Mercaptoethanol protects glutathione depleted cells

Buthionine sulfoximine depleted the glutathione (GSH) level of mouse lymphoma L1210A cells in culture to 6% of control and killed the cells within 48 hours in medium supplemented with fetal calf serum or bovine serum albumin. Mercaptoethanol or alpha-thioglycerol but not GSH or cysteine added to the medium protected the cells from the effect of GSH depletion. Horse serum was also protective, and this effect was removed by dialysis over 65 hours and could not be restored by adding GSH. Mercaptoethanol alone had a protective action in the dialyzed sera. The results suggest that mercaptoethanol may act independently and perform the functions of GSH.     

国产与进口牛血清促进Vero细胞生长的比较

 目的  通过对国产与进口牛血清促进Vero细胞生长的比较,筛选可替代进口血清用于轮状病毒疫苗生产中Vero细胞培养的国产牛血清。方法  以国产的3批胎牛血清和1批新生牛血清为待评血清,以进口胎牛血清为对照血清,制备细胞培养液。在T175细胞培养瓶（T瓶）和2层细胞工厂中连续传代培养Vero细胞各3代,观察每一代次的培养上清液、细胞形态和细胞汇合度,计算细胞收获量和与对照血清相比较的相对增长率。采用单样本t检验对细胞收获量与生产要求的细胞产量（T瓶：>1.50×10⁷ 个/ml;细胞工厂：>3.00×10⁸ 个/ml）进行比较。根据细胞相对增长率判断国产血清与进口对照血清促细胞生长活性的相近程度。结果  国产血清培养的Vero细胞生长状态均良好。T瓶培养时,国产血清组的细胞收获量为（1.56～9.30）×10⁷ 个/ml,与生产要求细胞产量的差异有统计学意义（t=2.44～3.76,P<0.05）,且t值均>0,说明符合生产要求。细胞相对增长率接近或超过100%。细胞工厂培养时,国产血清组的细胞收获量为（1.80～4.92）×10⁸ 个/ml,与生产要求产量的差异无统计学意义（t=－0.23～1.16,P>0.05）,但是只有1批胎牛血清和新生牛血清的细胞收获量均值>3.00×10⁸ 个/ml,且细胞相对增长率>90%。 结论  经与进口牛血清比较,国产的1批胎牛血清和1批新生牛血清可替代进口血清,用于轮状病毒疫苗生产中的Vero细胞培养。     

Growth medium for the evaluation of antiestrogenic compounds in MCF-7 cell culture.

The primary aim of the present study was to define a set of cell culture conditions that would be optimal for the evaluation of antiestrogens using estrogen dependent MCF-7 human breast cancer cells in culture. Therefore, the present study examined for the influence of media supplements which are known to alter the growth rate and estrogen receptor content of MCF-7 cells in culture. In this study, MCF-7 cell proliferation was evaluated using the hemocytometric trypan blue exclusion method. The results of this study clearly indicate that 1) charcoal-dextran stripped serum offers no advantage over low estradiol (less than 100 fM) calf serum which is available commercially, and 2) calf serum is as effective as fetal calf serum, in the presence or absence of insulin, in time-dependent growth of MCF-7 cells. Thus, growth media containing 5% non-stripped low estradiol (less than 100 fM) calf serum without insulin supplementation was found to be optimal for the evaluation of the antitumor activity of antiestrogenic compounds using MCF-7 cells in culture.     

甘草酸对血清和组织胺诱发的大鼠气道平滑肌细胞增生的影响

目的　观察甘草酸对血清和组织胺诱发的大鼠气道平滑肌细胞 (ASMC)增生的影响。方法　MTT法、细胞计数法和流式细胞术检测体外培养的ASMC的光密度、细胞数和细胞周期。结果　①在含 10 0g·L-1FCS的培养液中加入 6×10 -5mol·L-1的甘草酸可使A570 的增速增加 ,而加入 384×10 -5mol·L-1和 15 36× 10 -5mol·L-1却使A570 的增速减慢 ;在含 10 -2 mol·L-1组织胺的培养液中加入甘草酸使A5 70的增速减慢 ,浓度越大作用越明显。②在含 10 0g·L-1FCS的培养液中加入 6× 10 -5mol·L-1的甘草酸可促进细胞增生 ,而加入 384× 10 -5mol·L-1和 15 36× 10 -5mol·L-1则抑制细胞增生 ;在含 10 -2 mol·L-1组织胺的培养液中加入甘草酸 ,细胞增生受到抑制 ,浓度越大作用越明显。③在 10 0g·L-1FCS或 10 -2 mol·L-1的组织胺中培养 96h ,随着甘草酸浓度的增加 ,G1期细胞数增多 ,S期和G2 +M期细胞数减少。结论　①对FCS刺激引起的ASMC增生 ,低浓度甘草酸有促进作用 ,高浓度有抑制作用。②对组织胺刺激引起的ASMC增生 ,低浓度和高浓度甘草酸都有抑制作用     

Comparison of the cytotoxicity of the MEIC reference chemicals measured in protein free and in complete culture medium.

In order to investigate if a protein free cytotoxicity assay could improve the prediction of human acute toxicity, the cytotoxicity of 40 MEIC reference chemicals was measured by the neutral red uptake inhibition after 24h in protein free culture medium on rat hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Potassium cyanide, arsenic trioxide, mercuric chloride, hexachlorophene and pentachlorophenol were much more cytotoxic in PF medium, as was the case to a lower extent for 16 other chemicals. The cytotoxicity of 8 chemicals was only changed to a limited extent when tested in PF medium, suggesting that serum proteins do not strongly interact with their cytotoxicity. Eleven other chemicals were less cytotoxic in PF medium, maybe because of too poor physiological conditions. Although a large number of differences in cytotoxicity were observed in function of the medium used for the assay, a good correlation was observed between both series of data (r(2)=0.946). The correlation between the cytotoxicity in PF medium and the human acute toxicity is lower (r(2)=0.647) than that in complete medium (r(2)=0.746). The results show that further research is necessary in order to improve the in vitro/in vivo correlations by introducing protein-dependent considerations.     

The use of beta-cyclodextrin-containing culture medium for in vitro evaluation of immunomodulating agents

We have devised a new culture medium that is made of RPMI-1640 medium, 500 micrograms/ml beta-cyclodextrin (beta-CD) and 1% fetal calf serum (FCS) (beta-CD medium). Murine lymphocytes stimulated with sheep erythrocytes in vitro developed antibody-forming cells in beta-CD medium as efficiently as in 10% FCS-containing RPMI-1640 medium (10F medium). Immunostimulating agents (SA96 and levamisole) and immunosuppressive agents (hydrocortisone and D-penicillamine) showed similar immunomodulating effects on the antibody responses in both media. In addition, the enhancing effect of levamisole varied drastically depending on the lot of FCS used in 10F medium, but a clear enhancement was always observed in beta-CD medium containing any lot of FCS tested.     

Inhibitory regulation of serum factor(s)-caused ornithine decarboxylase induction by the protein kinase C system in A431 human epidermoid carcinoma cells

Replacement of the culture medium with fresh medium containing 10% fetal calf serum caused ornithine decarboxylase (ODC) induction in A431 human epidermoid carcinoma cells. Two peaks of ODC activity were observed at 5 and 14 hr after the medium replacement. The peak activity observed at 5 hr was more prominent than that at 14 hr. The first peak of ODC induction was suppressed by a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), in a concentration-dependent manner. The second peak, however, was not suppressed by TPA. Other potent protein kinase C activators, such as mezerein and 12-O-retinoylphorbol-13-acetate, also suppressed the first peak of ODC induction. Synthetic diacylglycerols, 1,2-dioctanoyl-sn-glycerol and 1-oleoyl-2-acetylglycerol, did not inhibit the serum factor(s)-caused ODC induction. Phorbol-13-acetate, an inactive phorbol ester, also failed to inhibit the ODC induction. The growth of A431 cells was slightly suppressed by TPA. In protein kinase C down-regulated cells, TPA failed to inhibit the serum factor(s)-caused ODC induction. These results suggest that the serum factor(s)-caused ODC induction in A431 cells is negatively regulated by the protein kinase C system, which may not be activated by exogenous diacylglycerols.     

人参二醇组皂苷对小细胞肺癌细胞增殖的影响及其机制

目的:探讨人参二醇组皂苷对小细胞肺癌细胞增殖的影响及其机制.方法:在小细胞肺癌细胞的培养液中加入不同浓度(50～250mg/L)的人参二醇组皂苷,培养一定时间后,通过测定其中氚标胸腺嘧啶脱氧核苷酸掺入量(3H-TdR),及以MTT法来评定小细胞肺癌细胞增殖程度.同时,测定细胞培养液中超氧化物歧化酶(S0D)、脂质过氧化物水平,以反映细胞氧化还原状态.结果:随着人参二醇组皂苷浓度的增加,3H-TdR及MTT测定的0D值逐渐减少,细胞增殖率逐渐增加,SOD水平逐渐增加,丙二醛(MDA)水平逐渐减少.当人参二醇组皂苷浓度为150,200,250mg/L时,各指标与对照组(未用药)相比均具有显著性差异(P＜0.05).结论:人参二醇组皂苷可增加S0D水平,减少活性氧自由基的产生,抑制小细胞肺癌细胞的增殖.     

小鼠肝细胞的简易高效高纯培养及鉴定

目的改进一种简易、高效的乳小鼠肝细胞培养法,并对所培养的细胞进行鉴定。方法原代培养采用改进的组织块贴壁方法,以少量培养基孵育1~2h。传代培养采用0·25%胰蛋白酶消化随后以小牛血清培养为单层细胞。整个过程无需离心。结果成功地用这种简便的方法获得了纯度较高的肝细胞并进行了传代和鉴定。结论使用此方法培养肝细胞简单、高效、快捷,适合大多数实验室培养非大规模肝细胞。     

促红细胞生成素和甲泼尼龙对损伤星形胶质细胞增殖的影响

目的研究在促红细胞生成素(erythropoietin,EPO)和甲泼尼龙(Methylprednisolone,MPSS)干预下对损伤星形胶质细胞增殖的影响,探讨EPO和MPSS联合应用在脊髓损伤中的作用。方法取自SD大鼠大脑原代培养星形胶质细胞,缺营养培养3h后分别在促红细胞生成素(erythropoietin,EPO)和甲泼尼龙(erythropoietin,EPO)的培养基中培养24、48小时后,MTT法检测星形胶质细胞增殖活性的变化。结果缺营养可抑制星形胶质细胞增殖,促红细胞生成素和甲泼尼龙对正常细胞增殖无明显影响作用,EPO和MPSS均促进损伤细胞增殖,两者联合减量应用对细胞增殖有明显影响作用。结论 EPO+MPSS(半量组)联合减量应用对星形胶质细胞增殖作用表达的升高作用优于单独应用甲泼尼龙。     

消化法培养大鼠脑血管平滑肌细胞

目的建立大鼠基底动脉平滑肌细胞培养的方法。方法取大鼠基底动脉,剪成约0.2mm的小段,用含胶原酶Ⅱ型(0.5g.L-1)、弹性酶(0.15g.L-1)、透明质酸酶Ⅳ-S型(0.5g.L-1)及脱氧核糖核酸酶Ⅰ型(0.1g.L-1)的消化液在37℃消化1h。而后用含20%FCS的DMEM/F12进行培养。结果原代培养3d后细胞可少量贴壁,1wk左右可传代,细胞传代成活率达97%。光镜和免疫细胞化学染色检测第4代细胞纯度为98%。结论与目前国内培养脑血管平滑肌细胞的方法相比,本方法操作简单,培养周期短,细胞纯度高。     

Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.     

Peptide stability in drug development: a comparison of peptide reactivity in different biological media.

Degradation kinetics for several peptides that bind to the major histocompatibility complex on antigen-presenting cells were determined in both human serum (HS; 25%) and synovial fluid (SF; 25%) from patients with rheumatoid arthritis to test whether therapeutic intervention of rheumatoid arthritis by direct intrasynovial injection is feasible (at least in terms of peptide stability). Controls consisted of enzymatically immature 10% fetal calf serum and peptidase-rich 5% liver homogenate (all diluted with RPMI-1040 tissue culture medium). Peptide half-lives ranged from approximately 4 to greater than 10,000 min, with most peptides showing half-lives of approximately 10-100 min. These studies show that, even though the populations of inflammatory and other cell types in SF and HS are different (and may, therefore, generate different peptidase profiles), the observed peptide stabilities in SF and HS are similar. This finding indicates that the effect of SF on peptide stability is similar to that of HS.     

3,4-二羟基苯乙酮抑制培养兔主动脉平滑肌细胞的增殖

传代SMC生长d 6加入DHAP(10～80μg/ml)介质。结晶紫染色法和[~3H]TdR参入试验表明SMC的生长受到DHAP的显著抑制,这一作用随剂量增加而加强。在细胞增殖周期的G_1期加药比S期加药产生更为明显的抑制作用,G_1期末加药抑制作用最大。DHAP的这一作用可能和降低SMC膜流动性有关。     

Uptake of persistent environmental chemicals by cultured human cells

Uptake of the persistent environmental chemicals 2,2',4,4',5,5'-hexachlorobiphenyl and 1,1,1-trichloro-2,2-di-(4-chlorophenyl)ethane (the insecticide DDT) by Chang liver cells, an established human cell line, has been investigated. Monolayer cells were incubated with culture medium to which the lipophilic model compounds had been added. The time course of uptake of either compound was biphasic, reaching equilibrium after about 5 hr of incubation. The ratio of DDT:hexachlorobiphenyl uptake was dependent on the presence of serum proteins. Increasing concentrations of serum proteins in the culture medium progressively inhibited uptake. Efflux from the cells was not entirely reversible: 10-20% of the chemicals were not released. Uptake was a linear function of the external concentration of the compounds. Absorptive binding to the outer cell plasma membrane could be determined by removing bound chemicals with fetal calf serum ("back exchange"). With this method, temperature-dependent translocation through the cell plasma membrane could directly be demonstrated. The effect of low temperature as well as the influence of metabolic inhibitors point out the contribution of energy-driven uptake pathways. Demonstration of LDL receptor-like binding protein on Chang liver cells facilitated estimation of the role of receptor-mediated uptake. This route of uptake proved to be of minor importance only, as was transport of the protein-bound chemicals via fluid pinocytosis. The results demonstrate that cellular endocytosis of plasma membrane-bound chemicals constitutes a major uptake pathway for lipophilic chemicals.     

Primary culture of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus).

Primary cultures of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus) were attempted. At first, six different cell types were obtained including potentially secreting epithelial-like cells. Nonepithelial cell cultures were later invaded by fibroblast-like cells. Cultures of epithelial-like gland cells were successfully maintained, after testing different culture conditions by varying the media, incubation temperature, use of dissociating agents and adhesion substrates. The best results were achieved using plates precoated with rattlesnake skin collagen and incubation in CMRL 1415 modified for snake gland cells plus 10% fetal calf serum at 30°C. The presence of venom could be demonstrated in the supernatant of five out of six epithelial-like gland cell cultures tested by ELISA, in the very first passages. After the third passage, however, venom amounts dropped to undetectable values. A total of 23 venom gland cell lines were obtained and are kept frozen in the laboratory; among them, five epithelial-like gland cell lines with up to 12 passages, that were continuously cultured for more than 30 weeks. The methodology described here was successfully applied to C. d. terrificus kidney cells culturing, developed to be used as negative control.     

胰蛋白酶对Madin-Darby犬肾细胞消化方法的研究

目的:考察不同胰蛋白酶及消化后是否弃去胰蛋白酶对Madin-Darby犬肾(Madin-Darby canine kidney,MDCK)细胞消化效果的影响,并评价培养基中添加胎牛血清对减轻胰蛋白酶细胞毒性的作用。方法:选用5种胰蛋白酶分别消化MDCK细胞,消化5 min后弃去胰蛋白酶为弃去组,不弃去为保留组。观察消化...     

Elevation of plasma endothelin concentrations during endotoxin shock in dogs.

The effect of endotoxin on the release of endothelin, a novel potent vasoconstrictor peptide, was examined in anesthetized dogs and in cultured endothelial cells. Administration of 2.63 mg lipopolysaccharide, E. coli 0111:B4/kg body weight caused shock in the animals and produced a long-lasting increase in the plasma immunoreactive endothelin-1 level that remained higher than the basal level (1.83 pg/ml as mean level) from 30 to 120 min after the injection, with a peak at 90 min (8.15 pg/ml as mean level). In vitro immunoreactive endothelin-1 in a culture medium, in which calf pulmonary artery endothelial cells were incubated in the presence of 10% fetal bovine serum, increased dose dependently with the concentration of added lipopolysaccharide between 0.01 and 10 micrograms/ml. These data indicate that plasma endothelin increases during endotoxin shock and that stimulation by endotoxin, per se, in the presence of serum participates at least partially in the mechanism for its release.     

Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests

Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.     

Extracellular requirements for the endocytosis of carcinogenic crystalline nickel sulfide particles by facultative phagocytes

Various culture medium components were examined for their effect upon the phagocytosis of carcinogenic crystalline and non-carcinogenic amorphous NiS by cultured fibroblastic cells using both a visual and radioactive assay for phagocytosis. Crystalline NiS was phagocytosed by cells in a simple salts/glucose maintenance medium to an extent similar to that observed in complex culture medium fortified with 10% fetal bovine serum (FBS), suggesting that serum proteins and other components in complex culture medium exert little influence upon the uptake of these heavy metal particles. Phagocytosis of crystalline NiS was shown to be highly dependent upon Ca²⁺since omission of Ca²⁺ from the salts/glucose medium substantially reduced phagocytosis, while readdition of Ca²⁺ stimulated uptake in a concentration-dependent manner. The uptake of the NiS particles was inhibited by trifluoperazine, a calmodulin antagonist, implicating intracellular Ca²⁺ in this phagocytosis process. Since the opposite surface charge of crystalline and amorphous NiS has been related to their different phagocytic uptake by cells whose primary function is not phagocytosis (facultative phagocytes), these results show that the culture medium components do not modify the surface charge of these particles in a way that significantly influences their uptake.