NMR-based Metabonomics Study of Patients with Cerebral Infraction Complication in Type 2 Diabetes Mellitus

目的应用氢谱核磁共振代谢组学(1H-nMr)方法研究2型糖尿病(T2dM)合并脑梗死患者血清中小分子代谢物代谢轮廓的变化,探讨2型糖尿病合并脑梗死的可能发病机制。方法选择T2dM合并脑梗死患者19例、单纯T2dM患者25例及健康对照者29例,利用1H-nMr方法检测血清的代谢轮廓,通过偏最小二乘判别分析方法,鉴别各组别间血清小分子代谢物的区别。结果从血清偏最小二乘判别分析得分图中可以区分3组患者,与T2dM合并脑梗死有关的特征代谢物包括极低密度脂蛋白、低密度脂蛋白、葡萄糖、乳酸、丙酮酸、3-羟基丁酸、n-乙酰糖蛋白、亮氨酸、缬氨酸、前列腺素d2、前列腺素E2、同型半胱氨酸、氧化三甲胺、甜菜碱等。结论脂类代谢、糖代谢、氨基酸代谢的紊乱可能在T2dM合并脑梗死的发病中起到重要作用。炎症因子、同型半胱氨酸的增加,肠道菌群失调引起的氧化三甲胺、甜菜碱的浓度改变可能也与T2dM合并脑梗死的发生有一定关系。Aim To investigate the possible mechanism of cerebral infarction in type 2 diabetes mellitus(T2DM) through identifying the changes of metabolic products in serum by applying the method of 1H-nuclear magnetic resonance(1H-NMR).Methods Nineteen patients with cerebral infarction complication in T2DM,25 T2DM patients without cerebral infarction and 29 healthy controls were included in our study.The micromolecular substances of each participant in serum were detected by 1H-NMR.The metabolic profile of each participant was obtained.The partial least square discriminant analysis(PLS-DA) was used to examine the difference of metabolic profile among groups.Results Based on the analysis of score plots of groups,the T2DM patients with cerebral infarction,T2DM patients without cerebral infarction and the group of healthy controls were clearly distinguished by applying PLS-DA.The characteristic metabolic products of patients with cerebral infarction complication in T2DM included very low density lipoprotein(VLDL),low density lipoprotein(LDL),glucose,3-hydroxy butanoic acid,lactate,pyruvic acid,glycoprotein,leucine,valine,prostaglandin D2,E2,homocysteine,trimethylamine oxide(TMAO) and betaine.Conclusions The results of our study suggested that the metabolic disorders of lipids,carbohydrate and amino acid may play essential role in the development of T2DM with cerebral infarction.The elevated level of prostaglandin D2,E2 and homocysteine might play a role in the occurrence of cerebral infarction in T2DM.The change of TMAO and betaine which were caused by enteric dysbacteriosis,also might be related to the occurrence of cerebral infarction in type 2 diabetes mellitus

Isolation of Egg Cells and Zygotes from Torenia fournieri

蓝猪耳(Toreniafournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构。用解剖和酶解-解剖两种方法都能分离出生活卵细胞。用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰。在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%)。用酶解-解剖方法也可分离出授粉14h后的合子细胞。Torenia fournieri is a special plant withembryo sac partly protruding through the micropyleof ovule, and its egg cell, two synergids and part ofthe central cell can be clearly observed using lightmicroscope (Plate I-1, 2). The feature of embryosac of T. fournieri makes it easy to isolate egg celland zygote, especially for physiologically mature andvigorous egg cell to study its fertilization mechanism.Egg cells of T. fournieri were isolated from em-bryo sacs 2 d after anthesis using enzymatic diges-tion or mechanical dissection after mild osmoticshock (Plate I-3, 4). About 5% egg cells (2–3 from50 ovules) could be mechanically dissected within2 h. When 0.1% cellulase and 0.1% pectinase wereadded into the solution, about 18% egg cells (8–10from 50 ovules) could be isolated within 2 h (PlateI-5). Although egg cells could be isolated by me-chanical dissection, they can be used in in vitro fer- tilization to probe fertilization mechanism withoutdeleterious effect of enzymatic action on the surfaceof egg cell. But it is easier to isolate egg cells byusing enzymatic digestion, and more egg cells canbe obtained for the research of molecular biology.Using enzymatic digestion method, the zygote ofTorenia fournieri was also isolated from the polli-nated ovules (Plate I-7–10). Basing on our success-ful isolation of mature sperm cells, the isolation ofegg cells of T. fournieri will make in vitro fertiliza-tion possible for the first time in a dicot plant.国家自然科学基金项目(No.30170060)资助。~