不同培养基对白假丝酵母菌生物膜芽管形成的影响

目的评估不同培养基对产生物膜白假丝酵母菌芽管产生的影响。方法收集临床产生物膜的白假丝酵母菌20株,购买3株可以产生物膜的质控菌株。比较23株白假丝酵母菌在5种不同培养基(人血清、胰蛋白胨大豆肉汤、脑心浸液肉汤、RPMI 1640、NaHCO₃溶液)的出芽菌株数量和出芽率。结果在23株产生物膜的白假丝酵母菌中,人血清和脑心浸液肉汤培养基中23株(100.0％)全部产生芽管,NaHCO₃培养基18株(78.3％)、胰蛋白胨大豆肉汤培养基16株(69.6％)和RPMI 1640培养基15株(65.2％)。芽管生成试验阳性的白假丝酵母菌中,在5% CO₂培养条件下的酵母细胞出芽率更高(孵育2 h,出芽率78.0%),芽管长度更长。结论 脑心浸液肉汤可以代替人血清作为芽管试验的诱导剂,人血清(5% CO₂)可以促进芽管的生成以及延伸芽管的长度。     

中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

小鼠淋巴细胞杂交瘤的电融合

本文报道我们用电场成功地诱导小鼠淋巴细胞与骨髓瘤细胞融合并筛选得到杂交细胞。 材料与方法 细胞对HAT敏感的SP2/0骨髓瘤细胞用RPMI 1640培养基(RPMI 1640培养液加15％小牛血清)培养,淋巴细胞取自活杀的小鼠脾。     

细菌产纤维素酶和脂肪酶固体培养基及接种和培养时间的研究

为准确鉴定和筛选产纤维素和脂肪酶细菌，通过平板扩散法测定不同氮源培养基对细菌产纤维素酶和不同碳源培养基对细菌产脂肪酶活性的影响，确定细菌产纤维素酶和脂肪酶的最佳培养基，利用最佳培养基研究不同琼脂含量、海水和淡水溶剂、菌种的培养时间及接种后的培养时间对细菌纤维素酶和脂肪酶活性的影响。结果表明，以蛋白胨为氮源的A培养基为细菌产纤维素酶最佳培养基，以Tween-20为碳源的培养基为细菌产脂肪酶最佳培养基；培养基中琼脂的最佳用量均为13‰；所有菌株在海水培养基上产生的纤维素酶活性比淡水培养基上高，但脂肪酶活性并非如此；鉴定和筛选产纤维素酶和脂肪酶细菌接种菌种的最佳培养时间分别为18 h和24~32 h，测定细菌产纤维素酶和脂肪酶的最佳培养时间分别为48~72 h和120 h。     

产蛋白酶毛霉菌株的初步筛选

对从农家自制豆酱中分离获得的 26 株毛霉菌株,采用酪素培养基平板透明圈法进行产蛋白酶活性初筛,获得了高产蛋白酶活力的菌株M-339、M-513和M-808.改变培养温度和培养基配比,采用Folin-酚法测定蛋白酶活力,分别获得了菌株M-339、M-513和M-808产蛋白酶活力的最佳培养条件.筛选出的菌株有望用于发酵食品的工厂化生产.     

用无血清培养基在填充床生物反应生产rHuEPO

在填充床生物反应器用含５％ＦＢＳ的ＤＭＥＭ：Ｆ１２培养基培养产重组人促红细胞生成素（ｒＨｕＥＰＯ）的细胞Ｃ２８￣１０ｄ后,使用自制的无血清生产培养基（ＳＦＭ－ｐ）生产ｒＨｕＥＰＯ。ＳＦＭ－ｐ培养基既能维持细胞生长,又能生产ＥＰＯ,也便于纯化分离ｒＨｕＥＰＯ。使用填充床生物反应器培养细胞,能维持培养２０￣２５ｄ,ｒＨｕＥＰＯ表达水平达１２￣２８．４ｍｇ／Ｌ之间,反应器的产率达到７１．０ｍｇ／Ｌ／ｄ,     

雷帕霉素对低氧下HL-60细胞的低氧调控信号分子表达的影响

摘要目的：通过小分子化合物氯（CoCt2）模拟的低氧环境,分析低氧下及其雷帕霉素（RPM）作用下人急性髓细胞白血病细胞HL．60的低氧调控信号分子表达的变化;方法：常规方法复苏、传代、培养HL-60细胞,培养细胞进入对数生长期后用于实验。低氧模拟组、低氧雷帕霉素处理组、常氧雷帕霉素处理组分别用含2001xmol／LCoCl2、2001xmol／LCOCl2／20nmol／LRPM、20nmol／LRPM的1640培养基处理生长状态良好的细胞,对照组细胞用1640培养基培养,各组置培养箱以37℃、5％CO2培养,并于处理后24h、48h、72h收集细胞用于检测;采用实时荧光定量PCR方法检测低氧诱导因子（HIF-1α）、内皮细胞生长因于（VEGF）、雷帕霉素靶蛋白（mTOR）及GAPDH在转录水平的表达;结果：①与各时段对照组相比,低氧模拟组HIF-1α表达随时间逐渐增加,72h明显上调;与常氧雷帕霉素处理组各时段比较,低氧雷帕霉素处理组HIF-1α表达早期（24h）相对下调,后期相对上调;②．与对照组比较,各处理组mTOR表达均下调,低氧雷帕霉素处理组在早期（24h）下调显著;与常氧雷帕霉素处理组比较,低氧雷帕霉素处理组mTOR各时段的表达均相对下调;③与对照组各时段相比,低氧模拟组VEGF的表达在早期显著上调,但后期呈下调;常氧雷帕霉素处理组各时段VEGF的表达下调,与其比较,低氧雷帕霉素处理组各时段均呈相对下调。结论：常氧和低氧下RPM作用HL-60细胞后VEGF、mTOR的mRNA均表达下调,RPM可在低氧环境下增强了这种下调表达作用。     

不同培养基对流式细胞仪分选外周血T细胞的影响

摘要 目的：通过探讨用于流式分选的T细胞体外扩增的无血清培养基，提高过继细胞的增殖能力和活性。方法：采用人外周血淋巴细胞分离管制备外周血单个核细胞，再用流式细胞分选仪从6例健康志愿者的外周血单个核细胞中分选CD3+T细胞到4种常用的培养基中：X-VIVO 15、KBM 581、TexMACS GMP和10 % FBS/1640，观察并记录培养细胞的状态和体外增殖能力。于第3天，第6天和第8天，通过胎盼蓝染色后进行活细胞计数。于第8天用凋亡试剂盒检测扩增细胞的凋亡情况，并用流式细胞分析仪检测细胞的免疫表型。结果：X-VIVO 15、TexMACS GMP和10 % FBS/1640作为流式细胞分选的接收液仅少量细胞碎片，而分选在KBM 581的细胞大量死亡，显著高于X-VIVO 15组（P<0.05）。X-VIVO 15中扩增的细胞数量最多，增殖检测结果显示活细胞在X-VIVO 15中快速增殖且细胞凋亡率显著低于KBM 581 和 TexMACS GMP（P<0.05）。4种培养基扩增的细胞主要呈现效应记忆型。其中，X-VIVO 15中效应记忆型T细胞比例显著高于TexMACS GMP（P<0.05）。TexMACS中效应细胞比例显著高于10 % FBS/1640（P<0.05）。结论：X-VIVO 15无血清培养基扩增流式分选的T细胞具有高增殖能力、细胞活性和记忆表型，适用于经流式分选后细胞的体外扩增。     

人胸腺上皮细胞培养及其分泌产物

本文通过降低培养基中血清含量,向RPMI 1640培养基中补加三碘甲腺原氨酸而获得一种人胸腺网状上皮细胞占优势生长的培养物。在此培养基中细胞经传代培养长达90天,仍维持正常形态特征。胸腺组织在培养14天后,新生细胞的突起形成网状结构,细胞化学检查和电镜观察表明具有丰富的分泌颗粒,囊泡及张力原纤维束和桥粒等上皮细胞特征。收集合并细胞培养液,经部分纯化后检查其生物活性,表现出具有促进玫瑰花结形成和降低胸腺细胞TdT活性的作用,说明培养细胞的分泌产物具有胸腺激素活性。根据形态学,细胞化学和生物活性检测结果,我们倾向于认为该培养物主要为网状上皮细胞。     

Effect of interleukin 2, interferon-gamma, and mitogens on the production of tumor necrosis factors alpha and beta

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant interleukin 2 and mitogens to secrete two distinct cytotoxic polypeptides, tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta), previously called lymphotoxin. Treatment of PBMC with recombinant human interleukin 2 (rIL 2) or mitogens in combination with recombinant human interferon-gamma (rIFN-gamma) resulted in augmented production of both TNF-alpha and TNF-beta. rIFN-gamma alone had no effect on production of either cytotoxic polypeptide. TNF-alpha was produced within 2 to 3 hr after induction and was the major cytotoxin produced by PBMC during the first 48 hr of culture, after which time TNF-beta became the predominant species. TNF-beta was first secreted into the media after 8 hr of induction. Enhanced levels of both TNF-alpha and TNF-beta were seen when the PBMC were separated into adherent and nonadherent cells. Both TNF-alpha and TNF-beta were induced in different tumor cell lines of hematopoietic origin. The results demonstrate that the production of TNF-alpha and TNF-beta can be enhanced by two lymphokines, IL 2 and IFN-gamma.     

Interleukin-4 receptors on human blood mononuclear cells

We have studied regulation of the expression of the interleukin-4 receptor (IL-4R) on human blood mononuclear cells (PBMC) using both 125I-IL-4 binding assay and flow cytometric analysis of biotinylated IL-4 (B-IL-4) binding. PBMC express approximately 300 high-affinity IL-4R per cell (Kd = 25-100 pM). Activation of PBMC for 60-80 hr by phytohemagglutinin (PHA) or concanavalin A (Con A) results in a 2- to 4.5-fold increase of IL-4R number without alteration of IL-4R affinity for IL-4. Binding of B-IL-4 showed that IL-4R expression is upregulated on virtually all PHA-stimulated PBMC, whereas it mostly concerns larger cells among Con A-activated PBMC. Reculture of PHA-blasts with 1 nM IL-4 further upregulates IL-4R expression to a level approximately 10-fold higher than observed on freshly isolated PBMC. Interestingly, IL-4 is able to reinduce high IL-4R levels on cells that have been deprived of IL-4 for 20 hr and IL-2 is almost as efficient. Finally, SDS-PAGE analysis of IL-4-binding molecules on unstimulated, PHA- and PHA/IL-4-activated PBMC revealed the same three peptides of MW 140-130, 80-75, and 70-65 kDa, as shown on human cell lines.     

Comparable growth of Tritrichomonas mobilensis in two commercially available culture media

The investigation reported herein was undertaken to determine which medium is more practical for the axenic laboratory culture of trichomonads. The growth of Tritrichomonas mobilensis was monitored in 2 different types of commercially available growth media. Although Roswell Park Memorial Institute (RPMI) 1640 medium is typically used as a mammalian cell culture medium, it was found to support the growth of trichomonads as well as the American Type Culture Collection (ATCC) medium 745 under similar conditions. Environmental variables, such as temperature and pH, known to affect the success of trichomonad cultures were controlled. The mean generation times (MGTs) of T. mobilensis in the log phase of growth were 5.1 and 4.9 hr for RPMI 1640 and ATCC medium 745, respectively. A stationary phase of zero growth was reached more quickly in the ATCC medium 745 cultures, and in both media a phase of rapid attrition followed this period of static growth. In assessing the practicality of the media, total cell amplification, as well as factors such as cost, ease of preparation, and storage capacity, were considered.     

Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.     

带有逆转录病毒的恶性T淋巴细胞株的建立

从一例患神经系统疾病病人的外周血淋巴细胞中建立了一株恶性Ｔ淋巴细胞株ＣＭ－１并研究了它的生物学特性。用过滤的ＣＭ－１细胞的培养上清,可命名多发性血管硬化症病人的淋巴细胞转化恶性Ｔ淋巴细胞,由此建立ＣＭ－２细胞株。用ＣＭ－１和ＣＭ－２细胞皮下接种裸鼠,都能使裸鼠产生弥漫性恶性淋巴瘤。电镜下见到了类似于Ｃ型逆转录病毒的颗粒,逆转酶活性检测阳性。血清学和基因检测表明ＣＭ－１和ＣＭ－２中不存在本室常用的其     

Monocyte-dependent,serum-borne suppressor of induction of lymphokine-activated killer cells in lymphocytes from melanoma patients

Summary Both phytohemagglutinin-induced cytotoxicity and recombinant-interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) activity against noncultured melanoma cells were significantly reduced when peripheral blood mononuclear cells (PBMC) from patients with metastatic melanoma were incubated in RPMI medium 1640 and 10% autologous human serum instead of 10% fetal calf serum, while serum from either healthy donors or patients with primary melanoma did not affect the level of cytotoxicity. The serum-mediated suppression was not restricted by major histocompatibility complex and was time-dependent. Addition of 10% human serum from the patients with metastatic melanoma [HS-Pt(m)] to the culture of PBMC with rIL-2 at the same time or 1 day after incubation significantly inhibited LAK activity. However, addition of 10% HS-Pt(m) 2 or 3 days after incubation did not inhibit LAK activity. Incubation of PBMC for 2 h with a high dose (10⁴ U/ml) of rIL-2 in the presence of 10% HS-Pt(m), followed by incubation in the absence of either rIL-2 or HS-Pt(m), did not affect LAK cell activity. These results suggest that HS-Pt(m) inhibits the early stage of LAK cell differentiation, rather than the binding of rIL-2 to PBMC or a later stage in the differentiation. In contrast to PBMC, monocyte-depleted peripheral blood lymphocytes exhibited comparable levels of LAK activity when cultured with rIL-2 either in 10% fetal calf serum, 10% human serum from healthy donors or 10% HS-Pt(m). Addition of purified autologous monocytes to the culture of monocyte-depleted peripheral blood lymphocytes with rIL-2 suppressed LAK cell induction when 10% HS-Pt(m) was present. Thus serum-mediated suppression of LAK cell induction is largely dependent on the presence of monocytes, which may produce a secondary inhibitor that acts on lymphocytes. Addition of indomethacin to the culture did not reverse this monocyte-dependent serum-mediated suppression in a majority of cases, suggesting that prostaglandin E₂ does not have a major role in the suppression.This work was supported in part by NIH grant RR5511-25 and grants from The Council for Tobacco Research USA Inc., The Meadows Foundation, the Erwin Zaban Melanoma Research Foundation, and the Gillson-Longenbaugh Foundation     

Inhibitory effect of extracellular histidine on cobalt-induced HIF-1alpha expression

Cobalt chloride (CoCl(2)) can mimic hypoxia in inducing hypoxia-inducible factor 1 (HIF-1). Several cultured cells were examined for susceptibility to CoCl(2) in DMEM, MEM and RPMI 1640 medium. Here we report that HIF-1α expression of mammalian cells by CoCl(2) was largely dependent on the culture medium. HIF-1α protein and hypoxia response element (HRE)-dependent reporter activity were strongly induced in RPMI 1640 but not in DMEM in several cultured cells including MCF-7, a human breast cancer cell line. Analysis of causal nutrients has revealed that histidine, which is contained richer in DMEM, acts as the inhibitory nutrient for cobalt-induced HIF-1α expression of MCF-7 cells in DMEM. D-Histidine also inhibited the HIF-1α activity at the same level as L-histidine, suggesting that sequestration of free cobaltous ion by chelation with histidine was the cause of the inhibition. These results demonstrate that selection of the culture medium must be considered with caution in cell culture experiments using CoCl(2) as a hypoxia-mimetic reagent.     

Temperature Effects on Legionella pneumophila Killing by and Multiplication in Phagocytes of Guinea Pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 × 10⁴ CFU) or a lethal dose (1.0 × 10⁵ CFU) of L. pneumophila elevated from 38.4±0.15 C to 40.2±0.42 C or 40.3 ± 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P<0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Hypoxia modulates cyclin and cytokine expression and inhibits peripheral mononuclear cell proliferation.

Previously, we found that hypoxia can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC). Here, we demonstrated that the cycle progression of hypoxic PBMC, cultured in the presence or not of a specific T cell activator such as phytohaemagglutinin (PHA), was delayed when compared with aerobic cultures. This delay was accompanied by a decrease of the expression of specific cyclins associated to cell cycle progression phases. Ribonuclease Protection Assay (RPA) studies reveal a decrease in the expression of cyclin A and B in PHA-stimulated PBMC kept for 40 hr under hypoxic condition (2% O(2)), when compared with aerobic cultures (20% O(2)). In concomitance, a decrease of cyclin D2 expression was present after 16 hr of hypoxic treatment. However, the decrease was transient and disappeared after 40 hr of hypoxic treatment. Furthermore, cyclin C expression was not affected by hypoxia. Hypoxia-induced cyclin modulation was accompanied by an increased synthesis of interleukin (IL)-2 and IL-4, analyzed by ELISA. By evaluating these results, it appears that hypoxia induces a growth suppressive state in mitogen-activated PBMC by inhibiting the synthesis of mitotic cyclins A and B. However hypoxic PBMC maintain their viability and capability of producing stimulatory cytokines, after mitogen treatment. This should be important in local hypoxia, usually associated with necrotic areas, in inflammation, and infections, where T lymphocyte capability of producing stimulatory cytokines is desirable.     

Constitutive production and characterization of interferon-gamma in a human T-lymphoblastoid cell line transformed by a human retrovirus

A human T-lymphoblastoid cell line, TCL-Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years. When cells were incubated in RPMI-1640 medium with 10% fetal calf serum for three days, IFN activity was detectable at a cell density of 6 X 10(4) cells/ml, whereas 2,000-16,000 units of IFN per ml were produced at 5-10 X 10(5) cells/ml. IFN production was also detected even in serumfree medium and as early as 2 hr after cultivation in fresh medium. IFN was inhibited by treatment of cells with either actinomycin D or cycloheximide, indicating the requirement of IFN-mRNA and protein for de novo synthesis. The molecular weight of the IFN was 45,000-60,000 as determined by Sephacryl S200 gel filtration. Two activity peaks corresponding to molecular weights of 22,000 and 39,000 were obtained by SDS-polyacrylamide gel electrophoresis. Analysis by isoelectric focusing revealed charge heterogeneity with four species at pIs of 6.0, 7.1, 8.6, and 9.3. Conventional IFN-gamma inducers, concanavalin A and 12-O-tetradecanoyl-phorbol-13-acetate, further enhanced the production of IFN in this cell line.