四物合剂干预顺铂作用卵巢颗粒细胞E_2分泌水平的实验研究

Experimental study of Siwu mixture intervening the secretion level of E_2 in ovarian granulose cell induced by cisplatin chemotherapy

目的探讨四物合剂对顺铂损伤后颗粒细胞分泌E2水平的影响。方法取10只22~24天SD雌性大鼠卵巢颗粒细胞培养,另取48只22~24天SD雌鼠随机分为生理对照组、模型组、阳性组、四物合剂大、中、小剂量组,每组8只,以生理盐水(生理对照组与模型组,0.5 mL/天),戊酸雌二醇片阳性组(0.4 mg/kg·d),四物合剂大剂量组(13.5 mL/kg·d)、中剂量组(6.75 mL/kg·d)、小剂量组(3.38 mL/kg·d)分别灌胃3天后取药理血清。颗粒细胞用含20%药理血清的细胞培养液分别培养24小时后检测指标。MTT法观察药理血清对顺铂损伤后原代颗粒细胞增殖的影响;放射免疫法观测四物合剂药理血清对顺铂作用颗粒细胞E_2分泌的干预效果;免疫组化法检测四物合剂药理血清对顺铂作用颗粒细胞CYP19a1基因表达和Smad2/3激活的干预效果。结果 MTT法检测结果表明:与模型组相比,阳性组、四物合剂各剂量组细胞吸光度均增强,四物合剂大剂量数值最高(P<0.01);放免法检测结果表明,四物合剂药理血清对E2的分泌有促进作用,且大剂量效果最明显(P<0.05);免疫组化法结果表明,四物合剂药理血清可以提高CYP19a1基因、Smad2/3蛋白、Psmad2/3蛋白的表达,其中大剂量作用明显(P<0.01)。结论四物合剂药理血清对顺铂作用后的颗粒细胞E_2的分泌水平有改善作用,但可能还不能起到完全修复的作用,还需进一步的实验观察。

Objective To explore the effect of Siwu mixture on secretion level of E2 in ovarian granulose cell induced by cisplatin chemotherapy. Methods Ovarian granulosa cells from ten SD female rat of 22 to 24 clays were taken to culture cells. 48 healthy SD female rats from 22 to 24 days were randomly divided into 6 groups： normal group, model group, positive medicine group, and the high ,medium and low dose of Siwu mixture group, 8 rats in each group. Rats were given corresponding drug by garage for 3 clays ： Physiological saline （ normal group and model group, 0. 5 mL/d ） , Estradiol valerate tablets （ positive medicine group, 0.4 mg/kg · d）, Siwu mixture high group（ 13.5 mL/kg · d）, Siwu mixture medium group（6.75 mL/kg · d）, Siwu mixture low group（3.38 mL/kg · d）. The ovarian granulosa cells were cultured by 20% pharmacological serum for 24 hours. MTT method was used to observe the effect of pharmacological serum on proliferation of primary granulosa cells after eisplatin injury. RIA was used to observe the intervention effect of pharmacological serum on E2 secretion. Mmunohistochemistry was used to detect intervention effect of serum pharmacological on CYP19al gene expression and activation of Smad2/3. Results MTT assay results showed that absorbance of positive medicine group and Siwu mixture groups were higher than the normal group, and the Siwu mixture high dose group was the highest（P〈0. O1 ） , RIA method results showed that Siwu mixture had a auxo-aetion of E2 , and high dose group was the most obvious （ P〈0.05 ）. Immunohistochemical method results showed that Siwu mixture could improve the expression of CYP19al gene and Smad2/3 and Psmad2/3 protein, the effect of high dose group was the most obvious （P〈0. 01 ,P〈0. 01 ,P〈0. 01 ）. Conehlsion Siwu mixture has the effect of improvement on the secretion of E2. But it may not be able to play the role of completely repair, and need for further experimental obse vation.