人参皂苷Rb1对体外培养大鼠雪旺细胞增殖、细胞周期及增殖细胞核抗原表达的影响

Effect of Ginsenoside Rb1 on proliferation, cell cycle and expression of proliferating cell nuclear antigen in rat Schwann cells in vitro

目的 观察人参皂苷Rb1（GRb1）对体外培养大鼠雪旺细胞（SCs）增殖、细胞周期及增殖细胞核抗原（PCNA）表达的影响.方法 取3～5d无特定病原体（SPF）级SD大鼠20只,雌雄不限,体质量25～30 g,分离双侧坐骨神经行SCs体外培养,培养细胞经S-100免疫荧光染色标记鉴定.取对数生长期SCs加入GRb1培养24 h,调整GRb1的终质量浓度为10 mg/L（B组）、20 mg/L（C组）、50 mg/L（D组）、100 mg/L（E组）、500 mg/L（F组）,对照组（A组）加入等量磷酸盐缓冲液（PBS）.细胞增殖检测法[细胞计数试剂盒（CCK-8）法]检测细胞增殖活性,碘化丙锭（PI）染色流式细胞仪检测细胞周期,实时定量聚合酶链反应（Real-time PCR）及Western blot法检测SCs内PCNA基因与蛋白的表达.结果 SCs经S-100免疫荧光标记染色提示阳性细胞率达90％以上,CCK-8检测结果显示GRb1在10～ 500 mg/L浓度范围内可以促进SCs增殖,流式细胞仪检测结果显示GRb1在10 ～500 mg/L浓度范围内可以促进SCs细胞周期进程,Real-time PCR及Western blot检测结果显示GRb1处理组SCs内PCNA基因和蛋白表达均高于对照组（P＜0.05）.结论 GRb1可以促进SCs增殖及细胞周期进程,并促进SCs内PCNA表达.

Objective To investigate the effects of Ginsenoside Rb1 （GRb1） on proliferation, cell cycle and expression of proliferating cellular nuclear antigen （PCNA） in Schwann cells （SCs）.Methods Twenty 3-5 days old specific pathogen free （SPF） SD rats were used, no matter male or female, weighing 25-30 g.The SCs were isolated from sciatic nerves of SD rats in vitro, and identified by S-100 immunofluorescence markers staining.SCs were divided into six groups： group A, GRb1 10 mg/L;group B, GRb1 20 mg/L, group C, GRb1 50 mg/L, group D, GRb1 100 mg/L, group E, GRb1 500 mg/L, and the control group treated by equal volume of phosphate buffer （PBS）.Cell counting kit-8 （CCK-8） assay was used to detect the cell proliferation, cell cycle was analyzed by propidium iodide （PI） staining and flow cytometry （FCM） analysis, and mRNA and protein levels were detected by real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting, respectively.Results S-100 immunofluorescence results showed that more than 95% cells were positive in culture.CCK-8 results showed that GRb1 10-500 mg/L could increase cell proliferation.PI staining results indicated that RBb1 could accelerate the cell cycle process.Real-time PCR and Western blotting results indicated GRb1 of 10-500 mg/L could increase PCNA mRNA and protein levels in SCs.Conclusion GRb1 could promote SCs proliferation, accelerate cell cycle process, and increase the expression of PCNA mRNA and protein.