刚地弓形虫速殖子培养上清液对人胃癌细胞BGC-823增殖和凋亡的影响

Effect of Culture Supernatant of Toxoplasma gondii on the Proliferation and Apoptosis of BGC-823 Cells

目的研究刚地弓形虫速殖子培养上清液对人胃癌BGC-823细胞增殖和凋亡的影响。方法取对数生长期的人胃癌BGC-823细胞(5×104个/ml)接种于细胞培养瓶中,分别加入不同浓度弓形虫速殖子(2×107/ml、4×107/ml和8×107/ml)培养24 h后的上清液,对照组加入等体积DMEM培养基。分别于培养24、48和72 h后使用CCK-8法检测BGC-823细胞增殖抑制率。于培养24 h后,加入Hoechst染料,荧光显微镜下观察细胞的凋亡情况,以及琼脂糖凝胶电泳检测细胞凋亡条带。流式细胞仪检测BGC-823细胞用弓形虫速殖子培养上清液培养24 h后的细胞周期,计算细胞增殖指数(PI),以及检测细胞凋亡相关蛋白p53和Bcl-2的表达情况。结果 CCK-8法检测结果显示,弓形虫速殖子浓度越高,作用时间越长,其培养上清对BGC-823细胞的增殖抑制作用越明显,8×107/ml速殖子培养上清液培养细胞72 h后,抑制率达34.3%。荧光显微镜下可见,实验组出现凋亡小体,且凋亡小体的数量随速殖子浓度的升高而增加。琼脂糖凝胶电泳结果显示,4×107/ml和8×107/ml弓形虫速殖子培养上清液培养24 h后的细胞均出现凋亡条带。流式细胞仪检测细胞周期结果显示,随弓形虫速殖子浓度的增加,其培养上清液所培养的细胞G0/G1期比例升高,S期比例降低,PI值随之降低,8×107/ml组的细胞PI值为0.36,显著低于对照组(0.60)(P<0.05)。速殖子培养上清液培养的细胞p53蛋白表达量上升,Bcl-2蛋白表达量下降,与对照组差异均有统计学意义(P<0.05)。结论弓形虫速殖子培养上清能够抑制人胃癌BGC-823细胞的增殖,并可能通过上调p53蛋白和下调Bcl-2蛋白表达,引起人胃癌BGC-823细胞的凋亡。

Objective To investigate the effect of culture supernatant of Toxoplasma gondii on the proliferation and apoptosis of human gastric cancer BGC-823 cells. Methods Toxoplasma gondii tachyzoites with seed counts of 2×l0^7/ml, 4×10^7/ml, and 8×10^7/ml harvested from infected mice were cultured for 24 h, and then the culture supernatant was col- lected. BGC-823 cells （5×104/ml） at mid-exponential phase were incubated with different concentrations of culture super- natants of Toxoplasma gondii taehyzoites. While in control group, the same volume of DMEM was given. At 24, 48 and 72 h after incubation, the measurement of tumor cell growth inhibition rate was performed by using CCK-8 kit. Cell apoptosis was observed under a fluorescence microscope after 24 h incubation. The DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Flow cytometric analysis was used to analyze cell cycle for cell proliferation index and the expression of p53 and Bcl-2. Results The culture supernatant of Toxoplasma tachy- zoites inhibited the proliferation of BGC-823 cells and the growth inhibition rates increased with the time and the con- centration. The highest rate reached at the 72nd hour when the concentration of Toxoplasma tachyzoites was 8x10^7/ml. Apoptotie bodies were found in experimental group. The amount of apoptotic body was positively associated with the tachyzoite concentration. DNA fragment in the treated cells after 24 h incubation was revealed by agarose electrophoresis. When the proportion of BGC-823 cells in Go/G1 phase increased, the proportion of cells in S phase decreased. Cell pro-liferation index decreased with the increase of the concentration of tachyzoites. The proliferation index （0.36） of the cells cultured with culture supernatant of 8xlOT/ml tachyzoites was significantly lower than that of control group （0.6,P〈0.05）. p53 protein expression was higher in experiment group than that of the control, whereas Bcl-2 protein expression in ex- periment group was lower than that of the control （P〈0.05）. Conclusion The culture supematant of Toxoplasma gondii can inhibit the proliferation of BGC-823 cells and cause apoptosis of BGC-823 cells, which may be related with up-regu- lating p53 expression and down-regulating Bcl-2 expression.