CAJ | 학술논문

目的构建携带Hsp47特异性siRNA的人源单链抗体融合蛋白,并探讨其诱导肝星状细胞凋亡的作用. 方法通过噬菌体展示技术得到单链抗体,采用分子克隆的方法将单链抗体ScFv和鱼精蛋白截短体tp基因融合,经转化,筛选,酶切连接测序后诱导表达并纯化蛋白,间接免疫荧光检测人原代肝星状细胞,人正常肝细胞7701对ScFv/tP融合蛋白的内吞效率;凝胶迁移阻滞实验检测ScFv/tP融合蛋白与DNA结合活性;Hsp47靶向性siRNA转染人原代肝星状细胞后,利用RT-PCR和Western blot分别检测细胞内Hsp47 mRNA和蛋白的表达情况;ScFv/tP融合蛋白介导Hsp47siRNA转染肝星状细胞后,采用流式细胞仪和Western blot分析其对细胞凋亡的影响,用CCK-8法分析其对细胞增殖的影响.两组间比较采用t检验,多组间比较采用单因素方差分析.结果所获得的ScFv/tP融合蛋白能够被人原代肝星状细胞内化进入细胞内,而不能被人正常肝细胞所内化,由ScFv/tP融合蛋白介导的Hsp47-siRNA能够降低人原代肝星状细胞Hsp47 mRNA及蛋白的表达水平,且转染后能够诱导肝星状细胞凋亡的发生. 结论人源单链抗体融合蛋白是一种良好的载体,能够转移靶向Hsp47特异性siRNA的诱导人原代肝星状细胞凋亡.
목적 구건휴대Hsp47특이성siRNA적인원단련항체융합단백,병탐토기유도간성상세포조망적작용. 방법 통과서균체전시기술득도단련항체,채용분자극륭적방법장단련항체ScFv화어정단백절단체tp기인융합,경전화,사선,매절련접측서후유도표체병순화단백,간접면역형광검측인원대간성상세포,인정상간세포7701대ScFv/tP융합단백적내탄효솔;응효천이조체실험검측ScFv/tP융합단백여DNA결합활성;Hsp47파향성siRNA전염인원대간성상세포후,이용RT-PCR화Western blot분별검측세포내Hsp47 mRNA화단백적표체정황;ScFv/tP융합단백개도Hsp47siRNA전염간성상세포후,채용류식세포의화Western blot분석기대세포조망적영향,용CCK-8법분석기대세포증식적영향.량조간비교채용t검험,다조간비교채용단인소방차분석.결과 소획득적ScFv/tP융합단백능구피인원대간성상세포내화진입세포내,이불능피인정상간세포소내화,유ScFv/tP융합단백개도적Hsp47-siRNA능구강저인원대간성상세포Hsp47 mRNA급단백적표체수평,차전염후능구유도간성상세포조망적발생. 결론 인원단련항체융합단백시일충량호적재체,능구전이파향Hsp47특이성siRNA적유도인원대간성상세포조망.
Objective To consauct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein,ScFv/tP,canying small interfering (si)RNA directed against the heat shock protein Hsp47,a collagenbinding glycoprotein,in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.Methods A single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences.Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein,internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining.The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells,the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Westem blotting; in addition,effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8,flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).Results Indirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells,as well as increased the cells' apoptosis remarkably.Conclusion The ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosls via blockade of Hsp47 expression.