摘要
目的:观察融合蛋白CTP-HBcAg18-27-Tapasin在抗原提呈细胞内的转导功能。方法:体外分离培养近交系BALB/c小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白细胞介素-4培养5d,再加入脂多糖诱导DC成熟。不同剂量的CTP-HBcAg18-27-Tapasin及RP-MI-1640培养液加入细胞培养介质中,激光共聚焦显微镜下观察免疫荧光在细胞内的分布及定位,并对荧光强度进行定量分析,进一步以West-ern blot观察不同组细胞中Tapasin表达的差异来检测CTP-HBcAg18-27-Tapasin的转导效率。结果:成功体外诱导培养并鉴定小鼠骨髓源性树突状细胞,免疫荧光法证实CTP-HBcAg18-27-Tapasin能够穿透树突状细胞膜进入细胞质,而不能进入细胞核,且荧光强度50μg/L CTP-HB-cAg18-27-Tapasin组依次高于10μg/L CTP-HBcAg18-27-Tapasin组和空白组,Western blot也证实CTP-HBcAg18-27-Tapasin能够穿透细胞膜进入树突状细胞,结果显示差异有统计学意义。结论:CTP-HBcAg18-27-Tapasin具有穿透树突状细胞膜定位于胞浆的能力。
ABSTRACT AIM： To observe the function of transduction of fusion protein CTP-HBcAg18- 27-Tapasin in antigen presenting ceils. METH- ODS： Bone marrow derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating fac- tor and recombinant interleutin-4 for 5 days fol- lowed by LPS added to induce DCs maturation. Then, different dosages of CTP-HBcAg18-27- Tapasin and RPMI-1640 were added to cell cul- ture medium. Subsequently, the distribution and localization of intracellular immunofluores- cence were observed by confocal microscopy with fluorescence intensity assaying quantitatively. Futhermore, the transduction efficiency of CTP- HBcAg18-27-Tapasin was evaluated by detecting the differences of expression of Tapasin using western blot in different group. RESULTS： DCswere cultured and identified successfully; Immu- nofluorescence confirmed the CTP-HBcAglS-27- Tapasin could penetrate the membrane into the cytoplasm of DCs but not into the nucleus. Flu- orescence intensity of 50 27-Tapasin group were μg/L CTP-HBcAg18- higher than 10 μg/L CTP-HBcAg18-27-Tapasin group and blank groups. Western blot also confirmed that CTP- HBcAg18-27-Tapasin can penetrate the cell membrane into the DCs, and the data showed the difference was statistical. CONCLUSION： CTP-HBcAg18：27-Tapasin has the ability of penetrating and locating in the cytoplasm of DCs.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2012年第1期25-29,共5页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
国家自然基金项目(31000414)
