Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13‐cis‐retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.     

比较胚胎和新生小鼠肠神经嵴干细胞体外增殖分化的实验研究

目的联合应用简化无血清培养基和连续传代法，从胚胎14～17d、新生1d、新生5d小鼠肠管分离培养肠神经嵴干细胞（gut neural crest stem cells，GNCSCs），观察其体外培养过程中增殖、分化的特点，用p75、GFAP、Peripherin、α-Actin作为特异性标志来鉴定GNCSCs及其分化的细胞系，比较胚胎和新生小鼠GNCSCs在生长速度、分化细胞的种类及数量上的差异。方法取3组小鼠的肠管，分离制成单细胞悬液，接种于DMEM／F12完全培养基贴壁培养，在连续传代培养中观察克隆球的形成；将克隆球接种于含血清培养基，观察其分化现象；用免疫细胞化学和免疫荧光法检测克隆球及其分化细胞系特异性标志物的表达。结果部分胚胎和新生小鼠肠管细胞在无血清培养基中连续传代培养形成克隆球。在血清刺激下克隆球可分化为多种形态的细胞。克隆球和分化细胞系的免疫染色均为阳性。结论胚胎和新生鼠肠管内存在具有自我更新能力的GNCSCs，且均可分化为神经元、神经胶质细胞和平滑肌三类细胞。新生鼠较胎鼠GNCSCs生长速度慢，分化的神经元数量减少，神经胶质细胞数量增加，平滑肌细胞无显著变化。     

碱性成纤维细胞生长因子对胚胎大鼠神经干细胞增殖分化的影响

目的探讨分离、培养及鉴定胚胎大鼠神经干细胞(NSCs)方法，观察碱性成纤维细胞生长因子(bFGF)对NSCs增殖、分化的影响。方法从大鼠胚胎脑组织中分离出NSCs，用bRGF和胎牛血清诱导其增殖和分化，予BrdU以标记分裂细胞，采用免疫细胞化学鉴定NSCs和分化神经细胞。结果大鼠胚胎脑NSCs在无细胞因子和胎牛血清的培养基中无新生细胞形成，但能在bFGF和血清诱导下形成克隆，并产生nestin和BrdU阳性细胞，贴壁后分化为神经元和星形胶质细胞。结论该方法从大鼠胚胎大脑分离出的细胞具有NSCs特性，即自我更新和多向分化潜能;bFGF是NSCs增殖必须的丝裂原。     

高氧、维甲酸对胎鼠肺角化细胞生长因子及其受体表达的影响

目的　探讨高氧、维甲酸 (RA)对胎鼠肺成纤维细胞 (LFs)和肺泡II型上皮细胞 (AECII)角化细胞生长因子 (KGF)及其受体 (KGFR)表达的影响。方法　原代培养胎鼠肺LFs及AECII ,待生长至亚汇合状态时 ,随机分为 :空气组 ,空气 +RA组 ,高氧组 ,高氧 +RA组。于培养 2、6、12、2 4、4 8(AECII)和 72h(LFs)时 ,采用半定量RT -PCR方法检测KGF和KGFR的mRNA表达。结果　与空气组比较 ,高氧 2h时 ,胎鼠LFsKGFmRNA表达即明显下降 ,6h时达极点 ,以后下降趋势逐渐减弱 ,至 4 8h后已无显著性差异 ;高氧 2、6、12和 2 4h时 ,其下降幅度分别为 3 0、5 2、2 3和 2 1倍 (P <0 0 5、0 0 1、0 0 1和 0 0 5 )。RA可上调高氧状态下胎鼠LFsKGFmRNA表达 ,与高氧组比较 ,高氧 +RA组在 2、6、12和 2 4h时KGFmRNA表达分别是高氧组的 2 4、3 4、1 7和 1 8倍 (P <0 0 5、0 0 1、0 0 1和 0 0 5 )。高氧对胎鼠AECIIKGFRmRNA表达影响较小 ,与空气组比较 ,仅在高氧 12h和 2 4h时其表达量分别是空气组的 2 3和 1 3倍 (P <0 0 5 ) ;RA对AECIIKGFRmRNA表达没有影响。结论　促进KGF的表达是RA发挥高氧肺损伤保护作用的重要机制之一     

Interkinetic nuclear migration in the mouse embryonic ureteric epithelium: Possible implication for congenital anomalies of the kidney and urinary tract

Interkinetic nuclear migration (INM) is a phenomenon in which progenitor cell nuclei migrate along the apico‐basal axis of the pseudostratified epithelium, which is characterized by the presence of apical primary cilia, in synchrony with the cell cycle in a manner of apical mitosis. INM is suggested to regulate not only stem/progenitor cell proliferation/differentiation but also organ size and shape. INM has been reported in epithelia of both ectoderm and endoderm origin. We examined whether INM exists in the mesoderm‐derived ureteric epithelium. At embryonic day (E) 11.5, E12.5 and E13.5, C57BL/6J mouse dams were injected with 5‐bromo‐2’‐deoxyuridine (BrdU) and embryos were killed 1, 2, 4, 6, 8, 10 and 12 h later. We immunostained transverse sections of the ureter for BrdU, and measured the position of BrdU (+) nuclei in the ureteric epithelia along the apico‐basal axis at each time point. We analyzed the distribution patterns of BrdU (+) nuclei in histograms using the multidimensional scaling. Changes in the nucleus distribution patterns suggested nucleus movement characteristic of INM in the ureteric epithelia, and the mode of INM varied throughout the ureter development. While apical primary cilia are related with INM by providing a centrosome for the apical mitosis, congenital anomalies of the kidney and urinary tract (CAKUT) include syndromes linked to primary ciliary dysfunction affecting epithelial tubular organs such as kidney, ureter, and brain. The present study showed that INM exists in the ureteric epithelium and suggests that INM may be related with the CAKUT etiology via primary ciliary protein function.     

Neural stem cells: properties and therapeutic potentials for hypoxic‐ischemic brain injury in newborn infants

Neural stem cells (NSCs) are defined by their ability to self‐renew, to differentiate into cells of all glial and neuronal lineages throughout the neuraxis, and to populate developing or degenerating central nervous system (CNS) regions. The recognition that NSCs propagated in culture could be reimplanted into the mammalian brain, where they might integrate appropriately throughout the mammalian CNS and stably express foreign genes, has unveiled a new role for neural transplantation and gene therapy and a possible strategy for addressing the CNS manifestations of diseases that hitherto had been refractory to intervention. An intriguing phenomenon with possible therapeutic potentials has begun to emerge from our observations of the behavior of NSCs in animal models of neonatal hypoxic‐ischemic (HI) brain injury. During phases of active neurodegeneration, factors seem to be transiently elaborated to which NSCs may respond by migrating to degenerating regions and differentiating specifically towards replacement of dying neural cells. NSCs may attempt to repopulate and reconstitute ablated regions. These ‘repair mechanisms’ may actually reflect the reexpression of basic developmental principles that may be harnessed for therapeutic ends. In addition, NSCs may serve as vehicles for gene delivery and appear capable of simultaneous neural cell replacement and gene therapy (e.g. with factors that might enhance neuronal differentiation, neurites outgrowth, proper connectivity, and/or neuroprotection). When combined with certain synthetic biomaterials, NSCs may be even more effective in ‘engineering’ the damaged CNS towards reconstitution. We have also cultured human NSCs or progenitors as neurospheres which were derived from fetal cadavers at 13 weeks of gestation, and transplanted them into HI‐injured immature brains to investigate their therapeutic potentials in this type of model.     

First experience of the AML‐Berlin‐Frankfurt‐Münster group in pediatric patients with standard‐risk acute promyelocytic leukemia treated with arsenic trioxide and all‐trans retinoid acid

Recently, studies in adults with acute promyelocytic leukemia (APL) showed high cure rates in low‐risk patients treated with all‐trans retinoid acid (ATRA) and arsenic trioxide (ATO), while toxicities were significantly reduced compared to the standard treatment with ATRA and chemotherapy. Here we report about first experience with 11 pediatric patients with low‐risk APL treated with ATRA and ATO. All patients stayed in molecular remission. All suffered from hyperleukocytosis. Two patients experienced reversible severe side effects. One suffered from osteonecroses at both femurs, seizures, as well as posterior reversible encephalopathy syndrome, the other patient had an abducens paresis.     

高压氧影响离体神经干细胞分化的量效及时效关系

目的：探讨不同压力和不同稳压时间下高压氧(HBO)对离体神经干细胞（NSCs）分化的影响,旨在找出HBO治疗的最佳压力与稳压时间。方法：取出生3日内新生Sprague-Dawley大鼠的脑皮质组织,在含B27、bFGF和EGF的DMEM/F12培养基中悬浮培养NSCs。将传至2~3代的NSCs随机分为不做处理的对照组（CON）和HBO处理组。HBO处理组根据所用压力和稳压时间分为6组：NSCs分别给予1个绝对大气压（ATA）、2 ATA、3 ATA 的HBO处理,每种压力再分别给予30和60 min两种稳压时间。HBO后继续培养24 h,7组细胞在同一时间行免疫荧光染色,荧光显微镜下观察NSCs分化为NSE阳性细胞的百分率。结果：NSCs分化为NSE阳性细胞的分化率从低到高依次为CON组（3.72±0.88%）,1ATA-30min组（3.85±0.44%）,1ATA-60min组（5.45±0.52%）,3ATA-30min组（6.08±0.57%）,2ATA-30min组（6.72±0.76%）,3ATA-60min组（7.89±0.62%）,2ATA-60min（9.17±0.50%）;除1ATA-30min组外,其余各HBO组与对照组比较,差异有统计学意义（P<0.01）;同一压力下60min组与30min组之间的差异及2ATA-60min组与其他各组之间的差异均有统计学意义（P<0.01）。结论：2 ATA压力下稳压时间为60 min的HBO促进NSCs向神经元分化的作用最强。［中国当代儿科杂志,2010,12（5）：368-372］     

Common nature in the effects of thalidomide on embryo‐fetal development in Kbl:JW and Kbl:NZW rabbits

The effects of thalidomide on the embryo‐fetal development (EFD) of rabbit fetuses and the sensitive periods (SP) for the various malformations were compared between Kbl:JW and Kbl:NZW rabbits to investigate possible strain differences. The post‐implantation loss rate and number of placental remnants were increased and the number of live fetuses was decreased in both of the strains in the EFD study and in Kbl:NZW at 300 mg/kg dosed on GD 7–8 in the SP study. In the external and skeletal examinations, head, limb and tail malformations were observed in both the strains in the EFD and SP studies at the same dose levels in the same dosing period. In the visceral examination, hydrocephaly, cardiovascular malformations, absent pulmonary intermedial lobe, diaphragmatic hernia and/or abnormal liver lobation were also observed in both of the strains in the EFD and SP studies at the same dose levels and in the same dosing period. Plasma concentrations of thalidomide were equivalent between the two strains in the SP study. There were strain differences in some parameters, such as the post‐implantation loss rate and the frequencies of malformations in forelimb and hindlimb and pulmonary intermedial lobe, but similar types of malformations or variations were induced at the same dose levels on the same dosing period in both strains. Therefore, it is concluded that there were no essential differences in sensitivity of the fetuses to thalidomide between Kbl:JW and NZW rabbits and both of the strains are useful to evaluate the teratogenic effects of thalidomide.     

外源性结缔组织生长因子对肾小管上皮细胞的转分化和胶原合成效应

目的：大量研究表明内源性结缔组织生长因子(CTGF)在肾小管间质纤维化(TIF)进程中发挥了重要作用,为进一步阐明外源性CTGF的作用机制,该实验探讨了重组人结缔组织生长因子(rhCTGF)刺激对人肾小管上皮细胞(HK2)转分化和胶原合成的作用。方法：rhCTGF 5 ng/mL刺激HK2细胞,用倒置显微镜观察细胞形态学变化,同时在刺激后0,3,6,12,24,48 h各点收集细胞,RT-PCR检测上皮细胞钙粘蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、胶原Iα1(Col Iα1)和胶原IVα1(Col IVα1)mRNA表达变化。结果：rhCTGF 5 ng/mL刺激使HK2细胞形态由椭圆形转为梭形,下调E-cadherin mRNA表达,上调-αSMA mRNA表达,但对Col Iα1和Col IVα1mRNA表达没有影响。结论：外源性CTGF能导致HK2细胞转分化,但不能促进其胶原合成。     

结核菌素皮试和全血γ干扰素测定试验的儿童结核感染诊断应用研究

目的 评价结核菌素（PPD）皮试和全血γ干扰素（IFN-γ）测定试验诊断儿童结核病的准确性。方法 选择2006年7月至2010年4月首都医科大学附属北京儿童医院住院临床诊断结核和呼吸系统疾病的患儿为研究对象。根据患儿所暴露的结核感染危险因素分为5组：A组：无结核病密切接触史的非结核病的呼吸系统疾病患儿;B组：有活动性结核病患者密切接触史的非结核病的呼吸系统疾病患儿;C组：无结核病密切接触史的临床诊断结核病患儿;D组：有活动性结核病患者密切接触史的临床诊断结核病患儿;E组：病原学或病理学确诊的活动性结核病患儿。患儿于入院当日行PPD皮试,入院后1~7 d采集外周静脉血行全血IFN-γ测定。以敏感度、特异度、阴性预测值、阳性预测值和似然比评价PPD皮试和全血IFN-γ测定对结核病的诊断价值。结果 125例患儿进入分析。A组40例,B组11例,C组29例,D组27例,E组18例。①PPD皮试取硬结≥10 mm为阳性判断标准时,诊断结核病的敏感度为77.0%,特异度为70.6%;取硬结≥15 mm为阳性判断标准时,诊断结核病的敏感度为50.0%、特异度为80.2%;全血IFN-γ测定的敏感度为85.1%、特异度为94.1%。②PPD皮试取硬结≥10 mm为阳性判断标准诊断结核病时,<3岁患儿PPD皮试的敏感度和特异度均显著低于≥3岁患儿,城区和郊区患儿的敏感度和特异度接近;全血IFN-γ测定诊断结核病的敏感度和特异度在不同年龄、居住地间差异无统计学意义。③全血IFN-γ测定阳性率与结核感染暴露因素的相关性优于PPD皮试（取硬结≥10或15 mm为阳性判断标准时）。结论 潜伏结核感染筛查时以硬结≥15 mm作为PPD皮试阳性判断标准,可提高诊断的特异度;临床疑似结核病的诊断以硬结≥10 mm作为PPD皮试阳性判断标准,可提高诊断的敏感度。全血IFN-γ测定诊断结核病的敏感度和特异度均较好。     

GM1和NGF对神经干细胞体外增殖的影响

目的：探讨单唾液酸神经节苷脂（GM1）、鼠神经生长因子（NGF）对神经干细胞（NSCs）体外增殖的影响。方法：①体外分离培养NSCs;②将含有表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)的NSCs完全培养基及不含bFGF、EGF的DMEM/F12培养基作为两种不同的培养介质,分别加入不同浓度的GM1和NGF,进行NSCs体外增殖研究;③以MTT法和细胞球计数观察NSCs增殖情况;以免疫组化法观察分化条件下药物对NSCs增殖的影响。结果：①在含有bFGF、EGF的NSCs完全培养基中,较高浓度GM1条件下,NSCs增殖明显（P<0.05）;②含血清的分化培养液中,随GM1浓度增加,NSCs增殖明显;随NGF浓度增加,神经元及胶质细胞比例增高。结论：高浓度GM1能促进NSCs增殖, NGF能促进NSCs分化。［中国当代儿科杂志,2009,11（10）：841-845］     

Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader‐Willi syndrome

Prader‐Will syndrome (PWS) is characterized by hyperphagia, growth hormone deficiency and central hypogonadism caused by the dysfunction of the hypothalamus. Patients with PWS present with methylation abnormalities of the PWS‐imprinting control region in chromosome 15q11.2, subject to parent‐of‐origin‐specific methylation and controlling the parent‐of‐origin‐specific expression of other paternally expressed genes flanking the region. In theory, the reversal of hypermethylation in the hypothalamic cells could be a promising strategy for the treatment of PWS patients, since cardinal symptoms of PWS patients are correlated with dysfunction of the hypothalamus. The genome‐wide methylation status dramatically changes during the reprograming of somatic cells into induced pluripotent stem cells (iPSCs) and during the in vitro culture of iPSCs. Here, we tested the methylation status of the chromosome 15q11.2 region in iPSCs from a PWS patient using pyrosequencing and a more detailed method of genome‐wide DNA methylation profiling to reveal whether iPSCs with a partially unmethylated status for the chromosome 15q11.2 region exhibit global methylation aberrations. As a result, we were able to show that a fully methylated status for chromosome 15q11.2 in a PWS patient could be reversed to a partially unmethylated status in at least some of the PWS‐iPSC lines. Genome‐wide DNA methylation profiling revealed that the partial unmethylation occurred at differentially methylated regions located in chromosome 15q11.2, but not at other differentially methylated regions associated with genome imprinting. The present data potentially opens a door to cell‐based therapy for PWS patients and, possibly, patients with other disorders associated with genomic imprinting.     

神经生长因子对儿童难治性癫(癎)脑神经细胞及早期生长反应基因1表达的影响

目的 探讨外源性神经生长因子(NGF)对儿童难治性癫(癎)脑(癎)性放电区神经细胞及早期生长反应基因1(Egr-1)表达的影响.方法 采用解离细胞即刻培养技术,将术中取下脑海马(癎)性放电区组织块解离后进行培养,设立对照组和NGF 12.5、50、100 ng/ml 3个不同浓度的加药组,孵育4 h后将其固定;通过免疫荧光细胞化学法,以双苯酰亚胺(Bb)染核,联合细胞特异性标志蛋白GFAP、MAP2标记星形胶质细胞、神经元,计算各组神经细胞总数,并利用RT-PCR技术半定量分析细胞中Egr-1mRNA表达情况.结果 外源性NGF作用4 h后,海马区存活的神经细胞总数及GFAP(+)的星形胶质细胞、MAP2(+)的神经元数较空白对照组升高,且与NGF剂量呈量效关系,随NGF浓度增高而增加(P<0.05);与此同时,神经细胞表达Egr-1mRNA也随NGF浓度增高而表达量增加,即AEgr-1/Aβ-actin值与NGF剂量呈量效关系(P<0.01);且存活的神经细胞数与其表达的Egr-1mRNA量呈正相关(r=0.780,P<0.01).结论 外源性NGF对(癎)性放电区损伤神经细胞有保护作用,其作用可能通过促进Egr-1基因的表达来实现.