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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 observe the effect of secretory leukocyte protease inhibitor (SLPI) on proliferation of dermal multipotent stem cells (DMSCs). Methods Enzymatic digestion method was used to separate, purify and amplify DMSCs in neonatal rats. Based on the successful construction of SLPI gene overexpression vector in the early stage, we selected the best interference vector to inhibit SLPI gene expression according to the design of si RNA of SLPI gene, and identified by RT-PCR and Western blot. The recombinant plasmid was transfected into DMSCs to observe its expression. The cell cycle was detected by CCK-8 assay and flow cytometry. The proliferation of DMSCs was observed by SPLI. RESULTS: The dermal derived MSCs were successfully isolated and the cell-derived DMSCs were successfully isolated. The eukaryotic vector expressing SLPI gene and si RNA expression vector of SLPI gene were successfully constructed and transfected into DMSCs (divided into overexpression group and interference group) Vector for the control group). The results of CCK-8 assay showed that the proliferation of the over-expression group was faster and the proliferation of the interference group was slower at 48, 72 and 96 h (P <0.05). At 72 and 96 h, the over-expression group and the control group The difference was statistically significant (P <0.05), indicating that the over-expression group proliferated faster, that SLPI gene can promote proliferation. Flow cytometry showed that there was significant difference between G1 and overexpression groups (P <0.05), but there was significant difference between S phase and G2 phase (P <0.05). Conclusion SLPI gene has a clear role in promoting proliferation of DMSCs, and promotes cell transformation from G1 to S and G2 phases, which is to promote proliferation by regulating cell cycle.