Teratogenic effect of bis-diamine on embryonic rat heart

ABSTRACT  Bis-diamine induces conotruncal anomalies including persistent truncus arteriosus, tetralogy of Fallot, interruption of the aortic arch, and ventricular septal defect in rat embryos when administered to the mother. Bis-diamine also induces extracardiac malformations including thymic hypoplasia, facial dysmorphism, forelimb anomalies and diaphragmatic hernia. However, the teratogenic mechanisms of this chemical in early developing rat hearts have not been fully established. Chimeric studies in chick and quail embryos demonstrated that the cranial neural crest cells reached the cardiac outflow tract, contributing to aorticopulmonary and truncal septation. Since an ablation of the cranial neural crest also produced the conotruncal anomalies, bis-diamine is proposed to disturb the normal migration of cardiac neural crest cells to the heart. Based on our data concerning cardiac anomalies induced by bis-diamine, we reviewed how the cardiac malformations were morphologically established in early developing rat hearts. Our data showed that 1) cardiovascular anomalies induced by bis-diamine are time- and species or strain- dependent. 2) bis-diamine reduces the number of neural crest cells migrating to participate in the conotruncal septation, 3) bis-diamine induces anomalous coronary arteries, thin ventricular walls and epicardial defects, and 4) some embryos cultured in the medium containing bis-diamine had extra-cardiac abnormalities including abnormal location of the otic placodes and delay in mid brain closure. Conclusively, bis-diamine does not appear to merely affect the cardiac development, but rather disturbs normal development of all the organs contributed to by neural crest cells.     

Participation of Neural Crest Cells in Cardiovascular Morphogenesis of Chick Embryos

Neural crest cells appear on top of the neural folds at closure of the neural tube. These cells are capable of migrating and differentiating into multiple tissues. In 1983, Kirby et al., using chimera methodology, reported that cranial neural crest cells participated in aorticopulmonary and truncal septation. Since that time, many experimental studies have been done in this field. In this paper, we report new experimental findings and review four areas of study on neural crest cells, i.e. their general characteristics, participation of neural crest cells in formation of the normal heart, cardiovascular anomalies due to ablation of the neural crest cells, and the relationship between neural crest cells and congenital malformation syndromes, especially the DiGeorge sequence.     

Immunohistochemical Distribution of HNK-1 and N-CAM in Rat Embryos Treated with Bis-Diamine

Bis-diamine is known to induce congenital anomalies including cardiac defects, thymic hypoplasia and snout defects in rat embryos. Cardiovascular lesions consist of persistent truncus arteriosus, tetralogy of Fallot and aortic arch anomalies, which are often found in DiGeorge syndrome. In the the present study, we investigated effects of bisdiamine on cardiac neural crest cells, which may be important to the teratogenecity. A single dose of 200 mg bis-diamine was administered to pregnant rats at 10.5 embryonic day (ED). Immunohistochemical studies were performed on embryos at 11.5, 12.5 and 13.5 ED using anti-HNK-1 and anti-N-CAM antibodies. The embryos at 11.5 and 12.5 ED showed the similar distributional patterns of either HNK-1 or N-CAM positive cells in control and bis-diamine treated embryos. N-CAM immunoreactivities in the splanchnic mesoderm were quite weakly detectable in the bis-diamine treated embryos at 12.5 ED. Closure of the neural tube was delayed in the treated embryos at this period. Immunohistochemistry of the control embryos at 13.5 ED demonstrated a continuous chain of N-CAM expression between the neural plexus near the foregut and the fourth aortic arch, while no apparent continuity of N-CAM positive tissue was observed in the bis-diamine treated embryos. These findings suggested that bis-diamine reduced the amount of neural crest cells which appeared to participate in the aortic arch formation or conotruncal septation. The primary effect of bis-diamine in the induction of various congenital anomalies including cardiovascular malformations may be an inhibition of the normal development of the neural tube and neural crest.     

高脂饮食诱导下肥胖易感大鼠与肥胖抗性大鼠间部分生理学指标差异的研究

目的：探讨高脂饮食诱导下肥胖易感（OP）大鼠与肥胖抗性（OR）大鼠在体重、食量、Lee′s指数、脂肪湿重、血脂及脂肪组织蛋白脂酶（LPL）基因表达等方面的生理学差异。方法：48只大鼠，以高脂饲料饲养1周后，按体重增殖从大到小百分排序，取第75百分位以上 OP组，第25百分位以下为OR组，继续高脂饲养4周；观察各大鼠体重变化、摄食量、Lee′s指数、脂肪湿重、血脂等，采用Northern Blot分析脂肪组织中LPL基因的mRNA表达水平。结果：1.OP大鼠的体重增殖、热能摄入、Lee′s指数及腹腔脂肪湿重均显著高于OR大鼠，但血脂水平两组间无差异；2.高脂饲养1周、5周时OP大鼠体重增值分别是OP大鼠的6.54、4.25倍，热能摄入仅为OR大鼠的1.13、1.15倍；3.OP大鼠脂肪组织中LPL基因的mRNA表达水平显著高于OR大鼠。结论：OP与OR大鼠间存在生理学差异，其原因除OP大鼠热能摄入较多因素外，OP大鼠体内较高的能量效能是其肥胖易感性较高的重要因素，而能量效能与脂肪组织LPL基因的表达水平可能存在密切关系。     

Simple in vitro migration assay for neural crest cells and the opposite effects of all‐trans‐retinoic acid on cephalic‐ and trunk‐derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all‐trans‐retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.     

Morphological and biochemical changes induced by arsenic trioxide in neuroblastoma cell lines

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in a variety of hematologic malignancies, but very little is known about its effects on solid tumors and especially on neuroblastoma cells that have self-differentiating characteristics. To demonstrate the growth inhibition induced in neuroblastoma cells (the SH-SY5Y and SK-N-AS cell line) and acute promyelocytic leukemia cells (HL-60) by arsenic trioxide (As₂O₃), the viable cell numbers were counted after trypan blue staining. Apoptosis was assessed by the cell morphology, by flow cytometry with annexin-V staining, and by Western blot analysis for the apoptosis-related proteins (bcl-2 and PARP). To decide the dose for the clinical application of As₂O₃, normal peripheral blood lymphocytes were also examined. The growth and survival of the SH-SY5Y and SK-N-AS cells were markedly inhibited by As₂O₃ treatment at a 3 μM concentration before the changes of the normal lymphocytes were observed. The apoptotic cells showed a shrunken cell nucleus, and an increase in the number and balloon-like swelling of the mitochondria at 72 h after the As₂O₃ was added. Apoptosis of the annexin-V-positive cell proportion in the neuroblastoma cell lines was increased with increasing the exposure time and the concentration of As₂O₃, just like the HL-60 cells. Bcl-2 downregulation and PARP degradation were also noted all the cell lines, but these changes were not statistically significant among the 3 cell lines. Taken together, these results indicate that As₂O₃ is an excellent candidate as a therapeutic agent for the treatment of neuroblastoma.     

需肌醇酶1介导的内质网应激通路参与高氧所致早产大鼠肺泡Ⅱ型上皮细胞凋亡

目的探讨需肌醇酶1(IRE1)介导的内质网应激通路与高氧暴露肺泡Ⅱ型上皮细胞(AECⅡ)凋亡的关系。方法原代培养早产大鼠AECⅡ,随机分为空气组和高氧组,建立高氧细胞损伤模型。在24、48及72h收集细胞,倒置相差显微镜观察细胞形态变化;AnnexinV/PI双染流式细胞术检测细胞凋亡;RT-PCR及Westernblot分别检测葡萄糖调节蛋白78(GRP78)、IRE1、X盒结合蛋白1(XBP1)及C/EBP同源蛋白(CHOP)m RNA及蛋白表达;免疫荧光检测CHOP表达。结果随着给氧时间延长,高氧组AECⅡ伸展呈不规则形,出现空泡样改变;高氧组AECⅡ凋亡率与同时间点空气组比较明显增加(P0.05);随着氧暴露时间延长,高氧组GRP78、IRE1、XBP1及CHOPm RNA及蛋白表达升高,且较同时间点空气组明显上升(P0.05);高氧组CHOP荧光强度高于同时间点空气组。高氧组CHOP蛋白表达与AECⅡ凋亡率、IRE1及XBP1蛋白表达呈显著正相关(r=0.97、0.85、0.88,均P0.05)。结论高氧所致AECⅡ凋亡可能是通过激活IRE1-XBP1-CHOP通路来实现。     

免疫球蛋白对戊四氮致惊大鼠海马神经元死亡及IL-1β和IL-1ra表达的影响

目的探讨免疫球蛋白对戊四氮致惊大鼠海马神经元死亡及IL-1β和IL-1 ra表达的影响。方法在W istar大鼠腹腔内注射戊四氮(pentylenetetrazol,PTZ)诱发大鼠惊厥发作,制作癫模型。将45只大鼠随机分为3组,正常对照组,PTZ静脉注射免疫球蛋白组(PTZ-IVIG),PTZ-生理盐水组(PTZ-NS)。经HE和TUNEL染色,在光镜下,观察神经细胞死亡,采用免疫组织化学方法检测神经细胞内IL-1β和IL-1 ra表达。结果海马CA1区IL-1β/IL-1 ra比值PTZ-IVIG组(0.5±0.1)较PTZ-NS组(1.9±0.5)低,两者比较差异有统计学意义(t=12.9,P<0.05)。PTZ-IVIG组海马CA1区凋亡细胞数较PTZ-NS组少,阳性细胞数前者为(16.4±3.3)个/1000μm2,后者为(41.7±3.5)个/1000μm2,两者差异有统计学意义(t=27.1,P<0.05)。PTZ-IVIG组海马CA1区坏死细胞数少,为(19.0±2.6)个/1000μm2,PTZ-NS组为(42.3±4.9)个/1000μm2,两者差异有统计学意义(t=20.9,P<0.05)。结论免疫球蛋白具有抑制惊厥所致的神经元死亡的作用,其作用可能机制为调节神经细胞内白介素1网络系统。     

Roles of progesterone on suppression of uterine decidualization and implantation failure induced by triphenyltin chloride in rats

ABSTRACT  In our previous studies, an administration of triphenyltin chloride (TPTCl) at 4.7 or 6.3 mg/kg on days 0–3 of pregnancy caused implantation failure, and the same doses of TPTCl on days 0–3 of pseudopregnancy caused a suppression of uterine decidualization correlated with a reduction in serum progesterone levels in rats. This study was conducted to determine the roles of progesterone on the TPTCl-induced suppression of uterine decidualization and implantation failure in rats. Although lower uterine weight was found in hormone-maintained ovariectomized rats given TPTCl at 4.7 or 6.3 mg/kg on days 0–3 and induced decidual cell response on day 4, no statistical significance in the uterine weight was detected between the control group and the TPTCl-treated groups. The pregnancy rate and number of implantations in the groups given TPTCl at 4.7 or 6.3 mg/kg on days 0–3 of pregnancy and progesterone on days 0–8 of pregnancy were significantly higher than those in the groups given TPTCl alone. No significant differences in these parameters were found between the control group and the groups given TPTCl and progesterone. It can be concluded that the TPTCl-induced suppression of uterine decidualization is mediated, at least partially, via the ovarian hormones, and that progesterone protects against the TPTCl-induced implantation failure.     

解偶联蛋白2与脂多糖诱导脓毒症大鼠心肌线粒体损伤的关系

目的探讨解偶联蛋白2(uncoupling protein 2,UCP2)与脂多糖诱导脓毒症大鼠心肌线粒体损伤的关系。方法通过腹腔注射脂多糖(LPS)建立脓毒症模型。将40只Sprague-Dawley(SD)雄性大鼠随机分为5组,每组8只:对照组(腹腔注射生理盐水)、脓毒症6 h组(LPS-6 h组)、脓毒症12 h组(LPS-12 h组)、脓毒症24 h组(LPS-24 h组)和脓毒症48 h组(LPS-48 h组)。在相应时间点留取大鼠血清和心脏组织,提取心肌线粒体,通过酶标仪检测肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)和活性氧(ROS)水平,流式细胞仪检测线粒体肿胀度和线粒体膜电位(MMP),采用蛋白质免疫印迹试验(Western blot)测定UCP2蛋白表达水平;电镜观察心肌组织线粒体形态学变化。结果与对照组比较,LPS各组血清CK、CK-MB水平、心肌组织ROS水平和线粒体肿胀度均明显升高(P0.05),峰值在LPS-24 h组;而LPS各组线粒体膜电位明显下降(P0.05),LPS-24 h组降至最低。Western blot检测发现LPS各组心肌组织UCP2的表达水平较对照组明显升高(P0.05),峰值在LPS-24 h组。电镜观察结果发现LPS大鼠心肌线粒体肿胀,线粒体膜部分破碎,有空泡形成,LPS-24 h组病变最严重。LPS大鼠心肌线粒体ROS水平、线粒体肿胀度与UCP2表达量呈正相关(分别r=0.796、0.893,P0.05);LPS大鼠心肌MMP与UCP2表达量呈负相关(r=-0.903,P0.05)。结论在脓毒症大鼠模型中,心肌和心肌线粒体都明显损伤,心肌组织UCP2的表达与线粒体损伤密切相关,推测UCP2可能在脓毒症心肌线粒体损伤中起重要作用。     

高氧诱导早产鼠慢性肺疾病肺超微结构变化及氧化应激反应的研究

目的　慢性肺疾病 (CLD)是早产儿吸入高浓度氧治疗后最常见的并发症 ,目前认为肺部氧化应激反应与CLD的发生密切相关。该文探讨高氧致早产鼠CLD发生中肺组织超微结构及氧化应激反应的动态变化。方法　高浓度氧致早产鼠CLD模型 (实验组 )和正常对照组各 4 0例为研究对象 ,应用分光光度计比色法在实验后1 ,3,7,1 4 ,2 1d动态测定肺组织超氧化物岐化酶 (SOD)活性及脂质过氧化产物丙二醛 (MDA)的含量 ,并同步观察肺组织超微结构的变化。结果　吸高氧后初期 (1～ 3d) ,Ⅱ型肺泡上皮细胞 (AEC Ⅱ )即出现线粒体、板层小体等细胞器的损伤 ;7d后 ,除细胞器结构破坏外 ,开始出现细胞核的异常 ,且随吸氧时间的延长上述改变逐渐加重。实验组肺组织SOD的活性虽逐渐增高 ,但与对照组比较 ,其差异无显著性 (P >0 .0 5 ) ;实验组MDA水平从吸高氧第3天起即明显高于对照组 (5 5 .9± 5 .5nmol/mgvs 2 2 .5± 4 .4nmol/mg) (P <0 .0 1 ) ,7d达高峰 94 .3± 1 2 .4nmol/mg ,持续 1周后逐渐下降 ,2 1d时仍高于对照组 (4 8.0± 7.5nmol/mgvs2 3.6± 5 .7nmol/mg) (P <0 .0 1 )。结论　AEC Ⅱ损伤是高氧诱导CLD的早期特征 ;肺部氧化应激反应与AEC Ⅱ损伤密切相关。     

高氧致慢性肺疾病新生大鼠肺泡损伤及肺泡上皮细胞的变化

目的 探讨高浓度氧致新生鼠肺损伤时肺泡形态学变化和肺泡上皮细胞动态变化.方法 通过吸入85%～90%高浓度氧气建立新生鼠慢性肺疾病组织模型.80只新生鼠分为实验组和对照组,实验组(n=40)吸人85%～90%氧气,对照组(n=40)吸入空气.分别留取1、3、7、14、21 d的肺组织,应用放射状肺泡计数(RAC)并且测量肺泡间隔厚度以判定肺泡发育情况,应用免疫组化及RT-PCR技术测定肺组织表面活性蛋白C(SPC)、水通道蛋白-5(AQP5)蛋白及mRNA表达.结果 在生后1 d和3 d,两组RAC和肺泡间隔厚度差异无显著性(P>0.05).在7 d和14 d,实验组的RAC明显低于对照组(P<0.01),肺泡间隔厚度高于对照组(P<0.01),21 d时肺泡间隔厚度升至最高,与对照组间比较差异有显著性(10.62±5.01 vs 3.62±0.88,P<0.001),RAC降至最低,与对照组间比较差异有显著性(3.57±1.24 vs10.47±0.88,P<0.001).高氧肺损伤实验组SPC 3 d时表达明显减少,7d后开始增多,14 d、21d明显高于对照组;而AQP5随着肺损伤的加重表达进行性下降.结论 高氧肺损伤可导致肺泡发育停滞,明显损伤肺泡上皮细胞,肺泡上皮细胞特异性标志物SPC、AQP5表达结果提示I型肺泡上皮细胞损伤严重,Ⅱ型肺泡上皮细胞虽然数量有所增加但其分化与转化能力明显下降.     

洛沙坦对高肺血流量大鼠肺血管平滑肌增殖及肺小动脉重构的抑制作用

目的观察血管紧张素受体拮抗剂对高肺血流量导致肺动脉高压肺血管重构的影响。方法采用腹主动脉-下腔静脉瘘制造大鼠容量负荷性肺动脉高压模型,并应用洛沙坦(losartan)进行干预,6周后观察肺动脉收缩压(PASP)、舒张压(PADP)、右室收缩压(PVSP)变化,心室重量变化,肺血管平滑肌细胞(VSMC)α-平滑肌肌动蛋白(α_actin)及增殖细胞核抗原(PCNA)免疫组化染色;比较各组α_actin积分光密度值(IOD)及肺血管VSMC的PCNA阳性率,比较无肌性动脉肌性化程度及肺小动脉血管厚度百分比(%WT)。结果手术组动物RVSP、PASP、PADP较对照组明显升高(P<0.05),手术 洛沙坦组RVSP、PASP、PADP明显低于手术组(P<0.05);手术组右心室(RV)/左心室加室间隔(LV S)、RV/体重(BW)、(LV S)/BW较对照组显著增加(P<0.001),而手术 洛沙坦组则较手术组显著降低(P<0.01)。与对照组比较,手术组α_actin染色IOD值显著降低(P<0.01),而手术 洛沙坦组IOD值则高于手术组(P<0.05),手术组细胞PCNA阳性率显著升高(P<0.01),而手术 洛沙坦组低于手术组(P<0.05);手术组与其他两组比较,管经50~100μm、101~150μm%WT及15~50μm血管肌性化百分比显著增加(P<0.01)。结论腹主动脉-腔静脉分流导致了肺血管重构和肺动脉高压的发生,洛沙坦能有效地抑制高肺血流量大鼠肺动脉VSMC增殖,减缓肺动脉高压肺血管重构进程。