检测细胞DNA断裂损伤效应的彗星实验法的改良

为了解决彗星实验过程中常出现的脱胶、细胞核分离操作繁琐、重复性低等问题,对彗星实验方法进行了改良,初步建立了彗星实验的快速操作流程。结果显示,通过对载玻片进行预处理,可确保凝胶悬挂均匀;采用改良机械法分离的细胞核浓度适中;以0.5%(w/v)涂层琼脂糖作为基层、以1.5%(w/v)低熔点包埋琼脂糖作为叠加层的"双层凝胶法",辅以"推片法"铺胶,操作便捷且不发生脱胶现象;细胞核膜经裂解处理后再进行电泳和荧光观察,彗星图像清晰,杂质少。应用改良后的彗星实验方法,操作简便,耗时更短,实验效果良好,可快速检测出细胞DNA损伤效应。     

植物活性物质对PM_(2.5)致细胞损伤的抗性作用

为研究植物活性物质对PM2.5致细胞损伤的抗性作用,研究了北京城区PM2.5对CHO(Chinese hamster ovary,中国仓鼠卵巢)细胞的损伤作用,并选择植物活性物质绿原酸、阿魏酸和荭草素探讨其对CHO细胞的保护作用.以CCK-8法确定PM2.5的试验剂量和3种活性物质的最佳保护剂量,然后通过彗星试验、微核试验、流式细胞术及Western Blotting分别检测PM2.5的损伤作用及植物活性物质的保护作用.结果表明:1由CHO细胞存活率可知,PM2.5的有效试验剂量为15μg/m L,绿原酸、阿魏酸和荭草素的最佳保护剂量分别为50、20、10μmol/L.2PM2.5可引起CHO细胞明显的DNA损伤、微核形成增加、细胞周期阻滞所致的细胞增殖指数降低,而经阿魏酸、绿原酸和荭草素预处理后,均可减轻PM2.5对细胞的损伤作用,起到了一定的保护作用.3PM2.5能引起CHO细胞中p53和caspase-3蛋白的表达增加,而阿魏酸、绿原酸和荭草素均可使其表达量有所降低.由PM2.5引起的细胞损伤经绿原酸、阿魏酸、荭草素作用后表达量降低.在3种植物活性物质中,以阿魏酸拮抗PM2.5损伤的效果最佳,绿原酸次之,荭草素相对较差.     

Cytogenotoxicity and histopathological assessment of Lekki Lagoon and Ogun River in Synodontis clarias (Linnaeus, 1758)

The cytogenotoxicity and histopathological alterations induced by xenobiotics in Lekki Lagoon and Ogun River on Synodontis clarias were investigated. Fish from these water bodies and a fish farm (control) were examined for micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood. Also gill, liver, kidney, and ovary were processed for histopathology using hematoxylin-eosin staining. Concentrations of cadmium, zinc, lead and copper in the water were determined. There was significant (p < 0.05) increase in micronucleated, binucleated, and immature erythrocytes in both gill and peripheral blood of S. clarias from the lagoon and river compared to the reference site. Loss and disorganization of the primary and secondary lamellae, multifocal degeneration, hemorrhages, cellular infiltration, congestions, vacuolations, atresia, and necrosis were common lesions in the examined tissues of fish from the lagoon and river. Cd, Zn, Pb, and Cu in water samples from the lagoon and river were higher than the reference site. Xenobiotics in Lekki Lagoon and Ogun River, mostly metals, induced deoxyribonucleic acid (DNA) and pathological damage in S. clarias.     

Effect of Tinospora cordifolia on the reduction of ultraviolet radiation‐induced cytotoxicity and DNA damage in PC12 cells

The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation‐induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL^?1 of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL^?1. In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL^?1 concentrations in serum‐deprived medium compared to control. To confirm the protective role against UV‐induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL^?1 of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm^?2, respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector.     

Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma

Objectives To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA). Methods The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples. Results Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series. Conclusions This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA. Copyright © 2007 John Wiley & Sons, Ltd.     

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19–25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART. Copyright © 2003 John Wiley & Sons, Ltd.     

阴离子粘土(LDH)对DNA吸附行为的研究

以4种阴离子粘土做吸附剂,研究了阴离子粘土对DNA的吸附行为.同时,采用XRD、FTIR、UV-vis等表征手段对吸附前后的材料进行研究.吸附结果显示,二元阴离子粘土对DNA的吸附量高于三元阴离子粘土;3:1型阴离子粘土对DNA吸附力强于2:1型阴离子粘土.4种材料对DNA的吸附均符合Langmuir、Freundlich两种吸附等温模型,且Langmuir吸附等温模型拟合度更高,说明阴离子粘土对DNA的吸附为单层吸附.XRD结果显示,吸附前后阴离子粘土基本结构并未发生改变,晶形完好,层间距未有明显变化,表明阴离子粘土对DNA的吸附仅发生在表面,DNA并未进入阴离子粘土层间结构中.UV-vis及电泳结果显示,吸附前后DNA的构型并未发生改变,阴离子粘土的吸附并未对DNA产生较大的影响.