人母乳细菌Streptococcus salivarius F286和S. parasanguinis F278对HT29细胞的细胞毒性

为了筛选到具有抗炎特性的有益菌,研究者通常将待测细菌的发酵液上清和热致死菌体与TNF-α刺激下的人类结肠腺癌细胞HT29共孵育,并测量细菌是否能够减少HT29细胞分泌的炎症因子。该测试的前提之一是待测细菌的发酵液上清或菌体不杀死或杀死<10%的HT29细胞。在前期的工作中,我们从人母乳中分离得到Streptococcus salivarius F286和S.parasanguinis F278两株菌。在研究这两株菌的抗炎能力之前,我们利用MTT法摸索不同浓度的S.salivarius F286和S.parasanguinis F278的发酵液上清和热致死菌体对HT29细胞的细胞毒性。实验表明,两株菌的发酵液上清的原液和稀释液对HT29细胞均没有细胞毒性;浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体、浓度5×10^5~2.5×10^6cfu/mL的F278热致死菌体对HT29细胞的细胞毒性低于10%,而浓度1×10^8cfu/mL的热致死F286和F278菌体分别杀死(23±5.3)%和(22±5.3)%的HT29细胞。因此,S.salivarius F286和S.parasanguinis F278的发酵液上清原液、以及浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体和5×10^5-2.5×10^6cfu/mL的F278热致死菌体可在HT29细胞模型中进行抗炎能力测试。本研究的方法可用于确定其他细菌在HT29细胞模型中进行抗炎能力测试的合理浓度范围。

The Cytotoxicity of Human Breast Milk Bacteria of Streptococcus salivarius F286 and S. parasanguinis F278 to HT29 Cells

To screen beneficial bacteria with anti-inflammatory property,the culture supernatant or heat-killed bacterium cells of the tested bacterial strains are co-cultured with human colon adenocarcinoma HT29 cells stimulated with TNF-α,and determined whether the inflammation factors secreted by HT29 cells are reuduced.One premise of this test is that the culture supernatant or heat-killed cells of the bacteria strains are able to kill less than 10%HT29 cells.In our previous work,Streptococcus salivarius F286 and S.parasanguinis F278 were isolated from the human breast milk.Prior to investigating the anti-inflammatory ability of the two strains,we used MTT assay to test the cytotoxicity of the culture supernatants and heat-killed cells of S.salivarius F286 and S.parasanguinis F278at different concentrations to HT29 cells.Both undiluted and diluted culture supernatants of the two strains had no cytotoxicity on HT29 cells;the cytotoxicity of heat-killed cells of F286 at concentrations of 5×10^5~7.5×10^6cfu/mL and of heat-killed cells of F278 with concentration of 5×10^5~2.5×10^6cfu/mL on HT29 cells was lower than 10%,while 1×10^8cfu/mL heat-killed cells of F286 and F278 killed(23±5.3)%and(22±5.3)%of HT29 cells,respectively.Consequently,it is feasible to conduct anti-inflammation assay with undiluted culture supernatants of S.salivarius F286 and S.parasanguinis F278,and 5×10^5~7.5×10^6cfu/mL heat-killed F286 cells and 5×10^5~2.5×10^6 cfu/mL heat-killed F278 cells in HT29 cell model.The method used in this study can be generalized to determine a reasonable range of concentrations of other bacteria to perform anti-inflammatory abilities tests by using HT29 cell model.