【作者】 刘坚；

【导师】 罗小平；

【作者基本信息】 华中科技大学 ， 儿科学， 2008， 博士

【摘要】 背景:神经管畸形的发生是由遗传因素与环境因素共同作用产生的。平面细胞极性(planar cell polarity, PCP)途径对脊椎动物神经胚形成非常重要,而PCP基因表达量及产物的不对称分布将影响PCP。该途径中基因的任何突变都可能导致神经管不能闭合,其表型与人类的颅脊柱裂相似。但是,环境因素是否影响以及如何影响PCP途径基因的时空表达到目前为止仍然不清楚。目的:研究维甲酸对胎鼠Vangl1及Vangl2基因表达的时空特征的影响。方法:全反式维甲酸橄榄油混悬液按120 mg/kg在BALB/C小鼠怀孕第9.5天(E9.5)(G1组)或E10.5(G2组)灌胃。两组的对照组(G1c、G2c)在相同时期仅用等量纯橄榄油灌胃。分别取E10.5、E11.5、E13.5、E15.5、E17.5、E19.5胎鼠,Vangl1及Vangl2基因表达量采用逆转录酶PCR(RT-PCR)检测,其时空表达采用全胚胎原位杂交(whole-mount in situ hybridization, WISH)检测。结果:活产NTDs的发生率及颅面部NTDs发生率G1组(100%、25.6%)均高于G2组(78.2%、5.7%),P均< 0.05。正常情况下Vangl1及Vangl2基因在整个神经胚形成过程中都有强表达。而G1组同期对照组比较,从胎鼠的脑到后神经孔Vangl1及Vangl2基因表达显著下调(P均< 0.05)。G2组则不同,二基因表达在全胚胎(从E11.5到E13.5)及神经管脊柱区(从E15.5到E19.5)显著下调并维持低表达水平,但在神经管的颅区(E15.5到E19.5)仅中度下调(P均< 0.05)。结论:结果提示,维甲酸诱导的神经管畸形的发生可能与Vangl1及Vangl2基因转录子下调有关。背景:神经管缺陷(neural tube defects, NTDs)是由环境危险因素与遗传危险因素间复杂的相互作用引起的。其潜在的发病机理仍然不清楚。从遗传和环境角度看,叶酸代谢相关基因都是NTDs的令人关注的候选基因。目前叶酸代谢相关基因间以及基因与环境间交互作用的研究较少,已有的文献间还存在相互矛盾。目的:通过对叶酸代谢相关基因的单核苷酸多态性(single nucleotide polymorphism, SNPs)与多重环境危险因子交互作用的关联分析,寻找NTDs致病基因及环境危险因素。方法:收集NTDs流产胎儿组织标本或者患者血标本(共117例)以及其双亲的血标本(共216例),记录围孕期补充叶酸、妊娠糖尿病、孕期服药史等情况。采用CEQ 8800系统进行多重SNP分析,对所有样本叶酸代谢相关的12个基因共28个SNPs测序。这些基因是:叶酸受体1及2、溶质载体家族19成员1、运钴胺蛋白II、亚甲四氢叶酸脱氢酶1、丝氨酸羟甲基转移酶1、5,10-亚甲基四氢叶酸还原酶、5-甲基四氢叶酸-同型半胱氨酸甲基转移酶、5-甲基四氢叶酸-同型半胱氨酸甲基转移酶还原酶、甜菜碱同型半胱氨酸甲基转移酶、胱硫醚β合酶和内皮一氧化氮合酶3。通过病例-双亲对照研究(case-parent control study)及传递/不平衡检验(transmission/ disequilibrium tests, TDT),分析SNPs与环境危险因子(孕期补充叶酸、妊娠糖尿病和孕期服药史)交互作用对NTDs发病的影响。结果:亚甲基四氢叶酸还原酶(MTHFR)rs1801133与NTDs的关联具有显著性意义(P < 0.05),而且环境风险因子(未补充叶酸、妊娠糖尿病)对NTDs的发生起增效作用;而甜菜碱同型半胱氨酸甲基转移酶(BHMT)rs3733890仅在未补充叶酸层与NTDs存在连锁不平衡(P < 0.05),基因型本身并不能单独导致疾病;而其他基因的SNPs与NTDs的发生没有显著性关联(P均> 0.05)。结论: MTHFR rs1801133是NTDs的危险因子,而BHMT rs3733890不是NTDs独立的危险因子。未来尚需要对更大的样本进行基因与基因、基因与环境交互作用的研究。背景:骨龄评估对内分泌障碍与生长落后等疾病的诊断与治疗、预测成人身高等有重要指导意义。常用的骨龄评估方法中,Greulich-Pyle法具有主观性,而Tanner-Whitehouse(TW3)法操作起来较为复杂。十多年来人们一直在探索自动化骨龄评估(automatic bone age assessment, ABAA),但兴趣区(regions of interest, ROI)分割与边界的识别仍然是目前限制ABAA的富有挑战性的难题。目的:应用两种新的算法即粒子群优化(PSO)及人工神经网络(ANN)以提高自动化骨龄评估的准确性和实用性。方法:建立基于目标的ROI。按照Tanner-Whitehouse (TW3)法将ROI分为RUS(包括尺桡骨及掌指骨)ROI及腕骨ROI。每个兴趣区提取5项特征(包括大小、形态及融合状态)输入人工神经网络(artificial neural networks, ANN)分类器,ANN建立在前馈的多层网络基础上,并以反向传播算法规则训练ANN以分别处理RUS及腕骨特征。约1,046份左手及腕的数字X光片被随机分成两部分,一半用以训练ANN,另一半用以ABAA,而之前全部采用修订的Greulich-Pyle法人工判读骨龄。结果:人工阅读骨龄与ABAA比较,RUS骨龄95%的可信区间是-0.010岁–0.084岁,腕骨骨龄95%的可信区间是-0.055岁–0.015岁。RUS及腕骨自动化骨龄评估结果与Greulich-Pyle法比较均具有很高的一致率(分别为97.9%与96.5%),变异系数相似(3.4与3.7),并且与人工阅读相比均无显著性差异(t=1.563与-1.123,P均> 0.05)。结论:PSO对图像分割与特征的提取更为有效和准确。该ANN经训练后能更全面地处理影像特征信息,准确判断骨龄。基于智能算法的ABAA系统成功地应用于骨龄0–18岁所有病例。更多还原

【Abstract】 Objective: This study reported the effects of retinoic acid upon the spatiotemporal expressions of Vangl1 and Vangl2 in mouse fetuses.Material and method: Single dose of 120 mg/kg body weight of all trans-retinoic acid suspended in olive oil was administered intragastrically to each pregnant BALB/C mice on embryonic day (E) 9.5 (group 1, G1) or E10.5 (group 2, G2); mice treated with pure olive oil on E9.5 or E10.5 served as control groups. The expressions of Vangl1 and Vangl2 in fetuses were investigated by reverse transcriptase PCR (RT-PCR) and their spatial and temporal expressions were detected by whole-mount in situ hybridization (WISH) on E10.5, E11.5, E13.5, E15.5, E17.5 and E19.5 respectively.Results: The study indicated that the incidence of NTDs in live birth and craniofacial NTDs rate were significantly higher in G1 (100% and 25.6%) than that in G2 (78.2% and 5.7%), both P < 0.05. Vangl1 and Vangl2 were strongly expressed throughout neurulation in embryos of control groups. G1 embryos exhibited a dramatic downregulation of Vangl1 and Vangl2 expressions from cranial region to posterior neuropore compared with the control group of G1 (all P < 0.05). In contrast, the both transcripts in G2 embryos were significantly downregulated and weakly expressed in whole embryos from E11.5 to E13.5 and in the spinal region of neural tube from E15.5 to E19.5, but moderately downregulated in the cranial region of neural tube from E15.5 Conclusion: Vangl1 and Vangl2 transcripts downregulation might be implicated in the occurrence of mouse NTDs induced by retinoic acid. Background: Neural tube defects (NTDs) are complex disease caused by multiple genes and environmental factors. Folic acid is a known environmental factor for NTDs. Folate metabolism pathway genes have been examined for association with NTDs, but most of these genes were studied individually, often with different populations providing conflicting results.Objective: Folate pathway genes SNPs in Chinese families with NTDs were investigated to discover the genetic and environmental factors that contribute to the cause of NTDs.Materials and Methods: We genotyped 28 SNPs in 12 genes in a total of 117 NTDs individuals from 115 family and 216 parental controls mainly Han Chinese from central China. Family-based association analysis was performed by case-parent control study and transmission/ disequilibrium test (TDT) to evaluate the association between SNPs and environmental risk factors: no peri-pregnant folate supplementation, gestational diabetes mellitus (GDM) and peri-pregnant taking medicine. These genes are: folate receptor 1 (FOLR1), folate receptor 2 (FOLR2), solute carrier family 19 member 1 (SLC19A1), transcobalamin II (TCN2), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), serine hydroxymethyltransferase 1 (SHMT1), 5,10-methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), betaine-homocysteine methyltransferase (BHMT), cystathionine-beta-synthase (CBS) and endothelial nitric oxide synthase 3 (NOS3).Results: MTHFR rs1801133 was significantly associated with NTDs, and environmental risk factors, no peri-pregnant folate supplementation and GDM, had potentiation on its contribution to NTDs. BHMT rs3733890 and no peri-pregnant folate supplementation conjointly led to NTDs, while genotype can not solely cause NTDs. Other SNPs in folate pathway genes had no significant association with NTDs.Conclusion: MTHFR rs1801133 is an independent genetic risk factor of NTDs, but BHMT rs3733890 is not a major risk factor. Background: Region of interest (ROI) segmentation and boundaries identification are still extremely challenging task for automatic bone age assessment (ABAA).Objective: New algorithms, particle swarm optimization (PSO) and artificial neural network (ANN) are proposed to improve the validity, accuracy and practicality of ABAA.Materials and Methods: The concept of object-based ROI was proposed. Thirteen RUS (including radius,ulna and short finger bones) ROIs and seven carpal ROIs were appointed respectively according to Tanner-Whitehouse (TW3) method. Five features including size, morphologic features and fusion stage of each ROI were extracted and input into ANN classifiers. ANNs were built upon feed-forward multilayer networks and trained with back-propagation algorithm rules to process RUS and Carpal features respectively. About 1,046 digital left hand-wrist radiographs were randomly utilized half for training ANNs and the rest for ABAA after manual reading by revised Greulich-Pyle method.Results: The 95% confidence intervals of the difference between ABAA and manual readings were -0.010 to 0.084 years for RUS bone age and -0.055 to 0.015 years for Carpal bone age. Both RUS and Carpal ABAA systems had no significant difference compared with manual method (t = 1.563 and -1.123, both P > 0.05), and both two systems had fairly high concordance rates (97.9% and 96.5% respectively) and similar coefficient variations (3.4 and 3.7 respectively).Conclusion: PSO method made image segmentation and feature extraction more valid and accurate, and the ANN models were sophisticated in processing image information. ABAA system based on intelligent algorithms had been successfully applied to all cases from 0 to 18 years of bone age.更多还原