绵羊乳腺上皮细胞的分离鉴定及炎性模型的建立

Isolation and identification of sheep mammary epithelial cells and establishment of inflammatory model

探索高效易行的绵羊乳腺上皮细胞原代培养方法并建立细胞炎性模型。分别采用组织块法、酶消化法和酶消化组织块法分离培养绵羊的乳腺上皮细胞,通过形态学观察、免疫荧光染色和RT-PCR等方法对细胞进行鉴定;用不同浓度的脂多糖(LPS)作用细胞后,通过CCK-8法检测细胞相对存活率;利用ELISA方法检测IL-8和TNF-α表达量;通过Western blot检测TLR-4蛋白表达量。结果显示,酶消化组织块法是最符合预期的方法,可在较短时间内获得活力旺盛的乳腺上皮细胞;细胞生长曲线呈"S"型,CK-18免疫荧光染色呈阳性,β-酪蛋白基因表达呈阳性。10 mg/L LPS处理12 h的细胞相对存活率明显升高(P<0.01),细胞上清液中IL-8和TNF-α表达量极显著上升(P<0.01),TLR-4蛋白表达量升高,表明细胞炎性模型建立成功。

To explore an efficient and feasible method for primary culture of sheep mammary epithelial cells and establish the inflammatory cell model,sheep mammary epithelial cells were isolated and cultured by tissue block method,enzyme digestion and enzymatic digestion of tissue blocks,respectively.The cells were identified by morphological observation,immunofluorescence staining and RT-PCR.The cells were stimulated with gradient concentrations of lipopolysaccharide(LPS),the relative survival rate of cells was detected by CCK-8 assay,the expressions of IL-8 and TNF-αwere detected by ELISA,and the expression of TLR-4 protein was detected by Western blot.The results showed that the enzymatic digestion of tissue blocks was the optimal method,and the mammary epithelial cells could be obtained in a short time.The growth curves of mammary epithelial cells showed"S"shape,CK-18 immunofluorescence staining andβ-casein gene expression were positive.The relative survival rate of cells stimulated by 10 mg/L LPS increased significantly(P<0.01),the expressions of IL-8 and TNF-αincreased significantly(P<0.01),the expression of TLR-4 increased,indicating that the inflammatory cell model was established successfully.