CAJ | 학술논문

目的探讨Qtracker体外标记兔成骨诱导分化后细胞的特点及可行性.方法抽取3个月龄健康新西兰大白兔骨髓,贴壁培养骨髓基质干细胞(BMSCs),传至第3代后向成骨细胞诱导,并做鉴定.Qtracker分别以1、2、4、8、16和32 nmol/10~6细胞的浓度标记成骨诱导分化后细胞,分别记为A、B、C、D、E、F组;未标记的细胞作为空白对照组(G组).分别利用荧光显微镜计数和流式细胞术两种方法枪测标记阳性率,台盼蓝拒染法检测标记后细胞存活率,甲基噻唑基四唑(MTT)法观察Qtracker染料对细胞增殖的影响.结果兔BMSCs经诱导后能向成骨细胞诱导分化.经Qtracker标记后,荧光显微镜下胞浆呈绿色荧光.随着标记浓度的增加,A、B、C、D、E、F组细胞标记阳性率逐渐增高,于8 nmol/10~6细胞的浓度标记时,在荧光显微镜下计数,标记率可达到(93.58±2.08)%;通过流式细胞仪检测,其标记率为(95.24±1.31)%,经两种方法测定,D、E、F组间标记率差异均无统计学意义(P>0.05),且与其他各组比较差异均有统计学意义(P<0.05);G组各时间点标记阳性率均为0.以不同浓度标记后各组细胞存活率均在96%以上,且各组之间差异无统计学意义(P>0.05).标记Qtracker后对细胞的增殖无影响(P>0.05).结论 Qtracker可用于兔成骨诱导分化后细胞的体外标记,在浓度为8 nmol/10~6细胞下可得到最佳的标记率,且其对成骨诱导分化后细胞的增殖无明显影响.
목적 탐토Qtracker체외표기토성골유도분화후세포적특점급가행성.방법 추취3개월령건강신서란대백토골수,첩벽배양골수기질간세포(BMSCs),전지제3대후향성골세포유도,병주감정.Qtracker분별이1、2、4、8、16화32 nmol/10~6세포적농도표기성골유도분화후세포,분별기위A、B、C、D、E、F조;미표기적세포작위공백대조조(G조).분별이용형광현미경계수화류식세포술량충방법창측표기양성솔,태반람거염법검측표기후세포존활솔,갑기새서기사서(MTT)법관찰Qtracker염료대세포증식적영향.결과 토BMSCs경유도후능향성골세포유도분화.경Qtracker표기후,형광현미경하포장정록색형광.수착표기농도적증가,A、B、C、D、E、F조세포표기양성솔축점증고,우8 nmol/10~6세포적농도표기시,재형광현미경하계수,표기솔가체도(93.58±2.08)%;통과류식세포의검측,기표기솔위(95.24±1.31)%,경량충방법측정,D、E、F조간표기솔차이균무통계학의의(P>0.05),차여기타각조비교차이균유통계학의의(P<0.05);G조각시간점표기양성솔균위0.이불동농도표기후각조세포존활솔균재96%이상,차각조지간차이무통계학의의(P>0.05).표기Qtracker후대세포적증식무영향(P>0.05).결론 Qtracker가용우토성골유도분화후세포적체외표기,재농도위8 nmol/10~6세포하가득도최가적표기솔,차기대성골유도분화후세포적증식무명현영향.
Objective To explore the feasibility of labeling in vitro rabbit osteoblasts with Qtracker and the features of Qtracker-labeled rabbit osteoblasts. Methods A healthy male rabbit, 3 months old, weighing 2 kg, was used in this study. After bone marrow was aspirated, bone marrow stromal cells (BMSCs) were isolated and cultured using the adherence method in vitro. The third passage of BMSCs was induced into osteablasts before incubation with Qtracker at concentrations of 1, 2, 4, 8, 16, 32 nmol/10~6 cells (Groups A, B, C, D, E, F respectively). Cells not labeled by Qtracker served as negative control (Group G). The following parameters were measured: induction, differentiation and determination of rabbit osteoblasts; the optimal mass concentration of Qtracker labeling by fluorescence microscopy and flow cytometry; the cell sur-vival rates at various concentrations of Qtraeker labeling by trypan-blue exclusion; Qtracker-labeled cell pro-liferation by MTr. Results The primary and the passage rabbit BMSCs were chiefly of fusiform shape. Rabbit BMSCs differentiated into osteoblasts following induction. The osteoblasts cytoplasm showed green fluorescence under fluorescence microscopy after being labeled by Qtracker. The mean labeling rate increased with the increased concentration of Qtracker, reaching up to (93.58±2.08) % after incubation at 8 nmol/ 10~6 cells by fluorescence microscopy, and (95.24±1.31) % by flow cytometry. There were no significant differences between Groups D, E, F(P>0.05), but significant differences were found between Groups A, B, C and Groups D, E, F (P<0.05). The labeling rate for Group G was 0. The cell survival rates were all above 96% (P>0.05) . No significant differences were found in the cell proliferation among various con-centrations (P>0.05). Conclusions Qtraeker can be used as a labeling marker for rabbit osteoblasts. When the concentration is at 8 nmol/10~6 cells, optimal labeling effect can be achieved. Rabbit osteoblasts labeled with Qtracker are of high efficiency and safety.