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

于建超; 王勤章; 王江平; 钱彪; 李应龙; 王新敏; 倪钊; 李强

摘要

目的：观察携带增强绿色荧光蛋白（GFP）的含人干细胞白血病（SCL）基因慢病毒载体转染体外培养的豚鼠膀胱 Cajal 样间质细胞（ICC）的效果。方法2014年10月—2015年8月，选取普通级成年健康豚鼠16只。构建含人 SCL 基因的慢病毒载体，并标定病毒滴度；采用随机数字表法选取健康豚鼠4只，采用颈椎脱臼方法将其杀死，体外培养豚鼠膀胱 ICC。设置不同感染复数（MOI）（0.5、1.0、5.0、10.0、50.0、100.0），利用含人 SCL 基因的慢病毒载体转染 ICC，在激光共聚焦显微镜下观察 ICC 生长状态并计算转染效率，确定最佳 MOI。采用随机数字表法将 ICC 分为正常对照组、空白质粒对照组与慢病毒转染组，其中正常对照组加入1％磷酸盐缓冲液（PBS），空白质粒对照组加入空慢病毒，慢病毒转染组按最佳 MOI 加入含人 SCL 基因的慢病毒载体，计算各组转染效率，确定最佳转染时间。采用反转录-聚合酶链式反应（RT-PCR）法检测 SCL mRNA 表达情况。上述实验均独立重复4次。结果经测定，病毒滴度为5×108 TU/ ml。倒置显微镜下发现一些特征性梭形细胞，其两侧有显著外凸分枝，细胞核较大，其外形规则，有序地实现细胞贴壁，而其他未贴壁细胞则呈现与 ICC 不一样的形态，胞体多为椭圆形，细胞两极无突起，整体形状扁平。激光共聚焦显微镜下可以发现酪氨酸蛋白激酶生长因子受体（c-kit）受体成功表达，证实培养的细胞是膀胱 ICC。观察各组 ICC 生长状态，当 MOI 为0.5、1.0、5.0、10.0时，细胞生长状态较好，无细胞死亡，当 MOI为50.0、100.0时，可见部分细胞死亡，细胞活性降低；MOI 为1.0、5.0、10.0、50.0、100.0时的转染效率高于 MOI为0.5时（P ＜0.05）；MOI 为5.0、10.0、50.0、100.0时的转染效率高于 MOI 为1.0时（P ＜0.05）；MOI 为10.0、50.0、100.0时的转染效率高于 MOI 为5.0时（P ＜0.05）；MOI 为50.0、100.0时的转染效率高于 MOI 为10.0时（P＜0.05）；综合 ICC 生长状态及转染效率，确定含人 SCL 基因慢病毒载体转染 ICC 的最佳 MOI 为10.0。正常对照组、空白质粒对照组 ICC 中无 GFP 表达；慢病毒转染组转染3 d 时 ICC 中 GFP 表达强度高于转染2 d、转染5 d 时，确定含人 SCL 基因慢病毒载体转染 ICC 的最佳转染时间为3 d。慢病毒转染组中扩增产物大小与目的片段大小1036 bp 一致，而正常对照组及空白质粒对照组均未见特异性条带表达，表明 SCL 基因成功导入 ICC。结论利用含人 SCL 基因慢病毒载体能高效转染体外培养的 ICC，并成功介导 SCL 基因在 ICC 中的表达，为下一步研究利用 SCL 基因进行体内转染实验奠定基础。%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.

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

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