人参总皂苷诱导红系血细胞增殖的信号转导(英文)

Signal transduction in erythropoiesis induced by total saponins of Panax ginseng

背景：研究证实多数造血生长因子通过JAKs-SWATs途径转导信号，调节血细胞的增殖与分化。人参总皂苷能够促进早期的造血干，祖细胞向红系细胞分化，具有类似造血生长因子的功效，在红系血细胞发生中造血细胞膜表面红细胞生成素受体起着关键性的作用。目的：通过红细胞生成素及其受体介导的JAK2／STAT5信号转导途径，分析人参总皂苷定向诱导红系血细胞发生的分子机制。设计、时间及地点：细胞学体外观察，于2006—05，2008—10在在重庆医科大学基础医学研究所、组织胚胎学教研室完成。材料：脐血细胞来源于正常足月妊娠产妇分娩脐带血，由重庆医科大学第一附属医院提供。人参总皂苷由重庆中药研究所惠赠，纯度95％以上，添加MI-1640培养液配成1g/L的工作液。方法：①集落形成实验：取5×10^6L^-1脐血单个核细胞，添加含马血清的RPMI-1640培养液，10，25，50，75，100mg／L人参总皂苷组分别加入对应终浓度的人参总皂苷，并设立空白对照组。在96孔板上培养，0．2mL/孔，14d计数早期红系祖细胞，6d计数晚期红系祖细胞。②取脐血单个核细胞悬液100uL（5×10^8L^-1），进行MTT检测。（3）分别将0，25，50，100mg／L人参总皂苷与脐血造血细胞1×10^9L^-1共培养24h，进行Westemblot检测。④取脐血造血细胞1×10^9L^-1，对照组加入含体积分数为10％马血清的RPMI-1640培养液，人参总皂苷组在常规培养体系中加入终浓度25mg／L人参总皂营。分别用5U／mL红细胞生成素诱导0，2，5，30min后终止反应，进行免疫沉淀检测。主要观察指标：人参总皂苷刺激红系造血祖细胞增殖的能力，红细胞生成素对脐血细胞增殖的影响，人参总皂苷对造血细胞红细胞生成素受体表达的影响，JAK2、STAT5蛋白酪氨酸磷酸化情况。结果：10～75mg/L人参总皂营均能提高脐血细胞形成早期红系祖细胞、晚期红系祖细胞的集落产率，以25mg/L提高幅度最为明显。2—50U/mL红细胞生成素均能促进脐血细胞的增殖，以5U/mL促进增殖效果最为明显。0—100mg/L人参总皂苷作用脐血细胞24h后，红细胞生成素受体的表达无明显变化。人参总皂苷作用造血细胞24h后，加入红细胞生成素继续刺激0～30min．JAK2和SWAT5的酪氨酸磷酸化程度均明硅增强。结论：人参总皂苷在促进造血细胞增殖的过程中，尽管对造血细胞红细胞生成素受体的表达无明显影响，但可能通过在红细胞生成素受体介导的信号转导过程中增强JAK2和SWAT5的酪氨酸磷酸化程度发挥作用。

BACKGROUND： Most of hematopoietic growth factor regulates proliferation and differentiation of blood cells through JAKs-STATs signal transduction pathway. Total saponins of Panax ginseng （TSPG） can promote in vitro differentiation of CD34＋ hematopeietic progenitor cells into erythroid cells, with similar effectiveness of hematopoietic growth factor. Erythropoietin receptor （EpoR） expression on the cell membrane of progenitor cells is critical during the erythroid differentiation process. OBJECTIVE; To investigate the molecular mechanism of TSPG to induce erythroid cells through erythropoiesis and its receptor-mediated JAK2/STAT5 signal transduction. DESIGN, TIME AND SETTING： An in vitro cytological observation. The study was performed at the Department of Histology and Embryology, Institute of Basic Medicine, Chongqing Medical University from May 2006 to October 2008. MATERIALS： Umbilical cord blood of normal full-term pregnancy was provided by the First Hospital of Chongqing Medical University. TSPG, purity 〉 95%, provided by Chongqing Institute of Traditional Chinese Medicine, was diluted in RPMI-1640 for work concentration of 1 g/L and degermed by positive pressure filtration. METHODS： （1）Colony-forming ability： 5 × 10^6/L mononuclear cells （MNCs） from human umbilical cord blood were incubated in RPMI-1640 culture solution containing horse serum, with various dilutions of TSPG （0 as blank control, 10, 25, 50, 75, 100 mg/L）. The MNCs were cultured on 96-well culture plate, with 0.2 mL in each well. Early erythroid cells were counted on day 14, and late erythroid cells on day 6. （2）100 μ L MNCs （5×10^8/L） was measured by MTT. G0, 10, 25, 50, and 100 mg/L TSPG were separately cultured with 1 × 109/L MNCs for 24 hours for Western blot determination. （4）1 ×109/L MNCs were harvested and cultured separately in RPMI-1640 culture solution containing 10% horse serum as control group and in TSPG （25 mg/L）- conditioned culture system as experimental group. 5 U/mL Epo was added for 0, 2, 5 and 30 minutes. Immunoprecipitation of JAK2/STAT5 was used for the effect of TSPG on Epo/EpoR-induced tyrosine phosphorylation of JAK2/STATs. MAIN OUTCOME MEASURES： Effect of TSPG on proliferation of erythroid progenitor cells from human umbilical cord blood; Effect of Epo on the proliferation of hematopoietic cells; Effect of TSPG on EpoR expression of the umbilical blood cells; tyrosine phosphorylations of JAK2 and STAT5. RERULTS： TSPG （10-75 mg/L） promoted the colony formation of BEU-E, CFU-E, and the preferential differentiation into erythroid lineage cells was most induced from 25 mg/L of TSPG. Using the colorimetric MTT assay, MNCs exhibited proliferative responses to Epo （2-50 U/mL） reaching maximum at 5 U/mL Epo. The addition of TSPG did not increase the expression of EpoR after MNCs were incubated in the presence of with or without TSPG for 24 hours. The pretreatment with TSPG for 24 hours enhanced Epo-induced tyrosine phosphorylation of JAK2 and STATs （STAT5a and STAT5b）. CONCLUSION： The Epo/EpoR-mediated signals are enhanced and JAK2 / STATs signals play an essential role in the effect of TSPG on erythropoiesis, whereas the expression level of EpoR remains unchanged.