【作者】 张龙；

【导师】 孙志伟；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2011， 硕士

【摘要】 孕期甲基汞暴露可致仔鼠脑发育受损,并且引起脑神经元细胞周期进程改变。CyclinD1和p21在其中可能发挥着重要的作用。本研究建立宫内暴露甲基汞的大鼠实验模型,检测仔鼠在不同时间和不同暴露剂量下大脑各脑区神经元细胞周期的变化情况,并且检测,Cyclin D1和p21的表达,以探索其在甲基汞所致仔鼠脑神经元细胞周期进程改变调控中的作用。1、甲基汞宫内暴露对神经细胞周期的影响随着染毒剂量增加：在大脑中,Go/G1期细胞百分率在逐渐降低, S和G2/M期细胞百分率逐渐升高。在1.5mg/kg剂量组出现G2/M期细胞阻滞；在3.0mg/kg剂量组出现S期细胞周期阻滞。在小脑中,细胞百分率在G0/G1期逐渐下降,在S和G2/M期逐渐升高。0.75mg/kg剂量组中出现S期细胞周期阻滞。在海马中,G0/G1和G2/M期细胞百分率都逐渐降低,S期细胞百分率有上升趋势。0.75mg/kg和3.0mg/kg剂量组出现S期细胞周期阻滞。孕期暴露甲基汞也显示出一定的时间效应。PND14出现G0/G1细胞比例降低,G2/M和S期细胞比例升高,而在PND30时,趋势与PND14相反,出现G0/G1期细胞比例升高,S期和G2/M期细胞比例降低。2、RT-PCR检测p21基因的表达采用RT-PCR方法检测p21wafl/ciPl mRNA的表达。在3.0mg/kg剂量组,大脑和海马p21wafl/ciPl mRNA表达,随着仔鼠出生后天数的增加p21wafl/ciPl mRNA水平表达逐渐降低。小脑p21wafl/ciPl mRNA表达,随着仔鼠出生后天数的增加,p21wafl/ciPl mRNA表达均逐渐增加。孕期暴露甲基汞仔鼠在出生后14天,大脑p21wafl/ciPl mRNA表达,甲基汞染毒各剂量组表达增加,0.75、1.5mg/kg剂量明显高于对照组；海马p21wafl/ciPl mRNA表达,各剂量染毒组均低于对照组。小脑p21wafl/ciPl mRNA表达,各剂量组低于对照组。3、免疫组织化学法检测Cyclin D1、p21、PKC和CaN的表达采用免疫组织化学法检测Cyclin D1和p21的表达。结果显示,在PND14,各脑区Cyclin D1蛋白表达随染毒剂量增加呈现逐渐升高的趋势,并且中、高剂量组Cyclin D1蛋白表达量与对照组比较,差异具有显著性(P<0.05); 3.0mg/kg剂量组各脑区Cyclin D1蛋白表达量随着时间的延长而逐渐升高,只是在PND30,小脑Cyclin D1表达下降,但与对照组没有显著性差异(P>0.05)。其它与对照组比较,除PND7外其它时间点各脑区差异均具有显著性(P<0.05)。p21蛋白表达与Cyclin D1蛋白表达趋势一致,随着时间的延长和染毒剂量的增加而呈现出时间依赖性和剂量依赖性。PKC和CaN蛋白均在染毒组中表达与在对照组中相比表达升高。4、Western-Blot法检测Cyclin D1蛋白的表达Western-Blot法检测结果与免疫组织化学法检测结果基本一致,Cyclin D1蛋白的表达呈现时间依赖性和剂量依赖性,随着时间的延长而表达升高,随着染毒剂量的增加,而表达升高。综上所述,宫内甲基汞暴露可以改变仔鼠脑神经元细胞周期进程。细胞周期调控蛋白Cyclin D1和p21随着宫内甲基汞的暴露呈现出时间依赖性和剂量依赖性,两者均随时间延长而表达增加,随甲基汞染毒剂量增加而表达增加。甲基汞在仔鼠脑中作用后,导致PKC高表达。PKC因此促进细胞周期G0/G1进入S期。CaN的高表达可能导致了CyclinD1的高表达。PKC和Cyclin D1表达上升都能导致G0/G1期细胞比例下降。另一方面,CaN可以抑制CDK4的基因转录水平,CaN的表达升高抑制了CDK4的蛋白表达,从而导致Cyclin D1/CDK4复合物产量降低。p21蛋白具有广泛的反应底物,能够在细胞周期进程中各期与CDKs结合,阻滞细胞周期在各期的进程。CDK4的表达受抑和p21蛋白的高表达共同抑制细胞周期从G0/G1期进入到S期。促进与抑制G0/G1期进入到S期的两种作用相互制衡,导致了染毒组G0/G1期细胞比例随染毒剂量的增加并未出现显著下降。而在S期和G2/M期,由于Cyclin D1主要在G0/G1期进入到S期时发挥作用,失去了Cyclin D1的制衡作用,p21导致了细胞在其它各期的阻滞。研究结果显示,甲基汞对细胞周期进程的影响也呈现出了时间效应。我们在实验中选取了PND1, PND14和PND30三个时间点。结果显示,PND30与PND1和PND14相比,细胞G0/G1期细胞比例出现了升高趋势,且在大脑中差异具有显著性,而S期和G2/M期则与之相反。PND30与PND14比较,S期和G2/M细胞比例下降。本研究结果提示,在PND30时,细胞周期较PND1,出现G0/G1细胞阻滞,而S和G2/M期细胞比例降低。出现这种情况的具体机制不清,可能的原因是PND30时,细胞凋亡,坏死等更加严重而导致p21的更高表达及抑制作用的增强。p21的G0/G1期阻滞作用超过了Cyclin D1的促进进入S期的作用,其具体的作用机制有待于进一步研究。更多还原

【Abstract】 Intrauterine methylmercury exposure could lead to offspring brain developmental damage and neuronal cell cycle alteration. Cyclin D1 and p21 might have important functions within this cell cycle regulation. In this study, rat experimental model was established of intrauterine methylmercury exposure. Offspring brain regions were determined cell cycle alterations at different time points and with different exposure dose. In the meantime, Cyclin D1 and p21 were also determined so as to investigate their role in the cell cycle regulation.1. Effect of intrauterine methylmercury exposure on neuronal cell cycleIntrauterine methylmercury exposure could lead to cell cycle alteration. As methylmercury exposure dose increased, in cerebrum, cell percentage in G0/G1 phase was decreased gradually, whereas it was increased in S and G2/M phase.1.5mg/kg dose group had G2/M cell cycle arrest; 3.0mg/kg dose group had S phase cell cycle arrest. In cerebellum, cell percentage in G0/G1 phase decreased gradually, and increased in S and G2/M phase.0.75mg/kg dose group had S phase cell cycle arrest. In hippocampus, cell percentage in G0/G1 and G2/M phase both decreased gradually, whereas S phase showed increase as exposure dose increased. 0.75 and 3.0mg/kg dose groups had S phase cell cycle arrest. Intrauterine methylmercury exposure showed also time effect. Cell percentage decreased at G0/G1 phase, and increased at S and G2/M phase on PND14. The trend went the countrary on PND30, where cell percentage increased at G0/G1 phase, and decreased at S and G2/M phase.2. p21 mRNA determinations by RT-PCRRT-PCR was applied to determine expression of p21Wafl/cipl mRNA. In 3.0mg/kg dose group, p2lWafl/Cipl mRNA expression in cerebrum and hippocampus decreased gradually as PND prolonged, whereas in cerebellum, p21Wafl/Cipl mRNA expression was increased.On PND 14 after intrauterine methylmercury exposure, p21Wafl/Cipl mRNA expression in cerebrum increased as methylmercury exposure dose increased where significant differences were observed in 0.75mg/kg and 1.5mg/kg dose group compared with control group. p21Wafl/Cipl mRNA in hippocampus showed lower expression in any dose group compared with control group. p21Wafl/Cipl mRNA expression in cerebellum showed the same trend as in hippocampus.3. Cyclin D1, p21, PKC and CaN determination by immunohistochemistryCyclin D1 and p21 were determined by immunohistochemistry. Results showed, on PND14, Cyclin D1 protein expression in every brain region increased gradually as exposure dose increased, where compared with control group, the medium and high dose group had significant differences compared with control group (P<0.05); of the 3.0mg/kg dose group, Cyclin D1 was determined increased as time prolonged, except that on PND30, Cyclin D1 expression in cerebellum decreased but had significant differences compared with control group (P>0.05).p21 expression showed similar situation to that of Cyclin D1. As time prolonged and exposure dose increased, p21 expression showed time and dose effect.PKC and CaN expression were identical, both of which increased in each exposure group.4. Cyclin D1 expressions by Western-BlotThe Cyclin D1 expression determined by Western-Blot was similar to that by immunohistochemistry. Cyclin D1 expression presented both time and dose effect. It increased as exposure dose increased and time prolonged.From the data above, we can see intrauterine methylmercury exposure could lead to offspring rat’s brain neuronal cell cycle alterations. Cyclin D1 and p21 showed dose and time effect after methylmercury intrauterine exposure in rat. Both proteins increased as time prolonged and exposure dose increased. PKC expressioin increased in offspring rat’s brain after intrauterine methylmercury exposure. This increased the capability of PKC to induce cell cycle proceeding from G0/G1 to S. The higher expression of CaN might be the reason that caused Cyclin D1 over expression. The higher expression of PKC and Cyclin D1 induced cell percentage decline at G0/G1. On the other hand, CaN can inhibit the transcription level of CDK4, which leaded to inhibition of Cyclin D1/CDK4 complex. p21 had a wide range of cyclins substrate to combine with, and posed arrest effect at any cell cycle phases. The higher expression of p21 and inhibition of CDK4 together prevented cell cycle from G0/G1 to S. The positive and negative effect balanced each other and resulted in G0/G1 cell percentage insignificantly decline. At S and G2/M phases, Cyclin D1 losed to balance the p21 effect, as Cyclin D1 mainly functioned at G0/G1 phase, which leaded to cell cycle arrest at these phases. The results also demonstrated time effect of methylmercury to cell cycle. PND1, PN14 and PND30 were chosen as the experiment time points. It showed cell cycle distribution on PND30 differred from PND1 and PND14. In fact, it was contratry to the case on PND14. Cell percentage increased at G0/G1 and decreased at S and G2/M. This indicated that G0/G1 phase cell cycle arrest on PND30. Possible reason might be that, as time prolonged, p21 showed higher expression and with higher activity as a response to more severe cell apoptosis, cell aging et al, the result of which leaded unbalanced and higher negative effect than the positive function by Cyclin D1, and finally induced cell cycle arrest at G0/G1 phase.更多还原