叶酸缺乏对胚胎停育患者绒毛印记基因甲基化的影响

目的 探讨叶酸缺乏对胚胎停育患者绒毛印记基因甲基化的影响.方法 选取2015年4月至2017年4月在陆军总医院263临床部妇科门诊224例胚胎停育患者(研究组)以及同期同比例自愿来陆军总医院263临床部人工流产孕妇(对照组),按照是否叶酸缺乏分为叶酸正常亚组和叶酸缺乏亚组,分析不同组别胰岛素生长因子2、父系表达基因3以及生长因子受体结合蛋白10甲基化状态.结果 对照组叶酸正常亚组IGF2、GRB10的甲基化指数均低于研究组叶酸缺乏亚组、叶酸正常亚组(t值分别为8.21、7.54、6.12、5.29,均P<0.05).对照组与研究组IGF2、GRB10异常甲基化率比较,差异有统计学意义(χ2值分别为6.11、6.34,均P<0.05).研究组组内叶酸缺乏亚组IGF2、GRB10异常甲基化率显著高于叶酸正常亚组(χ2值分别为5.02、5.44,均P<0.05);对照组叶酸正常亚组IGF2、GRB10异常甲基化率均显著低于研究组叶酸缺乏亚组(χ2值分别为6.17、6.43,均P<0.05).结论 叶酸缺乏可能导致IGF2、PEG3基因甲基化程度以及异常甲基化率增高,从而引起胚胎停育.     

早期胚胎发育与细胞凋亡

胚胎的发育过程是由众多基因精密调控的。在早期胚胎发育过程中,胚胎存在凋亡信号。植入前的胚胎已经存在凋亡现象,形态正常的胚胎也存在凋亡信号,而细胞凋亡的发生则是抗凋亡基因与促凋亡基因表达异常的直接结果。因此,研究早期胚胎发育过程中凋亡基因的表达变化,对于解释早期胚胎发育受阻机制显得尤为重要,对于提高辅助生殖技术的妊娠率有非常重要的意义。对凋亡相关因子BCL-2家族成员、抗凋亡因子热休克蛋白和生长因子进行了综述。     

叶酸对阿霉素诱导心脏毒性的拮抗作用研究

目的:探究叶酸对阿霉素(DOX)诱导心脏毒性的拮抗作用。方法:利用DOX(40μmol/L)与叶酸(75μmol/L)处理6hpf斑马鱼胚胎,72hpf时观察胚胎心包水肿情况,计录胚胎15s内心率,荧光显微镜下观察心脏结构,固兰染色观察心脏血细胞分布和量。利用DOX(5μmol/L)与叶酸(75μmol/L)共处理心肌细胞,免疫印迹试验检测切割后的半胱氨酸蛋白酶3(Caspase3)的水平。结果:DOX处理后胚胎表现出心包水肿、心率降低、心脏结构异常。叶酸与DOX共处理后胚胎心包水肿率降低至17%,低于DOX单处理组(P=0.0097)(n=25);心率恢复至37±1.6次/15s,高于DOX单处理组(P0.001)(n=10);叶酸抑制DOX引起的心脏结构异常,并且使固兰染色异常率降低至22.4%,低于DOX单处理组(P=0.0098)(n=35)。叶酸抑制DOX诱导的心肌细胞切割后的Caspase3水平升高。结论:叶酸对DOX诱导的心脏毒性具有一定的拮抗作用,其作用机制可能与抑制心肌细胞凋亡相关。     

连接蛋白Cx50 S258F突变体对斑马鱼晶状体发育的影响

目的:研究与先天性白内障相关的连接蛋白Cx50 S258F突变体对斑马鱼晶状体发育的影响。方法:将野生型和携带S258F突变的Cx50编码区分别构建到pcDNA3.1myc/his真核表达载体上,通过体外转录获得相应的RNA产物,将RNA产物分别显微注射到1细胞期的斑马鱼胚胎,通过显微镜观察斑马鱼晶状体表型,并利用全胚原位杂交技术分析过表达野生型和突变型Cx50对晶状体标志基因Cx23表达的影响。结果:与野生型胚胎相比,S258F突变型Cx50过表达引起斑马鱼胚胎白内障表型,过表达野生型Cx50也引起白内障表型,但是畸形数量明显少于突变型,野生型和突变型Cx50的过表达均下调了Cx23的表达。结论:连接蛋白Cx50S258F突变体可引起斑马鱼白内障表型,提示Cx50的S258F突变体是造成先天性白内障的原因。     

人食管癌顺铂耐药细胞系的建立及耐药相关基因的筛选

目的:建立人食管癌顺铂耐药细胞系Ec9706/cDDP,并初步探讨其耐药机制。方法:采用顺铂(cDDP)中等浓度、间歇冲击作用方法历时9mon诱导人食管癌细胞系Ec9706,建立相应耐药细胞系,采用MTT法测定耐药细胞系对cD-DP的耐药指数,用RT-PCR半定量方法测定亲本细胞及耐药细胞耐药相关基因如DNA聚合酶(polβ)、多药耐药相关基因(MRP)、谷胱甘肽转移酶(GST)、二氢叶酸还原酶(DHFR)、拓扑异构酶(TPO)和多药耐药基因(mdr1)等基因的mRNA表达水平。结果:人食管癌顺铂耐药细胞系Ec9706/cDDP的耐药指数为15.7,该细胞系中polβ、DHFR、GST基因mRNA的表达比亲本细胞显著增加,TPO的表达降低,而mdr1、MRP表达的增加无显著统计学意义。结论:成功建立了人食管癌顺铂耐药细胞系Ec9706/cDDP,其耐药性的产生与polβ、GST、DHFR、TPO等基因的异常表达有关,而与mdr1、MRP表达的关系不密切。     

甲基汞对大鼠胚胎细胞毒性和相关基因表达的影响

目的从细胞和基因水平揭示甲基汞的发育毒性作用机理。方法应用体外／体内大鼠模型、原位杂交等方法观察甲基汞（体外染毒剂量依次为０、０．０５、０．１０、０．２０、０．４０、０．８０和１．６０ｍｇ／Ｌ;体内染毒剂量依次为０、０．２、０．４、０．８、１．６和３．２ｍｇ／ｋｇ）对９．５天龄大鼠胚胎细胞行为和基因表达的影响。结果甲基汞能通过卵黄囊胎盘,但高浓度可抑制胎盘发育和血管分化;甲基汞对胚胎的发育毒性与其浓度间存在剂量反应关系,胚胎畸形的主要表现是神经管未闭和体位异常。甲基汞能激发胚胎细胞过度凋亡,明显抑制细胞ＤＮＡ和ＲＮＡ的合成,损害胚胎细胞超微结构。甲基汞能诱发热休克蛋白７０基因大量表达,抑制纤维连接蛋白基因和ｐ１６基因的表达;热休克蛋白７０基因、Ｃａ２＋、细胞凋亡与畸形的发生之间具有相关性。结论胚胎细胞行为和相关基因表达异常在甲基汞的发育毒性作用机理中起重要作用。     

微小RNAs对早期胚胎发育和着床的影响

微小RNAs(microRNAs,miRNAs)在早期胚胎发育和着床过程中发挥调控作用。miRNA的异常表达可通过引起滋养细胞过度凋亡、胎盘血管生成异常、抑制子宫内膜蜕膜化、母胎界面免疫反应等多种途径引起早期胚胎发育异常和着床失败。此外,miRNAs多态性通过抑制滋养细胞增殖及促进细胞凋亡从而引起胚胎丢失。现就miRNAs对早期胚胎发育和着床的影响进行综述。     

同型半胱氨酸对鸡胚早期心血管发育的影响

目的探讨同型半胱氨酸与胚胎早期心血管发育的关系,了解叶酸和维生素Ｂ１２是否能缓解同型半胱氨酸的心血管发育毒性。方法采用鸡胚孵化、电镜观察、甲基绿派洛宁染色、原位ＤＮＡ片段末端标记、叶酸和维生素Ｂ１２的干预试验等方法。结果同型半胱氨酸（０～１６．０μｍｏｌ／胚胎）能干扰２天和４天龄胚胎心脏发育和血管分化,其作用随着剂量的增加而增强,呈剂量反应关系;８．０μｍｏｌ／胚胎同型半胱氨酸诱发的心脏缺陷率分别为２４．１％和２５．０％,对卵黄囊血管的抑制率达６０７％。≥４．０μｍｏｌ／胚胎同型半胱氨酸能损伤心肌细胞及其细胞器结构,并抑制细胞ＤＮＡ和ＲＮＡ的合成。首次发现８．０μｍｏｌ／胚胎同型半胱氨酸可诱导早期心肌细胞发生过度凋亡（２．７％）,明显高于正常对照组（０．０８％）。叶酸能拮抗同型半胱氨酸的心血管毒性,其效果大于维生素Ｂ１２。结论同型半胱氨酸是一种新的能损害心血管发育的危险因素;叶酸能有效地拮抗同型半胱氨酸的发育毒性;细胞凋亡可能与心血管畸形的发生有关。     

大豆异黄酮配伍叶酸对神经管畸形胎鼠神经细胞中凋亡基因表达的影响

目的 :观察大豆异黄酮和叶酸的联合干预对神经管畸形胎鼠神经细胞凋亡基因表达情况的影响 ,并研究其干预机制。方法 :将大鼠孕鼠随机分成对照组、模型组、单独干预组和配伍干预组后进行不同的处理。孕 1 4 d时处死孕鼠取出胎鼠 ,将胎鼠的脑组织进行固定和包埋后进行免疫组化染色 ,在高倍镜下进行观察。结果 :环磷酰胺可以导致凋亡基因 p5 3和 bax表达的增强 ,而大豆异黄酮和叶酸的干预可以增强凋亡基因 Bcl- 2的表达而减弱 p5 3和 Bax的表达 ,且联合干预的效果较好。结论 :3种凋亡基因的表达可能联合参与了神经管畸形的发生机制 ,大豆异黄酮可能通过对神经管畸形胎鼠神经细胞凋亡基因表达的影响而增强叶酸对神经管畸形的干预作用。     

CdSe/ZnS量子点对斑马鱼胚胎发育的影响

目的研究Cd Se/Zn S量子点对斑马鱼胚胎发育的影响。方法以0、0.5、1、2、4、8和16 nmol/L Cd Se/Zn S量子点分别处理斑马鱼胚胎,于受精后24、48、72、96和120 h(简称hpf)5个时间点分别观察各自具有代表性的毒理学终点。结果在120 hpf时,Cd Se/Zn S量子点对斑马鱼胚胎的LC50为21.38 nmol/L(95%CI:17.21~26.57)。量子点Cd Se/Zn S对斑马鱼胚胎24 hpf时60 s内胚胎自主运动频率,48 hpf 60 s内心率、胚胎孵化率、死亡率等均有明显影响,高浓度8 nmol/L和16 nmol/L组Cd Se/Zn S量子点在120 hpf时可致斑马鱼胚胎发生心包水肿、肝脏变小、卵黄囊吸收延迟、肠道发育异常及肌肉变性等中毒症状。结论 8 nmol/L及以上浓度Cd Se/Zn S量子点对斑马鱼胚胎具有较强的发育毒性,暴露浓度和时间的增加,Cd Se/Zn S量子点可造斑马鱼胚胎死亡率升高。     

Relation between some folate-dependent metabolic pathways and dietary folate content in chicks

Responses of several folate-metabolizing pathways to dietary folic acid were studied in 2-week-old chicks. Oxidation of a histidine load to carbon dioxide was impaired in folate-deficient chicks. There was a curvilinear relation between oxidation and dietary folate, and maximum oxidation occurred with 2 mg supplemental folic acid/kg. Hepatic activities of glutamic acid formiminotransferase (EC 2.1.2.5) and glycine N-methyltransferase (EC 2.1.1.20) were not affected significantly (P greater than 0.05) by dietary folic acid. The activity of dihydrofolate reductase (EC 1.5.1.3) in erythrocytes was elevated in folate-deficient chicks. These studies show that the activities of two folate-dependent pathways can be used as biochemical criteria of folate status in chicks.     

10-Formyl-dihydrofolic acid is bioactive in human leukemia cells.

The bioactivity of 10-formyl-7,8-dihydrofolic acid and 10-formyl-folic acid was determined in human leukemia (CCRF-CEM) cells grown in a folate-depleted medium containing methotrexate. Excess 10-formyl-7,8-dihydrofolic acid, (but not 10-formyl folic acid) supported the growth of these cells, but it was less potent than5-formyl-5,6,7,8-tetrahydrofolic acid (a control). 10-formyl-7, 8-dihydrofolic acid (not 10-formyl folic acid) was active as substrate for aminoimidazole carboxamide ribotide transformylase and dihydrofolate reductase. This is the first experimental evidence that 10-formyl-7,8-dihydrofolic acid is a bioactive folate in mammalian cells. These experiments and several other lines of evidence in the literature suggest that 10-formyl-folic acid must be metabolized to bioactive folate by enteric bacteria before it can be utilized by the vertebrate host.     

Competitive inhibition of folate absorption by dihydrofolate reductase inhibitors, trimethoprim and pyrimethamine

Trimethoprim and pyrimethamine, inhibitors of dihydrofolate reductase (DHFR), cause folate deficiency in some patients. We investigated impairment of intestinal folate absorption by these drugs. By use of the in vivo intestinal-loop methods in rats, absorption of [3H] folic acid was significantly decreased in the presence of either drug. Kinetic studies using the influx chamber method demonstrated a pattern of competitive inhibition of folate transport. [3H] folic acid absorption from jejunal loops was determined 3-16 h after IV administration of methotrexate; this treatment abolished DHFR activity in the small intestine. In rats pretreated with methotrexate, luminal disappearance and systemic absorption of folic acid were significantly enhanced with respect to controls. Trimethoprim and pyrimethamine are weak competitive inhibitors of intestinal folate transport and folate absorption inhibition occurs at the site of membrane transport and appears to be unrelated to concurrent inhibition of DHFR activity in enterocytes.     

母体叶酸缺乏与胎儿21-三体综合征关系的研究

叶酸是人体必需的一种维生素,在DNA合成和甲基化方面起重要作用.研究显示孕妇在整个妊娠期血清叶酸水平均显著低于正常非孕妇女.叶酸代谢关键酶甲基四氢叶酸还原酶和甲硫氨酸合成酶还原酶基因多态性均可影响DNA甲基化而导致染色体不稳定和染色体在减数分裂时发生不分离.21-三体综合征是临床上最常见的常染色体疾病,其主要发生机理便是2l号染色体在减数分裂时发生不分离.故妊娠期母体叶酸缺乏可能通过影响DNA甲基化最终导致胎儿21-三体综合征的发生.     

Sparing effect of folic acid deficiency on the development of vitamin B12 deficiency in baboons fed a vitamin B12 deficient diet.

The effect of a vitamin B12 and folic acid deficient diet on juvenile and adolescent baboons (Papio cynocephalus anubis) was studied. The baboons developed clinical and hematological signs characteristics of folacin deficiency, although they were less severe in juvenile baboons. The signs disappeared when folic acid was replaced in the diet. The serum vitamin B12 levels increased in all baboons fed the vitamin B12 and folic acid deficient diet. When folic acid was added to the diet, the levels gradually decreased in adolescent baboons, liver vitamin B12 levels decreased to a lesser extent when fed a vitamin B12 and folic acid deficient diet than when fed a vitamin B12 deficient diet. In juvenile baboons fed a vitamin B12 and folic acid deficient diet, for 7 months and a vitamin B12 deficient diet for a further 11 months, liver vitamin B12 levels did not decrease at any time but were similar to those in baboons fed a vitamin B12 and folic acid supplemented diet.     

Dendrimer-based multivalent methotrexates as dual acting nanoconjugates for cancer cell targeting

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles, instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor, a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multivalent array of methotrexate and also demonstrates their dual biological activities by surface plasmon resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells.     

Folacin deficiency and requirement in the squirrel monkey (Saimiri sciuresus).

Acute folacin deficiency was studied in eight young squirrel monkeys (Saimiri sciureus). Half of the animals were fed a semipurified deficient diet (no added folic acid) and half were fed a control diet (0.84 mg of added folic acid per kilogram of dry diet). Monkeys fed the deficient diet lost weight and suffered from diarrhea and dehydration leading to the death of one of the animals after 6 weeks. Folacin deficiency also was studied in six older animals fed diets containing varying levels of added folic acid. Monkeys fed diets containing 0.14 or 0.27 mg of added folic acid per kilogram of dry diet slowly developed alopecia, a scaly dermatitis, and a mild macrocytic anemia. When these animals were fed the deficient diet, they lost weight rapidly, the alopecia and dermatitis worsened, excretion of formiminoglutamic acid in the urine increased, and a severe megaloblastic anemia with profound intramedullary hemolysis developed. Deficient monkeys had low plasma and red blood cell folacin values but maintained normal plasma vitamin B12 values. Repletion of the animals fed the deficient diet with injections of folic acid reversed both the hematological and physical deterioration. The folacin requirement for maintenance of body weight in these animals was 28 micrograms of total folacin per kilogram of body weight per day. More than 75 micrograms of total folacin per kilogram of body weight/day may be needed to assure growth and normal hematological parameters and bone marrow cytology.     

Methylenetetrahydrofolate reductase C677T polymorphism, folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 micromol/L. Viable cell growth was 25% greater in TT than in CC cells (P<0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P=0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P<0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P<0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P<0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P=0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid.     

Antagonism of hypervitaminosis A-induced anterior neural tube closure defects with a methyl-donor deficiency in murine whole-embryo culture

The interaction of a dietary excess of vitamin A (retinoid) and deficiency of methyl-donor compounds was examined in murine early-organogenesis embryonic development. Female mice were fed one of six diets from the time of vaginal plug detection until gestational d 8.0, when embryos were removed and grown in whole embryo culture for 46 h, using serum from rats fed the same diet for 36 d as the culture medium. The six diets were either methyl-donor deficient (designated -FCM: devoid of folic acid, choline and supplemental L-methionine, but having methionine as a component of the protein portion of the diet) or methyl-donor sufficient (designated +FCM: containing folic acid, choline and L-methionine supplementation), in combination with one of three concentrations of retinyl palmitate (0.016, 0.416 or 4.016 g/kg diet). The high dose of retinyl palmitate induced a failure of anterior neuropore closure and hypoplasia of the visceral arches, both of which were significantly ameliorated by simultaneous administration of the methyl-donor-deficient diet. The primary acidic retinoid detected in the rat serum was 9,13-di-cis-retinoic acid, although we hypothesize that teratogenic retinoids were formed by embryonic biotransformation of the retinyl esters to toxic metabolites. Biochemical measurements of metabolites in relevant pathways were performed. We propose that the amelioration of these malformations may be used to determine biochemical pathways critical for retinoid teratogenesis.