脱氧雪腐镰刀菌烯醇对小鼠胚胎干细胞的毒性作用

发布时间：2018-02-28 12:15

  本文关键词： 脱氧雪腐镰刀菌烯醇 小鼠胚胎干细胞 细胞增殖 细胞周期 基因表达 毒性　出处：《泰山医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的 利用小鼠胚胎干细胞(mouse embryonic stem cell, mESCs)为体外试验模型,探讨脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)对mES细胞增殖、周期的细胞毒性作用;对mES细胞阶段特异性胚胎表面抗原(stage-specific embryonic antigen 1, SSEA-1)、Nanog、Oct-4、Sox1及Casp-3基因表达情况的影响;对Notch信号通路的作用。为进一步筛选病原体和其他毒素对干细胞增殖、胚胎发育相关基因表达的作用机制提供理论依据。 方法 采用饲养层培养法培养mES细胞,不同浓度的DON(0,50,100,500,1000,2000ng/ml)分别作用于对数生长期的mES细胞24小时,噻唑蓝(MTT)法检测DON对mES细胞增殖的影响;利用差速贴壁法纯化mES细胞,检测DON对细胞周期的影响;免疫细胞化学法观察DON对SSEA-1、Nanog、Oct-4、Sox1、Casp-3基因表达情况;Trizol裂解细胞提取mES细胞总RNA,RT-PCR检测DON对转录因子Oct-4,Nanog基因的影响;用RIPA细胞裂解液提取mES细胞总蛋白,检测DON对Notch信号通路基因表达的影响。 结果 1.DON对干细胞增殖的影响: 将含有不同浓度DON的培养液加入对数生长期的mES细胞,继续培养24h后,观察DON对mES细胞增殖的作用,结果显示:随DON浓度的增加,吸光度值逐渐降低,DON浓度为50ng/ml、100ng/ml时,与空白对照组相比mES细胞增殖及存活率降低,但无统计学差异。当DON浓度为500ng/ml、1000ng/ml、2000ng/ml时,吸光度值明显降低,与0ng/ml、50ng/ml、100ng/ml组两两比较都有统计学意义(P0.01)。DON在2000ng/ml时,显微镜下观察mES细胞可见细胞克隆团比500ng/ml、1000ng/ml组减小更加明显,细胞密度逐渐降低,但无统计学意义。实验发现随DON浓度的增加,抑制mES细胞增殖的作用逐渐增强。 2.DON对细胞周期的影响: 流式细胞仪检测结果显示:DON作用mES细胞24h后,随DON浓度的增加,细胞增殖指数降低。5ng/ml、10ng/ml、50ng/ml、100ng/ml组结果与空白对照组相比区别不明显,500ng/ml及2000ng/ml组与0ng/ml、5ng/ml、10ng/ml、50ng/ml组两两比较均有统计学意义(P0.01)。2000ng/mlDON组作用mES细胞后比其它组S期降低显著,和500ng/ml组比较差异无统计学意义(P0.05)。试验表明DON影响mES细胞周期的分布,具有明显的抗增殖作用。 3.DON对基因表达的影响: 免疫细胞化学法检测结果显示:DON浓度为100ng/ml时,Nanog、Sox1、Oct-4的表达量与空白对照组相比变化不明显,而当DON浓度为500ng/ml时,上述基因表达量明显减少;DON在10ng/ml、100ng/ml及500ng/ml时,casp-3基因表达与空白对照组相比无明显变化,DON浓度为2000ng/ml组,可见其亮度增加,表达量增多;SSEA-1的表达在500ng/ml DON组,与空白对照组相比,其平均光密度值增加明显(P0.01),差异有统计学意义,1000ng/ml组与500ng/ml组相比,表达量略有增加,但无显著性差异(P0.05)。 RT-PCR检测发现500ng/ml DON组可抑制Oct-4,Nanog mRNA的表达,与空白对照组比较有统计学意义(P0.01)。随浓度的增加,Oct-4,Nanog的表达量降低,但1000ng/ml和500ng/ml组相比,无统计学差异(P0.05)。 4.DON对Notch信号通路的影响: Western blot检测显示:当DON浓度为10ng/ml时,NICD表达明显减少,提示Notch信号通路受到抑制,当DON浓度超过100ng/ml时,表达微弱,Notch信号通路抑制明显。Notch1受体Jag1、Notch1下游信号Hes1表达也随浓度增加减少明显。实验结果说明DON影响Notch信号通路,参与Notch信号通路的转导。 结论 1.DON对mES细胞有直接毒性作用,能抑制mES细胞的生长,影响细胞周期的分布,抑制细胞进入S期,使细胞停滞在G0/G1期,具有明显的抗增殖作用。 2.SSEA-1、Oct-4、Nanog是mES细胞的特异基因,其转录水平直接影响mES细胞的分化和发育。DON作用mES细胞后,SSEA-1表达量升高, Oct-4、Nanog基因表达下降,从而影响mES细胞的增殖及分化,异常的分化可使胚胎早期的正常发育受到损害。 3.Notch信号通路在胚胎发育、干细胞特性维持、细胞分化、增殖、凋亡等一系列活动中都起十分重要的作用。DON在低浓度(10ng/ml)时即可抑制Notch通路,影响mES细胞的特性维持、增殖及分化等,进而影响早期胚胎发育。
[Abstract]:objective
The use of mouse embryonic stem cells (mouse embryonic stem cell, mESCs) for in vitro model of deoxynivalenol (deoxynivalenol, DON) on the proliferation of mES cells, the cytotoxicity of mES cell cycle; stage specific embryonic antigen (stage-specific embryonic antigen 1, SSEA-1), Nanog, Oct-4, Sox1 expression and the effect of Casp-3 gene; Notch pathway. For further screening of pathogens and other toxins on stem cell proliferation, and provide a theoretical basis for the mechanism of expression of genes related to embryonic development.
Method
The feeder layer of cultured mES cells, different concentrations of DON (0,5010050010002000ng/ml) were added to mES cells in logarithmic growth time of 24 hours, thiazolyl blue (MTT) method was used to detect the effect of DON on proliferation of mES cells; using differential adhesion purified mES cells, to study the effect of DON on cell cycle; immune cells chemical method DON on SSEA-1, Nanog, Oct-4, Sox1, Casp-3 gene expression; mES cell extraction total RNA Trizol RT-PCR DON to detect cell lysis, transcription factor Oct-4, the effect of Nanog gene; mES cell total protein extraction with RIPA cell lysate, influence the detection of DON on the expression of Notch signaling genes.
Result
The effect of 1.DON on the proliferation of stem cells:
Cultured with different concentrations of DON into mES cells in logarithmic growth phase, continue to culture 24h, and observe the effect of DON on the proliferation of mES cells. The results show that with the increase of DON concentration, the absorbance value decreased gradually, the concentration of DON was 50ng/ml, 100ng/ml, compared to mES cell proliferation and decrease the survival rate and blank control group, but the difference was not statistically significant. When the concentration of DON is 500ng/ml, 1000ng/ml, 2000ng/ml, the absorbance value decreased significantly, and 0ng/ml, 50ng/ml, 100ng/ml 22 group comparison had statistical significance (P0.01) in.DON 2000ng/ml, under the microscope observation showed that cells mES cell clones than 500ng/ml, 1000ng/ml group decreased more obviously, cell the density gradually decreased, but there was no statistical significance. The experimental results showed that with the increase of DON concentration, inhibit the proliferation of mES cells increased gradually.
The effect of 2.DON on cell cycle:
Flow cytometry results showed that: DON mES cells after 24h, with the increase of DON concentration, the cell proliferation index decreased.5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml group results compared with the blank control group was no significant difference between 500ng/ml and 2000ng/ml group, and 0ng/ml, 5ng/ml, 10ng/ml, 50ng/ml 22 groups were statistically significant (P0.01).2000ng/mlDON group mES cells than the other group S decreased significantly, there was no significant difference compared with 500ng/ml group (P0.05). The test shows that the effect of mES on cell cycle distribution of DON, has obvious anti proliferative effect.
The effect of 3.DON on gene expression:
The detection results of immunocytochemistry showed that DON concentration of 100ng/ml, Nanog, Sox1, the expression of Oct-4 compared with the blank control group did not change significantly, but when the DON concentration is 500ng/ml, the gene expression decreased significantly; DON in 10ng/ml, 100ng/ml and 500ng/ml, casp-3 gene expression and the blank control group had no significant compared with the change of DON concentration in group 2000ng/ml, the brightness is increased, expression increased; the expression of SSEA-1 in the 500ng/ml DON group compared with the control group, the average optical density value increased significantly (P0.01), the difference was statistically significant, 1000ng/ml group compared with 500ng/ml group, the expression increased slightly, but not significantly the difference (P0.05).
RT-PCR detection showed that 500ng/ml DON group could inhibit the expression of Oct-4 and Nanog mRNA, which was statistically significant compared with the blank control group (P0.01). With the increase of concentration, the expression of Oct-4 and Nanog decreased, but there was no significant difference between 1000ng/ml and 500ng/ml group (Nanog).
The effect of 4.DON on the Notch signaling pathway:
Western blot detection: when the DON concentration is 10ng/ml, the expression of NICD was significantly reduced, suggesting that Notch signaling pathway was inhibited when the DON concentration exceeds 100ng/ml, the expression of weak Notch signal pathway significantly inhibited.Notch1 receptor Jag1, Notch1 expression of Hes1 downstream signaling also decreased with increasing concentrations significantly reduced. Experimental results show that the effect of DON Notch signaling pathway transduction of Notch signal pathway.
conclusion
1.DON has a direct toxic effect on mES cells. It can inhibit the growth of mES cells, influence the distribution of cell cycle, inhibit cell entry into S phase, make cells stagnate in G0/G1 stage, and have obvious anti proliferative effect.
2.SSEA-1, Oct-4, Nanog is the specific gene of mES cells, the transcription level of mES directly affects the differentiation and development of mES cells after.DON treatment, SSEA-1 expression increased, Oct-4 decreased, Nanog gene expression, thus affecting the proliferation and differentiation of mES cells, abnormal differentiation of the normal embryonic development of early damage.
The 3.Notch signaling pathway in embryonic development, stem cell maintenance, cell differentiation, proliferation, have played a very important role in the low concentration of.DON in a series of activities in apoptosis (10ng/ml) can inhibit the Notch pathway, maintain characteristics of mES cells, proliferation and differentiation, and effects of early embryonic development.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【引证文献】

相关期刊论文 前1条

1 武慧慧;于爱莲;;脱氧雪腐镰刀菌烯醇的细胞毒性作用[J];泰山医学院学报;2012年04期

相关硕士学位论文 前2条

1 武慧慧;脱氧雪腐镰刀菌烯醇对小鼠成骨细胞PAX1基因表达的影响[D];泰山医学院;2012年

2 孙豪;脱氧雪腐镰刀菌烯醇对小鼠成骨细胞RUNX2基因表达的影响[D];泰山医学院;2012年

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