双歧杆菌脂磷壁酸对结肠癌细胞抑制增殖和促凋亡的研究

The inhibitory and proapoptotic effects of Bifidobacterium bifidum lipoteichoic acid on colon carcinoma cells

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 and proapoptotic effects of Bifidobacterium bifidum lipoteichoic acid on human colon carcinoma cells HCT116. Methods MTF assay was used to explore the effects of LTA on the proliferation of HCT116 cells treated with different concentrations of LTA （20 μg/mL, 60 μg/mL and 100 μg/mL） and on the HCT116 control,. Flow cytometry assay was used to detect the cell cycle and apoptosis rate. Immunohistochemistry assay was used to detect the expression of bcl-2, NF-xBp65 and Cytochrome C. RT-PCR assay was used to detect the expression of TLR2 and TLR4. Results When HCT116 cells were treated with LTA （20, 60 and 100 μg/mL） , a significant reduction of the rate of cell proliferation was observed by MTT （P 〈 0.01 ） ; the cell cycle and apoptosis rate increased according to flow cytometry assay （ P 〈 0.05 ） ; the expression of bcl-2 and NF-xBp65 reduced while Cytochrome C increased according to hnmunohistochemistry （ P 〈 0. 05 ） ; and the up-regulated expression of TLR2 and TLR4 was showed by RT-PCR （P 〈 0. 05）. LTA had significantly inhibited HCTI 16 cell line proliferation in certain dose-time effect （ P 〈 0.05 ）. Conclusion LTA can significantly inhibit HCT116 cell proliferation and induce its apoptosis.