摘要
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.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2017年第1期32-36,共5页
Chinese Journal of Endemiology
基金
国家自然科学基金(81260409、81660523)