Wnt3a联合BMP9诱导C3H10T1/2细胞向心肌细胞样细胞分化

目的探讨Wnt3a单独以及联合骨形态发生蛋白9(BMP9)诱导C3H10T1/2细胞向心肌细胞样细胞分化的作用。方法利用HEK293细胞扩增重组腺病毒AdGFP、AdBMP9、AdWnt3a;分别转染C3H10T1/2细胞3周后,倒置显微镜和荧光显微镜观察细胞形态及绿色荧光蛋白表达;流式细胞术检测细胞转染效率;Westernblot法、免疫荧光技术及实时定量PCR(qRT-PCR)分别检测缝隙连接蛋白43(Cx43)、肌钙蛋白T(cTnT)、GATA结合蛋白4(GATA4)、心肌细胞增强因子2C(MEF2C)的表达。结果经HEK293细胞扩增可得到高滴度的重组腺病毒;转染C3H10T1/2细胞3周后,Wnt3a组Cx43、cTnT、GATA4、MEF2C的表达量与空白组比较无明显差异;Wnt3a联合BMP9组的Cx43、cTnT、GATA4、MEF2C基因的表达量显著高于空白组、GFP组和Wnt3a组,与单独BMP9组相比无显著性差异。结论Wnt3a不能单独诱导C3H10T1/2细胞向心肌细胞样细胞分化;Wnt3a联合BMP9可诱导C3H10T1/2细胞向心肌细胞样细胞分化。     

吲哚-3-甲醇酸性代谢产物辐射防护作用的研究

目的 探讨吲哚-3-甲醇(I3C)酸性代谢产物的辐射损伤防护作用.方法 采用细胞克隆形成实验检测细胞的存活分数;采用Western Blot方法检测蛋白表达水平.结果 采用正常纤维上皮细胞184A1,发现7种I3C酸性代谢产物表现出不同的辐射损伤防护效果,其中CTET(1μmol/L)、LTET(1μmol/L)、HI-IM(1 μmol/L)和3,3'-二吲哚甲烷(DIM)(0.3 μmol/L)4种代谢产物在细胞γ-射线照射前24 h预处理细胞均不同程度地提高了辐照细胞的存活分数,差异均有统计学意义(P＜0.05,P＜0.01),而CT(1μmol/L)、LTr-1(1 μmol/L)和ICZ(1μmol/L)3种代谢产物则对辐照细胞存活分数无影响(P＞0.05).进一步研究发现CTET、LTET、HI-IM和DIM可导致ATM、BRCA1和NBS1蛋白磷酸化水平的改变.同样,CT、LTr-1和ICZ对这些蛋白的磷酸化水平不产生影响.另外,HI-IM可明显降低辐射损伤诱导的细胞坏死和细胞凋亡.结论 CTET、LTET、HI-IM和DIM 4种I3C酸性代谢产物可明显降低184A1细胞的辐射敏感性,即具有辐射防护作用,其机制可能与其调节DNA损伤与修复蛋白磷酸化和细胞凋亡有关.     

BALB/c 3T3细胞转化实验的优化及其在致癌物协同作用研究中的应用

目的 优化BALB/c 3T3细胞转化实验,并应用于致癌物间协同致癌作用的研究.方法 从血清浓度、培养基类型和致癌物作用时间三个因素对BALB/c 3T3细胞转化实验进行优化.采用优化的实验方案,选择致癌物作用7 d后重新接种,再以含5%胎牛血清的DMEM/F12(1:1)培养基培养进行细胞转化实验,对致癌物间的协同作用进行检测,通过小鼠体内致瘤实验对转化灶细胞的恶性特征进行验证.结果 二乙基亚硝胺(DEN)与2、3、7、8-四氯二苯并二恶英间有较强的协同致癌作用.微囊藻毒素单独具有较强促细胞恶性转化能力,但这种促转化能力却受到DEN的抑制.实验诱发的转化灶具有Ⅱ型转化灶的特征,并可在体液与细胞免疫缺陷(SCID)小,鼠体内致瘤.结论 经优化的BALB/c 3T3细胞转化方案既充分模拟了致癌物联合作用的方式,又缩短了实验周期,可有效应用于致癌物间协同作用的研究.     

化学致癌剂3-4苯并吡诱发C_3H/10T 1/2细胞恶性转化的研究

本文用C_3H/10T(1/2)细胞作为靶细胞,研究了化学致癌剂3-4苯并吡(BP)诱发其恶性转化的发生。结果证明,在BP作用后7～9周出现转化灶。从转化灶中分离的细胞,失去了在培养中的接触抑制,能在软琼脂培养基中形成集落。接种于免疫抑制动物,能够形成肉瘤,表现出恶性细胞的生长特性。上述结果说明,C_3H/10T(1/2)细胞对研究致癌剂诱发体外培养细胞的恶性转化是有用的。染色体分析结果表明:恶性转化细胞的染色体数目和结构,都发生了明显的变化。说明化学致癌剂诱发的恶性转化,与其对细胞DNA的损伤有密切关系。     

Tim-3及其配体Galectin-9与病毒感染的关系

T细胞免疫球蛋白黏蛋白3(Tim-3)基因是Tim家族成员,与其配体半乳凝素-9(Galectin-9)结合可显著抑制T细胞的活化和增殖,并调节细胞因子的表达和分泌.Tim-3/Galectin-9参与免疫反应的调节及抗病毒免疫反应,阻断此通路可促进CD8+T细胞应答和对病毒的控制.因此,Tim-3/Galectin-9通路可能与病毒感染慢性化有关.本文对Tim-3及其配体的生物学特性、功能及在病毒感染中的研究近况进行综述.     

NR2F2促进小鼠MC3T3-E1前成骨细胞增殖

目的探讨NR2F2基因对小鼠前成骨细胞增殖的影响。方法用实时定量PCR检测mRNA表达量,MTS法检测细胞增殖,流式细胞术检测细胞周期,ELISA-Brdu法检测细胞DNA合成速度。结果在小鼠MC3T3-E1前成骨细胞增殖速度加快时,NR2F2基因的表达增高至对照的4.57±0.30倍(P<0.01)。过表达NR2F2基因促使MC3T3-E1细胞增殖速度加快,细胞数量增加(P<0.01),细胞周期中S期细胞比例明显升高,为对照组的2倍,G2/M期比例也有增加(P<0.05)。过表达NR2F2基因使MC3T3-E1细胞的Brdu掺入率增高,DNA的合成加速(P<0.01)。结论 NR2F2使S期细胞比例增加,促进小鼠前成骨细胞MC3T3-E1的增殖速度。     

内质网应激参与低浓度DNA加成性损伤剂诱发的细胞应答反应

目的：研究内质网应激在DNA损伤剂/致癌物诱发的细胞应答反应中的作用。 方法: 选择3种引起不同DNA损伤类型的DNA损伤剂/致癌物，烷化性DNA损伤剂N-甲基-N’-硝基-N-亚硝基胍（MNNG），大块加成性DNA损伤剂苯并［a］芘-7,8-9-二氢二醇-9,10-环氧化物（BPDE，环境致癌物苯并［a］芘在体内代谢形成的终致癌物）以及交联性DNA损伤剂丝裂霉素C（MMC）对人羊膜细胞FL系内质网应激反应的影响。采用SDS－PAGE 和免疫印迹法检测内质网应激蛋白GRP78/BiP, GADD153/CHOP的表达改变和定位于内质网的半胱天冬酶-12(caspase-12)的激活。 结果： 低浓度MNNG（0.25 μmol/L和1 μmol/L）和BPDE（5 nmol/L和50 nmol/L）均能引起FL细胞中GRP78/BiP和GADD153/CHOP的上调和caspase-12的激活；3种浓度的MMC（5 μmol/L、50 μmol/L和500 μmol/L）均引起GRP78/BiP的下调，不伴有GADD153/CHOP水平的改变和caspase-12的激活，更低浓度的MMC（5 nmol/L 和50 nmol/L）对此并无影响。 结论： 低浓度MNNG和BPDE可诱发暴露细胞的内质网应激，而MMC则导致在内质网应激反应诱发过程中起介导作用的GRP78/BiP蛋白的下调，从而可能改变细胞对内质网应激原的反应性。内质网应激在DNA损伤剂/致癌剂诱发的细胞应答反应中有一定作用。     

人趋化素样因子2对小鼠成纤维细胞BALB/c 3T3的促增殖和凋亡抑制作用

目的　探讨人趋化素样因子 2 (CKLF2 )对小鼠成纤维细胞BALB c 3T3增殖和凋亡的影响。方法　构建pcDI CKLF2真核表达载体 ;建立稳定表达人CKLF2的BALB c 3T3(3T3 CKLF2 )细胞系 ;以细胞计数和MTT法分析过表达人CKLF2后对 3T3细胞增殖的影响 ;以流式细胞术检测annexin Ⅴ阳性细胞为凋亡指标 ,分析 3T3 CKLF2细胞和 3T3 pcDI细胞对撤除血清诱导凋亡的反应情况。结果　成功地建立了稳定表达人CKLF2的BALB c 3T3细胞系 ,过量表达人CKLF2的 3T3细胞系增殖速度明显加快 ,同等接种量的细胞培养 40h以后 ,3T3 CKLF2细胞的数量 (1.0 7× 10 6 )比 3T3 pcDI细胞(2 .5 4× 10 5)及野生型 3T3细胞 (3 .0× 10 5)高 2～ 3倍 ;流式细胞术分析表明 ,撤除血清后 3T3 CKLF2的annexin Ⅴ阳性细胞数 (13.34% 48h、15 .44 % 96h)明显低于 3T3 pcDI细胞 (19.36 % 48h、39.2 % 96h)。结论　人CKLF2能够促进BALB c 3T3细胞的增殖和抵抗撤除血清诱导的细胞凋亡 ,这些研究为进一步探讨CKLF家族的生物学功能提供了实验依据。     

互隔交链孢酚增加NIH3T3细胞中DNA聚合酶β表达

目的 研究互隔交链孢酚(AOH)对小鼠胚胎成纤维细胞NIH3T3中DNA聚合酶β(DNA PoLβ)表达的影响.方法 采用半定量RT-PCR、细胞免疫化学和Western blot方法检测细胞中DNA POLβ mRNA和蛋白的表达.结果 与对照组相比,AOH可以引起NIH3T3细胞中DNA POLβ基因的mRNA水平和蛋白水平表达增高(并呈一定的剂量效应关系)(P0.05).结论 AOH可以引起NIH3T3细胞中DNA POLβ基因表达增高,可能有助于细胞应对AOH引起的DNA损伤.     

苯并(a)芘对神经细胞的毒性及细胞色素P4501A1的诱导表达

目的 探讨苯并(a)芘[B(a)P]对神经细胞毒性、细胞凋亡与细胞色素P4501Al( CYP1A1)诱导表达的关系.方法 选用新生1 ～3d的SD大鼠,分离大脑皮质,进行神经元培养,在细胞培养第5天左右,选取生长良好的同批次神经细胞,以苯并(a)芘分别对神经细胞染毒,使苯并(a)芘终浓度分别为0μmoL/L、10μmol/L、20μmol/L、40μ.mol/L.继续培养40h,应用cck8试剂盒检测神经细胞活力、Annexin V和PI双染法进行细胞凋亡的检测,应用RT-PCR法检测神经细胞CYP1A1mRNA的表达,免疫组织化学SABC法检测神经元CYP1A1蛋白的表达.结果 随着苯并[a]芘浓度的增加,神经细胞活力降低,早期凋亡率逐渐增高,细胞活力中高剂量组与对照组比较差异均有统计学意义,早期凋亡率仅高剂量组与对照组比较差异有统计学意义,趋势检验表明,细胞活力降低、凋亡率增高具有剂量依赖性.而且随B(a)P剂量的增高,CYP1A1mRNA及蛋白表达增多,有剂量-反应关系,CYP1A1基因和蛋白表达与神经细胞凋亡率的相关分析表明,神经细胞凋亡率与CYP1A1 mRNA表达呈正相关(r=0.831,P＜0.01);与CYP1A1蛋白表达呈正相关(r=0.780,P＜0.01).结论 苯并[a]芘可致神经细胞凋亡,神经细胞CYP1A1诱导表达是神经细胞损伤的关键因素.     

Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (+/-)7 beta-8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)anti-BPDE], and (+/-)7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)syn-BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose-response range of 0.005 micrograms/ml-0.25 micrograms/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor-initiating activity. For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 microgram/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 micrograms/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 x 10(5) bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7-methyl-dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6-methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.     

In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 micrograms/ml, did not induce transformation, whereas dioxane was very active in the induction of type III foci in the cultured BALB/3T3 cells.     

Lack of chloroform-induced DNA repair in vitro and in vivo in hepatocytes of female B6C3F1 mice

Chloroform has been shown to induce hepatocellular carcinomas in female B6C3F₁ mice when administered by gavage, but not when given in drinking water. When administered in corn oil at the carcinogenic doses of 238 and 477 mg/kg, chloroform induced necrosis and sustained regenerative cell proliferation in the liver. To investigate the mode of action of tumor induction in the target cells, the ability of chloroform to induce unscheduled DNA synthesis (UDS) was examined in the in vitro and in vivo hepatocyte DNA repair assays. In the in vitro assay, primary hepatocyte cultures from female B6C3F₁ mice were incubated with concentrations from 0.01 to 10 mM chloroform in the presence of ³H-thymidine. UDS was assessed by quantitative autoradiography. No induction of DNA repair was observed at any concentration. In the in vivo assay, animals were treated by gavage with 238 and 477 mg/kg chloroform in corn oil. Primary hepatocyte cultures were prepared 2 and 12 hr later, incubated with ³H-thymidine, and assessed for induction of UDS as above. No DNA repair activity was seen at either dose or at either timepoint. These negative results in the target organ are consistent with the concept that neither chloroform nor its metabolites are directly DNA reactive and that the carcinogenicity of chloroform is secondary to induced cytolethality and regenerative cell proliferation. © 1994 Wiley-Liss, Inc.     

Co-culture systems for assessing the stability and genotoxicity of reactive 1,2-dibromo-3-chloropropane (DBCP) metabolites

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by alkaline elution, at low concentrations (5-10 microM) in suspensions of hepatocytes and testicular cells isolated from rats. At higher concentrations (greater than or equal to 100 microM) DBCP caused DNA damage and increased the frequency of sister chromatid exchanges in Chinese hamster V79 cells. When DBCP (2.5-10 microM) was tested for its ability to cause unscheduled DNA synthesis (UDS) in monolayers of liver cells isolated from untreated rats, a clear positive response was obtained. No increase in UDS was detectable when liver cells isolated from PCB-pretreated rats were used. In contrast, DBCP (greater than or equal to 50 microM) was metabolized in hepatocytes from PCB-pretreated rats to products mutagenic to Salmonella typhimurium TA100 in co-culture with the hepatocytes, whereas control hepatocytes were substantially (40-fold) less active. No bacterial mutagenicity could be detected when the TA100 strain was co-incubated with isolated rat testicular cells and DBCP. In co-cultures of hepatocytes and V79 cells DBCP-induced DNA damage in V79 cells occurred at low concentrations (10 microM DBCP) compared to the concentration (100 microM) needed to induce DNA damage in the V79 cells incubated without hepatocytes. Testicular cells were not able to enhance DBCP-induced DNA damage in the V79 cells. The data indicate that the putative reactive DBCP episulphonium ion metabolite formed in the testicular cells presumably responsible for testicular cell DNA damage, is not capable of escaping the cell where it is formed. Other reactive DBCP metabolites generated in the liver cells seem to be able to interact with the DNA of neighbouring cells.     

分泌人可溶性FL基因转染细胞及其生物学活性分析

应用基因重组技术构建了人可溶性FL基因逆转录病毒载体pLXSN FL ,经转染PA317细胞和G4 18筛选抗性细胞克隆 ,获得重组病毒液。NIH3T3细胞进行病毒滴度测定后 ,选择适当滴度的重组病毒 (5× 10 5CFU/ml)感染ECV30 4细胞 ;应用聚合酶链反应 (PCR )及反转录聚合酶链反应 (RT PCR )检测感染ECV30 4细胞FL的DNA及mRNA表达 ;应用ELISA测定rhFL在ECV30 4中的表达水平 ,用荧光标记和流式细胞仪检测转基因上清对单个核细胞来源DC的表型影响及3 H TdR掺入法测定DC对T细胞的促增殖作用。结果表明 ,外源性rhFL基因整合到ECV30 4细胞染色体DNA并有效地转录和翻译 ,稳定表达水平为 82 4ng (10 6细胞 /2 4h )的人FL。转基因ECV细胞培养上清能有效诱导人PBMC来源DC的分化和增强DC对T细胞的激发作用。外源性FL基因可以转移到ECV30 4细胞并稳定表达 ,有助于体外研究内皮细胞与免疫细胞的相互作用     

Role of monocytes in anti-CD3-induced T-cell DNA synthesis: Effect of chloroquine and monensin on anti-CD3-induced human T-cell activation

Anti-CD3 monoclonal antibody (MoAb) induces proliferation of freshly isolated peripheral blood T cells only in the presence of monocytes/macrophages and requires binding of the Fc portion of antibody to monocytes/macrophages. In this investigation, we examined whether monocytes process anti-CD3 similar to any soluble antigen and present to T cells in context with HLA-DR to induce maximal DNA synthesis. Adherent monocytes were pulsed with anti-CD3 MoAb in the presence or absence of the lysozomotropic agents chloroquine and monensin, which are known to inhibit processing of soluble antigens, washed extensively, and then incubated with autologous T cells in the absence of soluble anti-CD3, and³H-thymidine incorporation and CD25 expression were measured. Both monensin and chloroquine inhibited anti-CD3-pulsed monocyte-induced T-cell DNA synthesis and CD25 expression in a dose-dependent manner. This inhibitory effect was not due to any loss in cell viability or the effect on the expression of HLA-DR on monocytes. Paraformaldehyde-fixed monocytes pulsed with anti-CD3 MoAb induced significantly less DNA synthesis, HLA-DR expression, and CD25 antigen expression on autologous T cells as compared to responses induced by unfixed anti-CD3-pulsed monocytes. The treatment of anti-CD3-pulsed monocytes with frame-work-specific anti-HLA-DR MoAb inhibited their capacity to induce T-cell DNA synthesis. These data suggest that monocytes, in addition to serving as the matrix for cross-linking, also process anti-CD3 MoAb and present to the T cells in the context of HLA-DR antigens to induce optimal DNA synthesis.     

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-α and interferon-γ

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), we investigated the effects of these two cytokines on death of luteal cells in vitro. Methods: Mouse luteal cells were cultured in serum-free medium with TNF-α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN-γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. Results:On day 3 of culture, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-γ (1,000 U/ml) and TNF-α (5,000 U/ml) did. On day 6, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-α alone at 5,000 U/ml did, and combinations of IFN-γ and TNF-α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-α. On days 3–6 of culture, combinations of IFN-γ and TNF-α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. Conclusions: The presence of IFN-γ modulates the ability of TNF-α to induce a reduction in the number of viable cells, although TNF-α alone at high concentrations can induce a reduction in the number of viable cells. © 1995 Wiley-Liss, Inc.     

Control of glucose-6-phosphate dehydrogenase deficiency on the formation of mutagenic and carcinogenic metabolites derived from benzo(a)pyrene

It has been observed that human lymphocytes (HL) and fibroblasts, isolated in vitro from donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD), show a great decrease in this enzymatic activity, the hexose monophosphate shunt, and the NADPH/NADP+ ratio. This effect is associated with a decreased sensitivity of G6PD-deficient cells to the benzo(a)pyrene (BaP) cytotoxic effect and to a decreased in vitro transformation of BaP-treated fibroblasts. Further, benzo(a)anthracene (BaA)-induced BaP hydroxylase activity is lower in G6PD-deficient cells, when measured in the presence of endogenous NADPH. It has been hypothesized that the NADPH level could be rate-limiting for the NADPH-dependent steps of BaP metabolic activation. To test this hypothesis, the formation of BaP metabolites was studied in normal and G6PD-deficient HL incubated with the carcinogen. HPLC profiles of organic-soluble metabolites revealed that both types of HL produced all the following known BaP metabolites: 9,10-, 4,5- and 7,8-dihydrodiols, quinones, 9- and 3-hydroxy and two peaks of more polar metabolites. There was a great decrease of the various metabolites in the deficient HL. A decrease of total water-soluble BaP metabolites also occurred. HL formed mutagenic metabolites for S. typhimurium TA100 (his-) when incubated with the rat liver S9 fraction. When intact HL substituted S9 fraction, a significant reversed mutation occurred only with normal HL. This could indicate that the NADPH pool is inadequate in G6PD-deficient HL for active BaP metabolism. Accordingly, deficient HL formed lower amounts of BaP:DNA adducts than control during incubation with BaP.     

微丝重组对细胞增值作用的研究

用罗丹明－鬼笔环肽（ｒｈｏｄａｍｉｎｅ－ｐｈａｌｌｏｉｄｉｎ）显示微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔｓ，ＭＦ）的荧光染色法对Ｇ期小鼠Ｃ３Ｈ１０Ｔ１／２成纤维细胞向Ｓ期过渡过程中，ＭＦ结构的变化及ＭＦ重组对ＤＮＡ合成的影响进行了研究，Ｇ期细胞在血清刺激１ｈ后ＭＦ解聚，３ｈ后重新组装，并恢复原来的分布。我们发现细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ，ＣＢ）使ＭＦ解聚并与蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ，ＰＫＣ）激活剂──佛波酯（ｐｈｏｒｂｏｌｅｓｔｅｒ，ＴＰＡ）同样可促进Ｇ期细胞提前进入Ｓ期，促进ＤＮＡ合成，但ＴＰＡ和ＣＢ的刺激作用必需依赖于血清的存在，而ＰＫＣ抑制剂Ｈ７则阻断Ｇ期细胞进入Ｓ期，ＭＦ稳定剂ｐｈａｌｌｏｉｄｉｎ对ＤＮＡ合成具有一定的抑制作用，实验结果表明，Ｇ期细胞向Ｓ期过渡的早期阶段存在ＭＦ重组过程，ＣＢ促使Ｇ细胞ＭＦ解聚并可促进ＤＮＡ合成，提示ＭＦ重组是Ｇ期细胞向Ｓ期过渡的一个重要的早期事件。