哈蟆油对大鼠卵巢颗粒细胞增殖与分泌的影响  

The effects of Oviductus Ranae on the proliferation and secretion of ovarian granulosa cells in rats

作　　者：彭娟[1] 邓虹珠[1] 马丹丹[1] 魏蕾初[1] 郑堰心[1] 梁磊[2] 

机构地区：[1]南方医科大学中医药学院,广东广州510515 [2]广州军区广州总医院药剂科,广东广州510010

出　　处：《时珍国医国药》2013年第3期532-535,共4页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金青年基金(No.81001701)

摘　　要：目的建立卵巢颗粒细胞的原代培养体系,探讨哈蟆油含药血清对大鼠卵巢颗粒细胞增殖及分泌的影响,并分析和选择哈蟆油含药血清对颗粒细胞的最佳作用浓度,为进一步体外实验奠定基础。方法采用SD雌性大鼠,皮下注射孕马血清促性腺激素,剖取双侧卵巢,采用机械分离方法释放卵巢颗粒细胞,分离培养。将体积分数为15%,30%,60%,120%,240%(大鼠灌胃浓度4.5 g/kg)的含药血清5个终浓度分别加入培养体系中,通过四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞增殖情况及药物的最大无毒浓度(TD0),观察其对卵巢颗粒细胞生长的影响。采用电化学发光法测定培养基中雌二醇(E2)的含量,分析哈蟆油含药血清对颗粒细胞雌激素分泌的影响。运用流式细胞术检测活细胞及凋亡细胞所占的比率。结果含药血清作用颗粒细胞后,呈现出不同程度的促增殖作用,含药血清最佳的作用浓度体积分数为15%～30%(大鼠灌胃哈蟆油剂量为4.5 g/kg)之间;哈蟆油含药血清可促进卵巢颗粒细胞分泌雌二醇,雌二醇含量分析显示雌二醇的分泌与细胞增殖情况存在正相关关系(Personr=0.406,P=0.01);流式细胞结果显示与空白含药血清比较,哈蟆油含药血清均能显著增加活细胞数目,抑制细胞无血清饥饿引发的凋亡(P值均小于0.01),起到细胞保护作用,其中体积分数为25%的含药血清作用效果更佳(P<0.01)。结论哈蟆油含药血清对离体培养的卵巢颗粒细胞增殖具有显著的促进作用,并存在一个最佳作用浓度范围,其调节机体性激素生成作用与促进卵巢颗粒细胞生长效应相关。Objective To establish the primary cultural system of ovarian granulosa cells in rats,and explore the effects of serum containing Oviductus Ranae on the proliferation and the estrogen（E2） production of the cells,to analyze and select the best optimal concentration for the next experiments in vitro.Methods Female SD rats were subcutaneously treated with pregnant mare serum gonadotropin（PMSG）.Then they were decapitated and the ovaries were aseptically removed,Granulosa cells were then released by mechanical method.Low_speed centrifugation separation were used for granulosa cells isolation,then the cells were incubated at 37℃in water_saturated environment of 5% CO2.The volume percent of 15%,30%,60%,120%,240% serum containing Oviductus Ranae was added into the granulosa cells cultivated for 24 h,48 h,72 h.Then measured the proliferation of the cells and TD0 of Oviductus Ranae by MTT.E2 concentration of Oviductus Ranae-containing serum on the cells was detected by the electrochemical luminescence detection kit.The rate of living cells and apoptotic cells were caculated with Flow cytometry.Results The serum containing Oviductus Ranae significantly improved the proliferation and E2 secretion of granulosa cells,the best optimal concentration was between 15% and 30%（the SD rats for irrigation the dose of Oviductus Ranae 4.5g/kg）.The positive correlations were demonstrated between E2 secretions and the proliferation of granulosa cells（Person r=0.406,P=0.01）,as well as the result of the Flow cytometry,the volume percent of 25% serum containing Oviductus Ranae was significantly protected granulosa cells（P0.01）.Conclusion The serum containing Oviductus Ranae could encourage the proliferation of rat ovary granulosa cells,whose mechanism involved may lie in its regulation of steroidogenesis.

关 键 词：哈蟆油 卵巢颗粒细胞 增殖 雌二醇 含药血清 MTT法 药物毒性 

分 类 号：R285.5[医药卫生—中药学]