摘要
背景:精原干细胞具有自我更新及多向分化潜能,并可将其体外定向诱导为特异性细胞,提示精原干细胞亦可能具有向成骨细胞转化的可能性,但有关这方面的研究尚未见报道。目的:观察小鼠精原干细胞在体外成骨细胞培养条件下的生物学特征变化。方法:取生后15-20 d小鼠胫骨骨髓,分离、培养骨髓基质细胞,5 d后丝裂霉素C处理制备成饲养层;取生后七至八天小鼠睾丸,经酶消化后制成单细胞悬液并接种于饲养层上。3 d后实验组和对照组分别用条件培养液和基本培养液进行诱导培养,通过倒置相差显微镜观察培养细胞生长状况及形态变化,并结合碱性磷酸酶染色、Ⅰ型胶原免疫荧光染色等方法进行结果检测。结果与结论:实验组精原干细胞在条件培养液中较对照组细胞贴壁生长快,条件培养3-6 d后见细胞生长迅速,呈集落样增长,细胞立体感增强,细胞团、簇的大小继续增加,细胞外基质增多,但未见明显细胞间桥。培养15 d后,对两组细胞进行碱性磷酸酶染色可见胞质、胞膜染为黑色,呈阳性。在荧光显微镜下可见实验组细胞浆内出现绿色荧光,呈阳性荧光反应;对照组细胞呈阴性反应,说明实验组细胞Ⅰ型胶原染色阳性,这与成骨细胞的生物学特性相似。提示精原干细胞在一定的诱导条件下具有成骨能力,有望为骨组织工程学研究提供种子细胞。
BACKGROUND：Spermatogonial stem cells are a kind of adult stem cells, which have self-renewal and differentiation potential, and can be differentiated into specific cells in vitro, suggesting that the spermatogonial stem cells may be possibly differentiated into osteoblasts. But the related research has not been reported. OBJECTIVE：To observe the biological characterization and osteogenic process of mouse spermatogonial stem cells cultured in vitro. METHODS：Spermatogonial stem cells were obtained from the testicle of mice aged 15-20 days, and were cultured on the feeder layer from bone marrow stroma cells in vitro. When cultured for 3 days, the cells were cultured in the conditioned medium （experimental group） and basic medium （control group）. The cells proliferation capability and osteogenic property were examined by phase-contrast microscope, alkaline phosphatase activity and type I col agen immunofluorescence staining. RESULTS AND CONCLUSION：Spermatogonial stem cells proliferated faster in the experiment group than in the control group. cells grew rapidly in colony-like shape in the conditioned medium at 3-6 days, the three-dimensional feeling enhanced, cellmass and clusters continued to increase in size, the extracellular matrix was increased in number and the cytoplasmic bridge was not obvious. After culture for 15 days, cells in the two groups were positive for alkaline phosphatase staining that the cytoplasmic membrane was dyed black. Under the fluorescent microscope, green fluorescence was visible in the experimental group, suggesting the cells in the experimental group was positive for type I col agen, but negative in the control group, which is similar with the biological characteristics of osteoblasts. These findings indicate that spermatogonial stem cells possess the osteogenic capability under induction conditions, which are expected to provide seed cells for bone tissue engineering.
出处
《中国组织工程研究》
CAS
CSCD
2014年第23期3702-3706,共5页
Chinese Journal of Tissue Engineering Research
基金
江西省吉安市2013年度科技计划项目([2013]18)
课题名称:小鼠精原干细胞体外培养及分化的研究~~
作者简介
胡红梅，女，1976年生，江西省抚州市人，汉族，2007年泸州医学院毕业，硕士，讲师，主要从事精原干细胞的分化研究。
通讯作者：李伟，硕士，副教授，井冈山大学临床医学院口腔系，江西省吉安市343000
