中国全科医学2016，Vol.19Issue(15)：1796-1802,7.DOI:10.3969/j.issn.1007-9572.2016.15.012
含人干细胞白血病基因慢病毒载体转染Cajal 样间质细胞的效果研究
Effect of Transfecting Human SCL Gene Lentiviral Vectors Into Cajal-like Cells
摘要
Abstract
Objective To transfect the lentiviral vector containing human stem cell leukemia( SCL)gene and enhanced green fluorescent protein(GFP)into Cajal - like cells cultured in vitro. Methods During October 2014 to August 2015,16 CV adult healthy guinea pigs were selected as study subjects. To construct human SCL gene lentiviral expression vectors and determine virus titers,4 healthy guinea pigs were selected by random number table method and killed by cervical dislocation method,Cajal - like cells were cultured in vitro by differential adherence method. Different MOI(0. 5,1. 0,5. 0,10. 0, 50. 0,100. 0)was set,the lentiviral vector containing human SCL gene was used to transfect into Cajal - like cells in vitro,the growth state of the Cajal - like cells was observed under laser scanning confocal microscope,and transfection efficiency was calculated,so as to determine the best MOI. The Cajal - like cells were divided into normal control group,empty plasmid control group and lentivirus transfection group by random number table method,cells in normal control group were treated with 1%phosphate buffered saline(PBS),cells in empty plasmid control group were treated with blank lentivirus,cells in lentivirus transfection group were treated with lentiviral vector containing the human SCL gene according to the best MOI,the transfection efficiency of each group was calculated respectively,the optimal transfection time was determined. The expression of SCL mRNA was measured by RT-PCR method. The above experiment was independently repeated 4 times. Results It was examined that virus titers was 5 × 108 TU/ ml. Some characteristic spindle cells were found under inverted microscope,both sides of the cells have significant convex branches,the cell nucleus was large and had regular shape,the cultured cells grew against the wall well,and other non - adherent cells presented some different shapes compared with Cajal - like cells,the overall shape of most cells was oval and flat,the two polar of cells had no protuberance. The c-kit receptor successfully expressed under laser scanning confocal microscope,it confirmed that the cultured cells were bladder Cajal - like cells. The growth state of cells in each group was observed,when MOI reached 0. 5,1. 0,5. 0 or 10. 0,the growth state was good,no cell died,when MOI reached 50. 0 or 100. 0,some cells died and cell activity decreased. The transfection efficiency when MOI reached 1. 0,5. 0,10. 0,50. 0 or 100. 0 was higher than that when MOI reached 0. 5( P < 0. 05). The transfection efficiency when MOI reached 5. 0,10. 0, 50. 0 or 100. 0 was higher than that when MOI reached 1. 0(P < 0. 05). The transfection efficiency when MOI reached 10. 0, 50. 0 or 100. 0 was higher than that when MOI reached 5. 0(P < 0. 05). The transfection efficiency when MOI reached 50. 0 or 100. 0 was higher than that when MOI reached 10. 0(P < 0. 05). According to Cajal - like cells growth state and transfection efficiency,to transfect lentiviral vector containing human SCL gene into Cajal - like cells,the best MOI was 10. 0. No GFP expression was found in cells in normal control group and empty plasmid group. The GFP expression intensity in Cajal - like cells in lentivirus transfection group 3 days after transfection was higher than that 2 days and 5 days after transfection,respectively, thus it was determined that to transfect lentiviral vector containing human SCL gene into Cajal - like cells,the optimal transfection time was 3 days. The amplified product size was 1 036 bp,and it was consistent with the size of target fragments in lentivirus transfection group,while the normal control group and empty plasmid control group showed no expression of specific bands, which showed SCL gene was successfully transmited into Cajal - like cells. Conclusion The lentivirus vector containing human SCL gene can efficiently transfect into Cajal - like cells in vitro,and can successfully mediated the expression of SCL gene in Cajal - like cells,the findings laid the foundations for transfection experiments using SCL gene in vivo.关键词
转染/干细胞白血病基因/慢病毒载体/Cajal样间质细胞Key words
Transfection/Stem cell leukemia gene/Lentiviral vector/Cajal-like interstitial cells分类
医药卫生引用本文复制引用
于建超,王勤章,王江平,钱彪,李应龙,王新敏,倪钊,李强..含人干细胞白血病基因慢病毒载体转染Cajal 样间质细胞的效果研究[J].中国全科医学,2016,19(15):1796-1802,7.基金项目
国家自然科学基金资助项目 （）
