摘要
目的探讨血小板衍生生长因子-BB(PDGF-BB)诱导兔骨髓间充质干细胞(BMSCs)的最佳干预时间及浓度,并鉴定其向成骨细胞分化的能力。方法体外分离培养新西兰大白兔BMSCs原代细胞,选用3代细胞用于PDGF-BB干预实验。将筛选好的细胞接种于96孔培养板中,分为空白组:加DMEM完全培养基;对照组:BMSCs+DMEM完全培养基;其余各组采用PDGF-BB干预浓度分别为10、25、50、100 ng/mL。分别于干预的24、72、120、168 h用CCK-8法检测每组浓度对细胞的增殖作用,筛选出PDGF-BB对BMSCs干预的最佳时间及浓度。诱导后的细胞用细胞免疫化学法鉴定骨钙蛋白(osteocalcin,OCN)、Runt相关转录因子2(Runx-2)、Ⅰ型胶原(COL-IA1),免疫荧光鉴定OCN。结果在不同的时间点PDGF-BB干预BMSCs的吸光度值改变不同,差异有统计学意义(F=441.351,P<0.01),表明不同浓度PDGF-BB干预BMSCs后,细胞增殖是不同的。25 ng/mL PDGF-BB组与其他5组比较差异均有统计学意义(P<0.01),干预72 h增殖率最高。诱导后的细胞免疫化学染色OCN骨钙素、Runx-2、COL-1均为阳性,OCN骨钙素的免疫荧光表达也是阳性,且阳性率>90%。结论25 ng/mL PDGF-BB组干预BMSCs在72 h是对细胞增殖作用最强,诱导后的细胞可以分化为成熟的成骨细胞,为下一步诱导后细胞移植治疗牵张成骨模型奠定了基础。
Objective To explore the best optimal intervention time and concentration of platelet-derived growth factor-BB(PDGF-BB)to induce rabbit bone marrow mesenchymal stem cells(BMSCs),to identify the ability to differentiate into osteoblasts.Methods Primary BMSCs of New Zealand white rabbit were isolated and cultured in vitro,and the 3rdpassage cells were selected for PDGF-BB intervention experiment.The selected cells were seeded into 96-well culture plates and divided into blank groups:complete DMEM medium;control group:BMSCs+DMEM complete medium;the remaining groups were treated with different concentration of PDGF-BB(10,25,50 and 100 ng/mL).CCK-8 method was used to detect the proliferation of cells in each group at 24,72,120 and 168 h,and the optimal time and concentration of PDGF-BB for BMSCs intervention were screened.The expression of OCN,Runx-2,and COL-1 were tested by cellular immunochemistry,and OCN was identified by immunofluorescence.Results The results showed that the absorbance value of PDGF-BB after intervention with BMSCs changed at different time points(F=441.351,P<0.01),indicating that the proliferation of cells was different after BMSCs were interfered with different concentrations of PDGF-BB.The results also showed that the difference between the 25 ng/mL PDGF-BB group and the other five groups was statistically significant(P<0.01),and the proliferation rate was the highest after 72 h of intervention.After induction,the cells were positive for OCN,Runx-2,and COL-1,and the immunofluorescence expression of OCN was also positive,and the positive rate was>90%.Conclusion It was concluded that the 25 ng/mL PDGF-BB group interfered with BMSCs demonstrated the strongest effect on the cell proliferation at 72 hours,and the induced cells could differentiate into mature osteoblasts.
作者
魏琴
买买艾力·玉山
张继秀
吴硕
马创
WEI Qin;Maimaiaili Yushan;ZhANG Jixu;WU Shuo;MA Chuang(Laboratory Animal Center of Xinjiang Medcial University,Urumqi 830011;Xinjiang Key Laboratory of Medical Animal Model Research,Urumqi 830011;Departmentof Orthopedics Center,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054;Vocational and Technical College of ChangJi of Xinjiang,Xinjiang ChangJi 831100,China)
出处
《新疆医科大学学报》
CAS
2021年第3期290-296,共7页
Journal of Xinjiang Medical University
基金
国家自然科学基金(81760397)
新疆维吾尔自治区高校科研计划(XJEDU2020Y022)。
关键词
骨髓间充质干细胞
PDGF-BB
细胞增殖
成骨细胞
bone marrow mesenchymal stem cells(BMSCs)
cell proliferation
platelet-derived growth factor-BB(PDGF-BB)
osteoblasts
