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目的建立叙利亚地鼠胚胎细胞(Syrian hamster embHo cell,SHE 细胞)混合培养模型,研究表没食子儿茶素没食子酸酯(epigallocatechin-3-Gallate,EGCG)对 SHE 癌前细胞生长、增殖和凋亡的作用,探讨 EGCG 的抑癌机制。方法将 SHE 癌前细胞和正常细胞分别按1:10 000、1:1000、1:100、1:10比例接种于6孔的培养皿中,培养7d,建立 SHE 细胞混合培养模型。以0μmol/L EGCG为对照组,分别选取浓度为0.5、1、5、10、50 μmol/L 的 EGCG,通过 SHE 细胞生长实验,原位细胞凋亡实验、原位细胞增殖实验和基因芯片检测 EGCG 对 SHE 正常细胞、SHE 癌前细胞和混合培养模型中SHE 癌前细胞的生长、增殖、凋亡及相关调控基因表达的影响。结果 SHE 癌前细胞与正常细胞的比例为1:100是合适的混合培养模型。0.5、1、5、10μmol/L 的 EGCG 促进了 SHE 正常细胞的生长,而浓度为50 μmol/L 的 EGCG 抑制了 SHE 正常细胞的生长。在1:200比例的 SHE 癌前细胞和正常细胞混合培养模型中,与对照组相比,浓度为5、10μmol/L 的 EGCG 抑制了不同大小的 SHE 癌前细胞克隆的生长。对照组中 SHE 癌前细胞的 DNA 合成指数和凋亡指数分别是39.3%和6.5%,5、10μmol/L EGCG 作用后 SHE 癌前细胞的 DNA 合成指数分别降至25.6%和24.8%,细胞凋亡指数分别升至12.65%和14.5%。EGCG 抑制了混合培养模型中 SHE 癌前细胞的生长和增殖,促进了其凋亡。EGCG 对于细胞凋亡的调控可能通过 p53、NF-κB、bel-2通道的基因表达来实现;EGCG 对于细胞增殖的调控可能通过阻滞细胞周期 G_1/S 期实现。结论 EGCG 可能是通过抑制癌前细胞的增殖和促进其凋亡进而抑制癌症。
Objective To establish a mixed culture model of Syrian hamster embryo cells (SHE) to study the effects of epigallocatechin-3-gallate (EGCG) on the growth, proliferation and apoptosis of SHE precancerous cells Role, to explore the tumor suppressor mechanism of EGCG. Methods SHE precancerous cells and normal cells were inoculated into 6-well petri dishes at a ratio of 1:10 000, 1: 1000, 1: 100 and 1:10, respectively, and cultured for 7 days. SHE cell mixed culture model was established. EGCG at concentrations of 0.5, 1, 5, 10 and 50 μmol / L were selected as the control group by 0 μmol / L EGCG. The apoptosis of the cells was evaluated by SHE cell growth assay, in situ cell apoptosis assay, in situ cell proliferation assay and DNA microarray Effects of EGCG on the growth, proliferation, apoptosis and related gene expression of SHE precancerous cells in normal SHE cells, SHE precancerous cells and mixed culture models. Results The ratio of SHE precancerous to normal cells was 1: 100 was a suitable mixed culture model. EGCG at 0.5, 1, 5 and 10 μmol / L promoted the growth of normal SHE cells, whereas EGCG at a concentration of 50 μmol / L inhibited the growth of normal SHE cells. In the 1: 200 SHE precancerous and normal cell mixed culture model, EGCG at a concentration of 5 and 10 μmol / L inhibited the growth of SHE precancerous cell clones of different sizes compared to the control group. The DNA synthesis index and apoptosis index of SHE precancerous cells in control group were 39.3% and 6.5%, respectively. The DNA synthesis index of SHE pre-cancerous cells decreased to 25.6% and 24.8% after 5 and 10μmol / L EGCG treatment, respectively The index rose to 12.65% and 14.5% respectively. EGCG inhibited the growth and proliferation of SHE precancerous cells in a mixed culture model and promoted their apoptosis. The regulation of EGCG on apoptosis may be through the gene expression of p53, NF-κB and bel-2 channels. The regulation of EGCG on cell proliferation may be through the arrest of cell cycle G_1 / S phase. Conclusion EGCG may inhibit cancer by inhibiting the proliferation of precancerous cells and promoting their apoptosis.