腺病毒介导的白介素10基因体外转导大鼠β_2m-/Thy-1^+骨髓源性肝干细胞  

Adenovirus-mediated IL-10 gene transfer of rat β_2m-/Thy-1^+ bone marrow-derived liver stem cells in vitro

作　　者：兰玲[1] 陈源文[1] 孙超[1] 孙巧玲[1] 李定国[1] 

机构地区：[1]上海交通大学医学院,新华医院消化内科,上海200092

出　　处：《胃肠病学和肝病学杂志》2008年第1期22-26,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金资助项目(30500236)

摘　　要：目的研究腺病毒介导的白介素10(interleukin 10,IL-10)基因(AdIL-10)体外转导β2m-/Thy-1+骨髓源性肝干细胞(bonemarrow-derived liver stem cell,BDLSC)的可行性。方法利用免疫磁珠细胞分选(magnetic bead cell sorting,MACS)方法分选大鼠β2m-/Thy-1+BDLSC,RT-PCR法鉴定其肝相关性表型的表达。AdIL-10转导BDLSC,根据腺病毒携带的绿色荧光蛋白表达率测定转导效率,采用RT-PCR法和ELISA法分别检测IL-10的基因表达和蛋白分泌水平。采用MTT法和免疫荧光染色法分别检测腺病毒转导对BDLSC增殖能力和肝细胞样表型白蛋白(albumin,ALB)表达的影响。结果MASC法能成功分选纯化BDLSC,其在基因水平表达肝细胞样表型。AdIL-10转导BDLSC的效率呈剂量依赖性,最佳转染复数为500pfu/cell。AdIL-10修饰BDLSC高表达IL-10基因,且持续高浓度分泌IL-10蛋白至胞外。AdIL-10转导72h后,BDLSC仍保持正常的增殖能力和ALB表达。结论BDLSC是AdIL-10转导的适宜载体,为AdIL-10修饰BDLSC这一新型结合方式用于慢性肝病的治疗奠定了实践基础。Objective To determine the feasibility of adenovirus-mediated IL-10 gene （AdIL-10） transfer of BDLSCs and transgene expression in vitro. Methods Rat β2m^-/Thy-1^＋ BDLSCs were isolated by magnetic bead cell sorting （MACS） , characterized for liver associated markers by RT-PCR, and transduced with AdIL-10. The transduction efficiency was observed using fluorescence microscope for the fluorescence of green fluorescent protein. The levels of IL-10 gene expression and protein secretion by BDLSCs were assessed by RT-PCR and ELISA, respectively. The proliferation ability and albumin （ALB） expression of BDLSCs after AdIL-10 transfer were detected by MTT assay and immunofluorescence staining, respectively. Results BDLSCs isolated by MACS possessed hepatocyte-like phenotype. The transduction efficiency with AdIL-10 was dose-dependent. And the optimal multiplicities of infection was 500 plaque forming units/cell. The high expression of IL-10 at the gene level in AdIL-10 transducted BDLSCs was confirmed. IL-10 secreted into culture medium presented persistently at a high-level of concentration. BDLSCs retained proliferation ability and ALB expression after AdIL-10 transfer for 72 h. Conclusion BDLSCs are suitable vehicles for AdIL-10 transfer, pointing out the potential utility of this novel combined strategy of AdIL-10 and BDLSCs for the treatment of chronic liver injuries.

关 键 词：骨髓源性肝干细胞 腺病毒载体 白介素10 基因转导 

分 类 号：Q813[生物学—生物工程] Q782