摘要
目的将间充质干细胞(MSCs)作为基因载体,利用基因转染技术构建高表达促血管生成素1(Ang1)基因的MSCs,回输体内治疗炎症性肺损伤模型,观察其肺内定位和修复作用。方法分离、培养和扩增MSCs至第四代,经流式细胞仪鉴定,得到纯度较高的MSCs。同时以三质粒共转染法在293T细胞中制备病毒颗粒Lenti-GFP-Ang1,并转染MSCs,通过实验确定转染的最佳MOI和最佳时间,通过RT-PCR检测转染后MSCs中Ang1的基因表达,确定转染成功。以脂多糖雾化吸入的方式诱导小鼠炎症性肺损伤模型,设未处理组为对照,设三组干预组,包括携带Ang1的MSCs组(MSC-Ang1组)、单纯Ang1组(Ang1组)和单纯MSCs组(MSCs组)。观察并记录各组生存天数,计算生存率并进行生存分析;免疫组化确定外源MSCs源性细胞在肺部的表现。结果经多重纯化后,流式细胞仪鉴定获得的干细胞为CD44(+)、Sca-1(+)、CD31(-)和CD45(-)的MSCs,并具有分化潜能。病毒载体构建亦通过鉴定成功。MSCs株经转染后高表达Ang1基因,当MOI=20时,MSCs的细胞活性及转染达到最佳效果,经荧光检测在第5d达表达高峰。生存率分析显示MSC-Ang1组生存率稍高于对照组,但差异无统计学意义(P=0.066)。移植后受损肺组织内可见表达绿色荧光蛋白的肺泡上皮样细胞和肺血管内皮样细胞。结论高表达目的基因Ang1的MSCs可通过基因技术有效构建,移植体内后,可在肺内检测到MSCs的转化修复。MSCs可作为治疗肺部疾病的基因运载工具。
Objective To determine if mesenchymal stem cells （MSCs） could be reconstructed as a vehicle for angiopoietin-1 （ Ang1 ） gene therapy in lung injury. Methods MSCs were obtained from adult male inbred mice and cultured to passage four. The cells were identified by fluorescence-activated cell sorting （FACS） analysis and cell differentiation detection. Lentiviral vectors contained GFP and Angl gene were conducted in 293T cells through three plasmids co-transfection method. Then MSCs were transduced with Angl gene efficiently through lentiviral vectors. The mRNA expression of Angl in MSCs was detected by RT-PCR before and after transfection. Also fluorescence from MSCs was detected by fluorescence microscope every day after transfection. Two hours after LPS inhalation, mice were infused via jugular vein with normal saline （NS group） , lentiviral vector carrying Angl （Angl group） , lentiviral vector carrying GFP （MSCs group）, and lentiviral vector carrying Angl/GFP （MSCs-Angl group）, respectively. Kaplan-Meier survival analysis was performed to compare the effects of MSCs-Angl on survival. And ectogenic MSCs origined lung cells were investigated in receipt mice. Results After passaged and purification, MSCs were confirmed to have the potential of differentiation. The lentiviral vectors carrying Angl and GFP were also identified. After transfection, the mRNA expression of Angl in MSCs was enhanced. Through the fluorescence microscope, MSCs get the most green fluorescence expression five days after the transfection when MOI was 20. Kaplan-Meier survival analysis showed that MSCs-Angl infusion had improved survival rates of lung injury rats compared with the control, but it did not reach statistical significance （ P = 0. 066）. Cells expressing GFP in lung tissues can be observed after MSCs were transplanted in vivo. Conclusions MSCs expressing Angl high can be constructed through lentiviral vector transfer, and MSCs-origined cells can be detected in receipt lungs after transplantation. So MSCs may serve as a vehicle for gene therapy in lung injury.
出处
《中国呼吸与危重监护杂志》
CAS
2009年第6期575-579,共5页
Chinese Journal of Respiratory and Critical Care Medicine
基金
国家自然科学基金(编号:30770930)
教育部博士点新教师基金(编号:200802471029)
上海市自然基金(编号:08ZR1421000)
