卷烟烟气致人淋巴细胞细胞毒性和遗传毒性的体外试验

目的 用不同体外试验评价卷烟烟气粒相物提取液(CSCs)致人外周血淋巴细胞的细胞毒性和遗传毒性.方法 分别以25、50、75、100和125 ug/ml的CSCs在加或不加肝脏微粒体酶(S9)系统下作用于人外周血淋巴细胞3 h,然后用CCK-8试验检测细胞毒性,用彗星试验检测DNA损伤,用hprt和TCR基因突变试验检测体细胞突变;以75ug/ml的CSCs作用于人外周血淋巴细胞3 h,分别给予30、60、90、120和240 min的修复时间,然后用彗星试验评价淋巴细胞的DNA修复情况.结果 细胞活性随剂量增加明显降低,100、125 ug/ml CSCs加S9组细胞活性明显高于不加S9组,差异有统计学意义(P<0.05,P<0.01);随CSCs暴露剂量增加,DNA损伤明显增加,并明显高于对照组,差异有统计学意义(P<0.01);各剂量加S9组DNA损伤明显低于不加S9组,差异有统计学意义(P<0.05,P<0.01).各剂量组TCR基因突变率明显高于对照组,差异有统计学意义(P<0.05,P<0.01).中高剂量组hprt基因突变率明显高于对照,差异有统计学意义(P<0.01),中、高剂量加S9与不加S9两组间TCR和hprt基因突变率均存在明显差异,差异有统计学意义(P<0.05,P<0.01).加与不加S9两组DNA损伤均可基本修复至正常水平,但两者修复速度存在差异.结论 CSCs可在体外诱发人外周血淋巴细胞的细胞和遗传损伤,但S9可降低CSCs毒性的效应,并可影响人淋巴细胞DNA损伤的修复速率.     

部分照射人离体血对淋巴细胞微核率的影响

目的 探讨60Co γ射线部分照射人离体血对淋巴细胞微核率的影响.方法 用2Cy 60Co γ射线37℃照射人离体周围血,以不同比例与未照射血混合后进行培养,44 h后加松胞素B,72 h收获,制片并分析微核率.结果 微核率随照射血比例的增加而增加,与单纯照射组相比,混合照射血的微核率高.1.0:1.0比例混合估算出的剂量为1.29 Gy,大于实际的1 Gy,0.5:1.0比例混合估算出的剂量为0.91 Gy,也大于0.50 Cy,而在1.0:0组,照射剂量与估算剂量基本一致.结论 微核率随照射血比例的不断增加而增加,为微核率作为估算局部照射剂量的生物学指标提供资料.     

人外周血淋巴细胞体外乙酸铅染毒后氧化应激损伤研究

目的 研究乙酸铅染毒人外周血淋巴细胞致氧化应激与DNA氧化损伤情况。
方法 分别用浓度为0 μmol/L、20 μmol/L、40 μmol/L和80 μmol/L的乙酸铅染毒人外周血淋巴细胞6 h、12 h、24 h, 应用2', 7'-二氯二氢荧光素二乙酸酯(DCFH-DA)染色分析和流式细胞仪检测染毒后细胞内活性氧类(ROS)水平, 高度水溶性四唑盐(WST-1)法检测染毒后细胞内总超氧化物歧化酶(T-SOD)活性, 酶联免疫吸附测定法(ELISA)试剂盒检测染毒后细胞8-羟基脱氧鸟苷(8-OHdG)水平, 并对ROS、T-SOD、8-OHdG三指标间的关系进行相关性分析。
结果 乙酸铅染毒6 h后, 各染毒组人外周血淋巴细胞ROS水平均高于对照组, 差异有统计学意义(P < 0.01);20 μmol/L染毒组人外周血淋巴细胞T-SOD和8-OHdG水平与对照组相比, 差异无统计学意义(P>0.05), 而40 μmol/L和80 μmol/L染毒组人外周血淋巴细胞T-SOD和8-OHdG水平均高于对照组, 差异有统计学意义(P < 0.01)。乙酸铅染毒12 h和24 h后, 各染毒组人外周血淋巴细胞ROS、T-SOD和8-OHdG水平均高于对照组, 差异有统计学意义(P < 0.01)。相关性分析显示, 细胞内ROS含量与T-SOD活性呈负相关(r_{6 h}=-0.865、r_{12 h}=-0.890、r_{24 h}=-0.801, P < 0.01), 与8-OHdG含量呈正相关(r_{6 h}=0.840、r_{12 h}=0.829、r_{24 h}=0.866, P < 0.01)。
结论 乙酸铅染毒人外周血淋巴细胞后会诱导细胞ROS生成, 抑制细胞内抗氧化酶SOD活性, 造成人外周血淋巴细胞氧化应激状态增强和DNA氧化性损伤。
     

杂色曲霉素对人外周血单个核细胞表面HLA-Ⅰ分子表达的影响

目的探讨杂色曲霉素(ST)对体外培养的人外周血单个核细胞表面(HPBMc)HLAⅠ分子表达的影响。方法采用流式细胞定量术(FCM)和免疫印迹(Westernblot)分析方法,研究不同浓度ST(0.125、0.25、0.5、1和2mgL)处理后人外周血单个核细胞表面HLAⅠ分子表达的变化。结果FCM定量分析结果表明,经不同浓度ST处理24小时后,与对照组相比,各组细胞HLAⅠ的平均荧光强度均降低,以较高浓度(0.5、1和2mgL)ST处理组降低更明显(P<0.05)。在0.125mgL到2mgL的浓度范围内,随ST处理浓度的升高,HLAⅠ荧光指数逐渐降低,两者呈明显的负相关(r=-0.841,P<0.01)。免疫印迹结果也表明,随ST浓度的增高,HLAⅠ分子降低越明显。结论提示在0.125mgL到2mgL的浓度范围内,ST抑制人外周血单个核细胞表面HLAⅠ分子的表达呈现出负的剂量-反应关系。     

杂色曲霉素对体外培养人外周血白细胞介素Ⅱ分泌影响的研究

杂色曲霉素是我国肿瘤高发区粮食中的优势污染霉菌毒素。为探讨杂色曲霉素 (ST)对人体免疫机能的影响 ,采用双抗体夹心ELISA方法对ST作用后人外周血单核细胞 (HPBMc)培养上清液中白血细胞介素Ⅱ (IL 2 )的分泌水平进行了检测。结果表明 ,不同浓度 ( 0 0 3 12 5～ 2mg L)ST处理 2 4h后 ,体外培养的HPB Mc的IL 2分泌均受到一定程度的抑制 ,其中以较低浓度ST( 0 0 3 12 5～ 0 12 5mg L)和较高浓度ST( 1～ 2mg L)抑制作用最明显 (P <0 0 5 )。在ST 1mg L作用 1～ 64h的时间范围内 ,ST对HPBMcIL 2的分泌总体表现抑制作用。ST处理后 8～ 64h ,随ST处理时间的延长 ,对IL 2分泌的抑制作用逐渐增强 (r =0 82 2 ,P <0 0 5 )。本研究结果提示 ,ST对HPBMcIL 2的分泌有一定的抑制作用     

Distribution of tocopherols in human plasma and red blood cells.

The content of various tocopherols was determined in normal plasma and red blood cells. alpha-Tocopherol concentrations ranged form 6.6 to 15.0 (mean 9.6) mug/ml in plasma and from 0.9 to 1.8 (mean 1.4) mug/ml in red blood cells. gamma-Tocopherol ranged from 0.7 to 2.7 (mean 1.6) mug/ml in plasma and fron 0.1 to 0.4 (mean 0.24) mug/ml in red blood cells. Only a minute amount (smaller than 0.3 mug/ml) of each of alpha-tocotrienol, beta-tocopherol, gamma-tocotrienol and delta-tocopherol was found in plasma but none in red blood cells. No delta-tochotrienol was detected in either plasma or red blood cells. Of the total tocopherols, alpha form accounted 83 per cent in plasma and 87 per cent in red blood cells and gamma from represented 13 per cent in each system. The recovery of added 14-C-alpha-tocopherol averaged 87 per cent and 69 per cent, respectively, for plasma and red blood cells. All alpha-tocopherol in the red blood cells was found to be localized in the membrane fraction.     

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Background

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes.

Aim

The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner.

Methods

In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1–10 μL/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1–10 μM). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes.

Results

In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 μL/mL WCE, 3.7-fold 1 μM PEITC) and SOD2 (12.1-fold, 10 μL/mL WCE, 7.3-fold, 10 μM PEITC), and increased SOD2 activity (1.9-fold, 10 μL/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact.

Conclusion

The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.     

Induction of metallothionein synthesis in human peripheral blood leukocytes

Metallothionein (MT), a low molecular weight, metal-binding protein, has recently been shown to protect murine mononuclear phagocytic cells from the cytotoxic effects of bacterial lipopolysaccharides (LPS), the endotoxic component of Enterobacteriaceae. MT appears to function intracellularly as an antioxidant since autolysis results from lipid peroxidation initiated by free radicals of O2. Since this activity is distinct from MT's capacity to specifically sequestrate heavy metals, we examined whether MT synthesis can be induced by direct membrane activation or through interaction with soluble leukocyte mediators. Normal human monocytes, polymorphonuclear neutrophils (PMN), and lymphocytes, isolated from heparinized whole blood, were incubated with and without LPS from Escherichia coli and Salmonella typhosa. MT in cell lysates was quantitated using a 203Hg-binding assay employing Sephadex G-10 "minicolumns." When incubated with monocytes, PMN, or lymphocytes, neither preparation of LPS (10-100 micrograms/ml) was capable of enhancing 203Hg-binding activity after 24 or 72 hr incubation. CdCl2 (2 micrograms/ml), however, increased binding activity in monocyte and lymphocyte cultures 4- and 15-fold, respectively. When monocytes and lymphocytes were cocultured with LPS, 203Hg-binding activity was not enhanced. Addition of human interleukin 1 (endogenous pyrogen) to these cultures had no significant effect. Leukocyte endogenous mediator (LEM), a product of LPS-activated PMN that possesses hypozincemic activity in vivo, did not induce MT synthesis. Collectively, these results demonstrate that leukocyte MT does not arise from direct LPS activation or from interaction with products secreted by LPS-activated cells. De novo synthesis of MT observed during endotoxemia and gram negative sepsis appears, therefore, to be induced by endogenously released corticosteroids.     

Cytogenetic observations in human peripheral blood leukocytes following in vitro exposure to THz radiation: a pilot study

Emerging technologies are considering the possible use of Terahertz radiation in different fields ranging from telecommunications to biology and biomedicine. The study of the potential effects of Terahertz radiation on biological systems is therefore an important issue in order to safely develop a variety of applications. This paper describes a pilot study devoted to determine if Terahertz radiation could induce genotoxic effects in human peripheral blood leukocytes. For this purpose, human whole blood samples from healthy donors were exposed for 20 min to Terahertz radiation. Since, to our knowledge, this is the first study devoted to the evaluation of possible genotoxic effects of such radiation, different electromagnetic conditions were considered. In particular, the frequencies of 120 and 130 GHz were chosen: the first one was tested at a specific absorption rate (SAR) of 0.4 mW g-1, while the second one was tested at SAR levels of 0.24, 1.4, and 2 mW g-1. Chromosomal damage was evaluated by means of the cytokinesis block micronucleus technique, which also gives information on cell cycle kinetics. Moreover, human whole blood samples exposed to 130 GHz at SAR levels of 1.4 and 2 mW g-1 were also tested for primary DNA damage by applying the alkaline comet assay immediately after exposure. The results obtained indicate that THz exposure, in the explored electromagnetic conditions, is not able to induce either genotoxicity or alteration of cell cycle kinetics in human blood cells from healthy subjects.     

脱氧雪腐镰刀菌烯醇对体外培养人外周血单核细胞增殖及肿瘤坏死因子-α分泌的影响

为探讨我国恶性肿瘤高发区居民饮食中的优势污染霉菌毒素 -脱氧雪腐镰刀菌烯醇 (DON)对人体免疫机能的影响 ,分别以流式细胞光度术 (FCM)、细胞计数、甲基噻唑基四唑 (MTT)比色、双抗体夹心EL ISA方法对 DON作用后人外周血单核细胞 (HPBM)增殖和肿瘤坏死因子 -α(TNF-α)分泌情况进行了研究。FCM检测结果表明 ,HPBM经 PHA预刺激 48h后 ,低浓度 DON(5 0～ 5 0 0 ng/ ml)处理 6 h的细胞增殖指数 (PI)较对照组明显降低 (19.80 %～ 2 6 .0 1%∶ 37.84% ) ;而高浓度 DON (10 0 0～ 2 0 0 0 ng/ ml)处理 2 4h的细胞 PI值则显著高于对照组 (35 .74%～ 34.37%∶ 2 2 .85 % )。细胞计数和 MTT比色法结果显示 ,在 PHA存在的情况下 ,DON可抑制 HPBM的增殖。双抗体夹心 EL ISA法检测结果显示 ,5 0 0和 10 0 0 ng/ ml的 DON可明显降低 HPBM TNF- α分泌 (P<0 .0 1)。研究结果表明 ,根据作用时间和作用浓度不同及有无 PHA刺激 ,DON对 HPBM增殖的影响有所不同 ,但主要表现为对细胞增殖的抑制作用 ,并抑制 TNF- α的分泌 ,从而对机体免疫机能造成负面影响     

脱氧雪腐镰刀菌烯醇对体外培养人外周血单核细胞增殖及肿瘤坏死 …

为探讨我国恶性肿瘤高发区居民饮食中的优势污染霉菌毒素－脱氧雪腐镰刀菌烯醇（ＤＯＮ）对人体免疫机能的影响，分别以流式细胞光度术（ＦＣＭ）、细胞计数、甲基噻唑基四唑（ＭＴＴ）比色、双抗体夹心ＥＬＩＳＡ方法对ＤＯＮ作用后人外周血单核细胞（ＨＰＢＭ）增殖和肿瘤坏死因子－α（ＴＮＦ－α）分泌情况进行了研究。ＦＣＭ检测结果表明，ＨＰＢＭ经ＰＨＡ预刺激４８ｈ后，低浓度ＤＯＮ（５０￣５００ｎｇ／ｍｌ）处理６ｈ的细     

职业接触铅工人血细胞参数分析

目的　了解职业接触铅对工人造血系统的影响以及血细胞参数的变化。方法　以 47名职业接触铅工人为接触组 ,以 15 0名非铅接触工人为对照组 ,将接触组与对照组的血细胞参数进行比对。尿铅测定采用双硫腙比色法 ;血细胞参数测定采用全自动血细胞分析仪。结果　接触组的血红蛋白浓度 (Hb)、血细胞比容 (Hct)、平均红细胞容积 (MCV)、平均红细胞血红蛋白量 (MCH)、平均红细胞血红蛋白浓度 (MCHC)、红细胞体积分布宽度 (RDW )、血小板平均容积 (MPV ,男 )、血小板比容 (Pct,男 )与对照组相比均有差异 ,(P <0 .0 5 ) ;两组的红细胞计数 (RBC)、血小板计数 (PC)、MPV(女 )、Pct(女 )、血小板分布宽度 (PDW )的差异无统计学意义 (P >0 .0 5 )。尿铅与接触铅工龄及各项血细胞参数的相关性分析 :尿铅与男性工人的Hb、Hct有弱的负相关关系 ,与RDW有弱的正相关关系 ;尿铅与女性工人的MPV有弱的正相关关系。贫血 :接触组贫血 10人( 10 /4 7) ,对照组贫血 1人 ( 1/15 0 ) ,差异有统计学意义 (P <0 .0 1)。结论　职业接触铅工人的多项血细胞参数发生改变 ,有贫血趋向。     

203例输卵管积液不孕患者病原体分析

目的 了解输卵管积液不孕的病原体感染状况,寻找该病的主要致病菌,为该疾病的临床合理治疗提供研究依据.方法 对203例输卵管积液不孕患者采集宫颈分泌物和输卵管积液标本,对标本进行病原体培养鉴定与药敏试验检测,将宫颈分泌物和输卵管积液的病原体培养与药敏试验结果进行比较分析.结果 203例输卵管积液不孕患者的宫颈分泌物和输卵管积液中培养出病原体628株,其中支原体属、衣原体属331株占52.71％,以沙眼衣原体为主,占27.39％,革兰阳性菌155株占24.68％,以金黄色葡萄球菌为主,占11.31％,革兰阴性菌125株占19.90％,以大肠埃希菌为主,占10.83％,真菌17株占2.71％,以白色假丝酵母菌为主,占2.39％;203例输卵管积液不孕患者的宫颈分泌物中培养出的沙眼衣原体、解脲脲支原体、二者混合感染的阳性率分别为43.84％、39.41％、13.79％,输卵管积液分别为40.89％、38.92％、12.81％,二者比较差异均无统计学意义.结论 临床治疗输卵管积液不孕时,应根据病原体检测结果合理使用抗菌药物;在制定治疗方案时应着重考虑沙眼衣原体、解脲脲支原体感染的治疗,还要重视对输卵管、宫颈及其他受感染部位的协同治疗.     

维生素C对脱氧雪腐镰刀菌烯醇诱导的单个核细胞凋亡及增殖抑制作用的影响

目的探讨维生素 C(vitamin C,VC)预处理对脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)诱导的人外周血单个核细胞(human peripheral blood mononuclear cells,HPBMC)凋亡及其相关基因表达和增殖抑制作用的影响。方法采用流式细胞术(flow cytometry,FCM)和蛋白印迹(Western blotting)方法研究不同剂量 VC 预处理后,DON 对 HPBMC 凋亡及相关基因 Bcl-2、Bax、Caspase-3蛋白表达影响的变化,同时观察 VC 预处理对 DON 引起的增殖抑制作用的影响。结果FCM 检测结果表明,终浓度为2000μg/L 的 DON 可诱导体外培养的 HPBMC 凋亡,其凋亡率为(28.82±1.67)%,25μmol/L VC 预处理可明显抑制 DON 诱导的体外培养 HPBMC 凋亡(22.39±1.05)%,P<0.05,而100μmol/L VC 预处理则可明显增高 DON 诱导的 HPBMC 凋亡(36.07±2.92)%,P<0.05。Western blotting 分析结果表明,25μmol/L VC 预处理可明显降低 DON 诱导的HPBMC Bax 和 caspase-3蛋白高表达,同时使 DON 抑制的 Bcl-2蛋白表达明显升高。100μmol/L VC预处理可明显促进 DON 诱导的 Bax 和 Caspase-3蛋白的高表达,但对 DON 抑制的 Bcl-2表达无明显影响。不同剂量 VC(25μmol/L 和100μmol/L)预处理均可降低 DON 对 HPBMC 增殖的抑制作用(P<0.05),但不同剂量 VC 之间没有显著差异。结论 25μmol/L VC 预处理可一定程度地抑制DON 诱导的 HPBMC 亡及凋亡相关基因的异常表达,而100μmol/L VC 则促进 DON 诱导的HPBMC 凋亡。不同剂量 VC 预处理可以明显降低 DON 对 HPBMC 的增殖抑制作用。     

Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests

A combined approach employing comet assay and micronucleus (MN) and sister chromatid exchanges (SCE) tests was utilized to assess the genotoxicity of two pesticides, imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitro-imidazolidin-2-ylideneamine] and RH-5849 [2'-benzoyl-1'-tert-butylbenzoylhydrazine], on human peripheral blood lymphocytes in vitro. No significant difference in the frequencies of MN and SCE from the negative groups (P>0.05) was observed at low dose levels (i.e., 0.05 mg/L for imidacloprid and 5mg/L for RH-5849). As the concentrations of imidacloprid and RH-5849 were increased to 0.1 and 25 mg/L, respectively, significant effects to the frequencies of MN and SCE (P<0.05) were achieved relative to those of the negative controls. MN and SCE frequencies increased similarly in a dose-related manner with both pesticides. With the comet assay, however, the distribution of DNA damage grades in all the pesticide-treated groups was significantly different from those in the control (P<0.01). DNA damage scores increased with the exposure levels of both pesticides, and linear dose-effect relationships were observed for both imidacloprid (r2=0.98) and RH-5849 (r2=0.92). The cytogenetic techniques and comet assay revealed potential adverse effects of both imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro. Combination of the comet assay and cytogenetic tests appears commendable to assess the potential risks of human exposure to the pesticides.     

Rabies-specific production of interleukin-2 by peripheral blood lymphocytes from human rabies vaccinees.

Cell-mediated immunity induced by rabies vaccination was studied in humans by the determination of specific interleukin-2 (IL-2) production in a large number of donors (postexposure immunized patients and pre-exposure immunized laboratory workers). Peripheral blood lymphocytes (PBL) from 35 donors were tested for IL-2 production after in vitro stimulation by different rabies and rabies-related viruses. IL-2 responses were compared to antibody recognition of these different virus serotypes by sera from the same individuals. IL-2 was produced by PBL from more than 85% of donors after stimulation with inactivated and purified rabies viruses (IPRV) prepared from either Pittman Moore (PM) or Pasteur Virus (PV) strains. IL-2 was also produced by 65 and 45% of donor PBL stimulated with IPRV from the European Bat Lyssavirus (EBL) and Mokola (Mok) rabies-related virus strains respectively. No correlation was found between the production of IL-2 by PBL and the levels of virus neutralizing antibody (VNAb). Moreover, 50, 25 and 35% of donors produced IL-2 after stimulation of their PBL with ribonucleoprotein (RNP) from PV-, EBL- and Mok-viruses, respectively. These results obtained with a large number of human rabies vaccinees and using an assay specific to T-cell activation confirm the significant cross-reactivity of T-cell responses directed against rabies and rabies-related viruses. This study shows that IL-2 production could be used for the study of cell-mediated immunity and T-cell memory induced in humans by rabies vaccination.