摘要
目的：探讨细胞因子信号转导抑制因子3（SOCS3）的激酶抑制区（KIR）对细胞内STAT3蛋白磷酸化的影响.方法：化学合成穿膜肽TAT与KIR的融合肽（TAT-KIR）,以异硫氰酸荧光素（FITC）进行氨基端标记;不同浓度融合肽加入培养的PC12细胞,荧光显微镜及流式细胞仪检测TAT-KIR的穿膜效果;以白细胞介素-6（IL-6）处理PC12细胞模拟炎症反应,MTT检测细胞活力/增殖情况,免疫印迹检测细胞内磷酸化STAT3 （P-STAT3）蛋白水平;对比阴性对照组（正常培养的细胞）、IL-6刺激组、TAT-KIR处理组及TAT-KIR＋IL-6处理组细胞的P-STAT3水平,分析SOCS3的KIR对STAT3磷酸化的影响.结果：加入不同浓度TAT-KIR后10 min,显微镜下可见细胞内有绿色荧光出现,并随浓度升高胞内绿色荧光强度增强,其中3 μmol/I融合肽30 min时其跨膜转运效率最高,可达99.94％;100 ng/ml IL-6可显著增加PC12细胞活力,并促进胞内STAT3磷酸化水平显著增高;与此相比,TAT-KIR＋IL-6组细胞内P-STAT3水平显著下降,但是TAT-KIR处理组P-STAT3水平与阴性对照组比较差异无统计学意义.结论：KIR可被TAT成功转入细胞内部,并有效抑制IL-6刺激引起的PC12细胞STAT3磷酸化水平的增高,对正常情况细胞STAT3磷酸化的影响不大.
Objective： To investigate the suppressing effect of kinase inhibitory region （KIR） of suppressors of cytokine signaling 3 （SOCS3） on the phosphorylation of STAT3. Methods： Fusion peptide TAT-KIR was constructed and labeled with fluorescein isothiocyanate （FITC）. KIR domain was delivered into PC12 cells through cell-penetrating peptide TAT and detected by fluorescence microscopy and flow cytometry. Inteleukin-6 （IL-6） was used to induce the inflammatory response of PC12 cells via JAK/STAT3 signalling pathway. After the treatment of IL-6, TAT-KIR and TAT-KIRq-IL-6, PC12 cell viability/proliferation and phosphorylation level of STAT3 were detected by MTT assay and Western blotting assay, respectively. Results： Fluorescent peptides inside PC12 cells were observed 10 min after addition of fusion peptides and the fluorescence was enhanced with the increase of the concentration of peptides. The maximum transcellular penetration was achieved at 30 min after addition of 3μmol/L fusion peptides. IL-6 at concentration of 100 ng/ml significantly promoted PC12 cell viability/proliferation and up-regulated the level of phosphorylated STAT3 （P-STAT3）. After treated with TAT-KIR＋ IL-6, the phosphorylation of STAT3 was significantly reduced, in comparison with the IL-6 treatment. However, there was no significant difference in the P-STAT3 level between the negative control and TAT-KIR treatment. Conclusion： KIR of SOCS3 was successfully deliverd into PC12 via TAT, and it significantly reduced the elevation of STAT3 phosphorylation induced by IL-6.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2014年第2期146-149,169,共5页
Chinese Journal of Anatomy
基金
国家自然科学基金面上项目（31070943,31271151）
