中药复方含药血清对人激素非依赖性前列腺癌细胞系PC-3增殖与凋亡影响的体外实验研究

The effect of serum containing compound recipe on proliferation and apoptosis of human prostate cancer cell line PC-3 in vitro

目的以临床常用的抗肿瘤中药组成复方,用血清药理学方法检测各复方含药血清对人激素非依赖性前列腺癌细胞PC-3增殖、细胞周期分布及凋亡的影响。方法以临床常用的中药组成三个复方,分别用三种中药复方水煎剂及等量生理盐水对成年雄性去势SD大鼠灌胃,每天2次,共5 d。在末次给药后60～90 min时间段内取下腔静脉血制成含药大鼠血清,同时制作空白对照大鼠血清。用含药和空白对照大鼠血清在体外培养激素非依赖性前列腺癌细胞PC-3,用MTT法检测培养4 h、24 h、48 h、72 h的细胞存活率;用流式细胞仪测定培养72 h的细胞周期分布;对培养72 h的细胞行Hoechst染色观察细胞凋亡情况。比较细胞存活率、细胞周期分布及凋亡情况的差异。结果中药一组的细胞存活率最低,且随着培养时间的增加而持续降低;中药二组的细胞存活率较中药一组高,但显著低于对照组,其细胞存活率在48 h时达到最低;中药三组的细胞存活率与对照组比较无显著差异。中药一组和中药二组的S期细胞比例显著低于对照组的S期细胞比例,而G0/G1期细胞比例显著高于对照组,G2/M期细胞比例与对照组比较无显著差异;中药三组的细胞各周期分布比例与对照组比较均无显著差异。中药一组的细胞中凋亡细胞明显增多,比例约为30%;中药二组中的凋亡细胞较中药一组的少,但显著多于对照组,比例约为10%;中药三组中凋亡细胞很少,与对照组相似。结论中药复方一和中药复方二在体外有显著的抑制激素非依赖性前列腺癌细胞PC-3增殖和诱导PC-3细胞凋亡的作用,可进一步用于动物荷瘤实验证实疗效。

Objective To identify a new compound recipe that inhibits proliferation and induces apoptosis of PC-3 cells. And then continue to animal experiment. Methods Three compound recipes were composed of commonly clinical used herbal medicine. The adult male castrated SD rats were gavaged respeetively with extracts of the three compound recipes and normal saline （NS） in the same volume, twice a day for 5 days. Between 60 to 90 minutes after the last administration, blood samples were taken from vena eava of the rats, and were prepared for drug-containing serum. Blank control serum was prepared simulta- neously. PC-3 cells were incubated in the culture medium with the drug-containing serum and blank con- trol serum respectively. The survival rate of the ceils that incubated for 4, 24, 48, 72 hours was measured by MTT assay. Cell cycle distribution of the cells that incubated for 72 hours was measured by flow cytometry. Cells that incubated for 72 hours were stained with Hoechst 33258 and then were observed under a flu-oreseence microscope. Statistical differences of survival rate, cell cycle distribution and apoptotic rate were compared. Results The survival rate of Group 1 was the lowest, and it decreased as the incubated time prolonged. The survival rate of Group 2 was higher than the survival rate of Group 1, but was significantly lower than the survival rate of the control group. And it achieved the valley value at 48 hours. There was no significant differences of the survival rate between Group 3 and the control group. The percentage of S- phase ceils in Group 1 and Group 2 was significantly lower than that in the control group, and the percent- age of G0/Gl-phase cells was higher than that in the control group significantly. There was no significant differences of the percentage of G2/M-phase cells between Group 1, Group 2 and the control group. There was no significant differences in percentage of cells in each cell cycle between Group 3 and the control group. The number of apoptotic cells in Group 1 was marked increased, and the ratio of which was approxi- mately 30%. The number of apoptotie cells was less in Group 2 than that in Group 1, but was significantly more than that in the control group. And the ratio of which was about 10%. There were rare apoptotic cells in Group 3, just like that in the control group. Conclusions The compound recipe 1 and 2 significantly inhibit proliferation and induce apoptosis of hormone independent prostate cancer cell line PC-3 in vitro, which need further investigation in animal model.