本文關(guān)鍵詞： 脫氧雪腐鐮刀菌烯醇 小鼠胚胎干細(xì)胞 細(xì)胞增殖 細(xì)胞周期 基因表達(dá) 毒性　出處：《泰山醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 利用小鼠胚胎干細(xì)胞(mouse embryonic stem cell, mESCs)為體外試驗(yàn)?zāi)Ｐ?探討脫氧雪腐鐮刀菌烯醇(deoxynivalenol, DON)對(duì)mES細(xì)胞增殖、周期的細(xì)胞毒性作用;對(duì)mES細(xì)胞階段特異性胚胎表面抗原(stage-specific embryonic antigen 1, SSEA-1)、Nanog、Oct-4、Sox1及Casp-3基因表達(dá)情況的影響;對(duì)Notch信號(hào)通路的作用。為進(jìn)一步篩選病原體和其他毒素對(duì)干細(xì)胞增殖、胚胎發(fā)育相關(guān)基因表達(dá)的作用機(jī)制提供理論依據(jù)。 方法 采用飼養(yǎng)層培養(yǎng)法培養(yǎng)mES細(xì)胞,不同濃度的DON(0,50,100,500,1000,2000ng/ml)分別作用于對(duì)數(shù)生長(zhǎng)期的mES細(xì)胞24小時(shí),噻唑藍(lán)(MTT)法檢測(cè)DON對(duì)mES細(xì)胞增殖的影響;利用差速貼壁法純化mES細(xì)胞,檢測(cè)DON對(duì)細(xì)胞周期的影響;免疫細(xì)胞化學(xué)法觀察DON對(duì)SSEA-1、Nanog、Oct-4、Sox1、Casp-3基因表達(dá)情況;Trizol裂解細(xì)胞提取mES細(xì)胞總RNA,RT-PCR檢測(cè)DON對(duì)轉(zhuǎn)錄因子Oct-4,Nanog基因的影響;用RIPA細(xì)胞裂解液提取mES細(xì)胞總蛋白,檢測(cè)DON對(duì)Notch信號(hào)通路基因表達(dá)的影響。 結(jié)果 1.DON對(duì)干細(xì)胞增殖的影響: 將含有不同濃度DON的培養(yǎng)液加入對(duì)數(shù)生長(zhǎng)期的mES細(xì)胞,繼續(xù)培養(yǎng)24h后,觀察DON對(duì)mES細(xì)胞增殖的作用,結(jié)果顯示:隨DON濃度的增加,吸光度值逐漸降低,DON濃度為50ng/ml、100ng/ml時(shí),與空白對(duì)照組相比mES細(xì)胞增殖及存活率降低,但無(wú)統(tǒng)計(jì)學(xué)差異。當(dāng)DON濃度為500ng/ml、1000ng/ml、2000ng/ml時(shí),吸光度值明顯降低,與0ng/ml、50ng/ml、100ng/ml組兩兩比較都有統(tǒng)計(jì)學(xué)意義(P0.01)。DON在2000ng/ml時(shí),顯微鏡下觀察mES細(xì)胞可見細(xì)胞克隆團(tuán)比500ng/ml、1000ng/ml組減小更加明顯,細(xì)胞密度逐漸降低,但無(wú)統(tǒng)計(jì)學(xué)意義。實(shí)驗(yàn)發(fā)現(xiàn)隨DON濃度的增加,抑制mES細(xì)胞增殖的作用逐漸增強(qiáng)。 2.DON對(duì)細(xì)胞周期的影響: 流式細(xì)胞儀檢測(cè)結(jié)果顯示:DON作用mES細(xì)胞24h后,隨DON濃度的增加,細(xì)胞增殖指數(shù)降低。5ng/ml、10ng/ml、50ng/ml、100ng/ml組結(jié)果與空白對(duì)照組相比區(qū)別不明顯,500ng/ml及2000ng/ml組與0ng/ml、5ng/ml、10ng/ml、50ng/ml組兩兩比較均有統(tǒng)計(jì)學(xué)意義(P0.01)。2000ng/mlDON組作用mES細(xì)胞后比其它組S期降低顯著,和500ng/ml組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。試驗(yàn)表明DON影響mES細(xì)胞周期的分布,具有明顯的抗增殖作用。 3.DON對(duì)基因表達(dá)的影響: 免疫細(xì)胞化學(xué)法檢測(cè)結(jié)果顯示:DON濃度為100ng/ml時(shí),Nanog、Sox1、Oct-4的表達(dá)量與空白對(duì)照組相比變化不明顯,而當(dāng)DON濃度為500ng/ml時(shí),上述基因表達(dá)量明顯減少;DON在10ng/ml、100ng/ml及500ng/ml時(shí),casp-3基因表達(dá)與空白對(duì)照組相比無(wú)明顯變化,DON濃度為2000ng/ml組,可見其亮度增加,表達(dá)量增多;SSEA-1的表達(dá)在500ng/ml DON組,與空白對(duì)照組相比,其平均光密度值增加明顯(P0.01),差異有統(tǒng)計(jì)學(xué)意義,1000ng/ml組與500ng/ml組相比,表達(dá)量略有增加,但無(wú)顯著性差異(P0.05)。 RT-PCR檢測(cè)發(fā)現(xiàn)500ng/ml DON組可抑制Oct-4,Nanog mRNA的表達(dá),與空白對(duì)照組比較有統(tǒng)計(jì)學(xué)意義(P0.01)。隨濃度的增加,Oct-4,Nanog的表達(dá)量降低,但1000ng/ml和500ng/ml組相比,無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 4.DON對(duì)Notch信號(hào)通路的影響: Western blot檢測(cè)顯示:當(dāng)DON濃度為10ng/ml時(shí),NICD表達(dá)明顯減少,提示Notch信號(hào)通路受到抑制,當(dāng)DON濃度超過(guò)100ng/ml時(shí),表達(dá)微弱,Notch信號(hào)通路抑制明顯。Notch1受體Jag1、Notch1下游信號(hào)Hes1表達(dá)也隨濃度增加減少明顯。實(shí)驗(yàn)結(jié)果說(shuō)明DON影響Notch信號(hào)通路,參與Notch信號(hào)通路的轉(zhuǎn)導(dǎo)。 結(jié)論 1.DON對(duì)mES細(xì)胞有直接毒性作用,能抑制mES細(xì)胞的生長(zhǎng),影響細(xì)胞周期的分布,抑制細(xì)胞進(jìn)入S期,使細(xì)胞停滯在G0/G1期,具有明顯的抗增殖作用。 2.SSEA-1、Oct-4、Nanog是mES細(xì)胞的特異基因,其轉(zhuǎn)錄水平直接影響mES細(xì)胞的分化和發(fā)育。DON作用mES細(xì)胞后,SSEA-1表達(dá)量升高, Oct-4、Nanog基因表達(dá)下降,從而影響mES細(xì)胞的增殖及分化,異常的分化可使胚胎早期的正常發(fā)育受到損害。 3.Notch信號(hào)通路在胚胎發(fā)育、干細(xì)胞特性維持、細(xì)胞分化、增殖、凋亡等一系列活動(dòng)中都起十分重要的作用。DON在低濃度(10ng/ml)時(shí)即可抑制Notch通路,影響mES細(xì)胞的特性維持、增殖及分化等,進(jìn)而影響早期胚胎發(fā)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.

【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 武慧慧;于愛(ài)蓮;;脫氧雪腐鐮刀菌烯醇的細(xì)胞毒性作用[J];泰山醫(yī)學(xué)院學(xué)報(bào);2012年04期

相關(guān)碩士學(xué)位論文 前2條

1 武慧慧;脫氧雪腐鐮刀菌烯醇對(duì)小鼠成骨細(xì)胞PAX1基因表達(dá)的影響[D];泰山醫(yī)學(xué)院;2012年

2 孫豪;脫氧雪腐鐮刀菌烯醇對(duì)小鼠成骨細(xì)胞RUNX2基因表達(dá)的影響[D];泰山醫(yī)學(xué)院;2012年

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本文編號(hào)：1547270