Salvianolic acid B maintains stem cell pluripotency and increase it’s proliferation rate by activating Jak2/Stat3 and the EGFR-ERK1/2 pathways

• Main Authors: Chia-Hui Liu, 劉佳慧
• Other Authors: Woei-Cherng Shyu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/p2bv2g

碩士 === 中國醫藥大學 === 免疫學研究所碩士班 === 100 === Stem cells provide hope for many diseases that currently lack effective therapeutic methods. In all type of stem cells, embryonic stem (ES) and induced pluripotent stem (iPS) cells are the most powerful cells that could differentiate into all three-layer cells. However, maintaining ES and iPS cells self-renewal in vitro must be need leukemia induced factor (LIF) -- an expensive reagent. Our goal is to find a cheaper pure compound that can maintain ES cells pluripotency. After testing Oct4 and Sox2 gene expression levels in 15 candidate compounds, 500μg/ml of Salvia miltiorrhizae triggers the up-regulation of Oct4 gene expression levels in MEF cells. Salvianolic acid B (Sal B), Salvia miltiorrhizae extract, also could up-regulate Oct4 and Sox2 gene expression levels in MEF cells. Viability analysis and BrdU analysis showed that Sal B not only did not cause cell death, but induced cell proliferation, especially in 0.01nM. We used ES cells treated with different concentrations of Sal B to test whether it is useful for maintaining stem cell pluripotency. Results indicated that compared to controls (did not contain LIF), Sal B -treated ES cells have higher expression levels of several stem cell markers in 0.01nM, including alkaline phosphatase, SSEA1, and Nanog. According to previous study, Sal B direct targeted to EGFR, and activated EGFR, then EGFR turn on Stat3 and ERK1/2. We demonstrated that Sal B could bind to LIF receptor by Docking analysis and EGFR to turn-on the Jak2-Stat3 signaling pathway and EGFR-ERK1/2 signaling pathways. We subsequently determined that phospholation-EGFR, phospholation-Jak2, phospholation-Stat3 and phospholation-ERK protein levels were increased after treated with Sal B by western blotting analysis . Cytokines associated with Jak2-Stat3 signaling pathway were also up-regulated.We add Jak2 antagonist AG490 and EGFR antagonist ZD1839 to comfirmed Jak2-Stat3 signaling pathway and EGFR-ERK1/2 signaling pathways. We subsequently determined that phospholation-EGFR, phospholation-Jak2, phospholation-Stat3 and phospholation-ERK protein levels were decreased after treated by Sal B and antagonist. In addition, after antagonist treatment, the stem cell marker expression were also down regulated in Sal B treated ES cells. Through viability assay, we determined that Sal B could increase cell proliferation. In conclusion, we determined that Sal B not only could replace LIF to maintain the pluripotency of ES cells but increase the proliferation rate. It may become a power reagent in stem cell research.