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目的探讨双歧杆菌脂磷壁酸(lipoteichoic acid,LTA)抑制人类结肠癌细胞株HCT116细胞增殖和促进凋亡情况。方法培养结肠癌细胞株HCT116和提取双歧杆菌LTA;实验分为4组,即LTA低剂量组(20μg/mL)、中剂量(60μg/mL)、高剂量(100μg/mL)和HCT116对照组(Control);用四甲基偶氮唑蓝法(MTT)检测LTA对结肠癌细胞增殖的抑制率,流式细胞仪检测LTA对结肠癌细胞周期分布变化和凋亡率,免疫组化分析bcl-2、Cytochrome C和NF-кBp65含量变化,RT-PCR检测TLR2mRNA和TLR4m RNA的表达。结果 MTT法测得LTA各组对结肠癌细胞HCT116均具有较好的抑制(P<0.01);流式细胞仪检得G0/G1期细胞显著增多(P<0.01),而G2和S期细胞减少(P<0.05),细胞凋亡率增加(P<0.05);免疫组化分析IOD/Area值,bcl-2和NF-кBp65表达显著下调,Cytochrome C显著上升(P<0.05);RT-PCR测得TLR2 mRNA和TLR4 mRNA的表达均上升(P<0.05)。结论 LTA具有明显的抑制结肠癌细胞增殖和促进凋亡作用,为研制高效、无毒的新型抗结肠癌药物奠定一定的基础。
Objective To investigate the inhibitory effect of lipoteichoic acid (LTA) on the proliferation and apoptosis of human colon cancer cell line HCT116. Methods The colon cancer cell line HCT116 and Bifidobacterium LTA were cultured. The experiment was divided into 4 groups: low dose LTA group (20μg / mL), middle dose (60μg / mL), high dose (100μg / mL) and HCT116 control group (Control). The inhibitory rate of LTA on the proliferation of colon cancer cells was detected by MTT assay. The changes of cell cycle distribution and apoptosis rate of LTA on colon cancer cells were detected by flow cytometry. The expression of bcl -2, Cytochrome C and NF-кBp65 were detected by RT-PCR. The expressions of TLR2mRNA and TLR4mRNA were detected by RT-PCR. Results MTT assay showed that all the LTA groups had a good inhibitory effect on HCT116 colon cancer cells (P <0.01). The number of cells in G0 / G1 phase was significantly increased by flow cytometry (P <0.01) (P <0.05). The expression of IOD / Area, bcl-2 and NF-кBp65 were significantly down-regulated and Cytochrome C was significantly increased (P <0.05) by immunohistochemistry. RT- PCR measured TLR2 mRNA and TLR4 mRNA expression were increased (P <0.05). Conclusion LTA has a significant inhibitory effect on the proliferation and apoptosis of human colon cancer cells, which lays a foundation for the development of a new anti-colon cancer drug with high efficacy and non-toxicity.