曲馬多引起胚胎發(fā)育缺陷的分子機制
發(fā)布時間：2018-08-03 10:17
【摘要】：目的母孕期藥物濫用是流產(chǎn)的重要因素之一。曲馬多是目前應用廣泛的止痛藥,具有鎮(zhèn)痛、抑制食欲、改善抑郁等多重功效。當前,曲馬多成癮和濫用有越來越年輕化的趨勢,報道顯示,越來越多的成癮者年齡在18-30歲之間,這個年齡階段正是女性的育齡期。臨床研究表明孕期曲馬多暴露大大增加女性流產(chǎn)率,但具體機制尚不清楚。本課題以非洲爪蛙為動物模型,發(fā)現(xiàn)胚胎在曲馬多暴露后造成包括細胞色素發(fā)育異常、胚胎體積變小、小頭等一系列的發(fā)育缺陷。由于色素細胞和顱面部與神經(jīng)嵴的發(fā)育相關,提示曲馬多可能干擾早期胚胎神經(jīng)嵴的發(fā)育引起胚胎缺陷。本課題進而進一步探討曲馬多對神經(jīng)嵴誘導和遷移的影響,并對相關分子機制等方面展開研究。研究結果將對了解該藥物的成癮原因、致畸原因和評估該類藥物在臨床應用中的潛在風險具有重大現(xiàn)實意義及科學價值。方法首先將卵裂期的爪蛙胚胎暴露于不同濃度的含曲馬多的MBS培養(yǎng)液中(1:200,1:400,1:500),在Olympus解剖顯微鏡下連續(xù)觀察胚胎的發(fā)育全過程,找出出現(xiàn)胚胎畸形的藥物濃度,并將實驗組和對照組胚胎在體視顯微鏡下進行攝影。對胚胎(45時期)的頭顱軟骨做阿辛藍染色,觀察顱面軟骨發(fā)育情況,并在體視顯微鏡下進行攝影�？陀^評價曲馬多是否對胚胎發(fā)育有影響,找出出現(xiàn)畸形胚胎的藥物濃度。同時用液相色譜法測定胚胎中曲馬多的藥物濃度,與人類曲馬多的血藥濃度比對。將暴露于不同濃度的含曲馬多的MBS培養(yǎng)液中的胚胎,培養(yǎng)至神經(jīng)板時期(14-15期)及尾牙期(25期),此時胚胎已完成誘導及開始遷移,收集此期的胚胎,并構建神經(jīng)嵴細胞誘導的相關基因探針(Foxd3、N-tubulin、Sox2、Slug、Twist),通過原位雜交方式檢測胚胎神經(jīng)板時期及尾牙期Wnt,Fgf,BMP,and Notch/Delta等信號通路的相關轉錄基因表達情況,研究曲馬多是否對神經(jīng)嵴誘導及遷移產(chǎn)生影響。體外實驗中,用U251細胞通過transwell實驗檢查體外曲馬多對細胞遷移的影響,觀察是否與胚胎中響應一致。用TUNEL檢測曲馬多暴露情況下,胚胎細胞凋亡的情況。并分別剝離17期神經(jīng)嵴,用Q-PCR檢測影響神經(jīng)嵴開始遷移時候的相關的粘附分子的表達在藥物暴露與對照組之間的差異情況。結果(1)早期胚胎在曲馬多暴露后造成包括細胞色素發(fā)育異常、胚胎體積變小、小頭等一系列的發(fā)育缺陷。(2)早期胚胎在曲馬多暴露引起的發(fā)育缺陷主要是抑制神經(jīng)嵴遷移所致。(3)曲馬多暴露后,鈣粘素依賴粘附分子表達的混亂可能是影響神經(jīng)嵴遷移障礙的原因。結論早期胚胎曲馬多暴露主要通過干擾胚胎神經(jīng)嵴的遷移引起發(fā)育異常。曲馬多對神經(jīng)嵴發(fā)育的干擾可能是通過干擾鈣粘素依賴的粘附分子的表達所致。由于臨床觀察發(fā)現(xiàn)曲馬多濫用易導致流產(chǎn)概率增加,而神經(jīng)嵴發(fā)育正是胚胎早期重要的生命事件,我們的發(fā)現(xiàn)為這一臨床觀察現(xiàn)象提供了可能的解釋。目的特應性皮炎(atopic dermatitis AD)是皮膚科最常見的瘙癢性疾病之一,其發(fā)病率已接近20%,本病以瘙癢為主要臨床表現(xiàn),易反復發(fā)作,不易治愈,患者常伴焦慮、抑郁等心理疾患,負面情緒會引起神經(jīng)遞質釋放,影響HPA軸,最終影響Th細胞表型,使得皮膚屏障功能受損,對瘙癢耐受性降低,加重心理疾病,惡化睡眠障礙,形成惡性循環(huán),臨床常用的抗組胺藥對于AD只有部分緩解瘙癢的臨床療效,結合國內外同行的研究報道與我們的研究基礎,認識到一方面氟西汀作為5-HT再攝取抑制劑能緩解焦慮、抑郁等精神癥狀,另外它能通過調節(jié)淋巴細胞的增殖,發(fā)揮對炎癥性皮膚病的影響,能打破AD的瘙癢-焦慮、抑郁惡性循環(huán),發(fā)揮雙重治療作用。我們希望通過研究氟西汀對特應性皮炎的治療效果及其作用機制,為特異性皮炎的治療提供新的思路和理論基礎。方法首先用2,4-二硝基氯苯(DNCB)丙酮溶液致敏激發(fā)BALB/c小鼠,建立特應性皮炎小鼠模型。以該模型為研究對象,研究5-HT再攝取抑制劑對特應性皮炎的影響及其作用的分子機制開展研究。通過觀察5-HT再攝取抑制劑(氟西汀)對特應性皮炎小鼠模型的臨床療效、SCORAD積分、抑郁、焦慮的變化情況詳細闡明5-HT再攝取抑制劑在特應性皮炎治療中的作用;通過對小鼠皮膚HE染色和甲苯胺藍染色檢測小鼠皮膚厚度和肥大細胞數(shù)量,通過檢測血漿、脾臟總Ig E、細胞因子(IL-2、IL-4、IL-13、IFN-r)m RNA水平的變化,探索5-HT再攝取抑制劑治療特應性皮炎的分子機制。結果(1)氟西汀可以減輕AD小鼠的臨床癥狀。(2)氟西汀可以明顯改善AD小鼠的抑郁及焦慮情緒。(3)氟西汀抑制了組織的炎癥反應,降低了AD小鼠真皮內肥大細胞數(shù)量。(4)氟西汀下調了AD小鼠血清中Ig E水平(5)氟西汀可以下調AD小鼠脾臟中IL-4/IL-13細胞因子水平,明顯逆轉Th1與Th2細胞相關因子的表達失衡。結論氟西汀可以改善AD的瘙癢癥狀,氟西汀可以通過下調IL-4/IL-13,以及抑制肥大細胞的產(chǎn)生,改善免疫失衡。氟西汀可能通過抑制心理應激引起的焦慮、抑郁從而改善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..
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R714.2
本文編號：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..
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R714.2
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