曲马多引起胚胎发育缺陷的分子机制
发布时间：2018-08-03 10:17
【摘要】：目的母孕期药物滥用是流产的重要因素之一。曲马多是目前应用广泛的止痛药,具有镇痛、抑制食欲、改善抑郁等多重功效。当前,曲马多成瘾和滥用有越来越年轻化的趋势,报道显示,越来越多的成瘾者年龄在18-30岁之间,这个年龄阶段正是女性的育龄期。临床研究表明孕期曲马多暴露大大增加女性流产率,但具体机制尚不清楚。本课题以非洲爪蛙为动物模型,发现胚胎在曲马多暴露后造成包括细胞色素发育异常、胚胎体积变小、小头等一系列的发育缺陷。由于色素细胞和颅面部与神经嵴的发育相关,提示曲马多可能干扰早期胚胎神经嵴的发育引起胚胎缺陷。本课题进而进一步探讨曲马多对神经嵴诱导和迁移的影响,并对相关分子机制等方面展开研究。研究结果将对了解该药物的成瘾原因、致畸原因和评估该类药物在临床应用中的潜在风险具有重大现实意义及科学价值。方法首先将卵裂期的爪蛙胚胎暴露于不同浓度的含曲马多的MBS培养液中(1:200,1:400,1:500),在Olympus解剖显微镜下连续观察胚胎的发育全过程,找出出现胚胎畸形的药物浓度,并将实验组和对照组胚胎在体视显微镜下进行摄影。对胚胎(45时期)的头颅软骨做阿辛蓝染色,观察颅面软骨发育情况,并在体视显微镜下进行摄影。客观评价曲马多是否对胚胎发育有影响,找出出现畸形胚胎的药物浓度。同时用液相色谱法测定胚胎中曲马多的药物浓度,与人类曲马多的血药浓度比对。将暴露于不同浓度的含曲马多的MBS培养液中的胚胎,培养至神经板时期(14-15期)及尾牙期(25期),此时胚胎已完成诱导及开始迁移,收集此期的胚胎,并构建神经嵴细胞诱导的相关基因探针(Foxd3、N-tubulin、Sox2、Slug、Twist),通过原位杂交方式检测胚胎神经板时期及尾牙期Wnt,Fgf,BMP,and Notch/Delta等信号通路的相关转录基因表达情况,研究曲马多是否对神经嵴诱导及迁移产生影响。体外实验中,用U251细胞通过transwell实验检查体外曲马多对细胞迁移的影响,观察是否与胚胎中响应一致。用TUNEL检测曲马多暴露情况下,胚胎细胞凋亡的情况。并分别剥离17期神经嵴,用Q-PCR检测影响神经嵴开始迁移时候的相关的粘附分子的表达在药物暴露与对照组之间的差异情况。结果(1)早期胚胎在曲马多暴露后造成包括细胞色素发育异常、胚胎体积变小、小头等一系列的发育缺陷。(2)早期胚胎在曲马多暴露引起的发育缺陷主要是抑制神经嵴迁移所致。(3)曲马多暴露后,钙粘素依赖粘附分子表达的混乱可能是影响神经嵴迁移障碍的原因。结论早期胚胎曲马多暴露主要通过干扰胚胎神经嵴的迁移引起发育异常。曲马多对神经嵴发育的干扰可能是通过干扰钙粘素依赖的粘附分子的表达所致。由于临床观察发现曲马多滥用易导致流产概率增加,而神经嵴发育正是胚胎早期重要的生命事件,我们的发现为这一临床观察现象提供了可能的解释。目的特应性皮炎(atopic dermatitis AD)是皮肤科最常见的瘙痒性疾病之一,其发病率已接近20%,本病以瘙痒为主要临床表现,易反复发作,不易治愈,患者常伴焦虑、抑郁等心理疾患,负面情绪会引起神经递质释放,影响HPA轴,最终影响Th细胞表型,使得皮肤屏障功能受损,对瘙痒耐受性降低,加重心理疾病,恶化睡眠障碍,形成恶性循环,临床常用的抗组胺药对于AD只有部分缓解瘙痒的临床疗效,结合国内外同行的研究报道与我们的研究基础,认识到一方面氟西汀作为5-HT再摄取抑制剂能缓解焦虑、抑郁等精神症状,另外它能通过调节淋巴细胞的增殖,发挥对炎症性皮肤病的影响,能打破AD的瘙痒-焦虑、抑郁恶性循环,发挥双重治疗作用。我们希望通过研究氟西汀对特应性皮炎的治疗效果及其作用机制,为特异性皮炎的治疗提供新的思路和理论基础。方法首先用2,4-二硝基氯苯(DNCB)丙酮溶液致敏激发BALB/c小鼠,建立特应性皮炎小鼠模型。以该模型为研究对象,研究5-HT再摄取抑制剂对特应性皮炎的影响及其作用的分子机制开展研究。通过观察5-HT再摄取抑制剂(氟西汀)对特应性皮炎小鼠模型的临床疗效、SCORAD积分、抑郁、焦虑的变化情况详细阐明5-HT再摄取抑制剂在特应性皮炎治疗中的作用;通过对小鼠皮肤HE染色和甲苯胺蓝染色检测小鼠皮肤厚度和肥大细胞数量,通过检测血浆、脾脏总Ig E、细胞因子(IL-2、IL-4、IL-13、IFN-r)m RNA水平的变化,探索5-HT再摄取抑制剂治疗特应性皮炎的分子机制。结果(1)氟西汀可以减轻AD小鼠的临床症状。(2)氟西汀可以明显改善AD小鼠的抑郁及焦虑情绪。(3)氟西汀抑制了组织的炎症反应,降低了AD小鼠真皮内肥大细胞数量。(4)氟西汀下调了AD小鼠血清中Ig E水平(5)氟西汀可以下调AD小鼠脾脏中IL-4/IL-13细胞因子水平,明显逆转Th1与Th2细胞相关因子的表达失衡。结论氟西汀可以改善AD的瘙痒症状,氟西汀可以通过下调IL-4/IL-13,以及抑制肥大细胞的产生,改善免疫失衡。氟西汀可能通过抑制心理应激引起的焦虑、抑郁从而改善AD。
[Abstract]:Drug abuse in maternal pregnancy is one of the most important factors for abortion. Tramadol is a widely used analgesic, which has the multiple effects of analgesia, appetite suppression and depression. Currently, the tendency of tramadol addiction and abuse is becoming more and more young. It is reported that more and more addicts are between 18-30 years of age and this age stage is positive. It is a woman's childbearing age. Clinical studies have shown that tramadol exposure during pregnancy greatly increases the abortion rate of women, but the specific mechanism is not clear. The relationship between craniofacial and neural crest development suggests that tramadol may interfere with the development of embryonic neural crest caused by embryonic defects. This topic further explores the effect of tramadol on the neural crest induction and migration, and studies the related molecular mechanisms. The results will be responsible for understanding the causes of the drug addiction and the causes of teratogenicity. It is of great practical significance and scientific value to evaluate the potential risk of this kind of drug in clinical application. Method first, the claw frog embryo in the cleavage stage was exposed to different concentrations of MBS culture containing tramadol (1:200,1:400,1:500). The whole process of embryo development was continuously observed under the Olympus anatomy microscope, and the embryo malformation was found. The drug concentration, and the experiment group and the control group of embryos were photographed under the stereoscopic microscope. The skull cartilage of the embryo (45 period) was stained with octyl blue, the development of the craniofacial cartilage was observed and photographed under the stereoscopic microscope. The objective evaluation of the effect of tramadol on the development of embryo was to find the drug concentration of the abnormal embryo. The concentration of tramadol in the embryo was measured by liquid chromatography and compared with the blood concentration of tramadol in human. The embryos were exposed to different concentrations of tramadol containing MBS culture. The embryos were cultured to the nerve plate period (14-15 phase) and the tail (25 phase). At this time the embryo had been induced and started to migrate, collect the embryo and construct the nerve. The related gene probes (Foxd3, N-tubulin, Sox2, Slug, Twist) induced by crest cells were used to detect the related transcriptional gene expression of Wnt, Fgf, BMP, and Notch/Delta in the embryonic neural plate and the tail teeth by in situ hybridization, and to investigate whether tramadol affects the neural crest induction and migration. In vitro experiments, U251 cells are used in the experiment. Transwell test was used to examine the effect of tramadol on cell migration in vitro and whether it was consistent with the response in the embryo. TUNEL was used to detect the apoptosis of fetal cells under tramadol exposure. The 17 stages of neural crest were stripped and Q-PCR was used to detect the expression of related adhesion molecules affecting the beginning of the migration of the neural crest. The difference between the control groups. Results (1) the early embryos were exposed to tramadol after exposure to abnormal cytochrome development, small embryo volume, small head and a series of developmental defects. (2) the development defects caused by tramadol exposure in early embryos were mainly caused by the inhibition of the migration of neural crest. (3) after tramadol exposure, cadherin adhered to adherence The confusion of molecular expression may be the cause of the disturbance of the neural crest migration. Conclusion the early embryo tramadol exposure is mainly caused by disturbance of the migration of the embryonic neural crest. The interference of tramadol on the neural crest development may be caused by the expression of the adhesion molecules interfering with the calcicin dependence. The clinical observation of tramadol Atopic dermatitis AD is one of the most common pruritus in Department of dermatology. The incidence of atopic dermatitis (atopic) is one of the most common pruritus in Department of dermatology. Its incidence is close to 20%. This disease is mainly pruritus. Clinical manifestations, easy to relapse, not easy to cure, patients often accompanied with anxiety, depression and other psychological disorders, negative emotions can cause neurotransmitter release, the impact of the HPA axis, and ultimately affect the phenotype of Th cells, the skin barrier function damage, itching tolerance, mental illness, worsening sleep disorders, the formation of a vicious cycle, clinical commonly used anti group Amines have only part of the clinical efficacy of AD in alleviating pruritus. Combined with our domestic and foreign research reports and our research basis, it is recognized that fluoxetine as a 5-HT reuptake inhibitor can relieve mental symptoms such as anxiety and depression. In addition, it can regulate the effects of lymphocyte proliferation on inflammatory dermatosis and can play a role in treating inflammatory dermatosis. We hope to provide a new idea and theoretical basis for the treatment of specific dermatitis by studying the therapeutic effect of fluoxetine on atopic dermatitis and the mechanism of action for the treatment of atopic dermatitis. Methods first, we use 2,4- two nitrochlorobenzene (DNCB) acetone (DNCB) acetone solution to stimulate BALB/c mice. A mouse model of allergic dermatitis was used to study the effect of 5-HT reuptake inhibitor on atopic dermatitis and the molecular mechanism of its effect. The clinical efficacy of 5-HT reuptake inhibitor (Fu Xiting) on the clinical efficacy, SCORAD score, depression and anxiety of the mice with atopic dermatitis was elucidated in detail by the 5-HT reuptake inhibitors. The role of inhibitors in the treatment of atopic dermatitis; by detecting the skin thickness and mast cell number of mice with HE staining and toluidine blue staining in mice, the changes in the RNA level of the total Ig E of the spleen, the cytokine (IL-2, IL-4, IL-13, IFN-r) m RNA levels were detected in the mice, and the molecular machine for the treatment of atopic dermatitis by 5-HT reuptake inhibitors was explored. Results (1) fluoxetine could reduce the clinical symptoms of AD mice. (2) fluoxetine could significantly improve the depression and anxiety of AD mice. (3) fluoxetine inhibited the inflammatory response of the tissue and reduced the number of mast cells in the dermis of AD mice. (4) fluoxetine lowered the level of Ig E in the serum of AD mice (5) fluoxetine could reduce the I in the spleen of AD mice. L-4/IL-13 cytokine levels significantly reverse the imbalance in the expression of Th1 and Th2 cell related factors. Conclusion fluoxetine can improve the pruritus of AD. Fluoxetine can improve the immune imbalance by reducing IL-4/IL-13 and inhibiting the production of mast cells. Fluoxetine may improve the anxiety by inhibiting psychological stress and depression and thus improve AD..
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.2
本文编号：2161430
[Abstract]:Drug abuse in maternal pregnancy is one of the most important factors for abortion. Tramadol is a widely used analgesic, which has the multiple effects of analgesia, appetite suppression and depression. Currently, the tendency of tramadol addiction and abuse is becoming more and more young. It is reported that more and more addicts are between 18-30 years of age and this age stage is positive. It is a woman's childbearing age. Clinical studies have shown that tramadol exposure during pregnancy greatly increases the abortion rate of women, but the specific mechanism is not clear. The relationship between craniofacial and neural crest development suggests that tramadol may interfere with the development of embryonic neural crest caused by embryonic defects. This topic further explores the effect of tramadol on the neural crest induction and migration, and studies the related molecular mechanisms. The results will be responsible for understanding the causes of the drug addiction and the causes of teratogenicity. It is of great practical significance and scientific value to evaluate the potential risk of this kind of drug in clinical application. Method first, the claw frog embryo in the cleavage stage was exposed to different concentrations of MBS culture containing tramadol (1:200,1:400,1:500). The whole process of embryo development was continuously observed under the Olympus anatomy microscope, and the embryo malformation was found. The drug concentration, and the experiment group and the control group of embryos were photographed under the stereoscopic microscope. The skull cartilage of the embryo (45 period) was stained with octyl blue, the development of the craniofacial cartilage was observed and photographed under the stereoscopic microscope. The objective evaluation of the effect of tramadol on the development of embryo was to find the drug concentration of the abnormal embryo. The concentration of tramadol in the embryo was measured by liquid chromatography and compared with the blood concentration of tramadol in human. The embryos were exposed to different concentrations of tramadol containing MBS culture. The embryos were cultured to the nerve plate period (14-15 phase) and the tail (25 phase). At this time the embryo had been induced and started to migrate, collect the embryo and construct the nerve. The related gene probes (Foxd3, N-tubulin, Sox2, Slug, Twist) induced by crest cells were used to detect the related transcriptional gene expression of Wnt, Fgf, BMP, and Notch/Delta in the embryonic neural plate and the tail teeth by in situ hybridization, and to investigate whether tramadol affects the neural crest induction and migration. In vitro experiments, U251 cells are used in the experiment. Transwell test was used to examine the effect of tramadol on cell migration in vitro and whether it was consistent with the response in the embryo. TUNEL was used to detect the apoptosis of fetal cells under tramadol exposure. The 17 stages of neural crest were stripped and Q-PCR was used to detect the expression of related adhesion molecules affecting the beginning of the migration of the neural crest. The difference between the control groups. Results (1) the early embryos were exposed to tramadol after exposure to abnormal cytochrome development, small embryo volume, small head and a series of developmental defects. (2) the development defects caused by tramadol exposure in early embryos were mainly caused by the inhibition of the migration of neural crest. (3) after tramadol exposure, cadherin adhered to adherence The confusion of molecular expression may be the cause of the disturbance of the neural crest migration. Conclusion the early embryo tramadol exposure is mainly caused by disturbance of the migration of the embryonic neural crest. The interference of tramadol on the neural crest development may be caused by the expression of the adhesion molecules interfering with the calcicin dependence. The clinical observation of tramadol Atopic dermatitis AD is one of the most common pruritus in Department of dermatology. The incidence of atopic dermatitis (atopic) is one of the most common pruritus in Department of dermatology. Its incidence is close to 20%. This disease is mainly pruritus. Clinical manifestations, easy to relapse, not easy to cure, patients often accompanied with anxiety, depression and other psychological disorders, negative emotions can cause neurotransmitter release, the impact of the HPA axis, and ultimately affect the phenotype of Th cells, the skin barrier function damage, itching tolerance, mental illness, worsening sleep disorders, the formation of a vicious cycle, clinical commonly used anti group Amines have only part of the clinical efficacy of AD in alleviating pruritus. Combined with our domestic and foreign research reports and our research basis, it is recognized that fluoxetine as a 5-HT reuptake inhibitor can relieve mental symptoms such as anxiety and depression. In addition, it can regulate the effects of lymphocyte proliferation on inflammatory dermatosis and can play a role in treating inflammatory dermatosis. We hope to provide a new idea and theoretical basis for the treatment of specific dermatitis by studying the therapeutic effect of fluoxetine on atopic dermatitis and the mechanism of action for the treatment of atopic dermatitis. Methods first, we use 2,4- two nitrochlorobenzene (DNCB) acetone (DNCB) acetone solution to stimulate BALB/c mice. A mouse model of allergic dermatitis was used to study the effect of 5-HT reuptake inhibitor on atopic dermatitis and the molecular mechanism of its effect. The clinical efficacy of 5-HT reuptake inhibitor (Fu Xiting) on the clinical efficacy, SCORAD score, depression and anxiety of the mice with atopic dermatitis was elucidated in detail by the 5-HT reuptake inhibitors. The role of inhibitors in the treatment of atopic dermatitis; by detecting the skin thickness and mast cell number of mice with HE staining and toluidine blue staining in mice, the changes in the RNA level of the total Ig E of the spleen, the cytokine (IL-2, IL-4, IL-13, IFN-r) m RNA levels were detected in the mice, and the molecular machine for the treatment of atopic dermatitis by 5-HT reuptake inhibitors was explored. Results (1) fluoxetine could reduce the clinical symptoms of AD mice. (2) fluoxetine could significantly improve the depression and anxiety of AD mice. (3) fluoxetine inhibited the inflammatory response of the tissue and reduced the number of mast cells in the dermis of AD mice. (4) fluoxetine lowered the level of Ig E in the serum of AD mice (5) fluoxetine could reduce the I in the spleen of AD mice. L-4/IL-13 cytokine levels significantly reverse the imbalance in the expression of Th1 and Th2 cell related factors. Conclusion fluoxetine can improve the pruritus of AD. Fluoxetine can improve the immune imbalance by reducing IL-4/IL-13 and inhibiting the production of mast cells. Fluoxetine may improve the anxiety by inhibiting psychological stress and depression and thus improve AD..
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.2
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