2, 3, 7, 8-四氯代二苯并二噁英(TCDD)和苯并芘(B[a]P)对原代培养鲫鱼肝细胞中卵黄蛋白原诱导的影响

通过测定原代培养鲫鱼(Carassius auratus)肝细胞中雌激素受体所介导的卵黄蛋白原(Vtg)生成以及芳香烃受体所介导的CYP1A1基因转录水平的变化, 建立了一种类雌激素体外实验模型. 实验结果表明, Vtg和Vtg mRNA的表达与己烯雌酚(DES)之间均有很好的剂量-效应关系, Vtg和Vtg mRNA均可作为指示类雌激素毒性的生物标志物. TCDD, B[a]P可显著抑制鱼肝细胞中DES诱导的Vtg和Vtg mRNA的表达, 呈明显的抗雌激素效应, 并同时激活了CYP1A1 基因的表达; 但0.1和0.2 pg/mL的TCDD和5 ng/mL的B[a]P对Vtg和Vtg mRNA表达却有一定的促进作用, 表明TCDD和B[a]P在不同的浓度下, 可能呈现出相反的毒性效应; b-naphthoflavone(β-NF)可抑制鱼肝细胞中DES诱导的Vtg和Vtg mRNA的表达, 并可激活CYP1A1基因的表达; Tamoxifen抑制了鱼肝细胞中Vtg和Vtg mRNA的表达, 但不能诱导CYP1A1基因的表达; 而TCDD诱导细胞CYP1A1基因的表达同样可被DES抑制; 上述实验结果说明鱼肝细胞中分别受雌激素受体和芳香烃受体所介导的途径之间存在某些相互作用关系, 这为研究类雌激素复合暴露下的致毒机理提供了新的实验证据.     

卵黄蛋白原的性质及热休克蛋白90对其合成代谢的调控

卵黄蛋白原(VTG)广泛存在于卵生脊椎动物和无脊椎动物的卵母细胞中,为卵母细胞的生长和胚胎的发育提供营养。VTG一般在卵巢、肝胰脏和(或)脂肪等组织中合成。VTGcDNA具有较强的保守性,为7 kB左右。VTG具有单体、二聚体等多种形式,其中二聚体有同源二聚体和异源二聚体两种形式。脊椎动物体内,热休克蛋白90(HSP90)通过和雌激素受体结合间接诱导VTG的合成,近年来的研究报道表明HSP90同样对无脊椎动物体内VTG合成也具有诱导作用,但其作用机制有待进一步研究。     

海河地区部分水体雌激素活性的初步调查

以野生鱼血清中的卵黄蛋白原(Vtg)为雌激素活性生物标记物, 对海河流域部分(子牙河, 入海口)水体中污染物的雌激素活性进行了初步调查, 并测定了水样中双酚A(BPA)、辛基酚(OP)和壬基酚(NP)的浓度. 结果显示, 在不同季节, 不同采样点的野生雌鱼和雄鱼血清中都有较高浓度的Vtg检出, 表明这些水体中存在一定程度的雌激素活性化合物污染. 采样点水体中均能检测到BPA, NP和OP, 但浓度远低于导致鱼Vtg生成的最低显效浓度.     

2,3,7,8-四氯代二苯并二英(TCDD)和苯并芘(B[a]P)对原代培养鲫鱼肝细胞中卵黄蛋白原诱导的影响

通过测定原代培养鲫鱼(Carassius auratus)肝细胞中雌激素受体所介导的卵黄蛋白原(Vtg)生成以及芳香烃受体所介导的CYP1A1基因转录水平的变化, 建立了一种类雌激素体外实验模型. 实验结果表明, Vtg和Vtg mRNA的表达与己烯雌酚(DES)之间均有很好的剂量-效应关系, Vtg和Vtg mRNA均可作为指示类雌激素毒性的生物标志物. TCDD, B[a]P可显著抑制鱼肝细胞中DES诱导的Vtg和Vtg mRNA的表达, 呈明显的抗雌激素效应, 并同时激活了CYP1A1 基因的表达; 但0.1和0.2 pg/mL的TCDD和5 ng/mL的B[a]P对Vtg和Vtg mRNA表达却有一定的促进作用, 表明TCDD和B[a]P在不同的浓度下, 可能呈现出相反的毒性效应; b-naphthoflavone(b-NF)可抑制鱼肝细胞中DES诱导的Vtg和Vtg mRNA的表达, 并可激活CYP1A1基因的表达; Tamoxifen抑制了鱼肝细胞中Vtg和Vtg mRNA的表达, 但不能诱导CYP1A1基因的表达; 而TCDD诱导细胞CYP1A1基因的表达同样可被DES抑制; 上述实验结果说明鱼肝细胞中分别受雌激素受体和芳香烃受体所介导的途径之间存在某些相互作用关系, 这为研究类雌激素复合暴露下的致毒机理提供了新的实验证据.     

2,3,7,8-四氯代二苯并二(口恶)英(TCDD)和苯并芘(B[a]P)对原代培养鲫鱼肝细胞中卵黄蛋白原诱导的影响

通过测定原代培养鲫鱼(Carassius auratus)肝细胞中雌激素受体所介导的卵黄蛋白原(Vtg)生成以及芳香烃受体所介导的CYP1A1基因转录水平的变化,建立了一种类雌激素体外实验模型.实验结果表明,Vtg和Vtg mRNA的表达与己烯雌酚(DES)之间均有很好的剂量-效应关系,Vtg和Vtg mRNA均可作为指示类雌激素毒性的生物标志物.TCDD,B[a]P可显著抑制鱼肝细胞中DES诱导的Vtg和VtgmRNA的表达,呈明显的抗雌激素效应,并同时激活了CYP1A1基因的表达;但0.1和0.2 pg/mL的TCDD和5 ng/mL的B[a]P对Vtg和Vtg mRNA表达却有一定的促进作用,表明TCDD和B[a]P在不同的浓度下,可能呈现出相反的毒性效应;β-naphthoflavone(β-NF)可抑制鱼肝细胞中DES诱导的Vtg和Vtg mRNA的表达,并可激活CYP1A1基因的表达;Tamoxifen抑制了鱼肝细胞中Vtg和Vtg mRNA的表达,但不能诱导CYP1A1基因的表达;而TCDD诱导细胞CYP1A1基因的表达同样可被DES抑制;上述实验结果说明鱼肝细胞中分别受雌激素受体和芳香烃受体所介导的途径之间存在某些相互作用关系,这为研究类雌激素复合暴露下的致毒机理提供了新的实验证据.     

Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp （Carassius auratus） hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in ritro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL)seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1μmol/L),and β-naphtho-flavone (β-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with β-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for β-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.     

克氏原螯虾(Procambius clarkii)卵黄蛋白的部分生化性质

通过电泳回收的方法对克氏原螯虾(Procambius clarkii)的卵黄蛋白进行了纯化,鉴定结果显示总分子质量为481 ku,具有6个亚基(198,176,132,111,92,82 ku);染色分析表明该蛋白是一种糖脂磷类胡萝卜素蛋白,亚基之间有1个二硫键交联.     

菊黄东方鲀卵黄蛋白原肽段vWD的真核重组表达及功能研究

河鲀的卵黄蛋白原可能参与河豚毒素（TTX）的体内转运过程。为了探讨该机理,建立了毕赤酵母真核表达系统,成功地表达了菊黄东方鲀（Takifugu flavidus）卵黄蛋白原的vWD结构域肽段（rTF_vWD）,并采用Biacore-表面等离子体共振（SPR）系统检测了rTF_vWD与TTX的亲和力,以及腹腔注射小鼠验证rTF_vWD对TTX毒力的中和作用。结果表明:rTF_vWD与TTX的平衡解离常数（KD）为3.1 mmol/L;将TTX与rTF_vWD共孵后肌肉注射小鼠,2 h内小鼠的死亡率显著下降,表明TTX与rTF_vWD结合后,TTX的毒性降低了。     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

双酚A对泥鳅卵黄蛋白原的诱导

研究了不同浓度[（0．1—500）μg／L）]、不同时间（7d,14d）BPA暴露对雄性泥鳅卵黄蛋白原的诱导效应。结果表明,雄性泥鳅血清Vtg含量随BPA暴露时间和暴露浓度的增加而显著增加。暴露14d后,诱导雄性泥鳅血清Vtg显著性增加的最低BPA浓度为1μg/L。