Small RNA interference-mediated gene silencing of TREK-1 potassium channel in cultured astrocytes

This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.     

三种常见肝细胞系免疫相关分子的表达

目的探讨三种常见肝细胞系Huh7、HepG2、HepG2.2.15免疫相关分子的表达。方法提取三种细胞的RNA并逆转录合成cDNA,用PCR方法检测细胞因子及其受体、MHC分子的表达情况。结果三种细胞均能表达Ⅰ型干扰素及干扰素受体、IL-7、IL-15等细胞因子,也能表达HLA-A、HLA-B、HLA-DP、HLA-DR及非经典的CD1d、MICB等MHC分子及其相关基因,HepG2、HepG2.2.15表达MICA。三种细胞均不表达IL-4、IL-5、IL-6、IFN-γ等细胞因子。与内参相比,IL-7及IL-15、HLA-A等的表达在HepG2和HepG2.2.15之间存在一定的差异。结论三种肝细胞系能表达Ⅰ型干扰素及干扰素受体、IL-7、IL-15、HLA等一系列的免疫相关分子。     

不同种小鼠乙型肝炎模型的建立及其感染状况研究

目的 探讨建立急性或慢性乙型肝炎病毒（HBV）感染小鼠模型的方法。方法 采用高压尾静脉注射pAAV/HBV1.2表达质粒,建立急性或慢性HBV感染小鼠模型,采用ELISA法检测HBV抗原,采用RT-PCR法检测免疫相关分子基因,使用FACS检测肝内淋巴细胞活化情况,采用酶联免疫斑点实验（ELISPOT）法检测脾脏相关免疫细胞特征。结果 成功建立急性和慢性高压尾静脉注射HBV感染小鼠模型;在急性BALB/c小鼠模型,血清HBsAg持续时间分别为（3.25±1.04） w,显著短于慢性C57BL/6小鼠组【（10.0±3.74）w,P<0.05）】;在BALB/c小鼠模型,注射2.5 μg病毒质粒小鼠血清HBsAg持续时间为（3.67±1.03） w,显著长于注射50 μg组【（2.33±0.52） w,（P<0.05）】;在高压尾静脉注射后第10 d,BALB/c小鼠脾脏出现了HBcAg特异性T淋巴细胞。结论 成功建立高压尾静脉注射HBV感染小鼠模型,发现宿主遗传背景、免疫应答状态和病毒剂量影响HBV感染小鼠模型的建立。