槟榔碱对大鼠体外胚胎肢芽发育的影响  

Effects of arecoline on the development of rat embryo limb bud cartilage in vitro

作　　者：朱思睿 杨艳伟[1] 赵曼曼 刘鑫磊 黄芝瑛[2] 周晓冰[1] 张河战[1] 李波[1] ZHU Si-rui;YANG Yan-wei;ZHAO Man-man;LIU Xin-lei;HUANG Zhi-ying;ZHOU Xiao-bing;ZHANG He-zhan;LI Bo(National Center for Safety Eoaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176.China;School of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou 510006,China)

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176 [2]中山大学药学院,广州510006

出　　处：《中国新药杂志》2021年第23期2157-2165,共9页Chinese Journal of New Drugs

基　　金：国家“重大新药创制”科技重大专项资助项目(2018ZX09201017-001);国家自然科学基金资助项目(81603210);中检院中青年基金课题(2020C1)。

摘　　要：目的:研究槟榔碱在体外培养下对大鼠胚胎软骨发育的影响,从而探究槟榔碱的胚胎致畸性。方法:取受孕d 14大鼠胚胎肢芽,前肢芽利用微团培养技术进行体外培养,根据半数细胞增殖抑制浓度(IC_(50)-P)和半数细胞分化抑制浓度(IC_(50)-D)及其比值判定受试物的致畸性;利用离体器官技术培养后肢芽,随机分为空白对照(0 mg·mL^(-1)槟榔碱)、低剂量(0.1 mg·mL^(-1)槟榔碱)、中剂量(1 mg·mL^(-1)槟榔碱)、高剂量(10 mg·mL^(-1)槟榔碱)、阳性对照(100 ng·mL^(-1)5-氟尿嘧啶)5个实验组,分别在体外培养24,72和120 h后收获,在体视镜下进行形态学评分后,石蜡包埋、切片、HE染色和甲苯胺蓝染色观察细胞和软骨形态,并通过免疫组化技术研究槟榔碱对在各组软骨细胞骨形态发生蛋白2(BMP-2)表达的影响。结果:在微团模型中根据欧洲替代方法验证中心(European Centre for the Validation of Alternative Methods,ECVAM)预测模型判定槟榔碱为胚胎中晚期发育毒性化学物,为非特异性弱致畸物;在整体肢芽器官模型中,1 mg·mL^(-1)(中剂量)及以上浓度的槟榔碱会对肢芽器官造成发育毒性,10 mg·mL^(-1)(高剂量)从给药24 h后便能产生显著毒性,各剂量组在72 h培养后发育状况出现了显著的剂量相关性;从病理切片结果可以看出,槟榔碱会进一步影响增殖软骨细胞和肥大软骨细胞的增殖分化,从而影响肢芽指掌与肢臂的发育;在BMP-2表达的免疫组化结果中,BMP-2的表达强度呈现与槟榔碱剂量相关性,槟榔碱的剂量越大,阳性表达程度越低,反映软骨细胞增殖分化放缓。结论:大于1 mg·mL^(-1)的槟榔碱在大鼠胚胎肢芽体外培养中有生长抑制作用,可初步认为槟榔碱对胚胎中后期发育具有弱致畸性,可能是通过影响BMP-2的表达来抑制软骨细胞的增殖分化。Objective: To study the effect of arecoline on rat embryo cartilage development in vitro, so as to explore the embryonic teratogenicity of arecoline. Methods: The embryonic limb buds of gestation day 14 timed-pregnant(GD14) rats were taken, and the forelimb buds were cultured in micromass(MM). The teratogenicity of arecoline was determined by the half-proliferation inhibitory concentration(IC_(50)-P), the half-differentiation inhibitory concentration(IC_(50)-D) and their ratios. The hindlimb buds were cultured in organ culture and randomly divided into five experimental groups, i.e., blank control(0 mg·mL^(-1)arecoline), low-dose(0.1 mg·mL^(-1)arecoline), middle-dose(1 mg·mL^(-1)arecoline), high-dose(10 mg·mL^(-1)arecoline), and positive control group(10 ng·mL^(-1)5-fluorouracil). They were cultured in vitro for 24, 72, and 120 h, respectively, and then harvested. Morphological scoring was performed under a stereo microscope, and followed by paraffin embedding, sectioning, HE staining and toluidine blue staining to observe the morphology cartilage cells. The effects of arecoline on the expression of bone morphogenetic protein 2(BMP-2) in each group were studied by immunohistochemistry. Results: In MM model, according to the ECVAM prediction model, it was determined that arecoline was a developmental toxic chemical and a non-specific weak teratogen. In the limb bud organ model, 1 mg·mL^(-1)(medium dose) and above concentrations of can cause developmental toxicity arecoline to limb bud organs. Arecoline at 10 mg·mL^(-1)(high dose) can produce significant toxicity after 24 h of administration. The developmental status of each dose group has a significant measurement correlation after 72 h of culture. It can be seen from the pathological section results that arecoline further affected the proliferation and differentiation of proliferating chondrocytes and hypertrophic chondrocytes, thereby affecting the development of limb buds, palms and limbs. In the immunohistochemical analysis, the expression intensity of

关 键 词：槟榔碱 胚胎毒性 致畸 替代实验 微团培养 离体器官培养 

分 类 号：R965[医药卫生—药理学]