叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子系统表达变化

目的：叶酸是一种水溶性B族维生素,在体内氨基酸与核苷酸代谢中起重要作用,是胎儿生长发育所必须的营养素。本文通过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变化。方法：雌性C57BL／6J小鼠叶酸缺乏组6只、正常对照组6只,分别饲以不舍叶酸和含2mg叶酸／kg的纯合饲料。四周后与雄鼠交配,于怀孕第13．5天（13．5dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用Real-timePCR法检测胎鼠肝脏组织中胰岛素生长因子I（IGFI）、胰岛素生长因子I受体（IGFIR）、胰岛素生长因子II（IGFII）、胰岛素生长因子II受体（IGFIIR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13．5dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR比率显著升高,差异有统计学意义（P〈0．05）;叶酸缺乏组胎鼠肝脏组织中IGFII和IGFIIRmRNA的相对表达水平均低于正常对照组（P〈0．05）,IGFI、IGFIR、IGFBP-1和IGFBP-3mRNA的相对表达水平两组间没有差异（P〉0．05）。结论：叶酸缺乏会导致小鼠孕中期胎鼠IUGR比率升高及胎肝IGFII和IGFIIRmRNA的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠I-UGR发生机制有关。     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

鱼类胰岛素样生长因子(IGF)系统的研究进展

鱼类胰岛素样生长因子(insulin—like growth factors,IGFs)是进化上相当保守的多肽,能促进组织细胞的增殖、分化和凋亡,对鱼类的生长和发育有重要的调节作用。IGF系统包括IGF—Ⅰ、IGF-Ⅱ、IGF—ⅠR、IGF-ⅡR和IGFBP家族。本文从IGF家族成员的分子结构、生理功能和表达调节等方面综述了有关鱼类IGF体系的研究进展。     

IGFs系统成分在维甲酸诱导的马蹄内翻足畸形中的作用

应用全反式维甲酸(All Trans Retinoic Acid,ATRA)构建Wistar大鼠先天性马蹄内翻足畸形(Congenital Crossfoot,CCF)动物模型,以ATRA和雌激素(雌二醇)分别或联合作用于MC-3T3-E1细胞株,应用Northern印迹杂交、RT—PCR和流式细胞仪检测CCF胎鼠的头盖骨组织和MC-3T3一E1细胞中胰岛素样生长因子-Ⅱ(IGF-Ⅱ)和胰岛素样生长因子结合蛋白-6(IGFBP-6)mRNA的表达以及细胞周期及增殖状态。研究发现,100～140mg／kg浓度的ATRA可以诱导大鼠骨骼畸形发育并产生CCF;IGFs家族基因中IGF-Ⅱ和IGFBP-6表达的调控与大鼠骨骼发育以及成骨细胞的增殖密切相关。     

妊娠糖尿病对仔鼠肺成熟的影响及吡格列酮的干预作用

摘要 目的：探究妊娠糖尿病（GDM）对仔鼠肺成熟的影响及吡格列酮对肺发育的干预作用。方法：将30只SD孕鼠分为对照组、GDM组和GDM+吡格列酮组（GDM+P组）,每组10只。GDM组和GDM+P组孕鼠通过腹腔注射链脲霉素（STZ,45 mg/kg）和高脂饮食饲养构建GDM孕鼠模型,GDM+P组大鼠建模后灌胃10 mg/kg的吡格列酮,对照组和GDM组孕鼠每天灌胃等体积生理盐水。分娩后,检测各组仔鼠的血糖和血浆胰岛素水平以及胎肺组织中的总磷脂量。通过苏木精伊红（HE）染色、油红O染色和透射电镜观察胎肺组织结构和形态变化。通过RT-PCR和Western blot检测胎肺组织中SP-A、SP-B、SIRT1和PPARγ的表达。结果：GDM组仔鼠的血糖水平与对照组无显著差异（P>0.05）,胰岛素水平明显高于对照组（P<0.05）。与对照组相比,GDM组仔鼠胎肺组织中的总磷脂含量降低（P<0.05）;胎肺组织中肺泡Ⅱ型上皮细胞(AECⅡ)数量和脂滴明显减少。与对照组相比,GDM组仔鼠胎肺组织中的SP-A、SP-B、SIRT1和PPARγ的mRNA和蛋白相对表达水平均降低（P<0.05）。吡格列酮干预显著逆转了GDM对仔鼠胰岛素、胎肺组织结构和形态变化的影响;GDM+P组仔鼠胎肺组织中的SP-A、SP-B、SIRT1和PPARγ的mRNA和蛋白相对表达水平相较GDM组均升高（P<0.05）。结论：GDM母鼠所生仔鼠存在肺发育延迟,吡格列酮干预可有效促进仔鼠的肺成熟。     

IGF系统的生物学功能及其与肿瘤的关系

胰岛素样生长因子（insulin-like growth factor,IGF)是体内重要的生长因子。它对组织细胞的增殖。分化,凋亡,机体的生长发育及肿瘤的发生发展起重要的调节作用。IGF系统的组成包括;IGF-Ⅰ,IGF-Ⅱ,IGF-ⅠR,IGF-ⅡR,IGFBP家族,IGF促增殖,促生长的活性主要由IGF-IR介导;IGF-ⅡR的主要作用是清除游离IGF-Ⅱ,调节IGF-Ⅱ的水平,IGFBP1-6是IGFs活性的调节因子。近年的研究表明它们也有不依赖IGF的生物活性。     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

IGF-Ⅱ、IGF-1R、IGFBP-3在结直肠癌中的表达及诊断价值研究

目的：探讨结直肠癌中IGF-Ⅱ、IGF-1R以及IGFBP-3等的阳性表达及诊断价值。方法：收集我院45例结直肠癌、33例炎性息肉、40例腺瘤以及35例正常肠粘膜组织予以免疫组织化学SP法进行IGF—Ⅱ、IGF—1R以及IGFBP-3检测,并统计结直肠腺瘤不同组织类型和结直肠癌不同Dukes分期的阳性表达差异。结果：结直肠癌组织中IGF—Ⅱ、IGF—1R以及IGFBP-3表达均呈高阳性率,并且显著高于其他3组（P〈0．05）。结直肠腺瘤管状、混合型、绒毛状等不同组织类型IGF-Ⅱ、IGF—1R以及IGFBP-3表达阳性率呈逐渐增高趋势,绒毛状腺瘤显著高于其他两型（P〈0．05）。45例结直肠癌中,Dukes分期A、B两期显著低于C期和D期,比较差异具有显著性（P〈0．05）。结论：IGF—Ⅱ、IGF-1R以及IGFBP-3可能在结直肠腺瘤发生、发展及进展为结直肠癌的过程中起一定作用,对结直肠癌早期诊断具有一定价值。     

营养状况对幼年鲤鱼肝脏IGF-Ⅰ mRNA表达的影响

通过室外喂养观察3种营养状况对幼年鲤鱼生长、血清生长激素（GH）水平和组织胰岛素样生长因子－Ⅰ（IGF-Ⅰ）mRNA表达的影响。一组鱼喂含40％酪蛋白的饵料（H组）,一组鱼喂含20％酪蛋白的饵料（L组）,饵料的总能量相同;另一组鱼先饥饿32天再投喂含40％酪蛋白的饵料。禁食后饥饿组鲤鱼体重和体长的生长受阻,在第16天饥饿组鱼血清GH水平明显升高,到第32天达到H组的4倍;而肝组织IGF－Ⅰ mRNA水平在第16天无明显下降,但是到第32天已降到不到H组的一半。鲤鱼其它组织饥饿32天未发现IGF－Ⅰ mRNA表达有明显变化。饥饿32天后开始投喂含40％酪蛋白的饵料,鲤鱼生长、肝组织IGF－Ⅰ mRNA的表达水平逐渐恢复,再投喂16天皆与H组无明显差异。再投喂过程中,GH水平也逐渐下降,第16天恢复正常。实验结果提示鲤鱼营养对肝组织IGF－Ⅰ mRNA的表达有调节作用。营养缺乏将导致鲤鱼肝组织IGF－Ⅰ mRNA的表达水平也相应恢复正常,从而导致鲤鱼生长恢复。同时,实验结果也提示,鲤鱼肝以外的组织IGF－Ⅰ mRNA表达不受营养调节。L组鲤鱼在喂养过程中,体重和体长的增长比H组低,但即使喂养40天,两组鱼体重和体长并无显著差异。两组鱼血清生长激素水平和各组织IGF－Ⅰ mRNA的表达水平到实验第32天也未发现有明显差异。推测在鲤鱼含正常能量和20％酪蛋白的饵料已能维持正常IGF－Ⅰ mRNA的表达。L组和H组鱼肝IGF－Ⅰ mRNA在喂养1个月后仍无显著差异也可能与实验时间较短有关。     

胰岛素样生长因子-Ⅰ受体和LH受体mRNA在黄体中的表达及调节

研究了大鼠卵巢黄体中胰岛素样生长因子-Ⅰ受体(IGF-ⅠR)与促黄体激素受体(LHR)的表达关系及调节. 在动情周期中, LHR阳性黄体能表达高水平的IGF-ⅠR mRNA. 动情期黄体的LHR表达水平降低, 而IGF-ⅠR表达仍较高. 假孕第1天黄体中LHR表达较低,第8天达最高水平, 第14天表达已降低到检测不到的程度. 与LHR表达不同, 在第1天假孕黄体中IGF-ⅠR的表达已较高, 此后至第14天在黄体中均检测到IGF-ⅠR信号. 孕鼠黄体同时表达LHR 和IGF-ⅠR mRNA. IGF-Ⅰ刺激LHR mRNA在黄体中的表达. 前列腺素(PG)F抑制IGF-ⅠR和LHR的表达. PGE2能消除PGF对IGF-ⅠR和LHR mRNA表达的抑制作用. 上述结果提示:IGF-Ⅰ可能通过其受体的表达变化参与调节黄体功能、维持黄体结构. PGF抑制IGF-ⅠR的表达可能是黄体萎缩的机制之一.     

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF₁₆₅) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF₁₆₅/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF₁₆₅-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF₁₆₅-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.     

Fetal growth as measured by ultrasound

Until the last decade, investigation of fetal growth has been based on data from premature births and abortuses. Advances in ultrasound scanning have expanded the precision and uses of this technology in studies of fetal growth. Most ultrasound studies of fetal growth are based on clinical populations and are reported in clinical journals. The present paper reviews features of ultrasound technology relating to fetal measurement. The most commonly measured dimensions and their associated measurement errors, distributions and uses are detailed. Ultrasound studies of intrauterine growth retardation and in utero fetal weight estimation are critically reviewed. Finally, a comparison between the fetal growth curve based on ultrasound measurement and on data from preterm or aborted fetuses is compared. Critical comments on sample design and statistical treatment of the data from ultrasound studies is included in each of the areas discussed.     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Malaria Transmission and Fetal Growth

In view of the known relation between infection of the maternal circulation of the placenta with Plasmodium falciparum and impaired fetal growth a study was made of the effect on birth weights of a malaria eradication campaign in the British Solomon Islands. Mean birth weights rose substantially within months of starting antimalarial operations. The increases between 1969 and 1971 averaged 252 g in babies of primigravidae and 165 g in all babies. The proportion of babies with birth weights of less than 2,500 g fell by 8% overall and by 20% among babies of primigravidae. The adverse effect of malaria transmission on fetal growth was apparently reversible if transmission of infection in the community was interrupted up to as late as the third trimester of pregnancy. The beneficial effects of malaria eradication operations on infant survival, child development, and social attitudes in developing countries are discussed.     

PLGA Microsphere-Mediated Growth Hormone Release Hormone Expression Induces Intergenerational Growth

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3–21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.     

建立胎儿生长受限动物模型的研究进展

动物模型法对于人类疾病病因学及病理生理学的研究有着独特的作用。将动物模型法引入对胎儿生长受限（fetal growth restriction,FGR）的探索,不仅可以让我们观察FGR患者及胎儿在不同孕期各自的表现,还可以让我们对发病胎儿在出生后不同年龄阶段的自然表现进行继续研究。人们在FGR动物模型的建立和利用方面已经积累了大量的宝贵经验,这为进一步阐明FGR的病因学及发病机制创造了条件。