HIF-1α转染方法的优化及其对间充质干细胞存活及分化功能的影响  

Method optimization of HIF-1α transfection and its effects on survival rate and differentiation of mesenchymal stem cells

作　　者：张扬[1] 辛畅泰[1] 

机构地区：[1]沈阳医学院附属奉天医院手外科,辽宁沈阳110024

出　　处：《基础医学与临床》2014年第3期365-371,共7页Basic and Clinical Medicine

摘　　要：目的探索应用脂质体作为载体将低氧诱导因子-1α(HIF-1α)转染至间充质干细胞(MSCs)的转染方法。方法应用脂质体作为载体将HIF-1α转染至P3代MSCs,荧光观察绿色荧光蛋白(GFP)表达,评估转染效率。比较在不同情况下细胞的转染效率。将转染的MSCs、空质粒转染的MSCs和未转染的MSCs置于缺氧环境下(CoCl2模拟缺氧),比较3种细胞HIF-1αmRNA、HIF-1α蛋白的表达。鉴定转染的MSCs向成骨细胞和脂肪细胞的分化能力,及在缺氧环境中的存活率。结果应用脂质体法可以成功将pcDNA3.0-HIF-1α-eGFP转染至MSCs。当细胞汇合度在80%以上,DNA与质粒的比为(μg/μL)1∶1.25,且转染时间为4 h时转染效率最高,为21%。转染HIF-1α的MSCs在缺氧环境中可被成功向脂肪细胞和成骨细胞诱导分化,HIF-1α转染可以提高缺氧时MSCs存活率(P<0.05)。在缺氧条件下pcDNA3.0-HIF-1α-eGFP瞬时转染的MSCs较空质粒(pcDNA3.0-eGFP)转染的MSCs和未转染的MSCs能高表达HIF-1αmRNA及HIF-1α蛋白(P<0.05)。结论应用脂质体作为载体可以将HIF-1α转染至MSCs,HIF-1α转染的MSCs在缺氧时高表达HIF-1αmRNA及蛋白。Objective To investigate a method of MSCs transfection with HIF-lα gene-recombined liposome. Methods HIF-lα was transferred into the third generation of MSCs by liposome. The expression of green floures ence protein （GFP） was observed and the efficiency of transfection was assessed. The three groups of cells, which were MSCs transfected with HIF-lα gene-recombined liposome, with control vector or with no transfection, were cultured in the hypoxia environment simulated through CoC12. The expressions of HIF-1α mRNA, HIF-1 αprotein of the different groups of MSCs were compared. Results pcDNA3. O-HIF-lα-eGFP could be transfected into MSCs by the liposome method successfully and the efficiency of the transfection is 21%. MSCs transfected with HIF-lα can be induced to differentiate to osteoblast and lipoblast successfully and HIF-lα transfection may increase MSCs sur vival rate under hypoxia condition （P 〈 0. 05 ）. The group of HIF-lα-transfected cells express much more HIF-lα mRNA and HIF-1 α protein than the other two （P 〈 O. 05 ）. Conclusions The study shows that the liposome can be used to transfect HIF-lα into MSCs successfully and MSCs transfected by HIF-lα gene-recombined liposome canstrongly express HIF-lα mRNA and HIF1α protein.

关 键 词：骨髓间充质干细胞 低氧诱导因子-1 脂质体 转染 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]