基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法的建立

目的：建立基于流式细胞术的生物标记物γ-H2AX自动化检测方法,并探讨此方法用于药物早期遗传毒性筛选和遗传毒性评价的前景。方法：试验分为不添加体外代谢活化系统（-S9）长时（24 h）处理组和添加体外代谢活化系统（+S9）短时（4 h）处理组,分别采用CCK-8方法选择4个不同浓度的依托泊苷（ETO）和环磷酰胺（CP）处理人成淋巴TK6细胞,24 h后收获细胞,采用连接荧光分子的γ-H2AX抗体和核酸染料SYTOX Green双色标记,使用高通量流式细胞仪分析组蛋白H2AX磷酸化（γ-H2AX）阳性的细胞比例。结果：与溶剂对照组比较,-S9 24 h处理组,不同浓度的依托泊苷诱导产生的γ-H2AX阳性细胞比例显著增加,量效关系明显（r=0.999,P < 0.05）;+S9 4 h处理组,不同浓度的环磷酰胺诱导产生的γ-H2AX阳性细胞比例亦显著增加,量效关系明显（r=0.988,P < 0.05）。结论：流式细胞仪可快速、准确地分析体外培养的TK6细胞中γ-H2AX的变化,本试验初步建立了基于流式细胞术的组蛋白γ-H2AX磷酸化检测方法。     

利用中国仓鼠肺细胞体外微核 试验评价三七提取液的遗传毒性

目的：评价传统中药三七提取液的遗传毒性。方法：采用体外胞质分裂阻断微核法检测三七提取液在加与不加代谢活化系统S9的两种条件下对中国仓鼠肺细胞微核率的影响,三七提取液的制备采用乙醇反复浸提法。根据中性红摄取法细胞毒性测定试验的结果,微核试验设3个三七染毒浓度组（0.625、1.250、2.500 mg/mL）、阳性对照组（+S9条件下为20 μg/mL环磷酰胺,-S9条件下为1 μg/mL丝裂霉素C）和阴性对照组（MEM培养基）。结果：三七提取液2.500 mg/mL浓度组,中性红摄取法细胞毒性测定试验中,吸光度值较阴性对照组显著降低（P < 0.05）,细胞存活率为80.3%;三七提取液0.625、1.250、2.500 mg/mL浓度组CHL细胞的微核率,在+S9条件下分别为17‰、16‰、16‰,在-S9条件下分别为17‰、15‰、16‰。与阴性对照组（+S9 16‰、-S9 15‰）比较,差异均无统计学意义（P > 0.05）。结论：在本研究条件下,体外微核试验结果表明,三七提取液不具有遗传毒性。     

甲基丙烯酸环氧丙酯的遗传毒性评价

目的：观察甲基丙烯酸环氧丙酯（GMA）诱发体外培养的中国仓鼠肺细胞（V79）微核发生变化情况,对其遗传毒性进行评价。方法：分别采用不同浓度（2.25、4.5、9.0、18.0、36.0 μg/mL）的GMA染毒V79细胞,设置空白对照组和DMSO溶剂对照组,其中染毒3 h处理组分别在加和不加体外活化系统（S9）条件下进行,染毒24 h处理组在不加S9条件下进行。分别计算细胞复制指数和微核细胞率。结果：GMA的浓度在2.25~36.0 μg/mL范围内,无S9的条件下,与空白对照组和DMSO溶剂对照组相比,GMA染毒3和24 h组的V79细胞复制指数均无明显变化,但微核细胞率明显升高,并呈浓度-效应关系;在有S9的条件下染毒3 h,各剂量组的GMA诱发微核细胞率的差异均无统计学意义。结论：在2.25~36.0 μg/mL浓度范围,GMA可致V79细胞的微核率升高,表明GMA可诱发遗传物质损伤,具有遗传毒性。     

多遗传学终点的遗传毒性试验组合的建立

目的：建立Ames波动试验、中国仓鼠肺成纤维（CHL）细胞体外微核试验和小鼠淋巴瘤细胞基因突变试验（MLA）方法,探讨其作为一套新的遗传毒性试验组合检测化合物诱变性的可能性。方法：在Ames波动试验中,用TA100沙门氏菌进行96孔板的Ames波动试验,非活化条件下以叠氮钠（SA）、活化条件下以环磷酰胺（CP）染毒处理,计数阳性孔数并进行卡方检验。在CHL细胞体外微核试验中,非活化条件下用丝裂霉素C （MMC）分别染毒处理4和24 h,活化条件下用CP染毒4 h,计数2000个细胞中含微核的细胞数,并进行卡方检验。在MLA中,小鼠淋巴瘤细胞经清除自发突变后,非活化条件下用MMC、活化条件下用CP分别染毒处理3 h,计算相对存活率（RS）、相对悬浮增长率（RSG）、相对总增长率（RTG）、平板效率PE0、PE2、突变率（MF）、小集落比例（SC）。2结果：Ames波动试验中SA和CP在各自试验条件下阳性孔数均较对照组显著增加（χ^2＞6.63,P＜0.01）,量效关系明显。CHL细胞体2外微核试验中,MMC和CP在各自试验条件下均引起微核率显著升高（χ^2＞6.63,P＜0.01）,量效关系明显。MLA中MMC和CP在各自＋/-试验条件下均引起L5178Y 3.7.2C-tk^＋/- 细胞的PE、RS、RSG和RTG呈剂量依赖性下降,MF呈剂量依赖性升高,高出溶剂对照的2倍以上。结论：此3种短期遗传毒性试验方法可互为补充、相互验证,其组合应用可提高检出化合物遗传毒性的准确性,有望得到进一步推广应用。     

草苔虫内酯的遗传毒性评价

目的：检测草苔虫内酯的遗传毒性。方法：采用Ames试验、体外培养中国仓鼠卵巢（chinese hamsterovary,crto）细胞染色体畸变试验和小鼠骨髓微核试验检测草苔虫内酯的遗传毒性。结果：Ames试验显示在每皿100、10、1、0．1g受试剂量下,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102及TAl535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示,在3．75、1．88和0．94g／ml 3个剂量组,在加S9条件下培养24h和不加S9培养24、48h的CHO细胞染色体畸变率与溶剂对照组相比差异有统计学意义（P〈0．05）。小鼠骨髓微核试验,在12．5、25、50g／kg3个剂量下对ICR小鼠的微核诱发率呈剂量反应关系,与溶剂对照组相比差异有统计学意义（P〈0．01）。结论：在本实验条件下,草苔虫内酯对鼠伤寒沙门氏菌无致突变性,对哺乳动物培养细胞染色体的致畸变作用为可疑阳性,对ICR／b鼠有诱发骨髓嗜多染红细胞微核的效应,提示草苔虫内酯对人体具有潜在的遗传毒性。     

胎盘免疫调节多肽遗传毒性和抗突变作用的研究

本语文应用体外培养人淋巴细胞并进行微核、染以体畸变的检测以及小鼬有髓微核实验的研究，评价胎盘免疫调节肽遗传毒性和抗突变效应，实验结果表明，胎盘免疫肽可显著抑制培养人淋巴细胞的自发和γ射线诱发的微核形成以及丝裂霉素Ｃ（ＭＭＣ）诱发的染色体畸变，并能明显抑制环磷酰胺诱发的小鼠骨髓多染性红细胞微核的增加，揭示了胎盘免疫调节肽具有抗突变作用。     

外周血微核试验检测可降解生物材料补片的遗传毒性

目的：用流式细胞术检测小鼠外周血中的网织红细胞微核(MN-RET)与成熟红细胞微核(MN-NCE),探究可降解生物材料补片的潜在遗传毒性风险。方法：实验设短周期和长周期给予受试物两种方案,小鼠分别腹腔注射介质对照液、样品试验液、环磷酰胺溶液(25、50 mg/kg),每日给药1次,短周期连续给予受试物3 d后采集外周血,流式细胞术检测外周血中的微核;长周期连续给予受试物14 d后采集外周血并摘取脾脏,流式细胞术检测外周血中的微核并分析脾脏细胞的凋亡情况,同时用HE对脾脏染色后进行组织病理学检查。结果：不同周期条件下样品试验组小鼠外周血中MN-RET百分率和MN-NCE百分率相较于介质对照组差异均无统计学意义(P>0.05);长周期给予样品试验组小鼠脾脏细胞凋亡率相较于介质对照组差异不显著(P>0.05),脾脏组织病理学检查无明显病理性改变。而长周期给予受试物后,与介质对照组相比,25和50 mg/kg环磷酰胺组晚期凋亡细胞率均显著升高(P<0.05或P<0.01),且50 mg/kg环磷酰胺组小鼠脾脏出现明显的白髓萎缩、淋巴细胞减少,红髓扩张、髓外造血增加等病理...     

体外双核细胞微核试验的应用研究

本实验用双核细胞微核试验(简称CB徽核试验)检测不同类型诱变剂对体外培养细胞微核率的影响。实验筛选了9株体外培养细胞,发现其中以BALB/c-3T3和CHL细胞的实验效果较好。丝裂霉素、硫酸镍、苯并[a]芘和香烟烟雾凝聚物等不同类型的诱变剂能直接或间接诱使细胞微核的形成。实验显示BALB/c—3T3细胞有部分代谢活化苯并[a]芘能力,CHL细胞则可能缺乏这种活性。实验还表明CB微核试验的稳定性和灵敏度都高于普通微核法,适用于检测多种类型的环境诱变剂的遗传损伤作用。     

Evaluation of genetic toxicity of bryostatin

目的:检测草苔虫内酯的遗传毒性.方法:采用Ames试验、体外培养中国仓鼠卵巢(chinese hamster ovary,CHO)细胞染色体畸变试验和小鼠骨髓微核试验检测草苔虫内酯的遗传毒性.结果:Ames试验显示在每皿100、10、1、0.1g受试剂量下,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102及TA1535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近.体外培养CHO细胞染色体畸变试验结果显示,在3.75、1.88和0.94 g/ml 3个剂量组,在加S9条件下培养24 h和不加S9培养24、48 h的CHO细胞染色体畸变率与溶剂对照组相比差异有统计学意义(P＜0.05).小鼠骨髓微核试验,在12.5、25、50 g/kg 3个剂量下对ICR小鼠的微核诱发率呈剂量反应关系,与溶剂对照组相比差异有统计学意义(P＜0.01).结论:在本实验条件下,草苔虫内酯对鼠伤寒沙门氏菌无致突变性,对哺乳动物培养细胞染色体的致畸变作用为可疑阳性,对ICR小鼠有诱发骨髓嗜多染红细胞微核的效应,提示草苔虫内酯对人体具有潜在的遗传毒性.     

乙双吗啉诱发人淋巴细胞染色体畸变与微核形成的研究

为了评价乙双吗啉的遗传毒性与潜在的致癌作用,应用终浓度为2.5～30μg/ml的乙双吗啉,体外处理培养人体外周血淋巴细胞,观察到中期染色体/染色单体的各类畸变和间期细胞的微核形成,其畸变细胞率与微核细胞率均呈剂量依赖性增加,可用线性回归描述;用上述浓度的乙双吗啉体外处理人外周血,放置18小时后直接制备富集淋巴细胞微核片,发现乙双吗啉可诱发G_0期淋巴细胞的微核形成,其微核细胞率亦呈剂量依赖性增加,同样可用线性回归描述。本实验三种短期测试法所检测的乙双吗啉最低浓度均为2.5μg/ml。这些实验结果一致地表明,乙双吗啉具有显著的遗传毒性,并可能具有潜在的致癌性,建议慎用于临床非肿瘤疾病的治疗。     

83—1除草剂及其主要代谢产物的遗传毒性研究

本文以Ａｍｅｓ试验和Ｖ７９细胞的ＨＰＲＴ位点的基因正向突变试验，微核试验及姐妹染色单体互换（ＳＣＥ）试验比较研究了新农药８３－１除草剂（ＤＣＮＰＡ）及其在哺乳动物体内的主要代谢产的２，４－二氯－６－氨基酚（ＤＣＡＰ）的致突变性和细胞遗传毒性，结果显示：ＤＣＮＰＡ在本研究的所有检测终点，无论加与不加大鼠肝Ｓ９均不能检出阳性反应，而经ＤＣＡＰ染毒的Ｖ７９细胞出现了具有剂量一反应关系的ＳＣＥ和微核率增加     

流式细胞术对小鼠外周血红细胞微核自动化检测的研究

本文报道双激光流式术对小鼠外周血嗜多染红细胞（ＰＣＥ）和成熟红细胞（ＮＣＥ）微核（ＭＮ）的自动化检测新方法。用噻唑橙染ＲＮＡ以区分ＰＣＥ和ＮＣＥ，用Ｈｏｅｃｈｓｔ３３３４２染ＤＮＡ以检测红细胞内ＭＮ。在用ＭＭＣ和ＣＯＬ进行的ＭＮ量效和时效关系研究中，其机检们和镜检结果吻合很好（ｒ＞０．９５），流式仪分选验证，证实其ＭＮ真实性在９５％以上，而检测速度比人工提高４０倍以上。本方法具有高速、精确、客观和完全自动化的特点，标志着流式仪对ＭＮ的自动化检测已达到实用化程度。     

The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability.

A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B-IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 x 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08-0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60-610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer.     

Usefulness of Tirapazamine as a Combined Agent in Chemoradiation and Thermo-chemoradiation Therapy at Mild Temperatures: Reference to the Effect on Intratumor Quiescent Cells

C3H/He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received one of six different DNA-damaging agents with or without mild temperature hyperthermia (40°C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of γ-rays. Immediately after irradiation, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with γ-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with γ-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.     

Usefulness of tirapazamine as a combined agent in chemoradiation and thermo-chemoradiation therapy at mild temperatures: reference to the effect on intratumor quiescent cells.

C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received one of six different DNA-damaging agents with or without mild temperature hyperthermia (40 degrees C, 30 min, MTH). These agents were adriamycin (ADM), mitomycin C (MMC), cyclophosphamide (CPA), bleomycin (BLM), cisplatin (CDDP), and tirapazamine (TPZ). After the drug treatment, the tumor-bearing mice were irradiated with a series of doses of gamma-rays. Immediately after irradiation, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cells was determined from the tumors that had not been pretreated with BrdU. MTH significantly increased the MN frequency of total cells in tumors irradiated with gamma-rays combined with CPA, BLM, CDDP or TPZ, and that of Q cells in tumors irradiated with gamma-rays combined with BLM or TPZ. The sensitivity difference in the MN frequency between total and Q tumor cells was significantly decreased by the combination with TPZ. TPZ combined with radiotherapy and TPZ combined with thermo-radiotherapy at mild temperatures appear to be promising modalities for sensitizing tumor cells in vivo, including Q tumor cells.     

体内碱性彗星试验与微核试验联合测定甲磺酸乙酯遗传毒性方法的建立

目的：建立彗星试验与微核试验结合的检测方法,应用其评价甲磺酸乙酯（EMS）对大鼠的遗传毒性。方法：试验设置阴性对照组（0.9%的生理盐水）及EMS低（50 mg/kg）、中（100 mg/kg）、高（200 mg/kg）剂量组,共4组,每组5只雄性SD大鼠,分别在0、24、48和69 h经口灌胃1次性给予受试物,在末次给药后3 h进行外周血采样,外周血经固定、洗脱、荧光染色后进行微核试验采用流式细胞术测定网织红细胞微核率;然后处死动物进行肝脏、肾脏和胃组织的取样,样品经单细胞悬液制备、制片、裂解、解旋电泳及染色后,应用Comet Assay IV软件进行彗星试验数据的收集。结果：彗星试验中,50、100和200 mg/kg EMS染毒组肝脏、肾脏和胃的尾DNA百分含量与阴性对照组相比均显著增加,且呈剂量反应关系（r=0.93,P<0.05）;微核试验中,EMS高剂量组骨髓毒性太大未收集到足够的细胞用于检测,仅EMS中剂量组外周血网织红细胞微核率较对照组显著增加（P<0.01）。结论：初步建立了大鼠多靶器官彗星试验方法;在同一试验中彗星试验和微核试验联合可节省动物数量,提高试验效率。     

Antimutagenicity and Antioxidative DNA Damage Properties of Oligomeric Proanthocyanidins from Thai Grape Seeds in TK6 Cells

Oligomeric proanthocyanidins (OPCs) are found mostly in red grape seeds. Many publications have reportedthat OPCs possess an excellent anti-oxidant effects. Since it could against cellular damage from reactive oxygenspecies (ROS) led to reduce the risk of chronic disease and cancers. We carried out this study on the ThaiOPCs to evaluate the mutagenicity/ anti-mutagenicity and anti-oxidative DNA damage effects in TK6 cells bymicronucleus (MN) and comet assays. In the MN assay, OPCs-treatment of TK6 cells at concentrations rangingfrom 10-200 μg/ml (4 and 24 h) did not cause micronucleus induction over the negative control group but revealeda significant reduction the micronucleus frequencies against the known mutagen (mitomycin C). In the cometassay, OPCs-treated TK6 cells at concentrations of 100, 250, 500, and 1,000 μg/ml could inhibit DNA damageinduced by H2O2 as indicated by 18.7, 36.4, 30.6, and 60.1%, respectively. Our results suggest that OPCs possessthe anti-mutagenic and anti-oxidative DNA damage effects in TK6 cells under the conditions of this assay.     

Effects of Bioreductive Agents, Tirapazamine and Mitomycin C, on Quiescent Cell Populations in Solid Tumors, Evaluated by Micronucleus Assay

Mice bearing transplantable solid tumors received 10 intraperitoneal administrations of 5-bromo-2'-deoxyuridine (BrdU) to label the proliferating (P) tumor cells, and were then irradiated with ⁶⁰Cogamma-rays or injected with cis -diamminedichloroplatinum (II) (cisplatin). The tumor cells were isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in the cells without BrdU labeling, which were regarded as quiescent (Q) cells in the tumor, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumor cell population was determined from tumors that were not pretreated with BrdU. Pretreatment with tirapazamine, a bioreductive agent, could enhance the sensitivity of tumor cells, including Q cells, to radiation more markedly than mitomycin C pretreatment as judged from an in vivo assay immediately after irradiation. Post-irradiation administration of tirapazamine produced a large post-irradiation radiosensitizing effect on both the total and Q tumor cell populations in vivo. Cisplatin treatment combined with tirapazamine demonstrated that tirapazamine also has a chemosensitizing potential for both the total and Q tumor cell populations. We confirmed that the sensitivity of Q cell populations to radiation and chemotherapy using cisplatin can be enhanced by combined treatment with tirapazamine.