组氨酸接枝聚介导的RNA干扰技术对大鼠肝脏主要组织相容性复合体Ⅱ类基因及其反式激活因子基因表达的抑制作用

The inhibiting effect on rat liver class Ⅱ major histocompatibility complex and its transactivator gene expression by histidine grafted poly vector-based RNA interference technology

目的 观察应用新型纳米载体组氨酸接枝聚（β-氨基酯）（HGPAEs）介导的RNA干扰技术对大鼠肝脏主要组织相容性复合体Ⅱ类基因（MHC-Ⅱ）及其反式激活因子基因（CⅡTA）表达的抑制作用.方法 构建HGPAEs及针对大鼠CⅡTA基因的短发夹RNA（shRNA）质粒,并将两者耦合成pCⅡTA-HGPAEs载体,分别设置生理盐水组、单纯HGPAEs组、pHK-HGPAEs阴性对照组和pCⅡTA-HGPAEs干预组,通过门静脉注射方法体内转染大鼠肝脏,分别采用荧光定量聚合酶链式反应（FQ-PCR）和Western blot检测转染后CⅡTA和MHC-Ⅱ基因的mRNA转录水平及蛋白表达水平.结果 成功构建载shRNA质粒的HGPAEs载体,体内转染大鼠肝脏后,pCⅡTA-HGPAEs组CⅡTA与MHC-Ⅱ基因的mRNA转录水平及蛋白表达水平显著下降（P＜0.01）,与生理盐水组比较,C ⅡTA及MHC-Ⅱ转录水平分别为（21.4±4.2）％和（26.3±5.8）％,蛋白表达水平分别为（21.6±7.6）％和（27.8±5.6）％.而生理盐水、HGPAEs、pHK-HGPAEs组转染后CⅡTA与MHC-Ⅱ基因mRNA转录水平及蛋白表达水平差异无统计学意义（P＞0.05）.结论 HGPAEs是一种较为理想的基因转运载体,应用RNA干扰技术经门静脉注射方法转染大鼠肝脏,可显著抑制CⅡTA和MHC-Ⅱ基因表达.

Objective To investigate the inhibitory effect on rat liver class Ⅱ major histocompatibility complex （MHC-Ⅱ） and MHC-Ⅱ transactivator （C Ⅱ TA） gene expression by histidine grafted poly （β-amino ester） s（HGPAEs） vector-based RNA interference（RNAi） technology.Methods According to C Ⅱ TA genetic information,short hairpin RNA （shRNA） sequence was designed and the HGPAEs vectors were constructed.Coupled HGPAEs and shRNA plasmid vectors targeting C Ⅱ TA into pC Ⅱ TA-HGPAEs vectors.Totally 4 groups were set respectively：control group,HGPAEs control group,pHK-C Ⅱ TA control group and pC Ⅱ TA-HGPAEs group,received liver transfection through portal vein injection.Real-time PCR and Western blotting were performed to detect the expression of C Ⅱ TA and MHC-Ⅱ in liver 3 days after transfection.Results pC Ⅱ TA-HGPAEs vectors were successfully constructed.Compared with control groups,the mRNA transcription levels and the protein expression levels of C Ⅱ TA and MHC-Ⅱ were significantly inhibited in pC Ⅱ TA-HGPAEs group （P 〈 0.01）.The mRNA expression levels ofC ⅡTA and MHC-]Ⅱ were （21.4 ±4.2）% and （26.3 ±5.8）%,and the protein expression levels were （21.6 ± 7.6） % and （27.8 ± 5.6） %.Conclusion HGPAEs vectors may be a promised gene carrier.The expression of rat liver C Ⅱ TA and MHC-Ⅱ can be obviously inhibited by pC Ⅱ TA-HGPAEs vectors.