【作者】 许莹；

【导师】 许榕仙； 胡志坚；

【作者基本信息】 福建医科大学 ， 营养与食品卫生学， 2008， 硕士

【摘要】 目的:应用分子生物学的技术,探讨微囊藻毒素对体外培养的肝细胞的损伤机制,并且对抗氧化营养素及B族维生素对微囊藻毒素染毒所致肝细胞的损伤的保护作用进行研究,为寻找肝脏保护与肝癌预防的有效方法提供参考。方法:1、购买BRL(大鼠肝细胞)细胞株,待增殖情况稳定,开始试验。2、MC-LR染毒肝细胞,24小时后MTT法检测细胞增殖情况。3、应用流式细胞仪检测毒素对细胞凋亡情况的影响。4、根据正交设计结果,BRL预先加入维生素作用,24小时后加入MC-LR染毒,检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化酶(GSHPX)。5、RT-PCR:结合正交设计实验,筛选出有效的保护组合,通过逆转录PCR技术,测定目的基因PCNA和UDG在mRNA水平上的改变。结果:1、与正常组细胞相比,MC-LR在浓度低于1μg/ml时,促进BRL增殖,浓度为0.5μg/ml时,细胞增长达到峰值;MC-LR浓度高于1.25μg/ml时,抑制细胞增殖。2、MC-LR染毒BRL后,细胞凋亡发生变化,当浓度在0.001～0.5μg/ml范围,凋亡率逐渐增加,1～2.5μg/ml凋亡率逐渐减少。3、与正常组细胞相比,不同浓度MC-LR作用下,PCNA表达量及UDG表达量差异有统计学意义。当MC-LR浓度在0.01～1.0μg/ml范围内,PCNA表达量随MC-LR浓度的增加而增加,在MC-LR浓度达到1.25μg/ml时,PCNA表达量开始下降;UDG表达的变化趋势与PCNA一致。4、正交分析结果显示:与没有维生素保护的染毒细胞相比,VitB6、VitB12、VC作用后,细胞内SOD、GSH-PX含量增加,差异有统计学意义,并且VitB12与VC存在协同效应。5、在高浓度MC-LR干预下,与没有维生素保护的染毒细胞相比,维生素B6、B12+VC作用后,PCNA表达量升高,差异有统计学意义。B6、B12、B12+VC作用后,UDG表达量增加,差异有统计学意义。在低浓度MC-LR干预下,与没有维生素保护的染毒细胞相比,维生素B6、B12作用后,PCNA表达量增加,差异有统计学意义; B6、VC、B12、B12+VC作用后,UDG表达量减少,差异有统计学意义。结论:1、MC-LR在不同浓度时对肝细胞增殖和凋亡的作用是不同的,在低浓度时有促进细胞增生的作用,凋亡率逐渐增加;在高浓度时却表现为抑制细胞生长或细胞毒作用的表现,凋亡率逐渐降低。2、MC-LR通过影响BRL的PCNA和UDG基因mRNA表达,启动BER修复途径,可能是BRL氧化损伤后的修复机制之一。3.维生素VitB6、FA、VitB12、VC对MC-LR致BRL损伤有保护作用,其中,维生素一阶交互作用的最佳保护组合为:VitB12+ VC。更多还原

【Abstract】 Objectives:To study the mechanism of microcystin-induced oxidative damage in vitro with molecular biology technologies, and the effect of vitamin B group & antioxidant nutrients protecting liver cells from microcystin injury, which would help us find an effective way to protect liver and prevent liver cancer.Methods:1、Purchase BRL cells, initiate the testing after stable proliferation.2、MC-LR treated liver cells, MTT was detected in cell proliferation after 24 hours.3、Analyze toxin impact upon apoptosis by FCM.4、According to results of orthogonal design, add vitamins for 24 hours in advances, then expose BRL to join MC-LR, measuring cells for ultra-oxide dismutase (SOD) and glutathione peroxidase (GSH-PX).5、RT-PCR: With the combination of orthogonal experimental design, select the effective protection portfolio by PCR technology and measure the PCNA and UDG changes at mRNA level.Results:1、Compared with normal cells, MC-LR at the concentration of less than 1μg / ml accelerate the BRL proliferation, the cell growth reached its peak at the concentration of 0.5μg / ml; MC-LR at the concentration of higher than 1.25μg / ml inhibited the cell proliferation. 2、The apoptosis changes when MC-LR’s takes effect on BRL, the apoptosis rate gradually increase in the 0.001 ~ 0.5μg / ml while at 1 ~ 2.5μg / ml the rate gradually reduced.3、Compared with normal cells, under different concentrations of MC-LR, PCNA expression and UDG expression had a significant difference. At the concentration of 0.01 ~ 1.0μg / ml, PCNA expression increased in accordance with the concentration increases of MC-LR. MC-LR in the concentration of 1.25μg / ml, PCNA expression began to decline. UDG follow with PCNA expression of change.4、Orthogonal analysis showed that: compared to the cells without the protection of the vitamin, after VitB6, VitB12, VC take effects, content of SOD and GSH-PX changed, and VitB12 synergies with VC.5、When without the protection of the vitamin, cells exposed in high concentrations of MC-LR intervention, PCNA expression increased and had a significant difference after vitamin B6, B12 +VC. After vitamin B6、B12、B12 +VC role, UDG expression increased and had a significant increase.Without the protection of the vitamin cells exposed in low concentration MC-LR, PCNA expression increased and had a significant difference after vitamin B6, B12’s effect; after B6, VC, B12, B12+VC effect, UDG expression decreased and had a significant difference .Conclusion:1、The effect of MC-LR in different concentrations on liver cell proliferation and on apoptosis is different, in low concentration to promote the role of cell proliferation, apoptosis rate increased gradually; in the high concentration when expressed as inhibiting cell growth or the performance of cytotoxicity , the rate of apoptosis reduced gradually.2、MC-LR changed the expression of PCNAmRNA and UDGmRNA, initiated BER repair channels, that may be the one of the repair mechanism for BRL oxidative damage.3. VitB6, VitB12, VC play a protection role in the MC-LR of BRL injury, and vitamin first order interaction for the best combination of protection: VitB12 + VC更多还原