摘要
目的观察肝细胞增殖时硫化氢对细胞周期蛋白D1（CyclinD1）和v21Cip／WAK-1的影响。方法肝穿刺获取慢性乙型肝炎患者肝组织，并分离培养肝细胞。所获肝细胞分为4组，前3组分别给予促肝细胞生长素（HGF组），炔丙基甘氨酸（PPG）和HGF（PPG＋HGF组），硫氢化钠（NariS）＋HGF（NaHs＋HGF组），剩余1组作为空白对照组。肝细胞培养24h后行台盼兰拒染实验及MTY比色法测定细胞增殖，利用去蛋白法检测细胞上清液中的硫化氢，免疫印迹（Western）检查细胞内p21Cip／WAK-1和CyclinD1变化情况。细胞洗涤后，提取各组细胞DNA，判定硫化氢对DNA的影响。结果培养后肝细胞的活性均超过90％。MTF比色法测定结果显示：与对照组比较，HGF组肝细胞增殖明显增强（0．713±0．207：0．411±0．113），且差异有统计学意义（t=6．943，P〈0．01）；与HGF组相比，PPG＋HGF组细胞增殖明显增强（0．965±0．329：0．713±0．207），差异有统计学意义（t=4．017，P〈0．05），NariS＋HGF组细胞增殖明显减弱（O．513±0．156：0．713±0．207），差异有统计学意义（t=5．328，P〈0．05）。Western实验结果显示，PPG＋HGF组p21cip／WAK-1表达增加而CychnDI表达降低，NaHS＋HGF组两者表达量呈相反趋势。HGF组和PPG＋HGF组DNA复制明显增加，而NaHS＋HGF组DNA无明显复制。结论硫化氢可能通过抑制细胞CyclinD1和增加p21Cip／WAK-1表达抑制肝细胞增殖。
Objective To observed the effect of hydrogen sulfide on cyclin D1 and p21Cip/WAK-1 in hepatocyte regeneration. Methods Liver tissue was obtained by liver biopsy form chronic hepatitis B （CHB） patients. The hepatocytes isolated from liver tissue were divided into 4 groups, the first 3 groups were stimulated by hepatocyte growth factor （ HGF group ）, propargyl glycine （PPG） and HGF （PPG ＋ HGF group）, sodium hydrosulfide （NAILS） and HGF（NaHS ＋ HGF group） respectively, the last one group was a control group. Trypan blue exclusion test of hepatocytes cultured for 24 h was conducted. The proliferation of hepatocytes was determined by MTT assay. Hydrogen sulfide in supernatant was detected using the deproteinized method. The p21Cip/WAK-1 and cyclin D1 in intracellular were determined by western blot. After washing ceils, DNA were extracted to determine the impact of hydrogen sulfide on DNA replication. Results Activity of cultured hepatocytes was over 90%. MTT assay results showed that： the cell proliferation of HGF group significantly increased （0.713 ± 0.207：0.411 ± 0.113, t = 6.943, P〈0.01）compared with control group. Compared with the HGF group, the cell proliferation of PPG ＋ HGF group significantly increased （0. 965 ±0.329： 0.713 ± 0.207, t =4.017, P 〈 0.05）, while that of NAILS ＋ HGF group significantly reduced （0.513 ± 0.156：0.713 ± 0.207, t = 5.328, P 〈0.05）. Western results showed that expression of p21Cip/WAK-1 in PPG ＋ HGF group increased and cyclin DI expression decreased, while the expression levels of p21Cip/WAK-1 and cyclin D1 in NAILS ＋ HGF group showed the opposite trend. The DNA replication in cells of PPG ＋ HGF and HGF groups significantly increased, and no replication of DNA was observed in Naris ＋ HGF group cells. Conclusions Hydrogen sulfide may inhibit hepatocyte proliferation by up regulation of p21Cip/WAK-1 and down regulation by cyclln D1.
出处
《国际流行病学传染病学杂志》
CAS
2013年第4期244-246,共3页
International Journal of Epidemiology and Infectious Disease
基金
杭州市卫生科技计划（2010B020）
