摘要
目的采用CCK-8法研究哈蟆油对体外培养大鼠卵巢颗粒细胞增殖的影响,对比分析哈蟆油含药血清对颗粒细胞的最佳作用浓度和时间,为进一步体外试验奠定基础。方法选用25日龄未成年雌性SD大鼠,腹腔注射孕马血清,48h后脱颈处死,收集卵巢颗粒细胞,在DMEM-F12培养液中培养,并通过HE染色和免疫荧光技术对颗粒细胞进行鉴定。25只SD大鼠分为正常对照组、阳性药对照组、哈蟆油低、中、高剂量组,灌胃给药7d后采血并分离血清。将体积分数为10%,20%,40%,80%各组不同浓度的含药血清加入体外培养的大鼠卵巢颗粒细胞体系中,采用CCK-8法检测各组24、48h和72h细胞增殖情况。结果哈蟆油显著提高了颗粒细胞的增殖能力,有一定的量效和时效关系,随着哈蟆油浓度的增加,其促增殖能力逐渐增强,在作用48h后,20%浓度体积分数的哈蟆油含药血清组最为显著(P<0.05)。结论体外建立大鼠卵巢颗粒细胞的培养方法,有助于深入研究哈蟆油等中药对卵巢的作用和机制。
Objective To optimize the effective methods of isolation,purification,culture in vitro and identification of SD rat ovarian granulosa cells,to research the effects of Oviductus Ranae on the proliferation of rat ovarian granulosa cells by CCK-8,and to contrastively analyze the best optimal action concentration and time of serum contsining Oviductus Ranae on granulosa cells to lay the foundation for further in vitro experiment.Methods Nonage SD rats aged 25 dwere selected and intraperitoneally injected by pregnant mare serum,then killed after 48 h.Ovarian granulosa cells were collected and cultured in the DMEM-F12 culture solution.The hematoxylin eosin（HE）staining and immunofluorescence technique were used to identify the ovarian granulosa cells.Twenty-five SD rats were randomly divided into the normal control group,positive medicine control group,and low,middle and high doses Oviductus Ranae groups.Blood was collected and serum was separated after 7dmediaction gavage.The volume percent of 10%,20%,40%,80% serum in each group was added into the in vitro medium system of ovarian granulosa cells culture.Then the cell proliferation situation at 24,48,72 hin each group was measured by CCK-8.Results Oviductus Ranae significantly increased the proliferation ability of granulosa cells in a certain dose-dependent relation.With the increase of Oviductus Ranae concentration concentration,its proliferation ability was gradually increased,after 48 haction,which in the Oviducthus Ranae-Comtaining serum group with the volume fraction of 20% was most significant（P〈0.05）.Conclusion Establishing in vitro cultural method of rat ovarian granulosa cells is conductive to further research the action and mechanism of Oviductus Ranae on ovary.
作者
零小妹
谭焱
季波
杨晶晶
吴新荣
梁磊
Ling Xiaomei Tan Yah Ji Bo Yang Jingjing Wu Xinrong Liang Lei(School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China Department of Pharmacy ,Guangzhou General Hospital of Guangzhou Military Region ,Guangzhou, Guangdong 510010, China First Department of Cadre Wards,Guangzhou General Hospital of Guangzhou Military Region, Guangzhou , Guangdong 51 0010, China)
出处
《重庆医学》
CAS
北大核心
2017年第13期1792-1796,共5页
Chongqing medicine
基金
广东省广州市珠江科技新星项目(2013J2200030)
广东省广州市科技计划项目(201509010012)
关键词
哈蟆油
卵巢颗粒细胞
增殖
含药血清
CCK-8
Oviductus Ranae
ovarian granulosa cells
proliferation
drug-containing serum
CCK-8
