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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(P>0.05)。(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(P<0.05),低和中剂量组与空白对照组比较差异无统计学意义(P>0.05)。(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(P<0.05),而低和中剂量与空白对照组比较差异无统计学意义(P>0.05)。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。
Objective To investigate the effects of Sangsheng decoction on the growth and development of mouse embryonic limb buds and the mRNA and protein expression of Tbx2 and BMP-2 in mouse embryos. Methods The supernatant was identified by HPLC. The forelimb buds of E13 (embryos on day 13) were randomly divided into low, medium and high dose groups and blank control group, and 0.5, 1 and 2 mg / ml different concentrations of Sanguineae Decoction and double distilled water suspension culture, limb buds were harvested 72 hours later, the limb bud length was measured, Alizarin blue staining with Neubert scoring system for evaluation of limb bud cartilage The mRNA and protein expression of Tbx2 and BMP-2 in limb buds were detected by real-time fluorescence quantitative PCR and immunohistochemistry respectively. Results (1) UPLC analysis, quercitrin peak area ratio of 38.45%, can be identified by the medicinal material for parasiticus. (2) Measurement of limb bud length: The length of the low, medium and high dose groups and the blank control group were 1091.18 ± 94.32,1107.07 ± 80.29,1118.76 ± 97.49 and 1080 ± 89.54μm, the difference between each dose group and the blank control group was no Statistical significance (P> 0.05). (3) Neubert score: The total score of low, medium and high dose group and blank control group were 223.24 ± 28.32,208.24 ± 28.32, 213.30 ± 30.59 and 201.7 ± 28.94 respectively. The high dose group had the highest score, compared with the blank control group, the difference was statistically significant (P <0.05). There was no significant difference between the low and medium dose groups and the blank control group (P> 0.05). (4) Real-time fluorescence quantitative PCR and immunohistochemistry results showed that each dose of Sanguine decoction increased Tbx2 and BMP-2 mRNA and protein expression levels, of which the high-dose group and the blank control group, the difference was statistically significant (P <0.05), while there was no significant difference between low and middle dose groups and blank control group (P> 0.05). Conclusions Sangsheng decoction does not show embryonic developmental toxicity on mouse embryonic limb buds. High doses of Tbx2 and BMP-2 mRNA and protein can promote the development of limb bud cartilage.