摘要
探索HIV P55-gag蛋白对骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)老化的影响,进一步阐明HIV P55-gag蛋白抑制BMSC成骨分化的机理。原代分离、培养、纯化BMSC,再将其分两组进行处理,一组为常规培养对照组(BMSC_(con)),另一组为添加HIV P55-gag蛋白培养组(BMSC_(gag));通过计算培养5、10、15及20天的BMSC_(con)和BMSC_(gag)的群体倍增水平,比较增殖能力;将培养20天的BMSC_(con)和BMSC_(gag)分别标记Brdu,免疫荧光法检测Brdu掺入率,比较两组细胞的增殖水平;通过检测细胞衰老生物标记β-半乳糖苷酶,比较两组细胞的衰老程度;Western检测两组细胞衰老相关蛋白P21的表达强度;将两组细胞诱导成骨分化,比较其成骨分化能力。在培养15天和20天时,BMSC_(gag)群体倍增水平明显低于BMSC_(con);BMSC_(gag)的Brdu掺入率明显低于BMSC_(con),分别为(26±4.1)%和(45±3.7)%;β-半乳糖苷酶染色检测显示,BMSC_(gag)的衰老细胞数明显多于BMSC_(con),分别为(21±4.7)%和(8±2.1)%;BMSC_(gag)的P21的表达强度明显强于BMSC_(con);成骨诱导后,茜素红染色和ALP染色均显示,BMSC_(gag)的成骨分化能力明显弱于BMSC_(con)。HIV P55-gag蛋白促进BMSC老化,进而抑制其成骨分化能力。
To explore the influence of HIV P55-gag protein on senescence of bone marrow mesenchymal stem cells, further clarify the mechanism of BMSC osteogenetic differentiation inhibited by HIV P55-gag protein.The BMSC were primary isolated, cultured and purified, then they were divided into two groups： one group was conventionally cultured （BMSCcon） and another was cultured with medium extra added HIV P55-gag protein （BMSCgag）.When the BMSCcon and BMSCgag were culture 5, 10, 15 and 20 d, the population doubling level（PDL） were calculated.BMSCcon and BMSCgag cultured 20 d were marked Brdu, then the Brdu incorporation rates were detected with immunofluorescence.The cellular senescence biomarker β-galactosidase of two group cells were detected.The expression intensity of senescence related protein P21 were detected with Western blot.Finally, the two group cells were induced to osteogenesis differentiation.At 15 d and 20 d, the PDL of BMSCgag were significantly lower than that of BMSCcon.The Brdu incorporation rates of BMSCgag were significantly lower than that of BMSCcon, （26±4.1）% and （45±3.7）% respectively.Aging cells of BMSCgag were significantly more than that of BMSCcon,（21±4.7）% and （8±2.1）% respectively.The P21 protein expression intensity of BMSCgag was significantly stronger than that of BMSCcon.Both the Alizarin red and ALP staining showed that osteogenetic differentiation ability of BMSCgag was significantly weaker than that of BMSCcon.The HIV protein P55-gag promotes human bone marrow mesenchymal stem cell senescence and then inhibits osteoblastic differentiation.
出处
《生物医学工程研究》
北大核心
2017年第1期52-56,61,共6页
Journal Of Biomedical Engineering Research
基金
国家科技部重大专项资助项目(2013ZX10001004-002-005)
关键词
HIV
P55-gag蛋白
骨髓间充质干细胞
老化
成骨分化
骨代谢
HIV
P55-gag protein
Bone marrow mesenchymal stem cell
Senescence
Osteogenetic differentiation
Bone metabolism
