12C6+束和Mitomycin C 诱导的DNA损伤效应

本文旨在分别研究重离子束及MMC在诱导细胞的DNA损伤效应中一些具体的分子机制，为治疗的进行以及相关辅助药物的开发提供理论依据。本文探索的重点有两个，第一个是重离子束辐射诱导的DNA损伤效应及p53在其中的激活，第二个是MMC诱导的DNA损伤效应及p53和BRCA1、H2AX等分子在其中的角色。 1. 12C6+离子束诱导HeLa细胞DNA损伤效应为了研究HeLa细胞经过12C6+ 束辐照之后的DNA损伤效应，及这个过程中p53激活的分子机制。我们运用中性单细胞电泳技术，检测了HeLa细胞经过4Gy 12C6+ 束辐照0h、3h、6h和12h之后DNA的损伤情况，以及0.5Gy、1Gy、2Gy和4Gy 12C6+ 束辐照0h后的DNA损伤情况。同时运用细胞生长实时监测仪监测了HeLa细胞在经过0Gy、0.5Gy和1Gy 12C6+ 束辐照之后的生长变化，并运用AO/EB双染检测了辐照24小时后的凋亡情况。另外，利用8mmol/L的caffeine（抑制ATM和ATR）和20μmol/L的wortmannin（抑制ATM和DNA-PK）处理HeLa细胞后再进行1Gy 12C6+ 束辐照，通过western blot检测p53的表达。结果显示，12C6+ 束辐照可造成HeLa细胞的DNA损伤，损伤随剂量升高而升高但随时间降低；并诱导HeLa细胞发生凋亡；而且辐照后p53表达升高，但经过caffeine或者wortmannin预先处理的细胞p53均没有显著升高。我们的结论是：12C6+ 束辐照可造成HeLa细胞的DNA损伤并诱导损伤修复及凋亡等效应，损伤效应相关的分子p53被激活，并且激活依赖于ATM。 2. MMC诱导的DNA损伤效应在这一部分研究中，首先，我们利用与上面相同的研究方法，探讨了p53在MMC诱导的DNA损伤效应中的激活情况，结果显示，MMC诱导的DNA损伤效应并不依赖于p53。另外，我们还探讨了， BRCA1在FANCD2的γ-H2AX依赖性转移中的作用。MMC可造成DNA的ICL（interstrand cross-link）损伤，ICL可通过FA(Fanconi Anemia)通路进行修复。FANCD2是FA通路的核心分子，在DNA产生ICL时被各种分子修饰然后转移到损伤部分，这个过程的涉及到ATR、γ-H2AX及BRCA1等，本文试图探讨BRCA1在其中的作用方式。研究中，我们监测了不同处理（包括对照、caffeine（可抑制ATR）、MMC及MMC ＋caffeine）的HCC1937(BRCA1缺陷型)和MCF7（BRCA1野生型）细胞的生长；并用Western blot检测MMC处理之后HCC1937细胞γ-H2AX的表达情况。结果表明，MMC和caffeine均可以抑制HCC1937的生长，但caffeine和MMC+caffeine的抑制效果是一样的；MMC和caffeine均可以抑制MCF7的生长，且MMC+caffeine处理比仅进行caffeine处理的抑制作用强；MMC处理之后，HCC1937的γ-H2AX表达显著升高。我们的结论是，在FANCD2的γ-H2AX依赖性转移中，H2AX的磷酸化并不依赖于BRCA1，不过，BRCA1和ATR应该参与一个相同的分子事件，可能是FANCD2的磷酸化。这个有待进一步的实验验证

草鱼体内肾细胞姐妹染色单体分化及交换的初步研究

本文确立了一个以草鱼体内肾细胞姐妹染色单体交换频率为指标的检测环境诱变或致癌物质的短期试验系统。采用硫堇-UV-Giemsa染色法,分析了草鱼体内肾细胞的SCD-2(注射BrdU后第二个细胞周期的中期分裂相的SCD)频率和SCE频率。用500微克/克体重BrdU体内标记5天,草鱼肾细胞SCD-2频率为8.58±0.22%;SCE频率为3.05±2.523 SCE_5/细胞。以丝裂霉素C(Mitomycin C,MMC)作为阳性对照,分析了化合物亚硝基胍(N-methyl-N~1-nitro-N-nitro

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.