邻苯二甲酸二丁酯诱导孕鼠尿道下裂模型后仔鼠睾丸细胞过氧化物酶6的表达

背景:前期实验发现在邻苯二甲酸二丁酯孕期暴露诱导的仔鼠睾丸发育中蛋白过氧化物酶6表达显著增高.目的:进一步验证邻苯二甲酸二丁酯胚胎期暴露对胎鼠睾丸蛋白过氧化物酶6表达的影响.方法:妊娠14~18 d按800 mg/(kg?d) 邻苯二甲酸二丁酯染毒孕鼠,妊娠21 d取出胚胎大鼠睾丸,提取蛋白,通过Western blotting及免疫组织化学方法检测睾丸细胞过氧化物酶6的表达,以正常雄性仔鼠睾丸为对照.结果与结论:Western blotting检测显示,实验组过氧化物酶6的相对表达量为0.205±0.020,对照组为0.110±0.020,实验组明显高于对照组(P < 0.05).免疫组织化学方法发现过氧化物酶6主要定位于胎鼠睾丸Leydig细胞中.说明过氧化物酶6可能通过发挥抗氧化作用及刺激Leydig细胞增殖功能抵抗邻苯二甲酸二丁酯的毒性作用,其功能有待进一步研究.     

增塑剂邻苯二甲酸二丁酯致小鼠睾丸组织的病理学改变

背景:目前国内外研究邻苯二甲酸二丁酯对生殖损害研究对象多为大鼠,且不同时间点下小鼠病理组织学未见,以小鼠为移植对象没有明确移植时间.目的:探讨邻苯二甲酸二丁酯致小鼠睾丸组织病理学改变,并找出其改变最大的时间点,为移植做准备.方法:妊娠balb/c小鼠20只随机分成3组,分别为正常对照组6只,玉米油对照组6只,DBP组8只.自妊娠12~21 d,分别经口给予邻苯二甲酸二丁酯和玉米油,分别在小鼠出生后4~8周每间隔1周观察仔代雄小鼠睾丸的组织病理学改变,找到变化最大的时间点.结果与结论:邻苯二甲酸二丁酯组染毒小鼠睾丸出现明显的生理、病理和电镜下的改变.邻苯二甲酸二丁酯可引起雄性仔鼠性分化异常,睾丸生精上皮损害和生精过程障碍,从而对雄性仔鼠生育力产生不利影响,在5,6周小鼠睾丸组织损害最大,可以作为移植变化最大时间进行选择.     

增塑剂邻苯二甲酸二丁酯致小鼠睾丸组织的病理学改变

背景：目前国内外研究邻苯二甲酸二丁酯对生殖损害研究对象多为大鼠,且不同时间点下小鼠病理组织学未见,以小鼠为移植对象没有明确移植时间。目的：探讨邻苯二甲酸二丁酯致小鼠睾丸组织病理学改变,并找出其改变最大的时间点,为移植做准备。方法：妊娠balb/c小鼠20只随机分成3组,分别为正常对照组6只,玉米油对照组6只,DBP组8只。自妊娠12～21d,分别经口给予邻苯二甲酸二丁酯和玉米油,分别在小鼠出生后4～8周每间隔1周观察仔代雄小鼠睾丸的组织病理学改变,找到变化最大的时间点。结果与结论：邻苯二甲酸二丁酯组染毒小鼠睾丸出现明显的生理、病理和电镜下的改变。邻苯二甲酸二丁酯可引起雄性仔鼠性分化异常,睾丸生精上皮损害和生精过程障碍,从而对雄性仔鼠生育力产生不利影响,在5,6周小鼠睾丸组织损害最大,可以作为移植变化最大时间进行选择。     

膜联蛋白5对大鼠睾丸间质细胞增殖过程中Ect2表达的影响

目的研究膜联蛋白5(annexin 5)对大鼠睾丸间质细胞增殖过程中上皮细胞转化序列2癌基因(epithelial cell transfor-ming sequence 2 oncogene,Ect2)表达的影响,探讨annexin 5影响睾丸间质细胞增殖的机制。方法原代培养大鼠睾丸间质细胞,不同剂量的annexin 5处理后,采用MTT法检测细胞增殖活力,流式细胞术检测细胞周期,RT-PCR检测Ect2的mRNA表达改变,western blot分析睾丸间质细胞中Ect2蛋白质表达变化。结果 MTT法检测结果显示,annexin 5对大鼠睾丸间质细胞增殖有明显的促进作用,且在2～3 d呈显著的剂量-时间依赖关系(P<0.01),在第5天细胞增殖作用已明显减弱。流式细胞分析发现,1 nmol/L annexin 5作用48 h时G2/M期细胞减少为24.49%,72 h时减少为16.43%,与对照组比较差异显著(P<0.05)。RT-PCR结果表明,0.1 nmol/L组和1 nmol/L组Ect2 mRNA表达[(0.77±0.06)和(0.85±0.04)]与对照组(0.67±0.06)比较,分别增加了14.9%(P<0.05)和26.9%(P<0.01),而10 nmol/L组Ect2 mRNA表达与对照组比较差异无统计学意义(P>0.05)。western blot结果表明,1 nmol/L annexin 5作用组的Ect2蛋白质表达比对照组增加了20.9%[(1.50±0.15)vs(1.24±0.07),P<0.05],而0.1 nmol/L组和10 nmol/L组与对照组比较差异无统计学意义(P>0.05)。结论 annexin 5对大鼠睾丸间质细胞增殖的促进作用是通过促进细胞周期由G2期向M期的转变来实现的。Ect2在基因和蛋白质水平均受annexin 5的影响,提示annexin 5调控睾丸间质细胞增殖可能通过增加Ect2的表达而实现。     

早期干预对宫内感染致脑损伤仔鼠脑组织NB-3表达的影响

目的探讨早期干预对仔鼠脑组织NB-3的表达及其运动功能的影响。方法孕17~18 d Wistar大鼠45只腹腔注射脂多糖(LPS)380μg/kg连续2 d(LPS组),另15只注射同等剂量的无菌生理盐水(NS组)。随机选取LPS组仔鼠80只,分为干预组40只(I组)和非干预组40只(NI组);随机选取NS组仔鼠40只为对照组。早期干预措施主要给予早期触摸与丰富康复训练。仔鼠出生后24 h内在LPS组和NS组随机取5只行脑组织HE染色。其余大鼠分别于出生后14 d、28 d行悬吊试验,1 d、7 d、14 d、21 d、28 d行脑组织免疫组织化学染色,观察NB-3的表达。结果脑组织NB-3的表达及悬吊试验评分三组间均有非常高度显著性差异(P<0.001)。结论宫内感染可致脑损伤鼠NB-3表达增加,早期干预使脑损伤鼠NB-3表达持续增加,并改善其运动功能。     

福尔马林致痛大鼠背根节内5-HT受体亚型mRNA的表达变化

目的:探讨外周5-HT受体亚型在外周伤害性感受中的作用.方法:用反转录PCR技术观察大鼠单侧足底皮下注射福尔马林致痛后背根节内5-HT1～7受体亚型mRNAs的表达变化.结果:在大鼠足底皮下注射福尔马林后1 h,注射侧腰段背根节内5-HT1A、5-HT1B、5-HT2A、5-HT3、5-HT4和5-HT7受体亚型mRNAs的表达水平显著升高,而5-HT1D、5-HT1F、5-HT2C、5-HT5A和5-HT6受体亚型mRNAs的表达在福尔马林致痛后无明显变化.在正常和福尔马林致痛大鼠的背根节内均未检测到5-HT1E、5-HT2B和5-HT5B受体亚型mRNAs的表达.结论:5-HT1A、5-HT1B、5-HT2A、5-HT3、5-HT4和5-HT7受体亚型可能参与了福尔马林诱导的炎性痛,且它们在外周伤害性信息的传递方面可能发挥不同的作用.     

急性心肌缺血期大鼠丘脑束旁核神经元5-HT1A受体mRNA表达的变化

目的：观察急性心肌缺血期大鼠丘脑柬旁核（parafascicular nucleus，Pf）神经元5-HT1A受体mRNA不同时点表达的变化，探讨5-HT1A受体在Pf痛觉整合和调制中的作用。方法：体重260～280g的健康成年雄性SD大鼠24只，随机分为4组，即非冠状动脉扎闭组（C组）、扎闭冠脉（coronary artery occlusion，CAO）lh组（CAO 1h组）、扎闭冠脉3h组（CAO 3h组）和扎闭冠脉6h组（CAO 6h组）。C片行原位杂交。杂交结果检测采用IDA-2000数码显微图像分析系统进行半定量分析。结果：CAO后1、3、6h大鼠Pf神经元5-HT1A受体mRNA的表达均较C组明显增强（P〈0．05），随缺血时间延长其表达有逐渐增加的趋势，在CAO后6h表达最强（P〈0、05）。结论：急性心肌缺血可诱发大鼠Pf神经元5-5-HT1A受体mRNA表达增强，5-HT1A受体参与急性心肌缺血伤害性刺激在Pf的调制。     

急性心肌缺血期大鼠丘脑束旁核神经元5-HT1A受体mRNA表达变化的研究

目的 观察急性心肌缺血期大鼠丘脑束旁核神经元5-HT1A受体mRNA不同时间点表达的变化，以探讨5-HT1A受体在丘脑束旁核痛觉整合和调制中的作用。方法 健康成年雄性SD大鼠24只，随机分为四组：对照组，即非冠状动脉扎闭（C组）组，扎闭冠状动脉（coronary artery occlusion，CAO）1h组（CAO1h组），扎闭冠状动脉3h组（CAO3h组）和扎闭冠状动脉6h组（CAO6h组）。对照组仅在开胸后冠状动脉左前降支下穿线不予结扎；CAO各组则扎闭冠状动脉左前降支。在预定的时点处死动物，取含有大鼠丘脑束旁核的脑片行原位杂交。杂交结果检测采用IDA-2000数码显微图像分析系统进行半定量分析。结果 CAO后1、3、6h大鼠丘脑束旁核神经元5-HT1A受体mRNA的表达均较对照组明显增强（P〈0．05），随缺血时间延长其表达有逐渐增加的趋势，在CAO后6h表达最强（P〈0．05）。结论 急性心肌缺血可诱发大鼠丘脑束旁核5-HT1A受体mRNA表达增强，5-HT1A 受体参与急性心肌缺血伤害性刺激在丘脑束旁核的调制。     

Donor exposure to the plasticizer di(2-ethylhexyl)phthalate during plateletpheresis

BACKGROUND: Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is contained in most PVC devices, including apheresis disposables. Because DEHP can be extracted from apheresis disposables as the blood passes through the apheresis device, DEHP exposure was determined in healthy donors undergoing plateletpheresis performed with commercially available apheresis systems. STUDY DESIGN AND METHODS: The study population consisted of 36 healthy PLT donors undergoing plateletpheresis with either continuous or discontinuous apheresis devices. Serum concentrations of DEHP were determined from peripheral blood obtained before and after plateletpheresis, with gas chromatography-mass spectroscopy. RESULTS: Plateletpheresis performed with standard collection disposables resulted in a median increase of 232 percent of serum DEHP compared to levels before apheresis, corresponding to a total amount of DEHP exposed during a single apheresis of a median of 6.46 (range, 1.8-20.3) microg per kg of body weight. Endogenous levels of triglycerides showed a positive correlation with the amount of DEHP released. Increase in serum DEHP was short-term as serum DEHP rapidly returned to levels obtained before apheresis within 3 hours after completion of the apheresis course. Donor exposure to DEHP led to no variation in liver cell function within 48 hours after plateletpheresis. CONCLUSION: Commercial plateletpheresis disposables release considerable amounts of DEHP during the apheresis procedure, but the total dose of DEHP retained by the donor is within the normal range of DEHP exposure of the general population.     

Transfusion-related exposure to the plasticizer di(2-ethylhexyl)phthalate in patients receiving plateletpheresis concentrates

BACKGROUND: Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that can leach from medical devices including storage bags for plateletpheresis concentrates (PCs). In this study, the DEHP exposure to patients receiving PCs was determined and several variables were evaluated to reduce DEHP load to PC recipients. STUDY DESIGN AND METHODS: In 12 patients, serum DEHP was assessed before and after PC transfusion. For in vitro investigations, PCs were produced either with donor plasma or with 65 percent additive solution (AS; T-Sol) and stored for 5 days. Washing of PCs was performed according to AABB standards. DEHP levels were determined by gas chromatography-mass spectrometry. RESULTS: Transfusion of PCs led to a significant increase in serum DEHP. DEHP levels in the PCs continuously increased during storage, although the accumulation of DEHP was less in PCs stored in the AS, T-Sol, than when stored in plasma. Storage-related accumulation of DEHP contributed to a major part of the total DEHP load in PCs stored for 5 days. Washing of PCs led to a reduction of DEHP load. CONCLUSION: Recipients of PCs are exposed to DEHP, although the total amount represents only a small percentage of the defined tolerable intake. Reduction of storage time, the storage of PC in T-Sol, or the exchange of the storage medium before transfusion are practicable means to reduce the DEHP load in PC.     

Effect of lipoprotein (a) on annexin A5 binding to cell membrane

BackgroundHigh blood lipoprotein (a) [Lp(a)] concentration is a risk factor for a thrombotic event. Annexin A5 is involved in anticoagulation on the endothelial surface. How Lp(a) affects the annexin A5 function is not clear. This study investigates annexin A5 binding on the cell membrane in the presence of Lp(a).MethodsLp(a) was isolated from human blood plasma by ultracentrifugation and annexin A5 protein was purchased commercially. The cell membrane was prepared from primary human umbilical vein endothelial cells (HUVEC) and cultured cell line HepG2 by sucrose density gradient centrifugation. Enzyme-linked immunosorbent assays (ELISA) were used to examine annexin A5 binding to the cell membrane in the presence of Lp(a). Flow cytometry was used to analyze the binding of fluorescence-labeled annexin A5 to phosphatidylserine (PS)-translocated intact cells in the presence of Lp(a).ResultsAnnexin A5 binding to the cell membrane was attenuated by a high concentration of Lp(a) in both HUVEC and HepG2 membrane surfaces. The phenomenon was also observed with annexin A5 surface labeling of HepG2 cells and flow cytometry analysis.ConclusionsThe results imply that Lp(a) interferes with annexin A5 binding to the procoagulant PS which translocates to the membrane surface under stress condition and therefore may increase the risk for thrombosis.     

Outcome of fetal talipes following in utero sonographic diagnosis.

OBJECTIVES: To examine the association between fetal talipes and other defects, and outcome in relation to postnatal surgery. METHODS: All cases of talipes presenting to the fetal medicine unit between 1993 and 1998 and cases of isolated talipes presenting to the ultrasound department between 1991 and 1998 were examined. The infants were followed-up to determine the number of cases that had structural or positional talipes and the number of cases requiring surgery. RESULTS: There were 76 cases, 59 of which attended the fetal medicine unit and 17 the ultrasound department. Postnatal follow-up details were available in 31 of the 40 live births. There were three neonates with unilateral talipes at birth who were thought to have bilateral talipes on prenatal ultrasound and one neonate had bilateral talipes at birth who had been thought to have unilateral talipes prenatally. In two (6.4%) neonates in whom talipes was not confirmed at birth the abnormality was diagnosed prenatally. Of the 29 neonates with confirmed talipes at birth, the defect was structural in 26 (90%) cases and positional in three. Surgery was necessary in 21 (72%) of the 29 cases and 18 (86%) of those undergoing surgery required only one operation. When live births with associated anomalies were excluded, there were 24 cases with confirmed isolated talipes and 18 (75%) required surgery. CONCLUSIONS: This study provides long-term outcome data which can be used to complement current prenatal counseling and shows that in cases of fetal talipes diagnosed prenatally, 90% have a structural rather than a positional deformity. For isolated talipes three quarters of children will require surgery and in the majority of cases only one operation on the foot is necessary. Parents should be made aware of the small possibility of a false-positive diagnosis and discrepancy between the ultrasound and postnatal diagnoses of laterality.     

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.     

A convenient separation method for di(2-ethylhexyl)phthalate by novel superparamagnetic molecularly imprinted polymers

Through a surface molecular imprinting technique and coating superparamagnetic Fe₃O₄ nanoparticles with molecularly imprinted polymers (MIPs), a novel magnetic molecularly imprinted polymer (MMIP) was successfully fabricated for the convenient separation of di(2-ethylhexyl)phthalate (DEHP) with methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross linker and 2,2-azobis(isobutyronitrile) as initiator. A magnetic non-molecularly imprinted polymer (MNIP) was also prepared for comparison purposes. The morphology structure and the magnetic properties of MMIP were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectrometry (FT-IR), X-ray diffraction, vibrating sample magnetometry (VSM) and thermo gravimetric analysis (TGA). The adsorption properties of MMIP and MNIP were investigated by static and dynamic adsorption experiments. The results show that the diameter of the synthesized MMIP microspheres is about 300–500 nm with good dispersibility in solvent. The prepared MMIP shows superparamagnetic properties with the maximum saturation magnetic intensity of 43.97 emu g⁻¹, and it can be conveniently separated using an external magnetic field. Compared with MNIP, MMIP has a higher adsorption capacity and better adsorption selectivity for DEHP, and the imprinting factor reaches 3.012. The regeneration adsorption experiment illuminates that the novel MMIP can be reused with good separation efficiency.

Through surface molecular imprinting technique and coating superparamagnetic Fe₃O₄ nanoparticles with molecularly imprinted polymers, a novel MMIP was successfully fabricated for the convenient separation of DEHP.     

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression

The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development.