无乳链球菌体外感染奶牛乳腺上皮细胞

Infection of Streptococcus agalactiae on mammary epithelial cells of dairy cow in vitro

用分离到的无乳链球菌(Streptococcus agalactiae,GBS)及标准菌株侵袭原代奶牛乳腺上皮细胞建立体外感染细胞模型,并进行细胞凋亡检测。同时,采用qRT-PCR方法检测细胞受到侵袭后白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子(TNF-α)等细胞炎性因子mRNA的转录水平。结果显示,标准菌株组(WL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组(FL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组与其标准菌株组的凋亡率差异也极显著(P<0.01)。qRT-PCR结果分析显示,标准株组TNF-α、IL-6及IL-8 mRNA转录水平与对照组相比,差异极显著(P<0.01);而分离菌株组TNF-αmRNA转录水平与对照组相比差异显著(P<0.05),IL-6及IL-8 mRNA转录水平差异不显著(P>0.05)。结果表明,与标准菌株相比,无乳链球菌分离株对奶牛乳腺上皮细胞侵袭和损伤能力较低,乳腺细胞的凋亡率也较低,当奶牛乳腺上皮细胞受到细菌侵袭的时候,细胞中IL-6、IL-8及TNF-α等炎性因子mRNA的转录水平都显著性提高,但在相同浓度下GBS标准株诱导细胞炎性因子的表达量显著高于GBS分离株。本试验结果为以后预防和控制GBS引起的隐性乳房炎提供试验依据。

In order to study the apoptosis of primary culture of bovine mammary epithelial cells induced by Streptococcus agalactiae(GBS),and to observe its effect on the gene transcription level of inflammatory factors in dairy mammary epithelial cells.In this experiment,Streptococcus agalactiae isolates and standard strains were used to invade bovine mammary epithelial cells,established an in vitro infected cell model,and tested for apoptosis.Meanwhile,qRT-PCR was used to detect the transcriptional level of inflammatory cytokines mRNA such as interleukin 6(IL-6),interleukin 8(IL-8) and tumor necrosis factor(TNF-α) after cell invasion.The results showed that the difference in apoptosis rate between the standard strain group(WL group) and the blank group was extremely significant(P<0.01);and the isolate group(FL group) was significantly different with the blank group(P<0.01);the difference between the isolate group and the standard strain group was also extremely significant(P<0.01).The results of qRT-PCR analysis showed that the transcript levels of TNF-α,IL-6 and IL-8 mRNA were extremely significant differences between the standard strain group and the control group(P<0.01),the transcript levels of TNF-α mRNA were significantly different between the isolates and the control group(P<0.05).There was no significant difference in IL-6 and IL-8 mRNA transcription levels(P>0.05). The results showed that compared with the standard strains,the S.agalactiae isolate had lower ability to invade and damage the mammary gland epithelial cells,and the apoptotic rate of the mammary gland cells was also lower.When the mammary gland epithelial cells were invaded by bacteria,the mRNA transcription levels of IL-6,IL-8,TNF-αand other inflammatory factors in the cells were significantly increased.However,at the same concentration,the expression level of inflammatory factors induced by GBS standard strain was significantly higher than that of GBS isolate.The results of this experiment provide experimental basis for the prevention and control of subclinical mastitis caused by GBS in the future.