王春霞 岳师师 毛炎佳

[摘要] 目的 探討补肾助孕汤对体外培养大鼠卵巢颗粒细胞生长中Smad3、Smad4的表达作用，为中药复方的研究提供实验数据支撑。 方法 用生长良好的大鼠颗粒细胞，调整细胞为1×104 cells/1 mL，用24孔细胞培养板，每孔滴入少许培养基，然后放入直径24 mm的盖玻片，让培养液与玻片粘合在一起，每孔再加入细胞悬液37℃ 5%CO2进行培养，在6 h、12 h、24 h进行观察培养细胞的贴片情况，用免疫组化SP法染色镜检。 结果 补肾助孕汤各剂量组用药后Smad3、Smad4的阳性率均高于对照组，Smad3：高剂量组用药6 h、12 h、24 h分别与对照组比较，差异具有统计学意义（P<0.05），中剂量组用药6 h、12 h、24 h分别与对照组比较，差异具有统计学意义（P<0.05），低剂量组用药12 h高于对照组，差异有统计学意义（P<0.05），各剂量组用药12 h最高，其高于用药6 h、24 h，差异有统计学意义（P<0.05）;Smad4：高剂量组和中剂量组用药6 h、12 h、24 h与对照组比较，差异具有统计学意义（P<0.05），低剂量组用药12 h高于对照组，差异有统计学意义（P<0.05）。 结论 补肾助孕汤能够促进体外培养的卵巢颗粒细胞的增殖分化，通过促进TGF-β1的含量上调Smad3、Smad4的表达，从而促进卵泡生长发育。

[关键词] 补肾助孕汤;卵巢颗粒细胞;Smad3;Smad4;信号通路

[中图分类号] R329          [文献标识码] A          [文章编号] 1673-9701（2020）15-0040-04

Study of the effect of kidney tonifying and gestation promoting decoction on rat ovarian granulosa cells Smad3 and Smad4

WANG Chunxia1   YUE Shishi2   MAO Yanjia2   JIANG Yaru2   LIU Yuchun1   YAN Sha1   LI Yongwei1

1.He'nan Province Hospital of Traditional Chinese Medicine， Zhengzhou  450002， China; 2.He'nan University of Chinese Medicine， Zhengzhou   450046， China

[Abstract] Objective To explore the effect of kidney tonifying and gestation promoting decoction on the expression of Smad3 and Smad4 in the growth of rat ovarian granulosa cells through in vitro culture， so as to provide experimental data support for the research of traditional Chinese medicine compound prescription. Methods Rat granulosa cells with good growth were chosen and adjusted to 1×104 cells/1 mL. A little culture medium was dropped into each well of 24-well cell culture plates， and then a cover glass with a diameter of 24 mm was put in to be bonded to the culture solution. Cell suspension 37°C 5% CO2 was added to each well for culture. The patch of cultured cells was observed at 6 h， 12 h， 24 h， and was given immunohistochemical SP staining and microscopic examination. Results The positive rates of Smad3 and Smad4 in each dose group of kidney tonifying and gestation promoting decoction were higher than those in the control group. Smad 3： High-dose group and the control group were compared at 6 h， 12 h， and 24 h of administration， with statistically significant difference（P<0.05）. Medium-dose group and the control group were compared at 6 h， 12 h， and 24 h of administration， with statistically significant difference（P<0.05）. The positive rate in low-dose group at 12 h of administration was higher than that in the control group， with statistically significant difference（P<0.05）. In all dose groups， the positive rate at 12 h of administration was higher than that at 6 h and 24 h， with statistically significant difference（P<0.05）. Smad4： High-dose group and medium-dose group at 6 h， 12 h， and 24 h of administration， were compared with the control group， with statistically significant difference（P<0.05）. The positive rate of low-dose group at 12 h of administration was higher than that of the control group， with statistically significant difference（P<0.05）. Conclusion Kidney tonifying and gestation promoting decoction can promote the proliferation and differentiation of ovarian granulosa cells in vitro， and regulate the expression of Smad3 and Smad4 up by increasing the content of TGF-β1， so as to accelerate the growth and development of follicles.

[Key words] Kidney tonifying and gestation promoting decoction; Rat ovarian granulosa cell; Smad3; Smad4; Signal pathway

卵巢颗粒细胞是卵泡的功能单位，其增殖、分化与卵泡的生长发育密切相关[1]。而颗粒细胞主要通过本身的分化、信号通路的转导以及分泌的细胞因子发挥作用。Smads蛋白是TGF-β信号通路唯一的底物蛋白[2]。补肾助孕汤是王春霞教授在长期临床实践中的经验方，通过多年临床观察对于恢复月经周期、促进排卵具有良好的临床效果，且临床观察中未发现并发症及副作用[3]。为了解其对卵泡生长发育的作用，本文用大鼠卵巢颗粒细胞进行实验研究，观察其对Smads蛋白的变化影响并分析作用，现报道如下。

1 资料与方法

1.1 实验细胞

大鼠卵巢颗粒细胞购于武汉原生原代生物医药科技有限公司，产品编号：RAT-CELL-0077，规格：5×105 cells/1 mL。

1.2 实验用药与耗材

补肾助孕汤（菟丝子，规格：10 g/袋，批号：1905006C、当归，规格：10 g/袋，批号：1906013C、枸杞子……