae-sensitive nasal allergy: the ciliary activity of the same ciliated cell in each culture did not change significantly. We then added 1 micrograms of D. farinae extracts to evoke an in vitro allergic reaction. Ciliary excitation was induced when D. farinae extracts were added to the blood from D. farinae-sensitive subjects but not when added to blood from subjects without sensitivity to D. farinae. The peak and plateau of such an event occurred after 20-30 min. Ciliary stimulations were correlated with D. farinae-induced histamine release from whole blood. Thus, in vitro allergic reactions stimulate activity in cilia from normal nasal mucosa.