用CHO细胞进行SCE检验方法探索

以姐妹染色单体交换(sister chromatid exchange,SCE)为指标,是继微核检测之后对各种化学物质的遗传毒性做进一步研究的有效方法。通过对化学物质进行SCE的数据统计,可以在一定程度上反映其致突变作用,是遗传毒性的敏感指标。试验中,应用CHO细胞株为主要材料,建立了以体外培养细胞SCE试验方法。在CHO细胞SCE试验中,在正式制片处理前3～4h加入秋水仙素;细胞通过65～70min低渗;第3次固定甲醇-冰乙酸比例为1：2;在加入5-溴脱氧尿嘧啶(BUda)后1～1．5个细胞周期后对细胞进行收获能取到最佳制片效果,得到良好的检测效果。     

mmc诱导绒毛细胞和淋巴细胞SCE的对比研究

用不同剂量的mmc分别诱导孕早期孕妇绒毛细炮和淋巴细胞SCE,发现除16ng对比组外,二 类细胞相同处理组间的mmc诱发的SCE无显著性差异。同时发现mmc剂量与二类细胞SCE频 率所形成的二条回归直线的斜率无显著性差异。这些结果表明在一定毒性剂量范围内,二类细胞SCE 对mmc的反应敏感性可能是相似的。对油胞PRI的分析结果提示,细胞PRI的改变会对其SCE 频率产生影响。     

餐洗剂对小鼠骨髓细胞SCE形成和Salmonella typhimurium基因回复突变的影响

本研究采用Salmonella typhimurium基因回复突变测试和小鼠骨髓细胞姐妹染色单体交换(SCE)分析,研究了四种餐洗剂及其主要成分LABS、Dispersol D、Alkanolamide的潜在诱变活性。结果表明四种牌号餐洗剂在高浓度时能诱发SCE形成,除TL牌餐洗剂加入S9代谢激活系统后未见诱变作用外,其他三种餐洗剂和三种主要成分都表现出致HisG46和HisD3052基因回复突变的能力。在相当于人日常使用量时,没观察出它们使SCE频率升高的现象,也不表现出致STY基因回复突变的能力。实验还观察了餐洗剂对小鼠骨髓细胞增殖的毒性效应,及其主要成分使细菌菌落异常生长的现象,并讨论了不同诱变效应之间可能存在的关系。     

石油化学工人的细胞遗传学研究——Ⅱ.姐妹染色单体交换(SCE)和细胞生长周期的研究

本文研究了180名某大型石油化工企业工人和180名对照组的SCE频率及其细胞Ⅰ、Ⅱ、Ⅲ周期的比率,结果表明,石化工人的SCE频率和细胞各周期比率与对照组无显著差异,即这两个地区由环境因子引起的这种细胞遗传效应差异不显著,表明它们有较相似的环境背景。但是,在石化企业中,两个污水处理车间工人的SCE频率明显地高于对照组和4个生产车间的工人,其细胞生长速率也明显地迟缓,揭示石化物质对人体有一定的细胞遗传效应,而且这种效应与人体所接触的石化物质浓度有关。     

职业性接触砷、铅工人外周淋巴细胞染色体畸变和姐妹染色单体交换率的变化

本实验以个旧云南锡业公司某矿坑下作业工人和某冶炼厂尿砷,尿铅超标工人为检查对象,初步探讨了污染与人类染色体损伤的关系,结果是使以上受检对象的外周淋巴细胞的染色单体断裂率、裂隙率、畸变细胞率和姐妹染色单体交换率都明显增高,与对照组相比差异显著。尿铅和尿砷超标工人外周淋巴细胞的染色体畸变和姐妹染色单体交换率分别与对照组相比,经统计学处理差异显著(P<0.05),且随着砷、铅在体内蓄积量的增加而有上升的趋势,表明砷、铅污染是引起人类染色体损伤的一个因素。     

Derivation of Induced Pluripotent Stem Cells from Human Peripheral Blood T Lymphocytes

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (“TiPS”) retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.     

Modulation of Apoptosis Induced by Tricyclic Antidepressants in Human Peripheral Lymphocytes

We have recently reported that the tricyclic antidepressants (TCAs) imipramine, clomipramine, and citalopram induce apoptosis in human peripheral lymphocytes. This system is well suited for studies on the pathophysiology/physiology of apoptosis regulation. Apoptosis was determined using both DNA gel electrophoresis and flow cytometric analysis. TCA-induced apoptosis in lymphocytes was monitored in the presence of the protein synthesis inhibitor cycloheximide (CHX), the RNA synthesis inhibitor actinomycin D (Act D), the antioxidant reduced glutathione (GSH), the nuclease inhibitor aurintricarboxylic acid (ATA), the cytokine interlukin-2 (IL-2), and the immunostimulator linomide. CHX and Act D failed to prevent and actually enhanced TCA-induced apoptosis in lymphocytes, indicating that protein and RNA syntheses are not required for this process. Exogenous IL-2, GSH, and ATA protected the lymphocytes from apoptosis induced by TCAs in a dose-dependent manner, whereas linomide had no effect on TCA-induced apoptosis under our in vitro conditions. Our data demonstrate that TCA-induced apoptosis in human lymphocytes shares many common features with other stimuli-induced apoptotic processes. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 115–123, 1998     

Transport of the Immunosuppressant 15-Deoxyspergualin in Human Peripheral Blood Lymphocytes

15-deoxyspergualin (DSG) is a potent immunosuppressive compound currently in clinical trials. In this study, we have characterized the uptake and intracellular localization of DSG in human peripheral blood lymphocytes (PBL′s). DSG is transported into human PBL′s and reaches an estimated maximum concentration of approximately 500μM in 6 hours. The majority of the [³H]-DSG remains in the cytoplasm of cells and that which is associated with the nucleus is only loosely associated. DSG was transported by HeLa cells, as well, suggesting uptake is not specific for hematopoietic cells. Positively charged amino acids and polyamines, which are structurally similar to DSG, were unable to compete for DSG transport suggesting that DSG is transported into cells via a pathway distinct from amino acids or polyamines.     

人外周血淋巴细胞体外诱导培养前后细胞表型与细胞毒活性相关性研究

为了分析体外细胞因子诱导培养CIK细胞过程中细胞表型的变化与其杀瘤活性的相关性及为临床过继免疫治疗提供实验依据,本研究采用体外诱导方法扩增培养正常人外周血淋巴细胞及单个核细胞,应用流式细胞术测定培养前、培养第7天和第14天的CD3~+等15种不同表型细胞百分率的变化,用CCK-8试剂检测第7天和第14天的细胞毒活性。结果显示,扩增培养后T细胞活化表型的表达和细胞毒活性在第7天最强,与其细胞表型CD3~+CD25~+、CD3~+CD28~+、CD3~+CD25~+CD28~+、CD3~+CD4~+呈正相关(P<0.05),与CD3~+CD45RA~+CD45RO~+呈负相关(P<0.05)。本研究表明测定培养细胞活化相关表型可以间接监测其杀瘤能力,为临床CIK细胞过继免疫治疗的应用提供实验依据。     

利用猪血清短期培养人体外周血淋巴细胞的研究

用猪血清代替小牛血清作人体外周血淋巴细胞短期培养,进行染色体分析及淋巴细胞转化的研究。在94例姐妹染色单体差别染色的培养中,培养液分别加入小牛血清与猎血清,细胞有丝分裂指数分别为3.91％和3.78％;21例常规法培养中,分裂指数分别为9.10％和9.21％;5例淋巴细胞转化试验,小牛血清和猪血清培养的转化率分别为69.52％和69.59％;16例阉割后公、母猪血清加入培养基中,细胞分裂指数(SCD法)分别为2.96％和3.14％。以上各项对照试验,均无显著性差异(P>0.05)本研究证实,猪血清可用于人体外周血淋巴细胞短期培养。     

人腺病毒55型感染肺部病变与外周血淋巴细胞关系研究

目的:探讨人腺病毒55型感染肺部病变与外周血淋巴细胞改变的关系及致病意义。方法:以50例经胸部CT证实为腺病毒肺炎的患者为研究对象(肺炎组),调查其外周血细胞及T淋巴细胞亚群变化情况,并与同期腺病毒55型感染未出现肺部病变的患者30例(非肺炎组)对照;亚组分析50例肺炎组中多肺叶病变与单肺叶病变患者之间T淋巴细胞变化的差异。结果:与非肺炎组患者相比,肺炎组患者急性期外周血淋巴细胞比例明显降低、单核细胞比例明显升高(P0.01);肺炎组CD3+T淋巴细胞比例、CD3+CD4+T淋巴细胞比例及CD4/CD8比值较非肺炎组均有明显降低(P0.01)。亚组分析显示,肺炎组患者肺部病变范围不同,T淋巴细胞亚群的变化亦有差异,多肺叶病变组患者CD3+CD8+T淋巴细胞比例较单肺叶病变组明显升高、CD4/CD8比值较单肺叶病变组明显降低(P0.05)。结论:HAdv-55感染引起肺炎病变时,宿主存在明显的细胞免疫功能受损,且与肺部病变程度有一定正相关。     

Variation in Chromatin Structure of the rDNA Transcribed Region in Human Peripheral Blood Lymphocytes

The rDNA transcribed region (TR) was tested for its accessibility for RsaI recognizing 15 TR sites, DNase I, and photoinducible arylazide (N-(4-azido-2-hydroxybenzoyl)-N,N"-diaminoheptane acetate) in isolated nuclei, and for the arylazide in intact cells. Arylazide readily entered the cells and did not appreciably affect the chromatin structure. Its photolysis products efficiently modified DNA in accessible sites. Single-strand breaks made by DNase I were not transformed into two-strand ones in rDNA TR, suggesting the necessity of denaturing electrophoresis for such an analysis. About 70% of all rDNA copies proved poorly accessible for endonucleases and arylazide, the accessibility being higher in their 18S and 5.8S rRNA gene regions than in the regions of the external transcribed spacers (ETSs) and the 28S rRNA gene. Proteinase K disrupted this structure, and the corresponding copies were extracted from nuclei. This explained whyin situ hybridization occasionally fails to reveal rDNA in the nucleolar fibrillar center (FC) on electron microscopic preparations. In other rDNA copies, TR (excluding 5"-ETS) was accessible for nucleases and arylazide. These copies were not extracted from nuclei treated with proteinase K. Some of their RsaI sites were protected by tightly bound proteins. Seven such regions were identified in TR. Possible association of the molecular structure, nucleolar location, and functional state of rDNA is discussed.     

Proteome Dynamics in Biobanked Horse Peripheral Blood Derived Lymphocytes (PBL) with Induced Autoimmune Uveitis

Equine recurrent uveitis is the only spontaneous model for recurrent autoimmune uveitis in humans, where T cells target retinal proteins. Differences between normal and autoaggressive lymphocytes were identified in this study by analyzing peripheral blood derived lymphocytes (PBL) proteomes from the same case with interphotoreceptor retinoid binding protein induced uveitis sampled before (Day 0), during (Day 15), and after uveitic attack (Day 23). Relative protein abundances of PBL were investigated in a quantitative, label‐free differential proteome analysis in cells that were kept frozen for 14 years since the initial experiment. Quantitative data could be acquired for 2632 proteins at all three time points. Profound changes (≥2‐fold change) in PBL protein abundance were observed when comparing Day 0 with 15, representing acute inflammation (1070 regulated proteins) and Day 0 with 23 (cessation; 1571 regulated). Significant differences applied to proteins with functions in integrin signaling during active uveitis, involving “Erk and pi‐3 kinase are necessary for collagen binding in corneal epithelia,” “integrins in angiogenesis,” and “integrin‐linked kinase signaling” pathways. In contrast, at cessation of uveitic attack, significantly changed proteins belonged to pathways of “nongenotropic androgen signaling,” “classical complement pathway,” and “Amb2 integrin signaling.” Several members of respective pathways were earlier shown to be changed in naturally occurring uveitis, underscoring the significance of these findings here and proofing the value of the induced model in mimicking spontaneous autoimmune uveitis. All MS data have been deposited to the ProteomeXchange consortium via the PRIDE partner repository (dataset identifier PXD005580).