摘要
目的观察分泌型白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor,SLPI)对真皮多能干细胞(dermal multipotent stem cells,DMSCs)的促增殖作用。方法酶消化法分离、纯化、扩增新生大鼠DMSCs。在前期成功构建SLPI基因过表达载体基础上,根据SLPI基因si RNA的设计挑选出最佳抑制SLPI基因表达的干扰载体,RT-PCR、Western blot鉴定。重组表达载体转染DMSCs,观察其表达,运用CCK-8法和流式细胞术检测细胞周期,观察SPLI基因促进DMSCs的增殖作用。结果成功分离获得真皮来源、具有细胞干性的DMSCs;并成功构建SLPI基因过表达真核载体及SLPI基因si RNA表达载体,转染DMSCs(分为过表达组和干扰组,并以转染空载体者为对照组)。CCK-8检测显示,48、72、96 h,过表达组增殖较快,干扰组增殖较慢,两组比较差异有统计学意义(P<0.05);72、96 h,过表达组与对照组比较差异有统计学意义(P<0.05),说明过表达组增殖较快,即SLPI基因有促进增殖作用。流式细胞检测表明,过表达组G1期与干扰组比较差异有统计学意义(P<0.05),S期、G2期与对照比较差异有统计学意义(P<0.05)。结论 SLPI基因具有明确的促DMSCs增殖作用,促进细胞从G1向S和G2期转变,即通过调控细胞周期发挥促增殖作用。
Objective To determine the effect of secretory leukocyte protease inhibitor （SLPI） on promoting proliferation of dermal muhipotent stem cells （DMSCs）. Methods DMSCs were isolated from neonatal rats by enzyme digestion, and then purified and amplified for further research. Based on successful construction of SLPI overexpression vector, SLPI gene siRNA expression vector was picked out according to RT-PCR and Western blot identification. DMSCs were transformed with recombinant expression plasmids of SLPI gene, and the effect of SPLI gene on promoting the proliferation of DMSCs was observed using CCK-8 assay and flow cytometry. Results DMSCs were successfully isolated from the dermis. The eukaryotic expression vector and siRNA expression vector of SLPI gene were constructed. The transformed DMSCs were divided into 3 groups： overexpression group, interference group, and control group （transformation with blank vector）. The results of CCK-8 assay showed that, in 48, 72 h and 96 h after transformation, the proliferation of DMSCs in the overexpression group was faster than that in the interference group （ P 〈 0. 05 ） , and in 72 and 96 h after transformation, DMSCs in the overexpression group were significantly different from those in the control group （P 〈 0. 05 ）, which suggested that SLPI gene might promote the proliferation of DMSCs. The results of flow cytometry showed that the cells in the G1 phase in the overexpression group were significantly different from those in the interference group （P 〈 0.05 ） , meanwhile, the cells in the S phase and G2 phase were significantly different from those in the control group （P 〈 0. 05 ）. Conclusion SLPI gene plays a role in promoting the proliferation of DMSCs, and promotes cell cycle transformation from G1, phase to S phase and G2 phase, which suggests that SLPI ,nay promote proliferation of DMSCs via regulating cell cycle.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第4期356-360,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(30370562
81071562)~~
关键词
分泌型白细胞蛋白酶抑制因子
重组表达载体
真皮多能干细胞
增殖
secretory leukocyte protease inhibitor
recombinant expression vector
dermalmultipotent stem cells
proliferation
