基于胚胎干细胞实验模型评价黄芩苷的胚胎毒性  

Evaluation of embryotoxicity of baicalin based on embryonic stem cell test system

作　　者：张崴[1,2] 宋殿荣[1] 王雅楠[1] 朱泽[3] 

机构地区：[1]天津中医药大学第二附属医院妇科,天津300150 [2]天津中医药大学,天津300193 [3]天津医科大学微生物教研室,天津300070

出　　处：《中国药理学与毒理学杂志》2012年第6期864-869,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：国家中医药管理局中医药行业科研专项(200707011)~~

摘　　要：目的应用胚胎干细胞实验模型体系评价黄芩苷的胚胎毒性。方法分别将胚胎干细胞D3和胚胎成纤维细胞(BALB/c 3T3)与黄芩苷20,40,60,80和100 mg·L-1共培养,MTT法检测细胞活性,分别计算黄芩苷对胚胎干细胞D3和3T3细胞增殖半数抑制浓度IC50(D3)和IC50(3T3)。利用悬滴-悬浮-贴壁方法,体外培养胚胎干细胞向心肌细胞分化,实时定量PCR方法检测心肌细胞特异表达肌球蛋白重链(β-MHC)基因的表达,计算胚胎干细胞D3定向分化半数抑制浓度,即ID50(D3)。利用胚胎毒性统计公式,预测黄芩苷的胚胎毒性。结果不同浓度黄芩苷作用10 d后,胚胎干细胞D3和3T3细胞存活能力随着黄芩苷浓度增加缓慢下降,黄芩苷对胚胎干细胞D3和3T3细胞增殖均有一定程度的抑制作用,其IC50(D3)和IC50(3T3)分别为135.9和63.3 mg·L-1。体外胚胎干细胞经悬滴-悬浮-贴壁培养可分化为能够表达β-MHC基因的心肌样细胞,黄芩苷2,5,10,20和40 mg·L-1对胚胎干细胞定向分化为心肌细胞的抑制率分别为29.5%,46.8%,59.6%,61.7%和69.0%,黄芩苷对胚胎干细胞分化有一定程度的抑制作用,其体外心肌细胞定向分化的ID50(D3)为7.25 mg·L-1。根据胚胎毒性计算公式,计算得黄芩苷具有弱胚胎毒性。结论黄芩苷具有弱胚胎毒性。OBJECTIVE To assess embryotoxicity of baicalin using embryonic stem （ES） cell test in vitro. METHODS ES D3 cells and BALB/c 3T3 cells were cultured respectively with baicalin 20, 40, 60, 80 and 100 mg·L-1. Cell morphology was observed with a phase contrast microscope and absorbance of the resulting colored solution at 570 nm was measured by microplate reader. According to the concentration-effect curve, 50% inhibition of cell growth （IC50） and viability were obtained in ES cell line D3[ ICso（ D3 ） ] and in 3T3 cells [ IC50（ 3T3 ） ]. ES cells were cultured in baicalin with using hanging drop-suspension-attachment method, then cardiac myoblasts specific genes myosin heavy chain （[β-MHC） in differentiation of embryonic stem cell were detected by real time Q-PCR, the growth inhibitory rate was calculated with quantitative analysis, according to the concentration-effect curve, 50% inhibition of ES cells differentiation into cardiac myoblasts [ ID50 （D3）] obtained. Baicalin embryotoxicity potential was predicted using statistics formula.RESULTS ES D3 cells and BALB/c 3T3 cells viability decreased slowly with the increase in baicalin concentration after they were cultured in different concentration of baicalin for ten days, which showed that baicalin had a certain degree of inhibition on ES D3 cell and BALB/c 3T3 cell proliferation. The half-maximal proliferation-inhibition concentration （IC50） of baicalin on ES cell line D3 [IC50（D3） ] and 3T3 cells[ICs0（3T3）] was 135.9 mg·L-1 and 63.34 mg·L-1. ES cells in vitro by hanging drop-suspension-adherent culture could differentiate to expression of β-MHC gene in myocardial cells. With baicalin 2, 5, 10, 20 and 40 mg·L-1, the ability of ES cells differentiate into myocardial cells gradually decreased, the inhibitory rate was 29.5%, 46. 8%, 59. 6% , 61.7% and 69.0%, respectively, and this indicated that baicalin had a certain degree of inhibitory effect on ES cells differentiation. The half-maximal differentiation-inhibition concentrati

关 键 词：胚胎干细胞 黄芩苷 毒性作用 

分 类 号：R285[医药卫生—中药学]