亚砷酸钠诱导大鼠肝星状细胞的激活及对DNA甲基转移酶1、3a mRNA表达的影响

Activation of rat hepatic stellate cells and changes of DNA methyltransferase 1, 3a mRNA expression levels induced by sodium arsenite

目的观察不同剂量亚砷酸钠对大鼠肝星状细胞（HSC-T6）纤维化的影响，及其纤维化后DNA甲基转移酶（DNMT）1、DNMT3a mRNA的表达情况。方法HSC-T6细胞分别暴露于终浓度为0（对照）、5（低剂量）、15（中剂量）、25（高剂量）μmol／L的亚砷酸钠培养基中培养24、48、72h。收取细胞和细胞培养上清液。采用酶联免疫吸附试验（ELISA）测定细胞培养上清液中纤维化因子Ⅰ型胶原（COL-1）、Ⅲ型胶原（COL-3）、α平滑肌肌动蛋白（α-SAM）的含量，实时荧光定量PCR（qRT—PCR）检测细胞DNMT1、DNMT3a mRNA的表达。结果不同染砷时间（24、48、72h）对HSC-T6细胞COL-1、COL-3、α-SMA含量的影响差异均有统计学意义（F=249．574、328．493、3157．436，P均〈0．01）；不同染砷含量（低、中、高剂量组）对HSC-T6细胞COL-1、COL-3、α-SMA含量的影响差异均有统计学意义（F=3946．521、1006．399、13025．770，P均〈0．01）。染砷24、48h，高剂量组DNMT1 mRNA的表达（4．33±0．24、2．34±0．43）高于对照组（1．00±0．00、1．00±0．00，P均〈0．05）；低、中、高剂量组DNMT1 mRNA的表达随染砷时间延长而下降（P均〈0．05）。染砷48、72h，高剂量组DNMT3a mRNA的表达（2．23±0．50、5．02±0．23）高于对照组（1．00±0．00、1．00±0．00，P均〈0．05）；中、高剂量组，染砷72h DNMT3a mRNA的表达（3．80±0．14、5．02±0．23）均高于24h（3．03±0．12、0．42±0．15，P均〈0．05）。结论亚砷酸钠对HSC-T6细胞具有明显的活化和促纤维化作用。肝纤维化后DNMT1、DNMT3a mRNA的表达水平均上调。

Objective To observe the activation of rat hepatic stellate cells （HSC-T6） and the changes of methyhransferase （DNMT）I, DNMT3a mRNA expression with different doses of sodium arsenite stimulation. Methods HSC-T6 cells were exposed to a final concentration of 0 （control）, 5 （low dose）, 15 （medium dose） and 25 （high dose） μmol/L sodium arsenite in culture medium for 24, 48 and 72 h, cells and cell culture supernatant were harvested. Enzyme-linked immunosorbent assay （ELISA） kit was used to measure fibrosis factors contents of type I collagen （COL-l）, type Ⅲ collagen （COL-3） and alpha smooth muscle actin （α-SMA） and quantitative real-time PCR was used to detect the expression levels of DNMT1 and DNMT3a mRNA. Results Different arsenic exposure time （24, 48, 72 h） had a significant effect on COL-1, COL-3 and α-SMA contents in HSC-T6 cells （F = 249.574, 328.493, 3 157.436, all P 〈 0.01）; Different arsenic exposure content （low, medium, high dose groups） had a significant effect on COL-1, COL-3 and α-SMA contents in HSC-T6 cells （F = 3 946.521, 1 006.399, 13 025.770, all P 〈 0.01）. After arsenic exposure for 24 and 48 h, the expression levels of DNMT1 mRNA in high dose group （4.33 ± 0.24, 2.34 ± 0.43） were higher than those of control group （1.00 ± 0.00, 1.00 ± 0.00, all P 〈 0.05）. At the same arsenic exposure levels （low, medium or high dose）, the expression level of DNMT1 mRNA was declined with prolongation of sodium arsenite stimulation time （all P 〈 0.05）. After arsenic exposure for 48 and 72 h, the expression levels of DNMT3a mRNA in high dose group （2.23 ± 0.50, 5.02 ± 0.23） were higher than those of control group （1.00 ± 0.00, 1.00 ± 0.00, all P 〈 0.05）. The expression levels of DNMT3a mRNA in medium and high dose groups at 72 h （3.80 ± 0.14, 5.02 ± 0.23） were higher than those of 24 h （3.03 ± 0.12, 0.42 ± 0.15, all P 〈 0.05）. Conclusion HSC-T6 cells are obviously activated with pro-fibrotic effect; the expression levels of DNMT1 and DNMT3a mRNA are both up regulated in HSC-T6 cells after being exposed to sodium arsenite.