基于HMME的PDT体外灭活HL60实验研究

目的:探讨血卟啉单甲醚(HMME)介导的光动力疗法(HMME-PDT)对HL60细胞的作用及PDT前后HL60细胞表面超微结构的变化。方法:CCK-8法检测光敏剂浓度和光照剂量对HL60细胞抑制率的影响,荧光分光光度计监测PDT过程中光敏剂荧光强度随时间的变化,Fluo 3-AM荧光探针检测不同浓度HMME作用后HL60细胞内Ca2+变化,原子力显微镜观测PDT作用前后不同扫描范围HL60细胞表面的超微结构图。结果:细胞灭活率呈光敏剂浓度-光剂量依赖关系,当HMME为50μg/mL,光照剂量为24 J/cm2时,灭活效率达到70%;随着光照时间的增加,光敏剂的荧光强度不断减弱,下降速率也逐渐变慢;随HMME作用浓度增加,钙离子浓度显著升高;HMME-PDT作用后HL60细胞表面结构出现明显变化。结论:HMME-PDT能有效灭活HL60细胞,光敏剂浓度和光剂量是影响PDT疗效的重要因素,PDT过程中伴随有光漂白现象的发生,细胞凋亡和钙离子浓度增加呈正相关,PDT作用前后细胞出现明显萎缩,细胞膜粗糙度增加。     

Beta-carboline derivatives: novel photosensitizers that intercalate into DNA to cause direct DNA damage in photodynamic therapy

Novel 1,3,9-trisubstituted beta-carboline derivatives were found to exhibit DNA photocleavage properties under visible light irradiation in a cell-free system, which could be reduced by antioxidant vitamin E. Their photo-cytotoxicity to human tumor cell line HeLa was confirmed, in which apoptosis only contributed a small part to the cell death, and necrosis was the dominating outcome of HeLa cells in photodynamic therapy (PDT) using beta-carboline derivatives. Different from other clinical PDT drugs, beta-carboline derivatives were demonstrated to be able to distribute in the nucleus and intercalate into DNA, and consequently cause direct DNA damage by photochemical reaction products in PDT, which was proved by the distinct DNA tails in the comet assay and the considerable amount of DNA damaged cells quantified by flow cytometry. This mechanism could be the explanation for the delay of cell proliferation at DNA synthesis and mitosis.     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.     

Proteins of BrdU-dependent hamster cell lines as characterized by two-dimensional gel electrophoresis.

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was greater than 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels. Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

Proteins of BrdU-Dependent Hamster Cell Lines as Characterized by Two-Dimensional Gel Electrophoresis

Cell lines which exhibit the BrdU-dependent phenotype (B4 and HAB) were studied with respect to BrdU-induced alterations in genetic expression by two-dimensional gel electrophoresis. A comparison of the proteins from the HAB cells, in which the DNA is 100% substituted by BrdU, to those of the unsubstituted parent line (3460) showed 55 protein alterations; the synthesis of 15 increased while that of the other 40 decreased. When 3460 cells were grown in BrdU such that their DNA was > 50% substituted, 27 protein changes could be detected; of these, the synthesis of 10 increased while that of 17 decreased. A comparison of all these changes in the various cell lines showed six which were common to the BrdU-substituted cell lines. The proteins from another Syrian hamster cell line, BHK.-21 (C-13) and those of HAB cells grown in thymidine or BrdC were also examined on two-dimensional gels.
Although BrdU has a dramatic effect on many cellular functions, relatively few changes in the pattern of protein synthesis could be detected in these cell lines, perhaps reflecting the specialized action of this analogue on particular cellular functions.     

Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine.

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell-associated fluorescence of several DNA-specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease influorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin-, olivomycin-, or chromomycin-stained cells, decreased that of propidium iodide-stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen-stained cells. Changes in relative fluorescence of cell associated dyes are due to changes in the amounts of dye bound to cells with BrdU-substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.     

绿盲蝽危害对枣树叶片生化指标的影响

绿盲蝽已在棉区爆发成灾,并逐渐向枣树等北方果树转移危害,已对我国北方果树生产造成严重威胁。绿盲蝽刺吸危害后,能诱导植物产生一系列生化反应。通过生物化学方法研究绿盲蝽不同危害程度对枣树叶片生化指标的影响,探讨受害枣树叶片对绿盲蝽危害的应激反应的变化规律。结果表明,受害枣树叶片内可溶性糖含量随着危害程度的加重先升高,后降低;蛋白质含量随受害程度的加重而呈现降低趋势;游离氨基酸含量随受害程度的加重逐渐升高。受害后枣树叶片内3种防御性酶随受害程度的加重发生不同程度的变化,超氧化物歧化酶(Superoxide Dismutase,SOD)的活性随着危害程度的加重先升高后下降;过氧化物酶(Peroxidase,POD)的活性总体升高;不同受害程度枣树叶片内过氧化氢酶(Catalase,CAT)的活性之间以及它们与未受害枣树叶片内过氧化氢酶(CAT)的活性之间均未达到显著性差异水平。总之,随着绿盲蝽危害程度的加重,枣树叶片内可溶性糖、蛋白质、游离氨基酸含量以及防御性酶活性发生了不同程度的变化,枣树叶片内除过氧化氢酶(CAT)的活性变化外,可溶性糖、游离氨基酸和蛋白质的含量变化以及超氧化物歧化酶(SOD)和过氧化物酶(POD)的活性变化与绿盲蝽危害胁迫有明显的关系,说明枣树叶片对绿盲蝽的危害胁迫产生了应激反应,且叶片内除过氧化氢酶(CAT)外的其他生化指标与绿盲蝽的不同危害程度有一定的相关性。研究结果为理解枣树受绿盲蝽危害后的应激反应机制提供了材料,对绿盲蝽可持续治理具有重要指导意义。     

大血藤叶片生化成分的动态变化

对自然条件下大血藤叶片生化成分的动态变化进行了研究。结果表明;大血藤叶片可溶性糖,淀粉和总糖含量的动态变化有相同的趋势。在叶片生长季节早期含量较低。随着叶片的生长发育,其含量逐渐上升。至8月达到最大值后逐渐下降,叶片总糖含量与日均净光合速率的季节变化呈极显著的正相关,在整个生长季节中,可溶性糖含量比淀粉高,大血藤叶片总蛋白含量季节性变化呈单峰曲线,在展叶初期含量较低;随后迅速上升,至8月达到最大值后逐渐下降,叶片总糖含量与日均净光合速率的季节变化呈极显著的正相关。在整个生长季节中,可溶性糖含量比淀粉高。大血藤叶片总蛋白含量季节性变化呈单峰曲线,在展叶初期含量较低;随后迅速上升,在6月初达到最大值后逐渐下降,落叶前降至最低占,可溶性蛋白质含量的季节性变化曲线与总蛋白含量基本相似,DNA含量在叶生长初期大幅度上升,至6月达到高峰后迅速下降,以后基本趋向稳定,RNA在叶生长初期有所上升,峰值也出现在6月,以后缓慢下降,到9月降至最低值,在落叶前又有所回升,总核酸含量的季节变化与RNA变化相似,RNA/DNA比值在生长季节中出现3次高峰,大血藤叶片总黄酮含量季节性变化呈“双峰”型,第1高峰期在开花期的5月。第2高峰期在秋季的9月份。     

Effect of dithiocarbamate pesticide zineb and its commercial formulation,azzurro. IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 microg/ml) or azzurro (100.0 microg/ml) for 80min, washed and reincubated in pesticide-free medium for 0-12h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6h of incubation. After 12h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0-12h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5-12h repair time for both pesticides. At 12h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0-12h repair period was observed.     

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.     

太湖新银鱼卵巢发育不同阶段个体生化组成和能量密度研究

对丹江口水库太湖新银鱼雌鱼性腺发育不同阶段个体的生化组成和能量密度变化进行研究, 以分析性腺发育的物质和能量来源。卵巢8-11月间基本处于Ⅱ期, 之后迅速发育; 1-3月主要为Ⅳ期或Ⅴ期, 4月开始以Ⅵ期个体为主。卵巢发育的Ⅱ期和Ⅲ期, 太湖新银鱼全鱼中的生化组成和能量密度随卵巢发育而显著增高。当发育至Ⅳ期时, 全鱼和去性腺鱼体中的干物质、粗脂肪、粗灰分相对含量和能量密度仍明显增加, 而粗蛋白相对含量在全鱼当中有所减少。由Ⅳ期发育到Ⅴ期时, 全鱼中的各生化组成无明显变化, 能量密度显著增加; 去性腺鱼体中的粗蛋白相对含量显著增加, 粗脂肪显著减少, 其他生化组成和能量密度无明显变化。产后Ⅵ期, 全鱼中的粗脂肪、粗灰分相对含量和能量密度显著下降, 干物质和粗蛋白相对含量则显著上升。性腺发育成熟前(Ⅳ期前), 去性腺鱼体中的总能量和粗蛋白绝对含量明显增加, 而性腺中能量和粗蛋白绝对含量增加不明显, 粗脂肪含量的增加明显。由Ⅳ期发育到Ⅴ期时, 全鱼中的总能量、粗蛋白和粗脂肪绝对含量均略有增加, 主要是由于性腺中能量、粗蛋白和粗脂肪含量的增加, 而去性腺鱼体中能量、粗蛋白和粗脂肪绝对含量均明显减少。产后Ⅵ期全鱼中的总能量、粗蛋白和粗脂肪绝对含量均急剧降低。上述结果初步表明太湖新银鱼卵巢发育成熟前在鱼体中储存物质和能量, 在卵巢发育成熟时这些储存的物质和能量可能向卵巢转移, 成为卵巢发育成熟的主要物质和能量来源。       

铝胁迫对海莲幼苗保护酶系统及脯氨酸含量的影响

为探讨Al~(3+)胁迫对海莲的影响,研究了10～50 mmol/L Al~(3+)处理下海莲幼苗叶片和根系的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)的活性以及可溶性蛋白质、丙二醛(MDA)和游离脯氨酸(Pro)含量的变化。结果表明,海莲幼苗能耐受50 mmol/L的Al~(3+)胁迫处理,具有较高的耐铝性。但在50 mmol/L Al~(3+)处理时,海莲幼苗叶片和根系的质膜系统膜脂过氧化加重,MDA含量增加;细胞活性氧代谢失衡。在保护酶系统中,Al~(3+)处理促进了叶片中APX和POD活性的提高,降低了CAT的活性,SOD的活性呈下降趋势;海莲根部POD和SOD活性均显著提高,而CAT活性下降。25～50 mmol/LAl~(3+)处理下,海莲叶片和根部可溶性蛋白质含量均显著下降;Pro的含量在叶片和根均有显著增加。     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

U2OS cells lacking Chk1 undergo aberrant mitosis and fail to activate the spindle checkpoint

Chk1 is a conserved protein kinase originally identified in fission yeast, required to delay entry of cells with damaged or unreplicated DNA into mitosis. The requirement of Chk1 for both S and G2/M checkpoints has been elucidated while only few studies have connected Chk1 to the mitotic spindle checkpoint. We used a small interference RNA strategy to investigate the role of Chk1 in unstressed conditions. Chk1 depletion in U2OS human osteosarcoma cells inhibited cell proliferation and raised the percentage of cells with a 4N DNA content, which correlated with accumulation of giant polynucleated cells morphologically distinct from apoptotic cells, while no increased number of cells in G2 or mitosis could be detected. Down-regulation of Chk1 also caused accumulation of cells in the last step of cytokinesis, and of tetraploid cells in G1 phase, which coincided with activation of p53 and increased levels of p21. In addition, Chk1-depleted U2OS cells failed to arrest in mitosis after spindle disruption by nocodazole and showed decreased protein levels of Mad2 and BubR1. These studies show that U2OS cells lacking Chk1 undergo abnormal mitosis and fail to activate the spindle checkpoint, suggesting a role of Chk1 in this checkpoint.     

Preferential binding of human XPA to the mitomycin C-DNA interstrand crosslink and modulation by arsenic and cadmium

The Xeroderma Pigmentosum A (XPA) protein is involved in the DNA damage recognition and repair complex formation steps of nucleotide excision repair (NER), and has been shown to preferentially bind to various forms of DNA damage including bulky lesions. DNA interstrand crosslinks are of particular interest as a form of DNA damage, since these lesions involve both strands of duplex DNA and present special challenges to the repair machinery, and mitomycin C (MMC) is one of several useful cancer chemotherapy drugs that induce these lesions. Purified XPA and the minimal DNA-binding domain of XPA are both fully capable of preferentially binding to MMC-DNA interstrand crosslinks in the absence of other proteins from the NER complex. Circular dichroism (CD) and gel shift assays were used to investigate XPA-DNA binding and to assess changes in secondary structure induced as a consequence of the interaction of XPA with model MMC-crosslinked and unmodified DNAs. These studies revealed that while XPA demonstrates only a modest increase in affinity for adducted DNA, it adopts a different conformation when bound to MMC-damaged DNA than when bound to undamaged DNA. This change in conformation may be more important in recruiting other proteins into a competent NER complex at damaged sites than preferential binding per se. Arsenic had little effect on XPA binding even at toxic concentrations, whereas cadmium reduced XPA binding to DNA to 10-15% that of Zn-XPA, and zinc addition could only partially restore activity. In addition, there was little or no change in conformation when Cd-XPA bound MMC-crosslinked DNA even though it demonstrated preferential binding, which may contribute to the mechanism by which cadmium can act as a co-mutagen and co-carcinogen.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.