曲馬多引起胚胎發(fā)育缺陷的分子機(jī)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(yīng)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(dāng)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(jī)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(xì)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(xì)胞和顱面部與神經(jīng)嵴的發(fā)育相關(guān),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(jìn)而進(jìn)一步探討曲馬多對神經(jīng)嵴誘導(dǎo)和遷移的影響,并對相關(guān)分子機(jī)制等方面展開研究。研究結(jié)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(yīng)用中的潛在風(fēng)險具有重大現(xiàn)實意義及科學(xué)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(jìn)行攝影。對胚胎(45時期)的頭顱軟骨做阿辛藍(lán)染色,觀察顱面軟骨發(fā)育情況,并在體視顯微鏡下進(jìn)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(dǎo)及開始遷移,收集此期的胚胎,并構(gòu)建神經(jīng)嵴細(xì)胞誘導(dǎo)的相關(guān)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(guān)轉(zhuǎn)錄基因表達(dá)情況,研究曲馬多是否對神經(jīng)嵴誘導(dǎo)及遷移產(chǎn)生影響。體外實驗中,用U251細(xì)胞通過transwell實驗檢查體外曲馬多對細(xì)胞遷移的影響,觀察是否與胚胎中響應(yīng)一致。用TUNEL檢測曲馬多暴露情況下,胚胎細(xì)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(guān)的粘附分子的表達(dá)在藥物暴露與對照組之間的差異情況。結(jié)果(1)早期胚胎在曲馬多暴露后造成包括細(xì)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(dá)的混亂可能是影響神經(jīng)嵴遷移障礙的原因。結(jié)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(dá)所致。由于臨床觀察發(fā)現(xiàn)曲馬多濫用易導(dǎo)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(yīng)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(fù)發(fā)作,不易治愈,患者常伴焦慮、抑郁等心理疾患,負(fù)面情緒會引起神經(jīng)遞質(zhì)釋放,影響HPA軸,最終影響Th細(xì)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(jié)合國內(nèi)外同行的研究報道與我們的研究基礎(chǔ),認(rèn)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(diào)節(jié)淋巴細(xì)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(yīng)性皮炎的治療效果及其作用機(jī)制,為特異性皮炎的治療提供新的思路和理論基礎(chǔ)。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(yīng)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(yīng)性皮炎的影響及其作用的分子機(jī)制開展研究。通過觀察5-HT再攝取抑制劑(氟西汀)對特應(yīng)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(xì)闡明5-HT再攝取抑制劑在特應(yīng)性皮炎治療中的作用;通過對小鼠皮膚HE染色和甲苯胺藍(lán)染色檢測小鼠皮膚厚度和肥大細(xì)胞數(shù)量,通過檢測血漿、脾臟總Ig E、細(xì)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(yīng)性皮炎的分子機(jī)制。結(jié)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(yīng),降低了AD小鼠真皮內(nèi)肥大細(xì)胞數(shù)量。(4)氟西汀下調(diào)了AD小鼠血清中Ig E水平(5)氟西汀可以下調(diào)AD小鼠脾臟中IL-4/IL-13細(xì)胞因子水平,明顯逆轉(zhuǎn)Th1與Th2細(xì)胞相關(guān)因子的表達(dá)失衡。結(jié)論氟西汀可以改善AD的瘙癢癥狀,氟西汀可以通過下調(diào)IL-4/IL-13,以及抑制肥大細(xì)胞的產(chǎn)生,改善免疫失衡。氟西汀可能通過抑制心理應(yīng)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R714.2
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
1 蒙一純,ＰｈｉｌｉｐＢｒａｕｅｒ;早期雞胚神經(jīng)管和微核神經(jīng)嵴的細(xì)胞凋亡[J];解剖科學(xué)進(jìn)展;1997年03期
2 張建中;S—100蛋白在神經(jīng)嵴起源組織及其腫瘤中的分布[J];實用癌癥雜志;1988年02期
3 蔡良琬;神經(jīng)組織發(fā)育與再生的研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;1994年06期
4 于蕾;倪潔麗;馬俊青;陳文靜;;Rap1b在斑馬魚神經(jīng)嵴中表達(dá)情況的初探[J];口腔生物醫(yī)學(xué);2013年03期
5 Bahn CF ,王傳富;以神經(jīng)嵴起源為根據(jù)的角膜內(nèi)皮疾病分類[J];眼科新進(jìn)展;1986年02期
6 陸穎靜;陳樹寶;陸歐倫;;心臟神經(jīng)嵴受損與心臟畸形發(fā)生的關(guān)系[J];中華兒科雜志;1997年01期
7 劉積才;譚義書;;神經(jīng)嵴起源的少見腫瘤[J];腫瘤防治研究;1988年04期
8 張華;馮永;;PAX3基因?qū)ι窠?jīng)嵴發(fā)育的調(diào)控及其在Waardenburg綜合征發(fā)病中的作用研究[J];聽力學(xué)及言語疾病雜志;2014年02期
9 趙曉晴;心臟神經(jīng)嵴發(fā)育與心臟錐干部畸形[J];國外醫(yī)學(xué)(兒科學(xué)分冊);2004年01期
10 劉向陽;王曄;宋維亞;張臻;;Wnt1-Cre標(biāo)記心臟神經(jīng)嵴來源細(xì)胞在房室瓣分布的實驗研究[J];中華臨床醫(yī)師雜志(電子版);2012年22期
相關(guān)會議論文 前1條
1 藺海燕;劉芳;張傳森;張志英;孔征東;張喜;;移植了毛囊神經(jīng)嵴干細(xì)胞源性神經(jīng)元和施萬細(xì)胞的神經(jīng)移植體的體外特性[A];中國解剖學(xué)會2011年年會論文文摘匯編[C];2011年
相關(guān)博士學(xué)位論文 前3條
1 王曉鈺;高糖環(huán)境誘導(dǎo)的過度自噬抑制雞胚顱面部神經(jīng)嵴產(chǎn)生[D];暨南大學(xué);2015年
2 李彥希;曲馬多引起胚胎發(fā)育缺陷的分子機(jī)制[D];重慶醫(yī)科大學(xué);2016年
3 行勇軍;神經(jīng)嵴來源外胚間充質(zhì)干細(xì)胞在牙向分化中的潛能及相關(guān)機(jī)制的研究[D];第三軍醫(yī)大學(xué);2016年
，本文編號：2161430
本文鏈接：http://sikaile.net/yixuelunwen/fuchankeerkelunwen/2161430.html
最近更新
教材專著
