【摘要】 目的构建靶向IGFBP7的LV5病毒表达载体,并检测其表达效能及在黑素瘤细胞系中促凋亡的作用。方法以人igfbp7基因序列作为模版合成编码核酸序列,通过限制性内切酶NotⅠ和NsiⅠ酶切、T4 DNA连接酶连接,将igfbp7插入LV5-GFP,构建靶向IGFBP7的慢病毒质粒表达载体,筛选阳性克隆后与包装质粒通过脂质体共转染SK-MEL-28人黑素瘤细胞株,对照组转染LV5空载体。病毒感染SK-MEL-28,采用荧光显微镜观察感染的效率,Realtime PCR检测igfbp7m RNA的表达,流式细胞术检测对照组及处理组细胞凋亡率。统计数据分析LV5-NC-IGFBP7促黑素瘤凋亡的效9能。结果成功构建LV5-NC-IGFBP7,病毒转染后测定其滴度为1x10 UT/m L,细胞感染率在80%以上,Realtime PCR检测表明处理组igfbp7 m RNA表达高于对照组(P<0.01),但对照组及处理组凋亡率差异无统计学意义(P>0.05)。结论靶向IGFBP7的LV5-NC-IGFBP7病毒载体能有效促进igfbp7 m RNA高表达,但促恶性黑素瘤凋亡的效能有待提高。更多还原

【Abstract】 Objective To detect induction of apoptosis by IGFBP7 LV5-NC viral vector in melanoma cell line. Methods To construct lentiviral vector of LV5-NC-IGFBP7, divide SK-MEL-28 melanoma cell lines with control group and treatment group. The Control group was transfected LV5-NC empty vector, and the treatment group with LV5-NC-IGFBP7, igfbp7 m RNA expression was detected in two groups of cells with Realtime PCR, apoptosis rate of cell was detected in cells of two groups with flow cytometry, apoptosis promotion by LV5-NC-IGFBP7 in melanoma cells was statistically analyzed. Results Successful construction LV5-NC-IGFBP7, igfbp7 m RNA expression is higher in treatment group than the control group, but the apoptosis rate was not statistically significantly different between control and treatment groups. Conclusion Targeted IGFBP7 LV5-NCIGFBP7 viral vector can effectively promote the igfbp7 m RNA high expression, but its effect on promoting apoptosis with malignant melanoma is not obvious.更多还原