过量atRA对胎鼠腭间充质细胞增殖的影响及作用机制

发布时间：2018-07-21 18:39

【摘要】：目的探讨过量全反式维甲酸(all-trans retinoic acid,at RA)小鼠腭间充质细胞增殖的影响及其作用机制。方法取周龄为10周左右的SPF级C57BL/6J近交系小鼠(雌鼠40只,雄鼠20只),于前一天晚8时按雌雄比2:1进行合笼交配。以次日早晨观察到阴栓阳性的雌鼠标记为妊娠0天(gestation day 0,GD0),共获得30只孕鼠,随机分组后,每组15只。在GD10时,实验组以一次性灌胃给药的方式给予孕鼠100mg/kg的at RA,对照组孕鼠灌以等量玉米油。分别取两组GD13、14、15和16时灌胃时点的胎鼠标本,利用HE染色连续观察胎鼠腭板发育情况。我们建立GD13胎鼠腭突间充质细胞原代培养模型,利用生长状态较好的第三代胎鼠腭间充质(mouse embryonic palate mesenchymal,MEPM)细胞进行体外实验,根据at RA作用浓度,实验共分为5组(0.1μmol/L、0.5μmol/L、1μmol/L、5μmol/L、10μmol/L),并设空白对照组。at RA作用时间分别为:24h、48h和72h。利用台盼蓝染色、MTT法检测at RA对MEPM细胞活性的时间-效应和剂量-效应关系,由此结合RA的临床药理特点,选择适宜的时间和浓度,进行后续的实验。利用Real-time PCR检测过量at RA对Smad2、Smad7 m RNA表达的影响,Western blot方法检测过量at RA对MEPM细胞内蛋白Smad2、Smda7和p-Smad2蛋白表达的影响。应用统计软件SPSS17.0分析本实验结果。检验水准均为双侧α=0.05。结果HE染色实验结果显示:对照组GD13、14、15和16胎鼠双侧腭板完成上抬、接触及融合过程;而与对照组相比,实验组GD13胎鼠双侧腭板体积较小,且并未观察到双侧腭板上抬;GD14时,实验组胎鼠双侧腭板未上抬,可观察到发育明显延迟;GD15时,实验组双侧腭板仍未发生接触,最终在GD16时观察到实验组胎鼠两侧腭板形成腭裂。台盼蓝染色结果显示,随at RA作用时间及浓度增加细胞拒染率逐渐降低,提示随at RA作用时间及浓度增加,细胞活性逐渐下降;MTT实验结果显示:at RA浓度在0.1-10μmol/L范围内,随着培养时间延长,MEPM细胞吸光度值为递减趋势,MEPM细胞活性随着at RA浓度增加细胞活性逐渐下降,尤其是加药72小时后,细胞增殖明显受到抑制,并呈一定的剂量效应关系。我们利用SPSS 17.0分析得到at RA作用72小时对MEPM细胞的半数抑制浓度为:4.76μmol/L。实时定量Real-time PCR结果显示,与对照组比,实验组(5μmol/L at RA)可促进Smad7m RNA的表达(P0.05),而对Smad2 m RNA表达无影响(P0.05);Western blot实验结果显示,与对照组相比,5μmol/L at RA可下调p-Smad2蛋白的表达,促进Smad7蛋白表达,但对Smad2蛋白表达并无影响(P0.05)。结论(1)体外培养条件下,at RA在一定范围内可抑制小鼠胚胎腭间充质细胞增殖,呈剂量效应关系。(2)过量at RA通过下调TGF-β/Smad途径信号分子的正常表达影响小鼠胚胎腭间充质细胞增殖,这可能是at RA诱发腭裂的生物学机制之一。
[Abstract]:Objective to investigate the effect of excessive all-trans retinoic acidat RA on the proliferation of palatal mesenchymal cells and its mechanism. Methods SPF C57BL / 6J inbred mice (40 female and 20 male) aged about 10 weeks were selected for cage mating at 8 pm on the previous day. A total of 30 pregnant mice were randomly divided into three groups, 15 in each group. At the time of GD10, the experimental group was given the same amount of corn oil as the pregnant rat 100mg/kg at RAA by one dose of intragastric administration, while the control group was given the same amount of corn oil. The fetal mouse at the time of gavage of GD1314 and 16:00 were used to observe the development of palatal plate of fetal mice by HE staining. We established the primary culture model of GD13 fetal mouse palatal process mesenchymal cells (GD13), and used the third generation fetal mouse palatal mesenchymal cells (mouse embryonic palate mesenchymal The experiment was divided into five groups (0.1 渭 mol / L, 0.5 渭 mol / L, 1 渭 mol / L, 5 渭 mol / L, 10 渭 mol / L, respectively). Trypan blue MTT assay was used to detect the time-effect and dose-effect relationship of ATRA on MEPM cells. According to the clinical pharmacological characteristics of RA, appropriate time and concentration were selected for further experiments. Real-time PCR was used to detect the effect of excessive at RA on the expression of Smad2 Smad7 mRNA. Western blot was used to detect the effect of excessive at RA on the expression of Smad2 Smad7 and p-Smad2 proteins in MEPM cells. The statistical software SPSS 17.0 was used to analyze the results of this experiment. The test level was 0. 05 for both sides. Results the results of HE staining showed that the bilateral palatal plates in the control group were raised, contacted and fused with GD1314 and 16 fetal mice, while the bilateral palatal plates of the GD13 fetal mice in the experimental group were smaller than those in the control group, and the bilateral palatal plates were not observed when the bilateral palatal plates were lifted up and GD14 was not observed in the experimental group. The bilateral palatal plate was not raised in the experimental group, and the development of GD15 was delayed obviously, and the bilateral palatal plate was not contacted in the experimental group. Finally, cleft palate was observed in the bilateral palatal plate of the experimental group at GD16. The results of trypan blue staining showed that the cell rejection rate decreased with the increase of ATRA concentration and time, suggesting that the cell activity decreased with the increase of ATRA concentration. The results of MTT assay showed that the concentration of at RA was in the range of 0.1-10 渭 mol / L. The activity of MEPM cells decreased with the increase of ATRA concentration, especially after 72 hours, the proliferation of MEPM cells was inhibited in a dose-dependent manner. By SPSS 17.0 analysis, we obtained that the half inhibitory concentration of ATRA on MEPM cells was: 1: 4.76 渭 mol / L for 72 hours. The results of real-time PCR showed that 5 渭 mol / L atRA could promote the expression of Smad7m RNA (P0.05), but had no effect on the expression of Smad2 mRNA (P0.05). The results of Western blot showed that 5 渭 mol / L tat RA could down-regulate the expression of p-Smad2 protein compared with the control group. Promote the expression of Smad7 protein, but have no effect on the expression of Smad2 protein (P0.05). Conclusion (1) the proliferation of mouse embryonic palate mesenchymal cells can be inhibited in a dose-dependent manner under the condition of culture in vitro. (2) excessive atRA can affect the proliferation of mouse embryonic palate mesenchymal cells by down-regulating the normal expression of TGF- 尾 / Smad signaling molecule. This may be one of the biological mechanisms of cleft palate induced by at RA.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R782.2

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