层粘连蛋白对晚G1期人胃癌BGC-823细胞生长的促进作用

为研究层粘连蛋白（laminin,LN）促进肿瘤细胞生长作用,采用脉冲标记计数有丝分裂百分率(percentage labeling mitosis,PLM)法测得体外培养人胃癌 (BGC 823) 细胞周期时间为41 h,其中G1期时间为24.5 h. 分裂细胞脱离法获取分裂期细胞,继续培养23 h，在细胞运行进入G1晚期时,将其置于LN 0、0.11、0.55、1.10 μmol/L基质上孵育4 h; 细胞荧光光度计检测晚G1期细胞内Ca2+浓度、钙调蛋白、DNA含量. 结果显示,LN与其膜上受体结合后引起细胞内Ca2+浓度、钙调蛋白、DNA含量增加,尤以在0.55 μmol/L LN作用显著（P<0. 001）.蛋白质免疫印迹分析证明,cPKC α呈现表达,提示 LN与其受体结合可增强其细胞cPKC-α的活性;分析G1期细胞周期蛋白E(cyclinE)、细胞周期蛋白依赖性激酶CDK2表达水平，呈现逐渐增强的趋势; LN可诱导c-Myc蛋白呈现高表达,提示 LN与其受体结合增强与细胞增殖密切相关的基因表达;在LN作用前后的BGC 823细胞均未检测到Bax蛋白表达.结果提示,在人胃癌 (BGC 823)细胞G1/S期交界处,层粘连蛋白与其膜上受体结合引起细胞内Ca2+浓度升高,诱导钙调蛋白的释放，其含量增加,增强蛋白激酶C的活化,导致细胞内DNA含量增加、G1/S期细胞周期蛋白与CDK表达增强、诱导原癌基因c-Myc呈持续表达状态，而凋亡基因Bax不表达.

Promotion of Late-G1 Phase of Human Gastric Cancer(BGC-823) Cells with Laminin

To investigate the effect of laminin(LN) to promote the growth of tumour cells, we detected the cell cycle of human gastric cancer(BGC-823) cells by the percentage labeling mitosis(PLM). Results show that the cell cycle time of BGC 823 is 41 hours,and the duration of G1 phase is about 24.5 hours. After the M phase,cancer cells were incubated for 23 hours, and the late-G1 phase cancer cells were incubated on LN coated substrate with or without LN at the final concentration of 0,0.11，0.55，1.1 μmol/L for 4 hours. The content of cytosolic Ca2+, calmodulin(CaM) content and intracellular DNA of the late G1 phase cancer cells were detected using cytofluorophotometer. Results show that LN not only increased the concentration of the intracellular Ca2+, but also the content of the intracellular DNA and CaM. The effect of LN is very significant (P＜0.001) especially when its concentration is 0.55 μmol/L. Using Western blot analysis, we found that the expression of the cPKCα was increased in cancer cells, indicating that the binding of LN and its receptor could enhance the activity of cPKC α in the late G1 phase BGC-823 cancer cells. The expression of cyclin E and CDK2 were increased gradually in cancer cells. We didn’t find the expression of Bax before and after treatment of LN in BGC 823 cells. These results suggest that during the G1/S transition of human gastric cancer(BGC 823) cells,the binding of the LN and its receptor triggers the increase of Ca2+ concentration and induces the release of CaM. These effects may subsequently activate the protein kinase C,increase the intracellular DNA content,increase the expression of cyclin E and CDK2, and induce the persistent expression of proto-oncogene c-Myc. However, the combination of the extrinsic LN and its receptor didn’t induce the expression of apoptotic gene Bax.