养精胶囊通过PI3K-Akt-Cyclin D1通路促进小鼠精原干细胞的增殖  

Yangjing Capsule extract promotes proliferation of spermatogonia stem cells via PI3K-Akt-Cyclin D1 pathway

作　　者：蔡滨[1] 金保方[1] 孙大林[1] 邓伟民[1] Cai Bin;Jin Baofang;Sun Dalin;Deng Weimin(Andrology Department of Integrative Medicine,Zhongda Hospital,Southeast University,Nanjing 210009,China)

机构地区：[1]东南大学附属中大医院中西医结合男科,江苏南京210009

出　　处：《中国男科学杂志》2020年第6期24-29,共6页Chinese Journal of Andrology

基　　金：国家自然科学基金(No.81603623,No.81874472)。

摘　　要：目的研究养精胶囊促进小鼠精原干细胞(SSCs)增殖的分子机制。方法提取4~6 d雄性BALB/c小鼠睾丸中的SSCs,纯化并传代获得稳定的细胞。将不同浓度的养精胶囊水提物加入SSCs中,CCK-8检测细胞的增殖活性,流式细胞仪检测细胞周期,q RT-PCR以及Western blot检测Cyclin D各个亚型的表达。之后进行阻断实验,在加入养精胶囊前预先加入si RNA-Cyclin D1或者LY294002(PI3K阻断剂),同样的方法检测细胞的增殖活性和细胞周期,以及p-Akt/Akt、Cyclin D1的表达情况。结果0.01、0.1、1 mg/m L浓度的养精胶囊在24、36、48 h这三个时间点可以促进SSCs的增殖,1 mg/m L养精胶囊作用细胞48 h后,S期细胞比例升高22.7%,有统计学差异。1 mg/m L养精胶囊可以促进Cyclin D1的表达,而对Cyclin D2、Cyclin D3的表达无影响。阻断实验表明,预先加入si RNA-Cyclin D1或者LY294002可以阻断PI3K-Akt-Cyclin D1通路,降低SSCs的增殖活性,减少S期细胞比例,抑制p-Akt/Akt、Cyclin D1的表达。结论补肾填精中药养精胶囊可以通过PI3K-Akt通路作用于Cyclin D1,提高S期细胞比例,促进SSCs增殖。Objective To investigate the molecular mechanism of Yangjing Capsule(YC)promoting the proliferation of mouse spermatogonia stem cells(SSCs).Methods SSCs were extracted from testes of male BALB/c mice at 4~6 d,purified and passaged to obtain stable cells.YC extracts with different concentrations were used to treat SSCs,and the proliferation activity of cells was measured by CCK-8,cell cycle was detected by flow cytometry,and the expression of Cyclin D subtype was analyzed by q RT-PCR and Western blot.Then the blocking experiments were respectively carried out using si RNA-Cyclin D1 or LY294002(PI3 K blocker)before the treatment of YC,and the proliferation activity and cell cycle of cells,as well as the expression of p-Akt/Akt and Cyclin D1 were detected by the same methods.Results YC of 0.01,0.1 and1 mg/m L all promoted the proliferation of SSCs at the time points of 24,36 and 48 h.The proportion of S-stage cells increased by 22.7%after 1 mg/m L YC intervention for 48 h,showing a statistical difference.YC of 1 mg/m L promoted the expression of Cyclin D1 but had no effect on Cyclin D2 and Cyclin D3 expressions.Blocking experiments showed that pre-treatment of si RNA-Cyclin D1 or LY294002 blocked the PI3 K-Akt-Cyclin D1 pathway,reduced the proliferation activity of SSCs,decreased the proportion of S-phase cells,and inhibited the expressions of p-Akt/Akt and Cyclin D1.Conclusion Nourishing kidney-essence drug YC can target on Cyclin D1 through the PI3 K-Akt pathway,improve the proportion of S phase cells and promote the proliferation of SSCs.

关 键 词：养精胶囊 精原干细胞 细胞周期蛋白D1 细胞增殖 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R349.53[医药卫生—基础医学]