CAJ | 학술논문

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。3d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3-6d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。
배경：정원간세포구유자아경신급다향분화잠능，병가장기체외정향유도위특이성세포，제시정원간세포역가능구유향성골세포전화적가능성，단유관저방면적연구상미견보도。목적：관찰소서정원간세포재체외성골세포배양조건하적생물학특정변화。방법：취생후15-20 d소서경골골수，분리、배양골수기질세포，5 d후사렬매소C처리제비성사양층；취생후칠지팔천소서고환，경매소화후제성단세포현액병접충우사양층상。3d후실험조화대조조분별용조건배양액화기본배양액진행유도배양，통과도치상차현미경관찰배양세포생장상황급형태변화，병결합감성린산매염색、Ⅰ형효원면역형광염색등방법진행결과검측。결과여결론：실험조정원간세포재조건배양액중교대조조세포첩벽생장쾌，조건배양3-6d후견세포생장신속，정집락양증장，세포입체감증강，세포단、족적대소계속증가，세포외기질증다，단미견명현세포간교。배양15 d후，대량조세포진행감성린산매염색가견포질、포막염위흑색，정양성。재형광현미경하가견실험조세포장내출현록색형광，정양성형광반응；대조조세포정음성반응，설명실험조세포Ⅰ형효원염색양성，저여성골세포적생물학특성상사。제시정원간세포재일정적유도조건하구유성골능력，유망위골조직공정학연구제공충자세포。
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.