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目的观察补肾调经方对小鼠体外培养窦前卵泡成活数及卵母细胞骨形成蛋白Ⅱ型受体(bone morphogenetic protein receptorⅡ,BMPRⅡ)/激活素受体样激酶6-Smads(activin receptor-like kinase6-drosophila mothers against decapentaplegic proteins,ALK6-Smads)信号通路的影响,探讨其提高体外生长卵泡卵母细胞质量的作用机制。方法 65只12日龄健康雌性昆明小鼠,采用机械法分离其窦前卵泡,分别以正常大鼠血清、高、中、低剂量补肾调经方大鼠含药血清、高剂量补肾调经方大鼠含药血清+K02288孵育,分为对照组、补肾高、中、低剂量组(即补肾组)、抑制剂组,普通法体外培养。培养第6天比较补肾组与对照组窦前卵泡成活数,实时荧光定量PCR法检测卵母细胞中BMPRⅡ、ALK6、Smad1、Smad5、Smad8mRNA表达;细胞免疫荧光法检测上述指标和磷酸化的Smad1/5/8(phospho-Smad1/5/8,p-Smad1/5/8)蛋白表达水平。结果与对照组比较,补肾高剂量组窦前卵泡成活数增加,补肾各剂量组BMPRⅡ、ALK6、Smad5、Smad8 mRNA表达水平升高,上述指标及p-Smad1/5/8蛋白表达水平升高,补肾高、中剂量组Smad1 mRNA及其蛋白表达水平升高(P<0.05)。与补肾高剂量组比较,抑制剂组p-Smad1/5/8蛋白表达降低(P<0.05)。结论补肾调经方能增加体外培养窦前卵泡成活数,提高卵母细胞质量,其作用机制可能与调控卵母细胞中BMPRⅡ/ALK6-Smads信号通路有关。
Objective To observe the effect of Bushen Tiaojing Recipe on the survival of preantral ovarian follicles and the expression of bone morphogenetic protein receptor Ⅱ (BMPR Ⅱ) / activin receptor-like kinase 6 -drosophila mothers against decapentaplegic proteins, ALK6-Smads) signaling pathways to explore its mechanism of enhancing the quality of oocyte in vitro growth of follicles. Methods Sixty healthy female Kunming mice of 12 days old were used to separate their preantral follicles by mechanical method. The serum of rats, high, middle and low doses of Bushen Tiaogan Prescription, Drug-containing serum + K02288 incubated, divided into control group, kidney high, medium and low dose group (ie, kidney group), inhibitor group, common law in vitro culture. On the 6th day, the survival of preantral follicles in the kidney-invigorating group and the control group were compared. The mRNA expression of BMPRⅡ, ALK6, Smad1, Smad5 and Smad8 in the oocytes was detected by real-time fluorescence quantitative PCR. The above indexes and phosphorylated Smad1 / 5/8 (phospho-Smad1 / 5/8, p-Smad1 / 5/8) protein expression levels. Results Compared with the control group, the survival rate of preantral ovarian follicles increased in the high-dose tonifying kidney group, and the mRNA expression of BMPRⅡ, ALK6, Smad5 and Smad8 increased in each dose group. The above indexes and the expression of p-Smad1 / 5 / The levels of Smad1 mRNA and protein in Bushen high and middle dose groups increased (P <0.05). Compared with the high-dose kidney-tonifying group, the expression of p-Smad1 / 5/8 protein in the inhibitor group decreased (P <0.05). Conclusion Bushen Tiaojing Recipe can increase the survival number of preantral ovarian follicles cultured in vitro and improve the quality of oocyte, which may be related to the regulation of BMPRⅡ / ALK6-Smads signaling pathway in oocytes.