氟诱导原代大鼠肝细胞凋亡及bax基因表达研究
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摘要
本试验采用改良的肝脏原位胶原酶灌流法，成功地分离了大鼠肝细胞，并以原代培养的肝细胞为实验模型，研究不同浓度(0-400mg／L)的氟(氟化钠)在不同时间段(12，24h)对大鼠肝细胞的毒性及其诱导的细胞凋亡，研究结果表明：
1．采用改良的肝脏原位胶原酶灌流法能有效的分离实验所需的肝细胞，分离肝细胞数在2×10~7／只以上，活率在90％以上；
2．采用MTT法检测氟化钠对大鼠肝细胞增殖的抑制率，氟化钠处理12h的半数抑制率(IC50)为153.85mg／L，24h的IC50为114.43mg／L；
3．提取氟化钠处理的肝细胞的DNA进行琼脂糖凝胶电泳，可见有明显的梯状带产生，确定细胞发生凋亡，且具有时间和剂量依赖性；而对照组无梯状带出现：
4．Western blot检测显示氟化钠处理的肝细胞中有凋亡基因bax的表达，且有时间和剂量依赖性。
结论：氟化钠对大鼠肝细胞即使在低浓度下即有毒性作用，处理12h的IC50为153.85mg／L，24h的IC50为114.43mg／L；DNA电泳检测出现凋亡细胞特征性的梯状带，表明在低浓度下(25mg／L)氟化钠的毒性作用主要是诱导细胞凋亡，这将为完善饲料氟的监测和食品卫生中氟的添加剂量标准提供参考作用；Western blot检测显示凋亡细胞中bax表达量增加，它提示氟化钠通过上调bax的表达而诱导的肝细胞凋亡，为氟中毒分子毒理学机制提供了实验证据，为氟中毒的防治提供
新的思路。
Improved two -step collagenase perfusion method was employed to isolate rat hepatocytcs in this study ,and the primary culture hepatocytes were used as a model to investigate the role of sodium fluoride in cytotoxicity and induction of apoptosis with varying concentrations from 0 to 400mg/L for different periods(12, 24h) .The results were showed as follows:
1. Hepatocytes of rat were isolate efficiently by employed improved two-step collagenase perfusion,the number of cell over 2×107 per rat and the viability of hepatocytes up to 90%;
2. Antiproliferative effect of NaF in hepatocytes of rat were measured by the MTT assay and the concentration requied for 50% inhibition of growth (IC50) for 12h of treatment is 153.85mg/L, IC50 of 24h is 114.43mg/ L.
3. Agarose gels electrophorese analysis of DNA extracted from NaF-treated hepatocytes of rat revealed that fragmentation of nuclear DNA was induced and increased in a dose and time-dependent manner,but the control group DNA ladder cann't be observed. The results ensure that NaF induced apoptosis in hepatocytes of rat in low concentration.
4. Western blot analysis showed that the hepatocytes of rat exposure to 25-400mg/L of NaF increased the expression of bax gene in a dose and time-dependent.
Conclusion: The cytotoxicity of NaF can be obsevered even at low
concentration in hepatocytcs of rat,and the value of IC50 incubated by NaF for 12h is!53.85mg/L, IC50 of 24h is 114.43 mg/L. DNA ladder was observed by agarose gels electrophorese analysis and it showed that the toxicity of NaF is induce apoptosis at low concentration (25mg/L) . The result will offer some data to consummate the standard dosage of NaF added in feed and foodstuff. Western blot analysis revealed that the expression of bax increased when treated with NaF.lt suggest that NaF induced apoptosis through the up-regulation of Bax.This study will provide some experimental data to uncover the molecular toxicology
mechanism of fluoride , put forward a new way to prevention and cure the fluorosis.
1．采用改良的肝脏原位胶原酶灌流法能有效的分离实验所需的肝细胞，分离肝细胞数在2×10~7／只以上，活率在90％以上；
2．采用MTT法检测氟化钠对大鼠肝细胞增殖的抑制率，氟化钠处理12h的半数抑制率(IC50)为153.85mg／L，24h的IC50为114.43mg／L；
3．提取氟化钠处理的肝细胞的DNA进行琼脂糖凝胶电泳，可见有明显的梯状带产生，确定细胞发生凋亡，且具有时间和剂量依赖性；而对照组无梯状带出现：
4．Western blot检测显示氟化钠处理的肝细胞中有凋亡基因bax的表达，且有时间和剂量依赖性。
结论：氟化钠对大鼠肝细胞即使在低浓度下即有毒性作用，处理12h的IC50为153.85mg／L，24h的IC50为114.43mg／L；DNA电泳检测出现凋亡细胞特征性的梯状带，表明在低浓度下(25mg／L)氟化钠的毒性作用主要是诱导细胞凋亡，这将为完善饲料氟的监测和食品卫生中氟的添加剂量标准提供参考作用；Western blot检测显示凋亡细胞中bax表达量增加，它提示氟化钠通过上调bax的表达而诱导的肝细胞凋亡，为氟中毒分子毒理学机制提供了实验证据，为氟中毒的防治提供
新的思路。
Improved two -step collagenase perfusion method was employed to isolate rat hepatocytcs in this study ,and the primary culture hepatocytes were used as a model to investigate the role of sodium fluoride in cytotoxicity and induction of apoptosis with varying concentrations from 0 to 400mg/L for different periods(12, 24h) .The results were showed as follows:
1. Hepatocytes of rat were isolate efficiently by employed improved two-step collagenase perfusion,the number of cell over 2×107 per rat and the viability of hepatocytes up to 90%;
2. Antiproliferative effect of NaF in hepatocytes of rat were measured by the MTT assay and the concentration requied for 50% inhibition of growth (IC50) for 12h of treatment is 153.85mg/L, IC50 of 24h is 114.43mg/ L.
3. Agarose gels electrophorese analysis of DNA extracted from NaF-treated hepatocytes of rat revealed that fragmentation of nuclear DNA was induced and increased in a dose and time-dependent manner,but the control group DNA ladder cann't be observed. The results ensure that NaF induced apoptosis in hepatocytes of rat in low concentration.
4. Western blot analysis showed that the hepatocytes of rat exposure to 25-400mg/L of NaF increased the expression of bax gene in a dose and time-dependent.
Conclusion: The cytotoxicity of NaF can be obsevered even at low
concentration in hepatocytcs of rat,and the value of IC50 incubated by NaF for 12h is!53.85mg/L, IC50 of 24h is 114.43 mg/L. DNA ladder was observed by agarose gels electrophorese analysis and it showed that the toxicity of NaF is induce apoptosis at low concentration (25mg/L) . The result will offer some data to consummate the standard dosage of NaF added in feed and foodstuff. Western blot analysis revealed that the expression of bax increased when treated with NaF.lt suggest that NaF induced apoptosis through the up-regulation of Bax.This study will provide some experimental data to uncover the molecular toxicology
mechanism of fluoride , put forward a new way to prevention and cure the fluorosis.
引文
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