桑寄生水煎液对小鼠胚胎肢芽生长发育及Tbx2和BMP-2基因表达影响

发布时间：2018-03-01 17:17

本文关键词： 桑寄生水煎液肢芽 Tbx BMP-　出处：《毒理学杂志》2017年02期 　论文类型：期刊论文

【摘要】：目的探讨桑寄生水煎液对小鼠胚胎肢芽生长发育及T-框蛋白2(Tbx2)和骨形成蛋白2(BMP-2)mRNA和蛋白表达的影响,评价桑寄生的胚胎发育毒性。方法利用超高高效液相色谱仪鉴定药材桑寄生,常规法制成水提液;取E13(embryos on day 13)前肢芽随机分为低、中、高剂量组和空白对照组,分别加入0.5、1和2 mg/ml不同浓度的桑寄生水煎液和双蒸水悬浮培养,72 h后收获肢芽,测量肢芽长度后,阿利新蓝染色用Neubert评分系统进行评分以评价肢芽软骨的发育情况;采用实时荧光定量PCR和免疫组化法分别检测肢芽中Tbx2和BMP-2 mRNA和蛋白的表达水平。结果 (1)经UPLC分析,槲皮苷峰面积比为38.45%,可鉴定所用药材为桑寄生。(2)肢芽长度测量:低、中、高剂量组和空白对照组的长度分别为1091.18±94.32、1107.07±80.29、1118.76±97.49和1080±89.54μm,各剂量组与空白对照组比较差异无统计学意义(P0.05)。(3)Neubert评分:低、中、高剂量组和空白对照组总评分分别为223.24±28.32、208.24±28.32、213.30±30.59和201.7±28.94。高剂量组评分最高,与空白对照组比较差异有统计学意义(P0.05),低和中剂量组与空白对照组比较差异无统计学意义(P0.05)。(4)实时荧光定量PCR和免疫组化结果显示:各剂量桑寄生水煎液均上调了Tbx2和BMP-2 mRNA和蛋白的表达水平,其中高剂量组与空白对照组比较差异有统计学意义(P0.05),而低和中剂量与空白对照组比较差异无统计学意义(P0.05)。结论各剂量桑寄生水煎液对小鼠胚胎肢芽的生长发育未体现出胚胎发育毒性,高剂量可诱导Tbx2和BMP-2 mRNA和蛋白表达促进肢芽软骨的发育。
[Abstract]:Objective to investigate the effects of water extract of Mori parasiticus on the growth and development of mouse embryonic limb buds and the expression of T- box protein 2 (Tbx2) and bone morphogenetic protein 2BMP 2 (BMP-2) mRNA and protein, and to evaluate the embryonic developmental toxicity of Mori parasiticus. Methods the parasitism of Morus mulberry was identified by ultrahigh performance liquid chromatography (UHP-HPLC). The forelimb buds of E13 embryo on day 13 were randomly divided into low, middle and high dose groups and blank control group. The limb buds were harvested after 72 h suspension culture with different concentrations of 0. 5 and 2 mg/ml water solution and double distilled water, respectively. After the length of limb bud was measured, Alisin blue staining was used to evaluate the development of limb bud cartilage by Neubert scoring system. The expression levels of Tbx2 and BMP-2 mRNA and protein in limb buds were detected by real-time fluorescence quantitative PCR and immunohistochemistry respectively. Results: by UPLC analysis, the peak area ratio of quercetin was 38.45, which could be identified as mulberry parasite. The length of the high dose group and the blank control group were 1091.18 卤94.32n 1107.07 卤80.29v 1118.76 卤97.49 and 1080 卤89.54 渭 m, respectively. There was no significant difference between the high dose group and the blank control group in the score of Neubert: the total score of low, middle, high dose and blank control group was 223.24 卤28.32n 208.24 卤28.32n 213.30 卤30.59 and 201.7 卤28.94.The total score of the high dose group was the highest. Compared with the blank control group, the difference was statistically significant (P 0.05), but there was no significant difference between the low and middle dose groups and the blank control group (P 0.05). The results of real-time fluorescence quantitative PCR and immunohistochemistry showed that all the doses of the decoction were upregulated. The expression level of Tbx2 and BMP-2 mRNA and protein, There was significant difference between the high dose group and the blank control group, but there was no significant difference between the low and middle dose groups and the blank control group. Conclusion there is no significant difference in the growth and development of embryonic limb bud of mice treated with different doses of mulberry parasitic decoction. The toxicity of embryonic development, High dose could induce the expression of Tbx2 and BMP-2 mRNA and protein to promote the development of limb bud cartilage.
【作者单位】：遵义医学院珠海校区生物工程系;华南师范大学生命科学学院;香洲区人民医院妇产科;
【基金】：贵州省中医药管理局中医药、民族医药科学技术研究项目(QZYY2013-98)
【分类号】：R285.5

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