Effect of monochromatic light of low intensity on L929 skin fibroblast culture

Effects of exposure to low-intensity monochromatic red spectrum radiation in different modes were studied on L929 skin fibroblast culture. Radiometric and cytological study of cell culture before and after irradiation showed that monochromatic low-intensity radiation stimulated skin fibroblasts in L929 culture under certain conditions. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 689–691, June, 2006     

激活素A对L929成纤维细胞迁移、侵袭及细胞因子分泌的影响

目的探讨激活素A(activin A)对小鼠成纤维细胞系L929细胞迁移、侵袭及细胞因子分泌的影响。方法采用5-溴-2’-脱氧尿嘧啶核苷(5-bromo-2’-deoxyuridine,BrdU)掺入实验检测L929细胞增殖,酶联免疫吸附实验(ELISA)检测L929细胞培养上清中细胞因子水平,基质胶transwell侵袭实验分析L929细胞迁移、侵袭,实时细胞分析技术(RTCA)检测L929细胞黏附,Western blot分析细胞内信号蛋白表达水平。结果与对照组相比,激活素A处理对L929细胞增殖没有影响,对L929细胞分泌TNF-α、IL-1β及IL-6等促炎细胞因子也没有影响,但却显著促进纤维化诱导因子TGF-β1分泌。激活素A还能显著增加L929细胞穿透基质胶迁移、侵袭细胞数量(P<0.01),并促进L929细胞黏附,添加阻断剂FST可以显著减弱激活素A诱导的L929细胞侵袭和促进黏附作用。激活素A处理L929细胞可以显著增加p-ERK和p-JNK蛋白水平,但对p-Smad3蛋白水平没有明显影响。结论激活素A可以促进L929细胞分泌TGF-β1、诱导L929细胞侵袭和促...     

HBsAg S与GM—CSF融合基因在L929细胞中的表达

将乙肝病毒表面抗原（ＨＢｓＡｇ）主蛋白的编码基因Ｓ与人ＧＭ－ＣＳＦ基因融蛤 后,插入真核表达质粒ｐｃＤＮＡ１中,经质粒酶切鉴定及ＤＮＡ序列分析证明,目的基因插入ｐｃＤＮＡ１的ＣＭＶ启动子下降。以获得的重组质粒ｐＳＧＭ转染Ｌ９２９细胞,经ＲＴ－ＰＣＲ及细胞原位杂交证实目的基因的转录。     

Lack of effect of dexamethasone on growth of Orientia tsutsugamushi Gilliam in mouse L929 cells

Purpose

Previous studies and our own clinical experience suggest that concurrent corticosteroid treatment for severe rickettsial disease with multiorgan failure may improve the clinical course or reduce mortality. However, the use of corticosteroids as adjunctive treatment for rickettsial diseases is controversial. We attempted to determine the influences of corticosteroid on the growth of Orientia tsutsugamushi in vitro to justify and evaluate the clinical applicability of corticosteroid in rickettsial disease.

Materials and Methods

L929 cells were infected with Orientia tsutsugamushi Gilliam. Dexamethasone was added to the cells at final concentrations of 10¹ and 10⁷ pg/mL. Cultures were incubated at 35℃ and processed for flow cytometry on the 6th day after addition of dexamethasone.

Results

Observation on the 6th day after treatment with dexamethasone in infected cultures revealed that there was no difference in fluorescence intensity among the treatment wells. Treatment of the cells with dexamethasone at concentrations of 10¹ and 10⁷ pg/mL showed no influence on the growth of Orientia tsutsugamushi.

Conclusion

Our results to show that isolated corticosteroid does not enhance the replication of Orientia tsutsugamushi in vitro. Concurrent use of anti-inflammatory or immunosuppressive doses of corticosteroids in conjunction with antibiotics may not have detrimental effects on the course of scrub typhus.     

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. WhereasNP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors. Received: 10 January 2000     

小鼠Tim-2基因在L929细胞表达后促进铁蛋白的内吞

目的获取BALB／c小鼠T细胞免疫球蛋白域．黏蛋白域蛋白-2（T cell immunoglobulin and mucin domain containing molecules-2，Tim-2）基因，构建真核表达载体在L929细胞中表达并研究其功能。方法RT-PCR获取BALB／c小鼠Tim-2基因，T-A克隆后经测序鉴定后酶切，插入pEGFP-N1真核表达载体，脂质体转染L929细胞，荧光显微镜观察外源基因的表达，然后加入铁蛋白与转染细胞作用。结果外源基因在L929细胞中得到高效表达，荧光显微镜的结果显示，外源基因定位于细胞膜上，并能促进细胞对铁蛋白的内吞。结论Tim-2基因在真核细胞中表达后定位于细胞膜上，并促进细胞对铁蛋白的内吞。     

人B-CAM/Lu基因逆转录病毒载体的构建及其在L929细胞中的表达

目的 克隆人B-CAM/Lu基因,构建其逆转录病毒表达载体,获得稳定高表达B-CAM/Lu的L929细胞株.方法 RT-PCR技术克隆人B-CAM/Lu基因,并插入逆转录病毒载体pEGZ中,该重组逆转录病毒载体与pH456、pH460两个辅助病毒载体一起,共转染包装细胞293T,并感染L929细胞,经Zeocin筛选获得的细胞株通过RT-PCR、Western blot及间接免疫荧光鉴定细胞中B-CAM/Lu基因mRNA的转录和蛋白表达.结果 成功获得人B-CAM/Lu基因,测序正确,亚克隆至逆转录病毒载体pEGZ后,经转染筛选,获得的抗性L929细胞株通过RT-PCR和Western blot分析,检测到B-CAM/Lu mRNA的转录和目的 蛋白的表达,通过间接免疫荧光确定该蛋白定位表达在L929转基因细胞膜上.结论 成功克隆并构建了人B-CAM/Lu基因的重组逆转录病毒表达载体,获得稳定高表达B-CAM/Lu蛋白的L929细胞株,为将其作为免疫源,制备B-CAM/Lu单抗用于镰刀型红细胞病及一些肿瘤疾病的检测诊断打下了基础.     

Pseudolaric Acid B Induced Cell Cycle Arrest,Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis.Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot.Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence.Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC.     

不同培养基对人脐静脉内皮细胞体外培养效果的影响

目的研究不同培养基对人脐静脉内皮细胞体外培养生长的影响,探讨内皮细胞的最佳体外扩增培养条件。方法取健康产妇分娩后脐带,用胶原酶Ⅰ消化后得脐静脉内皮细胞,进行原代培养,并用免疫荧光染色的方法对细胞进行鉴定,传代培养时则分别采用RPMI-1640培养基和EGM-2培养基,倒置显微镜观察两种培养条件下细胞的生长状态,同时利用流式细胞仪检测其生长周期,对比培养效果。结果 EGM-2组内皮细胞生长良好,2d后贴壁生长的细胞可达90%。EGM-2组S期细胞比例为（29.07±1.48）%,RPMI-1640培养基组S期细胞为（17.58±3.49）%,二者比较差异有统计学意义（P〈0.01）。结论 EGM-2培养基更适合人脐静脉内皮细胞的体外传代扩增培养。     

Propagation and assay of hepatitis A virus in vitro

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived) and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37°C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.     

Stromal fibroblasts of hematopoietic organs and antibody formation in culture

Stromal mechanocytes of thymic, bone-marrow, and splenic origin obtained from monolayer cultures at the 3rd–6th passage, if added to suspension cultures of rabbit spleen cells by the method of Mishell and Dutton, have a significant effect on the accumulation of antibody-forming cells (AFC) by the 4th day in culture. Their action clearly depends on dose. Stromal mechanocytes of bone marrow origin, in doses of 2.1×10³–6.25×10⁵, caused inhibition of AFC formation in culture. Stromal mechanocytes of thymic origin in doses of 2.75×10³–8×10⁵ caused an increase in the number of AFC, whereas mechanocytes of splenic origin in doses of 2.1×10³–1.3×10⁴ had no significant effect, and in doses of 8×10⁴–6.25×10⁵ inhibited AFC formation. Many of the living cells and AFC were concentrated in the fraction of nonadherent cells.Laboratory of Immunomorphology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 49–51, July, 1978.     

三种比色法评价4种牙科金属材料对L-929细胞的毒性

背景：采用不同方法评价材料的细胞毒性可能会得出不同的实验结果。 目的：采用3种比色法评价镍铬合金、钴铬合金、3铬13及纯钛等牙科金属材料对小鼠成纤维细胞(L-929细胞)的细胞毒性。 方法：以镍铬合金、钴铬合金、3铬13及纯钛4种牙科金属材料的浸提液分别作用于体外培养的L-929细胞24,72 h。以体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阴性对照组,以0.7%丙烯酰胺+体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阳性对照组,分别采用MTT、CCK-8及结晶紫3种比色法检测上述材料的细胞毒性。 结果与结论：①4种材料浸提液中培养的细胞形态正常,胞内结构清晰,随着培养时间延长细胞大量增殖,与阴性对照组细胞形态无明显差异。阳性对照组细胞数量明显减少,形态完整性受破坏,形成大量细胞碎片。②培养24 h时,CCK-8比色法检测中钴铬合金组的细胞相对增殖率低于阴性对照组(P < 0.05),MTT及结晶紫比色法检测中钴铬合金组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05);培养72 h时,MTT比色法检测中4种牙科金属材料组细胞相对增殖率低于阴性对照组(P < 0.01),CCK-8及结晶紫比色法检测中4组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05),但材料细胞毒性均为0-1级。表明上述4种牙科金属材料细胞毒性均在临床应用的允许范围内,具有良好的生物安全性。     

CIK细胞的体外培养与细胞毒作用

CIK(cytokine-induced killer)细胞在体外可以快速扩增,具有高效的非MHC限制性杀瘤活性,被认为是抗肿瘤过继细胞免疫治疗的新希望。随着细胞操作及基因修饰技术的日益精进,人们正逐渐改进CIK细胞的体外培养和处理方法,以便进一步提高CIK细胞的增殖率及特异杀伤力,使其更好地应用于临床。     

Micronucleus induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-irradiation and cyclophosphamide

A cytokinesis-block micronucleus (MN) method for the simultaneous but separate measurement of chromosome damage in erythroid and myeloid bone marrow cells is described. MN induction in cytokinesis-blocked mouse bone marrow cells in vitro following in vivo exposure to x-ray or cyclophosphamide (CP) was investigated. Immediately after whole body irradiation with acute doses of either 0, 1, 2 or 4 Gy x-rays, or 2 hr after treatment with either 0, 12.5, 25, or 50 mg CP/kg body weight, bone marrow cells were collected and then cultured in medium supplemented with 3.0 μ/ml cytochalasin B for 24 hr. The binucleated cells were scored in erythroid, myeloid, lymphoid and other cells. The myeloid/erythroid (M/E) ratio was decreased by x-irradiation or CP treatment in a dose-dependent manner. The dividing index (DI; binucleated cells/binucleated + mononucleated cells; %) was decreased in both eryth-roid and myeloid cells in the same manner. Dose-dependent increases in MN frequency were observed following x-irradiation in both erythroid and myeloid cells. A similar dose-dependent MN induction was observed with CP. The MN frequency in myeloid cells was much greater than in erythroid cells (about 4-fold following 4 Gy exposure, and more than 10-fold after 50 mg/kg CP). Lymphoid and other cells were not suitable for scoring DI and MN frequency because of insufficient numbers of binucleated cells. These results suggest that micronuclei can be identified in both myeloid and erythroid cells and that myeloid cells are more susceptible to x-ray or CP-induced chromosomal damage than erythroid cells as expressed by MN induction. © 1994 Wiley-Liss, Inc.     

Electrochemical study of Type 304 and 316L stainless steels in simulated body fluids and cell cultures

The electrochemical corrosion behaviour of Type 304 and 316L stainless steels was studied in Hanks’ solution, Eagle’s minimum essential medium (MEM), serum containing medium (MEM with 10% of fetal bovine serum) without cells, and serum containing medium with cells over a 1-week period. Polarization resistance measurements indicated that the stainless steels were resistant to Hanks’ and MEM solutions. Type 304 was more susceptible to pitting corrosion than Type 316L in Hanks’ and MEM solutions. The uniform corrosion resistance of stainless steels, determined by R_p, was lower in culturing medium than in Hanks’ and MEM. The low corrosion resistance was due to surface passive film with less protective to reveal high anodic dissolution rate. When cells were present, the initial corrosion resistance was low, but gradually increased after 3 days, consistent with the trend of cell coverage. The presence of cells was found to suppress the cathodic reaction, that is, oxygen reduction, and increase the uniform corrosion resistance as a consequence. On the other hand, both Type 304 and 316L stainless steels became more susceptible to pitting corrosion when they were covered with cells.     

小鼠卵母细胞体外成熟培养系统的建立与优化

目的 探讨激素刺激时间、体外培养时间对小鼠卵母细胞核成熟及不同激活方案对卵母细胞孤雌激活、胚胎发育能力的影响。方法 孕马血清促性腺激素（PMSG）超排处理,在体外培养的不同时间点检测卵母细胞核成熟率（每个处理至少重复3次,3只/重复,以下实验相同）。
分别采用乙醇结合6-二甲氨基嘌呤（6-DMAP）法和SrCl₂ 法激活卵母细胞,胚胎培养液选用CZB［胎牛血清（FBS）或牛血清清蛋白（BSA）］两种,确定最佳激活方案。对不同时间点成熟的卵母细胞进行激活,确定最佳激活卵龄。研究缩短PMSG刺激时间对卵母细胞发育的影响。结果 将
PMSG刺激时间从46h缩短至24h,卵母细胞获得最高核成熟率（97.6% vs 91.9%）的培养时间由14h延长至16h;缩短PMSG刺激时间,核成熟率不受影响,但能显著降低激活率（91.2% vs 37.1%）和囊胚率（20.9% vs 0.0%）。 两种方法体内成熟卵母细胞激活率均高于90%,但囊胚率差异
显著(P<0.05)。卵母细胞体外培养至24~26h时,激活率（89.5%）和囊胚率（21.9%）均达到最高点。结论 建立了一种小鼠卵母细胞体外成熟培养系统,即PMSG超排处理46h、卵母细胞培养24h,CZB（10mmol/L SrCl2）激活2.5h后采用CZB（0.5%BSA）进行胚胎培养。     

A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures

Summary A rapid and sensitive spectrophotometric assay for determining viability in monolayer cultured cell lines, with specific applications in normal and drug resistant cell line determinations, is described. The assay involves conversion of the tetrazolium salt MTT by viable proliferating cells to an insoluble product, purple formazan. The chief advantage of this assay is that it requires fewer cells than standard cytotoxicity assays. In addition, it allows for multiple sample concentrations on a single 96-well plate which is then rapidly quantitated using an automated spectrophotometric microplate reader.