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

Effects of taxilli herba decoction on the development in mice embryonic limb bud and expressions of Tbx2 and BMP-2

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 explore the effects of Taxillus Herba decoction on the development in mice embryonic limb buds and expressions of T-box protein 2 and bone morphogenetic protein-2 mRNA and protein in order to evaluate the embryotoxicity of Taxillus Herba. Methods The quality of Taxillus Herba was evaluated by UPLC, then made to decoction by usual method. Limb buds were gained from mice embryos on day 13 and divided to low, middle, high and blank control groups randomly, then added 0. 5,1,2 mg/ml Taxillus Herba decoction and ddH2O to culture respectively. Harvesting the limb buds after 72 h, their lengths were measured. Using Alcian Blue to stain the limb buds, the development of cartilage was evaluated by Neubert score system. The expressions of Tbx2 and BMP-2 mRNA and protein were tested by real-time fluorescent quantitative PCR and immunohistochemistry respectively. Results ① The peak area ratio of quercitin is 38. 45 percent by UPLC analysis, and the herbal can be identified as Taxillus Herba. ② The lengths of limb buds of low, middle, high and blank control group are 1091.18 ± 94. 32,1107. 07 ± 80. 29, 1118. 76 ± 97. 49 and 1080 ± 89. 54/xm respectively. The different doses of Taxillus Herba groups have no significant difference compared with blank control group（ P 〉 0. 05 ）. ③ The Neubert scores of low, middle, high and blank control group are 223.24 ± 28.32,208.24 ± 28.32,213.30 ± 30. 59 and 201.7 ±28.94 respectively. The Neubert score of high group is the highest of all, and has significant difference compared with blank control group（ P 〈 0. 05 ）. The low and middle group have no significant difference compared with blank control group （ P 〉 0. 05 ）. ④ The results of real-time fluorescent quantitative PCR and immunohistochemistry manifest that each dose of Taxillus Herba group increases the expressions of Tbx2 and BMP-2 mRNA and protein. Compared with blank control group, the high group has significant difference（ P 〈0± 05 ） , but the low and middle groups have no significant difference （P 〉 0. 05）. Conclusion The different doses of Taxillus Herba have no evident embryotoxicity on the development of the limb bud. The high dose of Taxillus Herba decoction can induce the expressions of Tbx2 and BMP-2 mRNA and protein to promote the development of the cartilage.