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目的探讨UⅡ/UT系统在LPS刺激大鼠肝枯否细胞(Kupffer cell,KC)TNF-α和IL-1β表达和分泌中的作用。方法采用胶原酶灌注消化和密度梯度离心分离大鼠KC。细胞分组和处理方法:正常对照组urantide(-)LPS(-)、urantide或UⅡ处理组urantide/UⅡ(+)LPS(-)、LPS刺激组urantide(-)LPS(+)、urantide或UⅡ预处理组urantide/UⅡ(+)LPS(+);细胞内基因表达采用RT-PCR和real-time PCR方法检测,细胞培养上清液中蛋白质分泌水平采用ELISA分析方法检测。结果 LPS刺激后,KC内UⅡ、UT mRNA相对表达水平和细胞培养上清液UⅡ多肽水平显著升高,但urantide预处理组显著低于LPS刺激组(P<0.01)。Urantide处理组细胞UⅡ/UT mRNA和上清液UⅡ多肽水平与正常对照组之间无明显统计学差异(P>0.05);LPS刺激后(LPS刺激组、UⅡ预处理组和urantide预处理组),TNF-α和IL-1βmRNA表达和蛋白质分泌水平较正常对照组明显升高(P<0.01),但urantide预处理组较LPS刺激组和UⅡ预处理组显著降低(P<0.01),LPS刺激组与UⅡ预处理组之间无明显统计学差异(P>0.05)。UⅡ或urantide处理组KC上述前炎细胞因子的表达和分泌水平与正常对照组之间无明显统计学差异(P>0.05)。结论 UⅡ/UT信号系统可能在LPS刺激肝KC前炎细胞因子TNF-α和IL-1β的表达和分泌中起重要作用。
Objective To investigate the role of UⅡ / UT system in LPS-induced expression and secretion of TNF-α and IL-1β in rat Kupffer cells (KC). Methods Rat KC was isolated by collagenase digestion and density gradient centrifugation. The cells were divided into groups and treated by urantide (-) LPS (-), urantide or UII treated group urantide / UII (+) LPS (-), urantide (-) LPS The expression of urantide / UⅡ (+) LPS (+) in the untreated group was detected by RT-PCR and real-time PCR. The level of protein secretion in the cell culture supernatant was detected by ELISA. Results After LPS stimulation, the relative expression levels of UⅡ and UT mRNA and the level of UⅡ polypeptide in the supernatant of KCs increased significantly in KC group, but the levels in Urantide pretreatment group were significantly lower than those in LPS stimulation group (P <0.01). There was no significant difference between Urantide group and control group (P> 0.05). After LPS stimulation (LPS stimulation group, UII preconditioning group and urantide preconditioning group) (P <0.01). The levels of TNF-α, IL-1βmRNA and protein secretion were significantly increased in Urantide preconditioning group compared with those in LPS preconditioning group and UⅡ preconditioning group (P <0.01) There was no significant difference between UⅡgroup and pretreatment group (P> 0.05). UⅡ or urantide treatment group KC pro inflammatory cytokine expression and secretion levels and the normal control group no significant difference (P> 0.05). Conclusion UⅡ / UT signaling system may play an important role in LPS stimulating the expression and secretion of inflammatory cytokines TNF-α and IL-1β before hepatic KC.