孕期尼古丁暴露所致子代成骨细胞BMP-4/Smad信号通路的变化  

Changes of Osteoblast BMP-4/Smad Signaling Pathway Caused by Nicotine Exposure in Pregnancy

作　　者：王军建 刘鑫 刘方娜 姚满叶 邓宇[2] Wang Junjian;Liu Xin;Liu Fangna(Department of Orthopedics,Zhengzhou Children's Hospital,Henan 450018,China)

机构地区：[1]郑州儿童医院骨科,450018 [2]武汉大学中南医院骨科,430071

出　　处：《医学研究杂志》2018年第11期57-61,共5页Journal of Medical Research

基　　金：国家自然科学基金青年科学基金资助项目(81502817)

摘　　要：目的探究大鼠孕期尼古丁暴露对子代成骨细胞(OB) BMP-4/Smad信号通路的影响。方法将妊娠期大鼠分为对照组与实验组,对照组受孕第9天皮下注射2mg/(kg·d)生理盐水,实验组在受孕后第9天于皮下注射2mg/(kg·d)尼古丁,新生3天乳鼠处死后收集颅骨,采用钙钴法对OB碱性磷酸酶进行染色,反转录聚合酶链反应法(RT-PCR)测定OB细胞中骨形态发生蛋白4 (BMP-4)、Smad1、Smad5、Smad8、Runt相关转录因子2(Runx-2)、成骨相关转录因子(Osterix)、Smads泛素化调节因子1(Smurf1)表达,免疫印迹法(Western blot)检测OB中BMP-4、Smad1、Smad5、Smad8、Runx-2、Osterix、Smurf1蛋白表达。结果 ALP染色显示OB细胞胞体较大,细胞核染色呈蓝紫色,细胞质呈淡紫色,对照组ALP细胞阳性率为42. 69%±5. 16%,高于实验组(18. 67%±3. 19%),差异有统计学意义(P <0. 05)。RT-PCR结果显示实验组BMP-4、Smad1、Smad5、Smad8、Runx-2、Osterix m RNA表达量均低于对照组,Smurf1mRNA表达量高于对照组,差异有统计学意意义(P <0. 05)。Western blot法检测结果显示,实验组乳鼠OB中BMP-4、Smad1、Smad5、Smad8、Runx-2、Osterix蛋白表达量均低于对照组,Smurf1蛋白表达量高于对照组。蛋白条带灰度扫描结果显示,实验组乳鼠OB中BMP-4、Smad1、Smad5、Smad8、Runx-2、Osterix蛋白相对灰度值均低于实验组,Smurf1蛋白相对灰度值高于对照组,差异有统计学意义(P <0. 05)。结论大鼠孕期尼古丁暴露对OB具有一定的抑制作用,可能与上调Smurf1表达、抑制BMP-4/Smad信号通路和下调转录因子Runx-2、Osterix有关。Objective To explore the influence of nicotine exposure in pregnancy on osteoblast (OB)BMP -4/Smad signaling pathway.Methods Pregnant rats were divided into control group and experimental group,and the control group were injected with 2mg/ (kg ~d)salines ubcutaueously at 9th day in pregnancy,and the experimental group were injected with 2mg/(kg·d)nicotine.The skull was collected after offspring suckling mice killed at 3rd day.OB alkaline phosphatase was stained by using calcium cobalt method,and bone morphogenetic protein 4(BMP-4),Smadl,SmadS,Smad8,Runt related transcription factor 2(Runx-2),osteogenic associated transcription factor (Osterix)and Smads ubiquitination regulator 1(Smurf1)expression in OB cells were detected by using reverse transcription polymerase chain reaction method (RT-PCR).BMP-4,Smadl,Smad5,Smad8,Runx-2,Osterix,Smurfl protein expression in OB cells were tested by using Western blot.Results ALP staining showed that OB cells bodies were larger,the nuclei stained blue and purple,and cytoplasm was pale purple.The positive rate of ALP cells in the control group 42.69%+5.16%,higher than that of (19.67%±3.19%)in the experimental group,with statistically significant difference (P <0.05).The results of RT-PCR showed that BMP -4,Smadl,Smad5,Smad8,Runx -2,Osterix and mRNA expression levels in the experimental group were lower than those in the control group.The expression of Smurfl mRNA was higher than that of the control group,with statistically significant difference (P < 0.05).The results of Western blot showed that BMP-4,Smadl,Smad5,Smad8,Runx-2 and Osterix expression in OB of the observation group were lower than those in the control group.The expression of Smurfl protein was higher than that in the control group.The results expression levels of protein stripe gray scale scanning showed that the relative gray values of BMP-4,Smadl,Smad5,Smad8, Runx-2,OB and Osterix in the observation group were lower than those in the observation group,the relative gray value of Smurfl protein was higher than tha

关 键 词：尼古丁暴露 子代乳鼠 成骨细胞 BMP-4/Smad信号通路 

分 类 号：R726.8[医药卫生—儿科]