CAJ | 학술논문

目的研究胆固醇基修饰的普鲁兰(CHSP)纳米粒的体外HepG2细胞的摄取机制及亚细胞分布.方法采用异硫氰酸荧光素(FTTC)标记CHSP,透析法制备FITC标记的CHSP(FITC-CHSP)自组装纳米粒并进行表征.采用MTT法考察CHSP纳米粒对HepG2细胞的毒性.选用选择性的内吞途径抑制剂氯丙嗪、菲律宾菌素和阿米洛利来研究HepG2细胞摄取CHSP纳米粒的机制.最后,采用细胞免疫荧光法对内质网、高尔基体和溶酶体进行染色,使用激光扫描共聚焦显微镜(CLSM)观察不同的孵育时间CHSP纳米粒的亚细胞分布.结果成功合成了FITC-CHSP,且FITC-CHSP能在水介质中自组装成纳米粒,该纳米粒呈规则球形,平均粒径为(63.0 ±1.9) nm.MTT结果表明CHSP纳米粒对HepG2细胞无明显的细胞毒性.内吞抑制实验表明网格蛋白介导的内吞途径以及巨胞饮途径共同参与了CHSP纳米粒的入胞过程.纳米粒的亚细胞分布实验表明:在研究的孵育时间(4 h)内,并未发现CHSP纳米粒进入高尔基体和内质网;当纳米粒与HepG2细胞孵育30 min时,没有纳米粒定位于溶酶体中,随着孵育时间的延长,大量纳米粒分布于溶酶体中.结论 CHSP纳米粒有望成为细胞内递送治疗剂的一种通用载体.
목적 연구담고순기수식적보로란(CHSP)납미립적체외HepG2세포적섭취궤제급아세포분포.방법 채용이류청산형광소(FTTC)표기CHSP,투석법제비FITC표기적CHSP(FITC-CHSP)자조장납미립병진행표정.채용MTT법고찰CHSP납미립대HepG2세포적독성.선용선택성적내탄도경억제제록병진、비률빈균소화아미락리래연구HepG2세포섭취CHSP납미립적궤제.최후,채용세포면역형광법대내질망、고이기체화용매체진행염색,사용격광소묘공취초현미경(CLSM)관찰불동적부육시간CHSP납미립적아세포분포.결과 성공합성료FITC-CHSP,차FITC-CHSP능재수개질중자조장성납미립,해납미립정규칙구형,평균립경위(63.0 ±1.9) nm.MTT결과표명CHSP납미립대HepG2세포무명현적세포독성.내탄억제실험표명망격단백개도적내탄도경이급거포음도경공동삼여료CHSP납미립적입포과정.납미립적아세포분포실험표명:재연구적부육시간(4 h)내,병미발현CHSP납미립진입고이기체화내질망;당납미립여HepG2세포부육30 min시,몰유납미립정위우용매체중,수착부육시간적연장,대량납미립분포우용매체중.결론 CHSP납미립유망성위세포내체송치료제적일충통용재체.
Objective To investigate the cellular uptake mechanism and subcellular distribution of cholesterol-modified pullulan (CHSP) nanoparticles by human hepatocellular carcinoma (HepG2) cells.Methods Fluorescein isothiocyanate (FITC) was conjugated to CHSP,and FITC-labeled CHSP (FITC-CHSP) nanoparticles were prepared by dialysis.The cytotoxicity of CHSP nanoparticles against HepG2 cells was evaluated by MTT assay.Meanwhile,several selective endocytosis inhibitors (chlorpromazine,filipin and amiloride) were selected to study the potential endocytosis mechanism.To study the subcellular distribution of CHSP nanoparticles at different incubation times,immunofluorescence staining was used to identify the Golgi apparatus,endoplasmic reticula (ER) and lysosomes,followed by confocal laser scanning microscopy(CLSM) observation.Results Covalent conjugation with FITC yielded stably labeled CHSP,which was successfully formulated into nanoparticles (mean particle size (63.0±1.9) nm) by dialysis with an almost spherical shape.MTT assay clearly indicated that the CHSP nanoparticles did not show significant toxicity in HepG2 cells.In vitro experiments with endocytic inhibitors revealed that clathrin-mediated endocytosis and macropinocytosis were both involved in the internalization of CHSP nanoparticles.The subcellular distribution study demonstrated that CHSP nanoparticles were entrapped in the lysosomes at 1 h after incubation,while colocalization of nanoparticles with either the Golgiapparatus or the ER was not observed during the entire course of the study.Conclusions CHSP nanoparticles may serve as a versatile carrier for intracellular delivery of therapeutic agents.