胚胎早期氯胺酮暴露对心脏发育的影响及其可能的分子机制

发布时间：2018-05-05 18:35

本文选题：氯胺酮 + 药物暴露　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:探讨胚胎早期氯胺酮暴露对爪蛙心脏发育的影响。方法:将爪蛙胚胎从第8期暴露氯胺酮至第21期。(1)利用活体胚胎、心脏组织成像,免疫荧光(immunofluorescence)以及整体胚胎原位杂交(whole-mount RNA in situ hybridization,WMISH)观察胚胎及其心脏形态的变化,利用透射式电子显微镜(transmission electron microscope,TEM)观察胚胎心脏超微结构的改变。(2)计数胚胎心率及计算心室缩短分数(ventricular shortening fraction,VSF),以评估胚胎的心脏功能。(3)实时荧光定量PCR(Real-time Quantitative PCR,RT-qPCR)检测心脏发育相关基因mRNA的表达。(4)蛋白质印迹技术(western blot,WB)检测XMLC2(Xenopus Myosin Light Chain 2)及pH3(phospho-histone H3)蛋白的表达。结果:随氯胺酮暴露浓度升高,爪蛙胚胎的死亡率逐渐增加,体长逐渐缩短(P0.05)。当暴露浓度为0.5mg/ml时,氯胺酮可导致以心脏增大为主的胚胎畸形,其他畸形还包括:肠道缺陷、体轴弯曲及体长缩短等;还导致胚胎的心肌超微结构中Z线的连续性中断甚至部分缺失;pH3蛋白在胚胎心脏中的相对表达量增加(P0.05);胚胎的心率减慢(P0.05)及心室缩短分数下降(P0.05);XMLC2的mRNA及蛋白的相对表达量在所检测的四个时期均明显下调(P0.05)。结论:早期氯胺酮暴露对爪蛙胚胎有呈浓度依赖性的致死性。当暴露浓度为0.5mg/ml时,氯胺酮可导致以心脏增大为主的胚胎畸形,并引起心肌超微结构异常、心脏细胞增殖增加及心脏功能受损。氯胺酮可能通过下调XMLC2在mRNA及蛋白水平的表达,导致心脏增大、心肌超微结构异常及心脏收缩力的改变。这些结果为孕早期氯胺酮暴露可能引起的胎儿缺陷提供了一个新的理论依据。
[Abstract]:Objective: to investigate the effects of early embryonic ketamine exposure on cardiac development of Rana claw. Methods: Frog embryos were exposed to ketamine from stage 8 to phase 21. The morphologic changes of the embryos and their hearts were observed using in vivo embryos, cardiac tissue imaging, immunofluorescence fluorescence) and in situ hybridization of whole embryos, whole-mount RNA in situ hybridization (WMISH). Using transmission electron microscope (TEM) to observe the ultrastructural changes of the embryonic heart, count the heart rate of the embryo and calculate the ventricular shortening fraction of ventricular shortening fractionation, to evaluate the cardiac function of the embryo. 3) real-time fluorescence quantitative PCR(Real-time Quantitative PCR RT qPCR) was used to detect cardiac hair. The expression of mRNA and pH3(phospho-histone H3) were detected by Western blotblotWB). Results: with the increase of ketamine concentration, the embryo mortality and body length of Rana claw increased. When the exposure concentration is 0.5mg/ml, ketamine can lead to fetal malformation with heart enlargement, other malformations include intestinal defects, body axis bending and shortening of body length. It also caused the continuity of Z line in the myocardial ultrastructure of the embryo and even the partial deletion of the pH 3 protein in the embryonic heart. The relative expression of P0.05 protein in the embryonic heart increased; the embryo heart rate slowed down P0.05) and the ventricular shortening fraction decreased P0.05 / xmlC2 mRNA and protein phase. The expression of P0.05 was significantly down-regulated in the four periods. Conclusion: early ketamine exposure is a concentration-dependent lethal effect on the embryos of Rana claw. When the exposure concentration was 0.5mg/ml, ketamine could lead to fetal malformation, cardiac ultrastructure abnormality, cardiac cell proliferation and cardiac function damage. Ketamine may induce cardiac enlargement, myocardial ultrastructure abnormality and cardiac contractility by down-regulating the expression of XMLC2 in mRNA and protein. These results provide a new theoretical basis for possible fetal defects caused by ketamine exposure in early pregnancy.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R614

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