食蟹猴精原干细胞分离纯化及鉴定

doi:10.3877/cma.j.jssn.2095-5820.2015.04.010

中华临床实验室管理电子杂志 ›› 2015, Vol. 03 ›› Issue (04) : 234 -239. doi: 10.3877/cma.j.jssn.2095-5820.2015.04.010

所属专题：文献；

实验研究

食蟹猴精原干细胞分离纯化及鉴定

陈鑫苹¹^,²^,¹^,², 许邦发²^,², 罗奋华³^,³, 张培²^,², 周红桃²^,², 蔡俊宏²^,², 吴应积³^,³, 符生苗²^,^†^,²

收稿日期:2015-06-30 出版日期:2015-11-28
通信作者: 符生苗
基金资助:
海南省重大科技资助项目(ZDZX2013003); 海南省自然科学基金资助项目(310120)

Isolation, purification and identification of spermatogonial stem cells of Macaca fascicularis

Xinping1,2 Chen¹^,²^,¹^,², Bangfa Xu²^,², Fenhua Luo³^,³, Pei Zhang²^,², Hongtao Zhou²^,², Junhong Cai²^,², Yingji Wu³^,³, Shengmiao Fu²^,²^,^†

Received:2015-06-30 Published:2015-11-28
Corresponding author: Shengmiao Fu
About author:
Corresponding author: Fu Shengmiao, Email: smfu2000@126.com

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目的

掌握食蟹猴精原干细胞 (spermatogonial stem cells，SSCs) 体外培养生长特性，建立食蟹猴精原干细胞分离、纯化、培养及初步鉴定的方法。

方法

经手术获取2岁14天食蟹雄猴单侧睾丸，采用三步酶消化法分离获得单细胞悬液和差异贴壁法富集精原干细胞。将细胞培养于经丝裂酶素C处理的STO细胞层上，使用添加神经胶质细胞源性的神经营养因子(GDNF)、碱性成纤维细胞生长因子(bFGF)、GDNF家族受体α (GFRa1) 三种重要生长因子的无血清培养液体外培养，20d后采用CDH1标记分子免疫荧光染色和RT-PCR对培养的食蟹猴细胞进行SSCs初步鉴定。

结果

通过差异贴壁法分离纯化富集的SSCs，接种到丝裂霉素C处理的STO饲养层细胞上，第2天开始分裂增殖。用含有生长因子的培养基培养2 d细胞形成小集落，5 d后细胞集落明显。培养20 d后，SSCs呈葡萄串状细胞簇，符合SSCs的形态特征，这些细胞经CDH1标记分子免疫荧光染色和RT-PCR均呈阳性表达。

结论

本研究成功建立食蟹猴精原干细胞的分离纯化及鉴定体系。基于STO饲养细胞的添加GDNF、bFGF和GFRa1三种生长因子的无血清培养体系可用于食蟹猴精原干细胞培养，CDH1可作为食蟹猴精原干细胞鉴定的标志物。

关键词: 食蟹猴, 精原干细胞, 分离纯化, 免疫荧光染色

Objective

To establish a system for separation, enrichment, culture, and primary identification for the spermatogonial stem cells of Macaca fascicularis (MSSCs), and to explore its in vitro growth characteristics.

Methods

One testis was surgically removed from a two years and 14 days old male Macaca fascicularis; SSCs were obtained through the three-step enzyme digestion, then enriched by culturing the suspension of cells with differential adherence method. The enriched SSCs were then co-cultured over 20 days with mitomycin C-treated STO cells in serum-free medium with adding three important growth factors of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and GDNF family receptor α1 (GFRα1). The generation of SSCs were tentatively identified with CDH1 immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR).

Results

The enriched SSCs acquired through differential adherence method began to divide and propagate one day later after seeding into mitomycin C-treated STO cells. Small clones emerged after culturing in medium containing growth, and these clones enlarged to remarkable size five days later. After 20-day culture, clusters of grape like cells appeared which were consistent with morphology features of SSCs; immunofluorescence staining and RT-PCR demonstrated that these cells express SSCs marker of CDH1.

Conclusions

A system of separating, purifying, culturing, and identifying SSCs for Macaca fascicularis has been successfully established. Serum-free culture system basing on feeder of STO cells and media with adding three growth factors of GDNF, bFGF, and GFRa1 is suitable for culturing MSSCs. CDH1 can serve as a primitive marker for identifying MSSCs.

Key words: Macaca fascicularis, Spermatogonial stem cells, Isolation and purification, Immunofluorescence staining

图1 体外培养MFSSCs 集落形态演变 (×200)

图2 MFSSCs的CDH1抗体免疫荧光染色 (×400)

图3 体外培养MFSSCs的RT-PCR

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