摘要
目的探讨氟中毒对人肝癌细胞（HepG2）线粒体凋亡及其相关蛋白的影响。方法体外培养HepG2细胞24h，分别采用0（对照）、1、3、6、9mmol／L氟化钠（NaF）处理细胞24h（n=5）。四甲基偶氮唑盐（MTr）法检测细胞毒性作用；提取对照组和3mmol/L NaF处理组（氟处理组）细胞RNA和蛋白（n=6），实时荧光定量PCR法和蛋白免疫印迹法检测HepG2细胞中线粒体凋亡诱导因子（apoptosisinducingfactor，AIF）mRNA和蛋白的表达；蛋白免疫印迹法检测线粒体凋亡途径相关蛋白B淋巴细胞瘤-2（B—cellslymphoma．2，Bcl-2）与Bcl-2共沉淀的x蛋白质（Bcl．as．sociatedXprotein，Bax）、细胞色素C（cytochromeC）、含半胱氨酸的天冬氨酸蛋白水解酶（cysteinylaspartatespecificproteinase，caspase）9和3蛋白的表达。结果0、1、3、6、9mol／L氟处理组MTT法检测结果分别为0．307±0．031、0．333±0．028、0．230±0．011、0．178±0．001、0．152±0．003，组间比较差异有统计学意义（，=82．224，P〈0．01）。氟处理组AIFmRNA表达[（153．14±5．41）％]高于对照组[（100．00±4．70）％，t=-4．73，P〈0．05]；AIF、Bcl-2、cytochromeC（在细胞质中）、caspase9、caspase3蛋白表达[（152．16±47．30）％、（171．90±51．52）％、（458．00±19．48）％、（527．17±200．67）％、（432．70±64．27）％]均高于对照组[（100．00±48．86）％、（100．00±34．44）％、（100．00±116．59）％、（100．00±19．58）％、（100．00±137．16）％，t=-3．80、-3．96、-15．76、-4．67、-5．06，P均〈0．05]；而Bax、cytochromeC（在线粒体中）蛋白表达[（24．66±26．04）％、（72．99±45．34）％]均低于对照组[（100．00±44．01）％、（100．00±34．14）％，t=6．35、0．68，P均〈0．05]。结论线粒体凋亡相关蛋白以及线粒体细胞凋亡途径可能参与了氟化物诱导HepG2细胞凋亡发生的病理机制。
Objective To investigate the possible pathological role of mitochondrial apoptosis pathways and its factors in fluorosis-induced apoptosis of human hepatocellular carcinoma cell strain （HepG2）. Methods Under the stimulation of 1, 3, 6 and 9 mmol/L concentrations of NaF in vitro for 24 h （n = 5）, while normal control group was cultured under normal condition, the cytotoxicity was measured with MTT. The mitochondrial apoptosis inducing factor （AIF） was measured at both mRNA （n = 5） and protein levels （n = 6）, respectively, by real-time PCR and Western blotting. The mitochondrial apoptosis related factors, such as B-cells lymphoma-2 （Bcl-2）, Bcl-as- sociated X protein （Bax）, cytochrome C, caspase-9 and caspase-3 were measured at protein levels （n = 6）. Results After treated with 0, 1, 3, 6 and 9 mmol/L NaF for 24 h, the cell absorbance of HepG2 cells was 0.307 ± 0.031, 0.333 ± 0.028, 0.230 ± 0.011, 0.178 ± 0.001 and 0.152 ± 0.003, respectively, and the differences were statistically significant among groups （F = 82.224, P 〈 0.01）. After treated with 3 mol/L NaF for 24 h, the mRNA level of AIF was [（153.14 ±5.41）%] which was increased compared to the control group [（100.00 ± 4.70）%, t = - 4.73, P 〈0.05]. Under the same condition, the protein levels of AIF, Bcl-2, cytochrome C in cytoplasm, caspase-9 and caspase-3 were （152.16 ± 47.30）%, （171.90 ± 51.52）%, （458.00 ± 19.48）%, （527.17 ± 200.67）% and （432.70 ± 64.27）%, which were increased compared to those of the control groups [（100.00 ± 48.86）%, （100.00 ± 34.44）%, （100.00 ± 116.59）%, （100.00 ± 19.58）% and （100.00 ± 137.16）%, t = - 3.80, - 3.96, - 15.76, - 4.64, - 5.06, all P 〈 0.05], while the protein levels of Bax and cytochrome C in mitochondrion were （24.66 ±26.04）%, （72.99 ± 45.34）%, which were decreased compared to those of the control groups [（100.00 ± 44.01）%, （100.00 ± 34.14）%, t = 6.35, 0.68, all P 〈 0.05]. Conelllslon The mitochondrial apoptosis pathway and related factors may be involved in NaF-induced cell death in HepG2 cells.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2016年第8期547-551,共5页
Chinese Journal of Endemiology
基金
基金项目：国家自然科学基金（81160335）
科技部项目（2013BA105803）
贵州省科技厅国际合作项目（黔科合外G字[2011]7014号）
